EA043050B1 - WAYS TO INCREASE GRAIN YIELD - Google Patents

Description

Field of invention

The present invention relates to methods for increasing plant yield, and in particular grain yield, by reducing the level of expression and/or activity of OTUB1 and subsequently modifying the levels of at least one transcription factor like the SQUAMOSA promoter-binding protein (SBP domain) in plants. . Also described are genetically modified plants characterized by the above phenotype, and methods for growing such plants.

Prerequisites for the creation of the invention

Rice is an important food consumed by people all over the world. Rice grain size and shape are key agronomic traits that determine grain yield and appearance. In rice, the length, width, and thickness of a grain is associated with the size and shape of the grain. Several important genes affecting grain size and shape have been characterized in rice (Fan et al., 2006, Song et al., 2007, Shomura et al., 2008, Weng et al., 2008, Che et al., 2015, Duan et al., 2015, Hu et al., 2015, Wang et al., 2015 and Si et al., 2016), but the molecular mechanisms that determine grain size and shape have not yet been precisely determined.

Several factors that affect cell proliferation determine the grain size of rice. For example, the loss of function gene GRAIN SIZE 3 (GS3) produces a long grain as a result of an increase in the number of cells (Fan et al., 2006, Mao et al., 2010). GS3 encodes the putative γ subunit of the G protein. Similarly, a putative protein phosphatase (OsPPKL1) encoded by GL3.1/qGL3 limits cell proliferation and its loss-of-function mutant produces a long grain (Hu et al., 2012, Qi et al., 2012, Zhang et al. , 2012). In contrast, the putative serine carboxypeptidase encoded by GS5 and the transcription factor OsSPL16 stimulate mainly grain growth through their effect on cell number (Li et al., 2011b, Wang et al., 2012). The OsMKK4-OsMPK6 module affects grain growth by increasing the number of cells (Duan et al., 2014, Liu et al., 2015). The cell multiplication process also plays a very important role in determining grain size. A high level of expression of the transcription factor SPL13 gives a long grain as a result of an increase in cell length (Si et al., 2016). Growth regulatory factor 4 (OsGRF4), encoded by GS2, associates with transcription coactivators (OsGIF1/2/3), which leads to an increase in grain size, predominantly through an effect on cell size (Che et al., 2015, Duan et al., 2015, Hu et al., 2015, Li et al., 2016, Sun et al., 2016). These studies suggest that transcriptional regulation plays an important role in rice grain growth. A high level of expression of GW7/GL7/SLG7, which is likely involved in microtubule organization, gives rise to a long grain as a result of an increase in cell length and/or an increase in their number (Wang et al., 2015a, Wang et al., 2015b, Zhou et al. ., 2015). Thus, the processes of cell proliferation and cell multiplication have a coordinated effect on the grain size of rice.

The ubiquitin-proteasome pathway plays an important role in the growth of rice seeds and other plant species (Li and Li, 2014, Li and Li, 2016). Ubiquitin can be attached to target proteins through ubiquitination. Deubiquitinizing enzymes (DUB) such as otubain protease, ubiquitin specific protease, ubiquitin C-terminal hydrolase, Josephins and JAMM can cleave ubiquitin from ubiquitinized proteins (Nijman et al., 2005). In rice, a protein of unknown function encoded by qSW5/GW5 is putatively involved in the ubiquitin pathway, and disruption of qSW5 results in a wide grain (Shomura et al., 2008, Weng et al., 2008). It should be noted that the previously unidentified GSE5 gene at the qSW5/GW5 locus was recently described as a grain width limiting gene (Duan et al., 2017). GSE5 encodes a plasma membrane-associated protein containing IQ domains, and the low level of GSE5 expression in some indica cultivars and in most japonica cultivars results in a wide grain (Duan et al., 2017). RING type E3 ubiquitin ligase, encoded by GW2, restricts grain growth, and its loss-of-function mutant produces wide grains (Song et al., 2007). Similarly, its DA2 homologue in Arabidopsis limits seed growth through the maternal outer coat (Xia et al., 2013). DA2 and another E3 ubiquitin ligase BB/EOD1 physically interact with the DA1 ubiquitin receptor, resulting in suppression of seed and organ growth (Li et al., 2008, Xia et al., 2013). DA1 also has peptidase activity that can cleave ubiquitin-specific protease (UBP15/SOD2) (Du et al., 2014, Dong et al., 2017). Thus, the modification of proteins by ubiquitin plays an important role in determining the size of plant seeds.

Rice is eaten by more than half of the world's population. Despite significant progress in harvesting through the use of semi-dwarf plants and the use of heterosis ^1-3 , increasing rice yields, possibly exceeding those of existing elite varieties, is a major challenge for rice growers ⁴ . To go beyond the yield limits of existing rice varieties, an idiotypic method has been proposed and this method has been applied in rice production programs ^4-7 . Since the early 1990s, specialists from the International Rice Research Institute (IRRI) have developed a number of new type rice (NPT) varieties, where the structure of these plants differs from that of conventional varieties, i.e. these plants have larger panicles, less sterile shoots, and stronger stems. Although several NPT rice cultivars have been commercialized ^6,7 , however, the genetic basis of their phonotype has only been elucidated at the level of quantitative trait loci (QTL) ⁸ . The authors found that NPT1 encodes the OTUB1 gene; otubain-like protease with deubiquity

- 1 043050 lowering activity.

Regardless of this, in order to further reveal the mechanisms for determining the size and shape of the grain, the authors identified several mutants based on the grain size of rice (Duan et al., 2014, Fang et al., 2016). The authors have characterized the mutant with a wide and thick grain 1 (wtg1-1), which gives a wide, thick, short and heavy grain. WTG1 encodes OTUB1, i.e. the same gene that is present at the NPT locus in rice. Overexpression of WTG1 results in a narrow, thin, and long grain. Thus, the present inventors have identified the WTG1 otubain-like protease as an important determinant of grain size and shape, as well as other important agronomic traits, including overall crop yield.

Therefore, it is necessary to increase the grain yield of commercially valuable crops such as rice. This problem can be solved using the present invention.

The essence of the invention

The authors unexpectedly found that the OTUB1 gene (also known and referred to as NPT1, DEP5 and WTG1, where these terms can be used interchangeably) retains a quantitative trait of grain yield. In particular, the authors unexpectedly found that inhibition or abolition of the expression or deubiquitinase activity of this protein leads to an increase in meristem activity, an increase in the number of grains per panicle, an increase in grain mass and width, and, most importantly, an increase in grain yield.

In one of its aspects, the present invention relates to a method for increasing the grain yield of a plant, which method includes reducing the expression level of at least one nucleic acid encoding an otubain-like protease (OTUB1) and/or reducing the activity of an otubain-like protease. Preferably, said increase in grain yield includes increasing at least the number of grains, the number of grains per tassel, grain weight, grain width, grain thickness, and/or thousand grain weight.

The method may include introducing, through a single mutation, at least one nucleic acid sequence encoding an OTUB1 polypeptide and/or an OTUB1 polypeptide promoter. The mutation may be a loss-of-function or partial loss-of-function mutation and/or an insertion, a deletion, and/or a substitution. Preferably, the mutation is any mutation that can reduce the level of expression or activity of OTUB1.

If this method involves the introduction of a mutation, then such a mutation can be introduced by targeted modification of the genome, and preferably ZFN, TALEN or CRISPR/Cas9. Alternatively, in some embodiments, the mutation may be introduced by mutagenesis, and preferably by TILLING or T-DNA insertion.

In other embodiments of the invention, the method may include the use of RNA interference to reduce or abolish the expression of the OTUB1 nucleic acid.

In some embodiments of the invention, the specified yield was increased compared to the yield of the wild-type plant or control plant.

In some embodiments of the invention, the mutation results in a decrease or abolition of the deubiquitinase activity of OTUB1.

The present invention also relates to a genetically modified plant, part or cell thereof, wherein said plant contains at least one mutation in at least one nucleic acid encoding an OTUB1 polypeptide and/or an OTUB1 promoter.

The plant can be characterized by reduced or absent expression of the OTUB1 polypeptide. Alternatively or additionally, said plant can be characterized by a reduction or absence of OTUB1 deubiquitinase activity.

In some embodiments of the invention, the specified plant is characterized by an increase in grain yield, preferably if the specified plant is compared with a control plant or wild type plant. Said increase in grain yield includes an increase in at least the number of grains, the number of grains per panicle, grain weight, grain width, grain thickness, thousand grain weight, and/or decrease in grain length.

In some embodiments of the invention, said mutation is a loss-of-function or partial loss-of-function mutation. Preferably said mutation is an insertion, deletion and/or substitution. Preferably, the mutation is any mutation that can reduce the level of expression or activity of OTUB1.

In some embodiments of the invention, the mutation can be introduced through targeted modification of the genome, and preferably ZFN, TALEN or CRISPR/Cas9. Alternatively, the mutation may be introduced by mutagenesis, and preferably by TILLING or T-DNA insertion.

In some embodiments, the plant includes an RNA interference construct that reduces the level of expression of an OTUB1 polypeptide. In one example, the design for RNA interference contains SEQ ID NO: 210 or consists of SEQ ID NO: 210 or its variants, as defined in this application.

The plant part is preferably a grain or seed.

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The present invention also relates to a method for growing a plant with increased grain yield, which method includes introducing at least one mutation in at least one nucleic acid sequence encoding an OTUB1 polypeptide and/or an OTUB1 polypeptide promoter. The mutation may be a loss-of-function or partial loss-of-function mutation, and preferably an insertion, deletion, and/or substitution. Preferably, the mutation is any mutation that can reduce the level of expression or activity of OTUB1.

In some embodiments of the invention, the mutation is introduced by targeted modification of the genome, and preferably ZFN, TALEN or CRISPR/Cas9. Alternatively, the mutation is introduced by mutagenesis, and preferably by TILLING or T-DNA insertion.

The present invention also relates to a method of growing a plant with increased grain yield, which method includes introducing into said plant and expressing in said plant an RNA interference construct that reduces the expression level or abolishes the expression of an OTUB1 nucleic acid. In one example, the design for RNA interference contains SEQ ID NO: 210 or consists of SEQ ID NO: 210 or its variants, as defined in this application.

The methods of the invention may also include estimating the level of increase in at least grain yield, wherein said estimating includes determining an increase in at least the number of grains, the number of grains per tassel, grain weight, grain width, grain thickness, thousand grain weight, and/or decrease in grain length. . Preferably said increase is compared to a control or wild type plant.

The methods of the invention may also include assessing a decrease or lack of expression of an OTUB1 nucleic acid and/or assessing a decrease or absence of activity, and preferably deubiquitinase activity, of an OTUB1 polypeptide.

The methods of the invention may also include plant regeneration and screening for increased grain yield.

The present invention also relates to a plant, plant part or plant cell obtained or obtained by any one or more of the methods described above.

The present invention also relates to a method for identifying and/or selecting a plant that will produce an increased grain yield, preferably compared to a wild-type plant or a control plant, which method includes detecting in the plant or in the germplasm of the plant at least one polymorphism in the OTUB1 gene and/or in the OTUB1 promoter, and selecting said plant or progeny thereof. A polymorphism can be an insertion, a deletion, and/or a substitution. The method may also include introgression of a chromosomal region containing at least one polymorphism in the OTUB1 gene and/or OTUB1 promoter into a second plant or into the germplasm of that plant to obtain an introgressed plant or plant germplasm.

In one embodiment of any of the above aspects, the OTUB1 polypeptide may include, or consist of, the amino acid sequence of SEQ ID NO: 1, or a functional homologue or variant thereof. In another embodiment, the OTUB1 nucleic acid comprises, or consists of, a nucleic acid sequence that encodes for SEQ ID NO: 1. In a preferred embodiment, the nucleic acid comprises, or consists of, a nucleic acid sequence selected from SEQ ID NO: 2-5, or a functional variant or homologue thereof. In one example, a homologue may have a nucleic acid sequence that encodes an OTUB1 polypeptide as defined in SEQ ID NOs: 14-20, or a functional variant thereof as defined herein. Preferably, the OTUB1 homologue nucleic acid sequence is selected from SEQ ID NOs: 7-13 or a functional variant thereof as defined herein.

In other embodiments of any of the aspects described above, the OTUB1 promoter nucleic acid sequence may include, or consist of, SEQ ID NO: 6, or a functional variant or homologue thereof. In one embodiment of the invention, the homologue is selected from SEQ ID NOs: 21-27 or their functional variant as defined in this application.

The plant is preferably selected from rice, wheat, corn, soybean, sorghum, cabbage and barley. In one example, the plant is rice. In another example, the plant is wheat. In another example, the plant is corn.

The present invention also relates to a nucleic acid construct comprising a nucleic acid sequence encoding at least one DNA binding domain that can bind to at least one target sequence in the OTUB1 gene and/or promoter. In one example, the DNA binding domain sequence is selected from SEQ ID NOs: 28, 34, 38, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84 , 87, 90, 93, 96, 99, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142 and 146 or a sequence that is at least 90% identical to one of SEQ ID NO : 28, 34, 38, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 106 , 110, 114, 118, 122, 126, 130, 134, 138, 142 and 146.

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In one embodiment of the invention, the nucleic acid construct contains at least one protospacer element, where the protospacer element contains a DNA-binding domain. In one embodiment, the protospacer element sequence is selected from SEQ ID NOs: 29, 35, 39, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 82, 85, 88, 91, 94, 97, 100, 103, 107, 111, 115, 119, 123, 127, 131, 135, 139, 143 and 147 or a sequence that is at least 90% identical to SEQ ID NOs: 29, 35, 39, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 82, 85, 88, 91, 94, 97, 100, 103, 107, 111, 115, 119, 123, 127, 131, 135, 139, 143 and 147.

The construct may also include a nucleic acid sequence encoding a CRISPR RNA (cr-RNA) sequence, wherein said cr-RNA sequence contains a protospacer element sequence and additional nucleotides. The construct may also include a nucleic acid sequence encoding a transactivating RNA (tracr-RNA). In one embodiment, the tracr-RNA comprises or consists of SEQ ID NO: 30 or a functional variant thereof.

In another embodiment, the nucleic acid construct comprises a nucleic acid sequence encoding at least one single-stranded guide RNA (sssrRNA), wherein said ssrRNA contains a tracr RNA sequence and a cr RNA sequence or a protospacer sequence. In one embodiment, the sssrRNA sequence contains or consists of a sequence selected from 31, 36, 40, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144 and 148, or a sequence that is at least 90% identical to 31 , 36, 40, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 108, 112 , 116, 120, 124, 128, 132, 136, 140, 144 and 148.

In another aspect, the present invention relates to a nucleic acid construct comprising at least one nucleic acid encoding an ssrRNA molecule, wherein the ssrRNA molecule binds to a target sequence selected from SEQ ID NOs: 28, 34, 38, 42, 45, 48 , 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 106, 110, 114, 118, 122, 126, 130 , 134, 138, 142 and 146 or its variant. In a preferred embodiment of the invention, the ssrRNA nucleic acid sequence is selected from 31, 36, 40, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92 , 95, 98, 101, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144 and 148 or its variant.

In one example, the nucleic acid construct sequence is selected from SEQ ID NOs: 33, 37, 41, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, and 149, or a variant thereof, wherein said variant has a sequence that is at least 75%, more preferably at least 80%, even more preferably at least 90%, and most preferably at least 95% identical to SEQ ID NOs: 33, 37, 41, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145 and 149.

In some embodiments of the invention, the construct may be operably linked to a promoter, and preferably to a constitutive promoter.

In some embodiments, the nucleic acid construct also contains a nucleic acid sequence encoding a CRISPR enzyme. In one example, the CRISPR enzyme may be a Cas protein, and preferably Cas9 or a functional variant thereof.

In an alternative embodiment of the invention, the nucleic acid construct encodes a TAL effector. In some embodiments, the nucleic acid construct comprises a nucleic acid sequence encoding at least one DNA-binding domain that can bind to at least one target sequence in the OTUB1 gene and/or promoter. In one example, the DNA binding domain sequence is selected from SEQ ID NOs: 28, 34, 38, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142 and 146 or a sequence that is at least 90% identical to one of SEQ ID NOs: 28, 34, 38, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142 and 146, and also contains a sequence encoding an endonuclease or its DNA cleavage domain. The endonuclease may be Fokl.

In another aspect, the present invention provides a single-stranded guide molecule (ssr) RNA, wherein said ssrRNA comprises a crRNA sequence and a tracrRNA sequence, wherein the ssrRNA sequence can bind to at least one sequence selected from SEQ ID NOs: 28, 34, 38, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142 and 146 or a sequence that is at least 90% identical to one of SEQ ID NOs: 28, 34, 38, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142 and 146.

The present invention also relates to an isolated plant cell transfected with at least one nucleic acid construct as defined herein; or to an isolated plant cell transfected with at least one nucleic acid construct as defined herein; and a second nucleic acid construct, where said

- 4 043050 the second nucleic acid construct contains a nucleic acid sequence encoding a Cas protein, and preferably a Cas9 protein or a functional variant thereof. The second nucleic acid construct may be transfected before, during, or after transfection of the nucleic acid construct defined herein. In another aspect, the present invention relates to an isolated plant cell transfected with an ssrRNA molecule as defined above.

The present invention also relates to a genetically modified plant, where the specified plant contains the transfected cell defined in this application. The nucleic acid encoding the ssrRNA and/or the nucleic acid encoding the Cas protein may be integrated in a stable form.

The present invention also relates to a method for increasing the grain yield of a plant, which method includes introducing into the plant and expressing in the plant the nucleic acid construct or ssrRNA molecule described in this application, where preferably this increase is considered in relation to a control plant or a wild-type plant. The present invention also relates to a plant obtained or obtained by this method.

The present invention also relates to a method for producing a genetically modified plant, as defined in this application, where this method includes:

a) selection of the plant part;

b) transfecting at least one cell of the plant part of item (a) with a nucleic acid construct or sssrRNA as defined herein;

c) regenerating at least one plant derived from the transfected cell or transfected cells;

d) selecting one or more plants obtained in accordance with paragraph (c) and having a reduced level of expression of at least one OTUB1 nucleic acid in said plant.

In another aspect, the present invention relates to a method of modifying, and preferably increasing the levels of a transcription factor like a protein that binds to the SQUAMOSA promoter (SBP domain), which method includes increasing the level of expression or activity of UBC13 or reducing or abolishing the expression or activity of OTUB1 as described in this application.

Description of drawings

The present invention is also described in the following non-limiting drawings.

In FIG. 1 shows positional cloning of qNPT1. (a) Appearance of a mature plant. RIL52 was created by crossing Chunjiang06 (CJ06) x IR66167-27-5-1-6. Scale: 20 cm. (b) Panicle morphology. Scale: 5 cm. (c-e) Comparison of RIL52 with parents in terms of (c) number of grains, (d) number of shoots and (e) stem diameter. Data are presented as mean ± sr. sq. osh. (n=30). The presence of the same lowercase letters indicates a non-significant difference in means (P < 0.05). (f) QTL mapping for grain number, shoot number and stem diameter, (g) Positional cloning of qNPT1. The locus was mapped to a ~4.1 kb genomic region flanked by P139 and P143. The numbers between the lines indicate the number of recombinants between qNPT1 and the specified adjacent marker. The candidate gene was predicted to produce two alternative transcripts, (h) Sequence variants at the NPT1 locus in the promoter and coding regions indicated in g.

In FIG. 2 shows the abundance of OsOTUB1 transcript associated with tassel branching and grain yield, (a) OsOTUB1 transcript levels present in NIL plant organs. R: root; C: stem; LB: leaf blade; LS: leaf sheath; SAM: shoot apex meristem; YP0.2, YP6, YP12: juvenile panicles of medium length, 0.2, 6 and 12 cm, respectively. Relative expression levels are expressed as relative copies per 1000 copies of Actinl rice. Data are presented as mean ± sr. sq. osh. (n=3). (b) Plant morphology. Scale: 20 cm. (c) Panicle morphology. Scale: 5 cm. (d) Grain morphology. Scale: 2 mm. (e-j) Quantitative comparison of two NILs. (e) Time of formation of head panicles, (f) Plant height, (g) Number of shoots per plant, (h) Number of secondary branches per panicle, (i) Number of grains per panicle, (j) Weight of 1000 grains, (k) Photograph shoot apex meristems. Scale: 50 µm. (l) Photograph of a stem taken with a scanning electron microscope. Scale: 25 µm. (m) Stalk vascular system, (n) Total number of large and small vascular bundles presented in (m). (o) Total grain yield per plant. Data are presented as mean ± sr. sq. osh. (n=288). All phenotypic data were obtained from plants grown in irrigated rice fields under normal cultivation conditions. Student's t-test was used to calculate P values.

In FIG. 3 shows the OsOTUB1-OsSPL14 interaction regulating plant architecture, (a) BiFC assays. The N-terminus of YFP-tagged OsSPL14, the SBP-domain, or the deleted OsSPL14 variant was co-transformed into rice protoplasts along with the C-terminus of YFP-tagged OsOTUB 1.1. Panels (left to right): DAPI staining, YFP signal, differential interfering contrast agent, pooled channel. Scale: 10 µm. (b) Co-immunoprecipitation of OsOTUB1.1-GFP and

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OsSPL14. IB, Immunoblot; IP, immunoprecipitation. (c) Plant morphology. Scale: 20 cm. (d) Panicle morphology. Scale: 5 cm. (f) OsSPL14 transcript excess. Transcription versus transcription level in ZH11 plants was taken as 1. Data are presented as mean ± sr. sq. osh. (n=3). (f) Number of shoots per plant, (g) Number of grains per panicle, (h) Stem diameter. All phenotypic data were obtained from plants grown in the field under normal cultivation conditions. Data in (f-h) are presented as mean ± sr. sq. osh. (n=120). The presence of the same lowercase letters indicates a non-significant difference in means (P < 0.05).

In FIG. 4 shows that OsOTUB1 stimulates the degradation of OsSPL14. (a) Accumulation of OsSPL14 in ZH11 and ZH11-npt1 plants. Excess HSP90 protein was used as a loading control. (b) Treatment with proteasome inhibitor MG132 stabilizes OsSPL14. Total proteins were extracted from young panicles (<0.2 cm long) of ZH11 plants treated with 0 or 50 μM MG132. The immunoblot was probed with antibodies against OsSPL14 or against HSP90. (c) OsOTUB1 destabilizes OsSPL14. ZH11 and ZH11-npt1 young panicle lysates were co-incubated with GST-OsSPL14 in the presence or absence of His-OsOTUB 1. The lysates were harvested at various time points and immunoblotted to assess the accumulation of OsSPL14 and HSP90. (d) OsSPL14 ubiquitination. Young panicle protein extracts were immunoprecipitated with an anti-Myc antibody and then analyzed using anti-ubiquitin, ubiquitin K48 or ubiquitin K63 antibody chain conjugates. (e) Flag-OsSPL14 can be modified by K48 ubiquitin binding. Rice protoplasts were co-transfected with Flag-OsSPL14 and HA-ubiquitin (HA-labelled wild-type ubiquitin or K48R ubiquitin) and the ubiquitinized forms of Flag-OsSPL14 were immunoprecipitated with an anti-Flag antibody and then analyzed with an anti-HA antibody, (f) K63 -related ubiquitination of OsSPL14 was regulated by OsOTUB1. Rice protoplasts were co-transfected with Flag-OsSPL14 and HA-ubiquitin (HA-labelled wild-type ubiquitin, ubiquitin K48R, K63R, K48O, or K63O) in the presence or absence of Myc-OsOTUB1, after which the lysates were harvested and immunoblotted to assess OsSPL14 accumulation, and analyzed ubiquitinated forms of Flag-OsSPL14 are described in (e).

In FIG. 5 illustrates the phylogenetic analysis. The human OTUB1 sequence and its orthologues of mice (MmOTUB1), Arabidopsis thaliana (AtOTUB1), soybean (GmOTUB1), corn (ZmOTUB1), sorghum (SbOTUB1), barley (HvOTUB1), wild-type spelled wheat (TuOTUB1), and rice (OsOTUB1) were taken from www.ncbi.nlm.nih.gov. The numbers on the right indicate the position of the residues in each protein.

Identical residues are shown in dark dashed areas, conserved residues are shown in light dashed areas, and variable residues are not shaded.

In FIG. 6 shows the effect of functional OsOTUB1 on plant architecture and grain yield, (a) Mature plant morphology. Scale: 20 cm. (b) OsOTUB1 loss-of-function mutations generated by CRISPR/Cas9. (c) Time of head panicle formation, (d) Plant height, (e) Upper internode diameter, (f) Number of shoots per plant, (g) Panicle length, (h) Number of primary branches per panicle, (i) Number of secondary branches per panicle, (j) Number of grains per panicle, (k) Weight of 1000 grains. (l) Total grain yield per plant. Data are presented as mean ± sr. sq. osh. (n=288). All phenotypic data were obtained from paddy rice plants grown under normal cultivation conditions. The presence of the same lowercase letters indicates a non-significant difference in means (P < 0.05).

In FIG. 7 illustrates the expression analysis of pOsOTUB1::GUS. (a) GUS expression in five day old seedlings. Scale: 1 cm. (b) Plant root tips are shown in (a). Scale: 200 µm. (c) Cross section of the GUS-stained root elongation zone shown in (a). Scale: 200 µm. (d) Cross section of a stem. Scale: 100 µm. (e) Various stages of panicle formation. Scale: 1 cm. (f) Spikelet rind before fertilization. Scale: 1 mm.

In FIG. 8 shows the subcellular localization of OsOTUB1.1-GFP. (a) Expression of OsOTUB1.1-GFP in the zone of root elongation. Scale: 20 µm. (b) Expression of GFP in protoplasts isolated from leaf sheaths of ZH11 plants overexpressing OsOTUB1.1-GFP. Scale: 10 µm. Panels (left to right): GFP signal, differential interfering contrast agent (DIC), pooled channel.

In FIG. 9 shows the phenotype of ZH11 plants overexpressing OsOTUB1.1. (a) Reduced shoot count and dwarfism were observed in two independent transgenic lines. Scale: 10 cm. (b) Panicle size decreased. Scale: 5 cm. (c) Leaf necrosis was observed. Scale: 3 mm. (d) Apoptosis was induced in elongated leaves as analyzed by Evans blue staining. Scale: 3 mm. (f) OsOTUB1.1 transcript excess. in a young panicle. Transcription versus transcription level in ZH11 plants was taken as 1. Data are presented as mean ± sr. sq. osh. (n=3). (f) Plant height, (g) Number of shoots per plant, (h) Number of grains per panicle. Data are presented as mean ± sr. sq. osh. (n=60). All phenotypic data were obtained from paddy rice plants grown under normal cultivation conditions. The presence of the same lowercase letters indicates a non-significant difference in means (P < 0.05, f-h panels).

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In FIG. 10 shows that OsOTUBl has the activity of cleaving the tetramers of K48- and K63-bound ubiquitin. K48- and K63-linked ubiquitin tetramer cleavage activity (Tetraub) was analyzed using OTUB1, His-OsOTUB1.1 or OTUB1.2. The starting materials (Ub ₄ ) and their cleavage products [trimers (Ub ₃ ), dimers (Ub ₂ ) and monomers (Ubi)] are labeled on the left. Products were visualized by Western blot analysis using an anti-ubiquitin antibody.

In FIG. 11 shows the effect of expression of human OTUB1 or its orthologues on ZH11-npt1 plant architecture. (a) Plant morphology. Scale: 20 cm. (b) Panicle morphology. Scale: 5 cm. (c) Excess of OTUB1 transcript (or its orthologues) in young panicles compared to OsOTUB1 level in ZH11-npt1 plants. Data are presented as mean ± sr. sq. osh. (n=3). (d) Number of shoots per plant, (e) Number of grains per panicle, (f) Upper internode diameter. Data are presented as mean ± sr. sq. osh. (n=30). All phenotypic data were obtained from paddy rice plants grown under normal cultivation conditions. The presence of the same lowercase letters indicates a non-significant difference in means (P < 0.05, panels d-f).

In FIG. 12 shows that the interaction between OsOTUB1 and OsUBC13 regulates plant architecture, (a) Assays for the presence of the two components in yeast. (b) Inhibition assays using recombinant GST-OsOTUB1 and His-OsUBC13. (c) BiFC analyzes in rice protoplasts. Scale: 10 µm. (d) Morphology of transgenic ZH11 plants. Scale: 20 cm. (f) Cross section of upper internodes. Scale: 500 µm. (f) Effect of OsUBC13 on panicle branching. Scale: 5 cm. (g) Grain size and shape. Scale: 2 mm. (h) OsOTUB1 transcript excess in young panicle compared to ZH11. Data are presented as mean ± sr. sq. osh. (n=3). (i) Number of shoots per plant, (j) Number of grains per panicle, (k) Weight of 1000 grains. (l) Upper internode diameter. Data are presented as mean ± sr. sq. osh. (n=30). All phenotypic data were obtained from paddy rice plants grown under normal cultivation conditions. The presence of the same lowercase letters indicates a non-significant difference in means (P < 0.05, panels i-l).

In FIG. 13 shows that the SBP domain is required for the OsSPL14-OsOTUB1 interaction. (a) Two-component assays in yeast confirmed an interaction between the C-terminus of OsOTUB1 and OsSPL14. (b) Schematic representation of deleted and non-deleted variants of OsSPL14 proteins used in BiFC assays. (c) BiFC assays. Scale: 10 µm.

In FIG. 14 shows that OsOTUB1 interacts with rice SPL transcription factors. BiFC analyzes were performed using rice protoplasts where the C-terminus of YFP-labeled OsOTUB1.1 was co-transformed with the N-terminus of YFP-labeled OsSPL. Panels (left to right): YFP signal, differential interfering contrast agent (DIC), pooled channel. Scale: 10 µm.

In FIG. 15 shows that OsOTUB1 and OsSPL14 antagonistically regulate common target genes, (a) Number and overlap of OsSPL14-activated and OsOTUB1-inhibited target genes. RNA sequencing was performed using young panicles (<0.2 cm long) of NIL plants. (b) OsSPL14-regulated gene excess as measured in young panicle compared to ZH11. Data are presented as mean ± sr. sq. osh. (n=3).

In FIG. 16 shows the effect of the OsSPL14-OsOTUB1 interaction on OsSPL14 DNA binding affinity. Analysis for competitive binding to the OsSPL14-GST protein was performed using 10x, 20x, and 50x unlabeled probes containing GTAC-box motifs of the DEP1 gene promoter or using 1x, 2x, and 4x unlabeled Myc-OsOTUB1.1 fusion proteins, respectively.

In FIG. 17 shows the primer sequences used in the analysis for constructs and DNA transcripts.

In FIG. 18 shows that WTG1 affects grain size, shape and weight.

(a) A grain of Zhonghuajing (ZHJ) paddy rice and wtg1-1.

(b) Brown rice grain ZHJ and wtg1-1.

(c) Cross section of ZHJ and wtg1-1 brown rice grains. The red lines show the grain thickness.

(d) ZHJ grain length and wtg1-1 (n>50).

(e) ZHJ grain width and wtg1-1 (n>50).

(f) ZHJ grain width and wtg1-1 (n>50).

(g) Weight of 1000 grains of ZHJ and wtg1-1. The mass of three batches of samples (n=3) was measured. Values in (d-g) mean mean ± cf. sq. osh. **P < 0.01 compared to parental line (ZHJ) by Student's t-test.

Scale: 3 mm (a-c).

In FIG. 19 shows that WTG1 affects panicle size, panicle shape, and grains per panicle.

(a, b) ZHJ plants (a) and wtg1-1 (b). Plants grown in an irrigated paddy field were dug up and planted in pots to obtain a photograph of the entire plant.

(c, d) Elongated leaves of ZHJ (c) and wtg1-1 (d).

(e) Panicles ZHJ (left) and wtg1-1 (right).

(f) Plant height ZHJ and wtg1-1 (n>10).

- 7 043050 (g, h) Length (g) and width (h) of ZHJ and wtg1-1 leaves (n>10).

(i) Tassel length ZHJ and wtg1-1 (n>10).

(j) Distance between each primary branch and panicle neck (n > 10).

(k,l) Number of primary panicle branches (k) and number of secondary panicle branches (l) (n>10).

(m) Number of grains per panicle (n>10).

Values in (f-m) mean ± cf. sq. osh. **P < 0.01 compared with ZHJ by Student's t-test.

Scale: 10 cm (a, b); 1 cm (s, d); 5 cm (e).

In FIG. 20 shows that WTG1 encodes an otubain-like protease with deubiquitinizing activity.

(a) WTG1 gene. Open boxes show 5' and 3' untranslated regions. The shaded boxes show the coding sequences. Also shown are the start codon (ATG) and stop codon (TAG), wtg1-1 contains a m.p.o. deletion at the exon-intron junction of the fourth intron.

(b) The dCAPS1 marker was expressed based on the wtg1-1 mutation. The restriction enzyme Hpy1881 was used to hydrolyze the PCR products.

(c) RT-PCR analysis of WTG1 in ZHJ and wtg1-1 panicles. The wtg1-1 mutation resulted in modification of WTG1 splicing.

(d, e) WTG1 protein (d) and mutated wtg1-1 protein (e). The WTG1 protein contains an otubain domain. The mutated wtg1-1 protein has an N-terminal region, part of the otubain domain, and an unrelated peptide (green frame).

(f) Paddy rice grain ZHJ, wtg1-1 and gWTG1; wtg1-1. gWTG1; wtg1-1 means that the genomic sequence of the WTG1 gene has been turned into a wtg1-1 mutant.

(g) Brown rice grain ZHJ, wtg1-1 and gWTG1; wtg1-1.

(h) Cross section of brown rice grains ZHJ, wtg1-1 and gWTG1; wtg1-1. The red lines show the grain thickness.

(i-k) Grain length (i), grain width (j) and grain thickness (k) ZHJ, wtg1-1 and gWTG1; wtg1-1 (n>50).

(l) WTG1 has deubiquitinizing activity in vitro. MBP-WTG1 cleaves HisUBQ10 in vitro, and MBP, MBP-WTG1 ^wtg1-1 and MBP-WTG1 ^{D68E; C71S; H267R} do not cleave His-UBQ10. Anti-His and anti-MBP antibodies were used to detect His-UBQ, and His-UBQ10, MBP, MBP-WTG1 ^wtg1-1 and MBP-WTG1 ^{D68E were digested; C71S; h267R} , respectively.

Values in (i-k) mean ± cf. sq. osh. **P < 0.01 compared with the parental line (ZHJ) according to Student's t-test.

Scale: 3 mm (f-h).

In FIG. 21 shows the expression and submolecular localization of WTG1.

(a) WTG1 expression in roots and leaves of young seedlings and in developing panicles ranging in size from 1 cm (P1) to 15 cm (P15) was analyzed by quantitative real-time RT-PCR. Values are given as mean ± cf. sq. osh. for three duplicates.

(b-d) WTG1 expression was examined using proWTG1:GUS transgenic plants. GUS activity is shown in 8-day-old seedlings (b), in the skin of developing spikelets (c), and in developing panicles (d).

(e-g) Subcellular localization of GFP-WTG1 in pro35S:GFP-WTG1 root cells. GFP-WTG1 GFP fluorescence (e), DAPI staining (f), and merged images (g) are shown.

(h) Subcellular fractionation and immunoblot analyses. Pro35S:GFP-WTG1 leaves were used to isolate the cytoplasmic protein fraction (C) and the nuclear protein fraction (N). Immunoblot analysis was performed using an anti-GFP antibody. Bip, a luminal binding protein, was used as a cytoplasmic marker. Histone H4 was used as a nuclear marker.

Scale: 5 mm (b), 2 mm (c), 10 mm (d) and 10 µm (e-g).

In FIG. 22 shows that WTG1 overexpression results in a narrow, thin, and long grain.

(a) Paddy rice grain of transgenic lines Zhonghuajing (ZHJ) and proActin:WTG1.

(b) Brown rice grain of ZHJ and proActin:WTG1 transgenic lines.

(c) Cross section of grain of transgenic plants ZHJ and proActin:WTG1. The red lines show the grain thickness.

(d-f) Length (d), width (e), and thickness (f) of ZHJ and proActin:WTG1 grains (n>40).

(g) WTG1 expression level in ZHJ and proActin:WTG1 transgenic lines. Three duplicates were evaluated.

Values in (d-g) mean mean ± cf. sq. osh. **P < 0.01 compared to parental line (ZHJ) by Student's t-test.

Scale: 3 mm (a-c).

In FIG. 23 illustrates the identification of the wtg1-1 mutation by the MutMap method. Whole genome sequencing revealed one deletion in the LOC-Os08g42540 gene, which has an SNP/INDEL=1 index.

In FIG. 24 shows the phylogenetic tree of WTG1 and its homologues. WTG1 homologues were semi

- 8 043050 by searching the database (https://blast.ncbi.nlm.nih.gov). The phylogenetic tree of WTG1 homologues was constructed by joining adjacent ends using the MEGA7.0 program. The numbers in the notes mean the percentage of 1000 bootstrap duplicates.

In FIG. 25 shows the alignment of WTG1 and its homologues.

Proteins of Oryza sativa (MSULocus: LOC_Os08g42540), Caenorhabditis elegans (C. elegans, NP_506709), Homo sapiens (OTUB1, AK000120; OTUB2, AK025569), Mus musculiis (Mus musculus, NP 080856), Drosophila melanogaster, (D. melanogaster, NP 609375), Chlamydomonas reinhardtii (C.reinhardtii, CHLREDRAFT 55169), Zea mays (Zea mays, ACG38232), Hordeum vulgare (Hordeum vulgare, BAJ96717), Triticum urartu (Triticum urartu, EMS61506), Arabidopsis thaliana (A. thal iana, At1 g28120 ) and Glycine max (Glycine max, XP 014623731) was used for alignment. Red triangles show conserved amino acids in the putative cysteine-protease catalytic triad, and red boxes show conserved sequences in the otubaine-like domain.

In FIG. 26 shows that WTG1 overexpression affects cell proliferation in the spikelet rind.

(a, b) Mean length (a) and width (b) of outer epidermal cells in the lower lemma of transgenic ZHJ and proActin:WTG1 plants. More than 100 cells were evaluated (n>100).

(c) Number of outer epidermal cells along the grain length of the lower lemma. Twenty grains (n=20) were used to count the number of cells.

(d) Number of outer epidermal cells across grain width. Twenty grains (n=20) were used to count the number of cells.

Values in (a-d) are given as mean ± cf. sq. osh. **P < 0.01 compared to parental line (ZHJ) by Student's t-test.

In FIG. 27 shows the primers used in example 2.

In FIG. 28 shows OsOTUB1 RNAi silencing. (a) OsOTUB1 RNAi silencing has a ZH11-npt1 like phenotype. Scale: 20 cm. (b) OsOTUB1 expression levels of transgenic plants, (c) Transgenic plant stem thickness. Scale: 5 mm. (d) Panicle architecture of transgenic plants. Scale: 5 cm. (e) Grain shape of transgenic plants. Scale: 5 mm. (f) The shape of the elongated leaves of transgenic plants. Scale: 5 cm.

In FIG. 29 shows the plant phenotype with reduced levels of OTUB1 expression by RNAi. (a) Number of primary branches per panicle. (b) Number of secondary branches per panicle, (c) Number of grains per panicle, (d) Tassel length, (e) Plant height, (f) Total grain yield per plant.

Detailed description of the invention

The present invention is described in more detail below. In the following sections, various aspects of the invention are defined in more detail. Each aspect thus defined may be combined with any other aspect or aspects unless otherwise noted. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

The present invention can be practiced, unless otherwise noted, using standard methods used in the fields of botany, microbiology, tissue culture, molecular biology, chemistry, biochemistry, recombinant DNA technology and bioinformatics, which are known to specialists in this field. Such methods are described in detail in the literature.

As used herein, the terms nucleic acid, nucleic acid sequence, nucleotide, nucleic acid molecule, or polynucleotide include DNA molecules (eg, cDNA or genomic DNA), RNA molecules (eg, mRNA), naturally occurring, mutated, synthetic DNA or RNA molecules, and DNA analogs. or RNA obtained using nucleotide analogues. These sequences may be single stranded or double stranded. Such nucleic acids or polynucleotides include, but are not limited to, structural gene coding sequences, antisense sequences, and non-coding regulatory sequences that do not encode mRNA or protein products. These terms also include gene. As used herein, the term gene or gene sequence is used in a broad sense and means a DNA nucleic acid associated with a biological function. Thus, genes may include introns and exons, as in a genomic sequence, or may contain only a coding sequence, as in cDNA, and/or may include cDNA in combination with regulatory sequences.

As used herein, the terms polypeptide and protein are synonymous and refer to amino acids in polymer form of any length linked to each other by peptide bonds.

Aspects of the present invention include recombinant DNA techniques and exclude variants that are based solely on cultivating plants by traditional breeding methods.

Ways to increase yield

Accordingly, in its first aspect, the present invention relates to a method for increasing plant yield, which method includes reducing or abolishing the expression of at least one nucleic acid encoding an otubain-like protease (referred to here as OTUB1 (protein that binds to ubiquitin aldehyde and containing ovarian tumor domain

- 9 043050

1)), and/or reducing or abolishing the activity of the OTUB1 polypeptide in said plant. Preferably, the present invention relates to a method for increasing grain yield. OTUB1 may also be referred to here as NPT1, DEP5 or WTG1 and these terms may be used interchangeably. In one embodiment of the invention, the method may reduce, but not abolish, the expression and/or activity of OTUB1.

The term yield in general terms means the measurable achievement of economic value, usually associated with a specified crop, in a particular area and in a particular period of time. Individual plant parts directly contribute to the yield depending on their number, size and/or weight. Alternatively, the actual yield is the crop yield per square meter per year, which is determined by dividing the total output (including harvested and rated product) by the square meters planted.

The term yield increase as defined herein may include any or at least one of the following and may be determined by evaluating one or more of: (a) increased biomass (mass) of one or more plant parts, above ground parts (harvested ) or increased root biomass, increased root volume, increased root length, increased root diameter, or increased biomass of any other part of the harvested part. The increased biomass can be expressed as g/plant or kg/hectare; (b) increased seed yield per plant, which may include one or more of: increased seed biomass (mass) per plant or individual part thereof, (c) increased seed filling rate, (d) increased seed filling rate, (e) increased yield index, which can be expressed as the ratio of the yield of harvested parts, such as seeds, to the total biomass, (f) increased germination/germination efficiency, (g) increased number or size or mass of seeds or pods or pods or grains, ( h) increased seed volume (which may result from changes in composition (i.e., total content and composition of lipids (also referred to here as oil)), proteins, and carbohydrates), (i) increased (individual or average) seed area, (j) increased (individual or average) seed length, (k) increased (individual or average) seed width, (l) increased (individual or average) seed perimeter, (m) increased growth or branching, for example, inflorescences with a large number of branches, ( n) increased fresh weight or grain fullness, (o) increased head weight, (p) increased thousand-kernel weight (TKW), which can be calculated from the number of filled seeds counted and their total weight, and can result from an increase in seed size and/ or seed weight, (q) reduced number of barren shoots per plant, and (r) stronger or stronger culms or stems. All parameters are given in relation to the wild-type plant or to the control plant.

Preferably, the increased yield includes at least one increase in the number of grains, the number of grains per spike or tassel, the weight of the grain, the width of the grain and the thickness of the grain, the weight of a thousand grains. The yield is increased compared to that of the control plant or wild type plant. So, for example, the yield was increased by 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, 25, 30 , 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95% compared to the yield of the control plant or wild type plant. In one of the embodiments of the invention, the yield can be increased by 20-50%, and more preferably by 5-15% or more compared to the yield of the control plant. The increase in grain yield can be assessed by determining one or more of the parameters: number of grains, number of grains per panicle, grain weight, grain width and grain thickness, thousand grain weight, and/or number of fruiting shoots per plant. One skilled in the art can determine any of the above grain yield parameters by known methods.

As used herein, the terms seed and grain may be considered here as synonyms. The terms enhancement, enhancement, or enhancement are also used interchangeably herein.

The term reduction means a decrease in the levels of expression and/or activity of OTUB1 by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% or more compared to the level in the wild-type plant or control plant. The term reduction may or may not include the abolition of expression, and preferably it does not include such a abolition. The term abolition of expression means that no expression of OTUB1 is detected or no functional OTUB1 polypeptide is produced. Methods for determining the level of expression and/or activity of OTUB1 are well known to those skilled in the art. Such a reduction can be measured by any standard method known to those skilled in the art. For example, a decrease in expression levels and/or levels of at least OTUB1 may be indicative of protein and/or nucleic acid levels and may be determined by any known method such as, but not limited to, any form of gel electrophoresis or chromatography (e.g. , HPLC). In one embodiment, the mutation reduces or abolishes the deubiquitinase activity of OTUB1. Accordingly, the method may include assessing the deubiquitinase activity of the protein by standard methods, such as using a fluorescent deubiquitinase substrate.

In a preferred embodiment of any of the aspects of the invention described here, expression and/or

- 10 043050 OTUB 1 activity is reduced but not cancelled.

The term at least one mutation means that if the OTUB1 gene is present as more than one copy or homologue (with the same or slightly different sequence), then at least one mutation is present in at least one gene. Preferably all genes are mutated.

In one of the embodiments of the invention, the method includes introducing at least one mutation, preferably in an endogenous gene encoding OTUB1 and/or in the OTUB1 promoter. Preferably, said mutation is a mutation in the coding region of the OTUB1 gene. Alternatively, the specified mutation is a mutation in the intron sequence or in the 5'-UTR. In another embodiment of the invention, at least one mutation or structural modification can be introduced into the OTUB1 promoter such that the OTUB1 gene is not expressed (i.e., expression is abolished) or is expressed at a reduced level, as defined in this application. In an alternative embodiment of the invention, at least one mutation may be introduced into the OTUB1 gene such that the modified gene does not express a full-length (ie, expresses a truncated) OTUB1 protein or does not express a fully functional OTUB1 protein. Thus, the activity of the OTUB 1 polypeptide can be considered as reduced or abolished, as described in this application. In any case, the mutation may result in the expression of OTUB1 with no in vivo biological activity, with its significant decrease or change. Alternatively, OTUB1 may not be expressed at all.

In one embodiment, the OTUB1 gene sequence comprises or consists of it, a nucleic acid sequence as defined in any of SEQ ID NOs: 2-5, or a functional variant or homologue thereof, and encodes a polypeptide as defined in SEQ ID NO: 1, or its functional variant or homologue.

The term OTUB1 promoter means a region extending at least 2.5 kb. above the ATG OPC OTUB1 codon. In one embodiment, the OTUB1 promoter sequence comprises, or consists of, the nucleic acid sequence as defined in SEQ ID NO: 6, or a functional variant or homologue thereof. Examples of promoter homologues are shown in SEQ ID NOs: 21-27.

In the above embodiments, endogenous nucleic acid may mean a native or natural sequence in the genome of a plant. In one of the embodiments of the invention, the endogenous sequence of the OTUB1 gene contains any of SEQ ID NO: 2, 3, 4 or 5 and encodes the amino acid sequence defined in SEQ ID NO: 1 or its homologues. The scope of the present invention also includes functional variants (defined in this application) and homologs of the sequences identified above. Examples of homologues are shown in SEQ ID NOs: 7-27. Accordingly, in one embodiment, the homologue encodes a polypeptide selected from SEQ ID NOs: 14-20 or contains or consists of a nucleic acid sequence selected from SEQ ID NOs: 7-13.

The term functional variant (or variant) as used herein with reference to any of SEQ ID NOs: 2-210 means a variant sequence or a portion of a sequence thereof that retains the biological function of the full length unmodified sequence. A functional variant also includes a variant of the gene of interest that has sequence modifications that do not affect function, such as non-conserved residues. Also included within the scope of the present invention is a variant that is substantially identical to the wild-type sequences presented here, ie. has only some sequence modifications, eg in non-conservative residues, and is biologically active. Modifications in a nucleic acid sequence that result in the production of a different amino acid at that position without affecting the functional properties of the encoded polypeptide are well known to those skilled in the art. So, for example, the codon of the amino acid alanine, i.e. hydrophobic amino acid may be replaced by a codon encoding another less hydrophobic residue such as glycine or a more hydrophobic residue such as valine, leucine or isoleucine. Similarly, modifications that result in the substitution of one negatively charged residue for another, for example, aspartic acid for glutamic acid, or one positively charged residue for another, for example, lysine for arginine, can also, as expected, produce a functionally equivalent product. Also, nucleotide substitutions that alter the N-terminal and C-terminal portions of the polypeptide molecule should not be expected to alter the activity of the polypeptide. Each of the proposed modifications is within the skill of the ordinary person and has been determined not to alter the biological activity of the encoded products.

In one embodiment, the functional variant has a sequence with a common identity of at least 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 , 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65 , 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 , 91, 92, 93, 94, 95, 96, 97, 98 or at least 99% compared to the unmodified nuk sequence

- 11 043050 leic acid or amino acid sequence.

As used herein, the term homologue also means an OTUB1 promoter or an orthologue of the OTUB1 gene derived from plants of other species. A homologue may have, in order of increasing preference, a sequence whose common identity is at least 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or at least 99% compared to the amino acid sequence represented by SEQ ID NO: 1 or any of the nucleic acid sequences represented by SEQ ID NO: 2 or 6. B one of the embodiments of the invention, the overall sequence identity is at least 37%. In one embodiment, the overall sequence identity is at least 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99%, and most preferably 90, 91, 92, 93, 94, 95, 96, 97, 98 or at least 99 %.

Also within the scope of the present invention are functional variants of the OTUB1 homologues defined above.

OTUB1 (ubiquitin aldehyde binding protein containing ovarian tumor domain 1) encodes an otubain-like protease. As discussed above, OTUB1 may also be referred to herein as NPT1, DEP5, or WTG1, and these terms may be used interchangeably.

OTUB1 is characterized by a number of conserved domains including, but not limited to, an otubain-like domain.

In another embodiment of the invention, the sequence of the otubain-like domain is shown below:

SEQ ID NO: 211

PYVGDKEPLSTLAAEFQSGSPILQEKIKLLGEQYDALRRTRGDGNCFYRSFMFSY

LEH

ILETQDKAEVERILKKIEQCKKTL ADLGYIEFTFEDFF SIFIDQLES VLQGHES SIGAEELL ERTRDQMVSDYVVMFFRFVTSGEIQRRAEFFEPFISGLTNSTVVQFCKASVEPMGEE SDHVHIIALSDALGVPIRVMYLDRS SCD AGNIS VNHHDF SPEANS SDGAAAAAEKP YITL LYRPGHYDILYP

Amino acids highlighted in bold in SEQ ID NO: 211 form a catalytic triad, or SEQ ID NO: 212

SPILQEKIKLLGEQYDALRRTRGDGNCFYRSFMFSYLEHILETQDKAEVERILKKI

EQCK

KTLADLGYIEFTFEDFF SIFIDQLES VLQGHES SIGAEELLERTRDQMVSD YVVMFFRF VTSGEIQRRAEFFEPFISGLTNSTVVQFCKASVEPMGEESDHVHIIALSDALGVPIRVM YLDRSSCDAGNISVNHHDFSPEANSSDGAAAAAEKPYITLLYRPGHYDILYPK

Accordingly, in one embodiment of the invention, the OTUB1 nucleic acid sequence (coding) encodes an OTUB1 protein containing at least one SBP domain and/or an otubain-like domain as defined below, or a variant thereof, wherein said variant has the sequence , whose common identity is at least 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 or at least 99% compared to the sequence of SEQ ID NO: 211 or 212. In a preferred embodiment of the invention, the OTUB1 polypeptide is characterized by the presence of at least one otubain-like domain and has a sequence that is at least 75 % homologous to SEQ ID NO: 211 or 212. In another embodiment, the OTUB1 protein contains a catalytic triad and preferably contains SEQ ID NO: 211 or SEQ ID NO: 212 and/or a variant thereof as defined above, wherein the sequence of this variant contains at least least aspartate and cysteine (preferably in positions 4 and 7) and/or histidine in SEQ ID NO: 211 (preferably in the first position).

Two nucleic acid or polypeptide sequences are said to be identical if the sequence of nucleotides or amino acid residues, respectively, in the two sequences are the same when aligned for maximum correspondence, as described below. The terms identical or percent identity with respect to two or more nucleic acids or polypeptide sequences refers to two or more sequences or subsequences that are the same or have a certain percentage of the same amino acid residues or nucleotides when compared and aligned for maximum match across the comparison window, as was determined using one of the following sequence comparison algorithms or by manual alignment and visual observation. If the percentage is correct

- 12 043050 sequence accuracy is used in relation to proteins or peptides, it is believed that the positions of residues that are not identical are often distinguished by conservative amino acid substitutions, where amino acid residues have been replaced by other amino acid residues with similar chemical properties (for example, charge or hydrophobicity) a , therefore, do not change the functional properties of the molecule. If the sequences differ by conservative substitutions, then the percent sequence identity can be adjusted upward to correct for the conservative nature of the substitutions. Techniques for such correction are well known to those skilled in the art. For sequence comparisons, typically one sequence is chosen as a reference sequence against which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are determined, if necessary, and algorithm parameters of the sequence program are calculated. The program's default settings may be used, or alternative settings may be selected. The sequence comparison algorithm then calculates the percent sequence identity for the test sequences compared to the reference sequence based on the program parameters. Non-limiting examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms.

Suitable homologues can be identified by comparing sequences and conserved domain identifications. Those skilled in the art are aware of prediction programs that can be used to identify such sequences. The function of a homologue can be identified as described herein and thus a person skilled in the art can confirm the function, for example if it is overexpressed in a plant.

Thus, the nucleotide sequences of the invention and the sequences described herein can also be used to isolate corresponding sequences from other organisms, and in particular other plants, such as crops. Thus, methods such as PCR, hybridization, and the like. can be used to identify such sequences based on the homology of these sequences with the sequences described here. The topology of sequences and domains with a characteristic structure can also be taken into account in the identification and isolation of homologues. Sequences can be distinguished based on the general identity of their sequences or their fragments. In hybridization methods, all or part of a known nucleotide sequence is used as a probe that selectively hybridizes to other relevant nucleotide sequences present in a population of cloned genomic DNA fragments or cDNA fragments (i.e. genomic DNA or cDNA libraries) of a selected plant. These hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group or any other detectable marker. Methods for obtaining probes for hybridization and construction of cDNA and genomic libraries are generally known in the art and are described in Sambrook, et al., (1989) Molecular Cloning: A Library Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, New York).

Hybridization of such sequences can be carried out under conditions of a certain stringency. By stringent conditions or stringent hybridization conditions is meant conditions under which a probe will hybridize to a target sequence to a detectably greater extent than to other sequences (eg, at least 2-fold compared to background). The stringency conditions depend on the sequence and will differ in different cases. By adjusting the conditions for the stringency of hybridization and/or washing, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, the stringency conditions can be adjusted so that it allows for some inconsistency in sequence, but so that lower degrees of similarity can be detected (heterologous probing). Typically, the probe is less than about 1000 nucleotides in length, and preferably less than 500 nucleotides.

Typically, stringency conditions may be conditions where the salt concentration is less than about 1.5 M Na ions, and typically about 0.01 to 1.0 M Na ions (or other salts) at pH 7, 0-8.3, and the temperature is at least about 30°C for short probes (eg, 10 to 50 nucleotides) and at least about 60°C for long probes (eg, more than 50 nucleotides). The duration of hybridization is usually less than about 24 hours, and most often from about 4 to 12 hours. Rigidity conditions can also be achieved by adding destabilizing agents such as formamide.

In another embodiment, the variant used herein may include a nucleic acid sequence encoding an OTUB1 polypeptide as defined herein.

- 13 043050 description, where the specified sequence is capable of hybridizing under the stringency conditions defined here with the nucleic acid sequence defined in SEQ ID NO: 2-5.

In one of the embodiments of the invention, the method includes reducing or canceling, and preferably reducing the expression of at least one nucleic acid encoding an OTUB1 polypeptide or reducing or canceling the activity of an OTUB1 polypeptide, as described in this application, where this method includes the introduction of at least one mutations in at least one OTUB1 gene and/or promoter, wherein said OTUB1 gene contains or consists of:

a. a nucleic acid sequence encoding a polypeptide defined in one of SEQ ID NOs: 1 and 14-20; or

b. a nucleic acid sequence as defined in one of SEQ ID NOs: 2-13; or

c. nucleic acid sequences whose common identity is at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 , 95, 96, 97, 98 or at least 99% compared to sequences (a) or (b); or

d. a nucleic acid sequence encoding an OTUB1 polypeptide as defined herein and capable of hybridizing under the stringency conditions defined herein to a nucleic acid sequence according to any of (a) to (c), and wherein the OTUB1 promoter comprises or consists of:

e. a nucleic acid sequence as defined in SEQ ID NOs: 6 and 21-27;

f. nucleic acid sequences whose common identity is at least 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 , 95, 96, 97, 98 or at least 99% compared to sequence (e); or

g. a nucleic acid sequence capable of hybridizing under the stringency conditions defined herein to a nucleic acid sequence according to any of (e)-(f).

In a preferred embodiment of the invention, the mutation that is introduced into the endogenous OTUB1 gene or promoter to silence, reduce or inhibit the biological activity and/or expression levels of the OTUB1 gene or protein may be selected from mutations of the following types:

1. missense mutation, which is a modification of the nucleic acid sequence, leading to the replacement of one amino acid by another amino acid;

2. nonsense mutations or stop codon mutations, which are a modification in the nucleic acid sequence that results in the introduction of a premature stop codon and thus termination of translation (forming a truncated protein); where in plants translation termination codons can be selected from TGA (UGA in RNA), TAA (UAA in RNA) and TAG (UAG in RNA); and thus any nucleotide substitution, insertion and deletion results in one of these codons for translation of the mature mRNA (in-frame) and stops translation.

3. insertion mutation of one or more nucleotides or one or more amino acids due to the addition of one or more codons to the nucleic acid coding sequence;

4. deletion mutation of one or more nucleotides or one or more amino acids due to the deletion of one or more codons in the nucleic acid coding sequence;

5. frameshift mutations that result in translation of a nucleic acid sequence in a different reading frame downstream of the mutation. A frameshift mutation can occur for various reasons, such as an insertion, deletion, or duplication of one or more nucleotides.

6. splice site mutation, which is a mutation resulting in an insertion, deletion, or substitution of nucleotides at a splice site.

As used herein, the terms insertion, deletion, or substitution may mean an insertion, deletion, or substitution of at least one, two, three, four, five, six, seven, eight, nine, or ten nucleotides. In one particular embodiment, said mutation may include at least one of the following substitutions:

C to T at position 1135 SEQ ID NO: 2 or 5;

G to C at position 1462 SEQ ID NO: 2 or 5; and/or

G to C at position 1798 SEQ ID NO: 2 or 5.

In another additional or alternative embodiment of the invention, said mutation is the insertion of one amino acid, and preferably T, at position 2234 of SEQ ID NO: 2 or 5.

In another additional or alternative embodiment of the invention, said mutation is a deletion of at least four nucleotides, and preferably four nucleotides, underlined in SEQ ID NO: 3. This mutation is a mutation in the exon-intron splicing sequence, resulting in a mutated CDS sequence containing the fourth intron sequence as defined in SEQ ID NO: 153 (the fourth intron is underlined). As a result, the wtg mutation leads to premature termination of the predicted protein (as described in SEQ ID NO: 154).

In another additional or alternative embodiment of the invention, said mu

- 14 043050 The change may be to replace G with A at position 1824 of SEQ ID NO: 155.

Generally speaking, it will be apparent to those skilled in the art that at least one mutation as defined above, resulting in an insertion, deletion, or substitution of at least one nucleic acid or amino acid, compared to the sequence of the OTUB1 promoter or wild-type OTUB1 nucleic acid or protein, can influence the biological activity of the OTUB1 protein. Preferably, said mutation abolishes or reduces the deubiquitinase activity of OTUB1.

In one embodiment, the mutation is introduced into an SBP domain and/or an otubain-like domain. Preferably, said mutation is a loss-of-function or partial loss-of-function mutation, such as a premature stop codon or an amino acid substitution in a highly conserved region, which has been predicted to play an important role in protein structure formation. In another embodiment of the invention, the mutation is introduced into the OTUB1 promoter and such mutation is at least a deletion and/or insertion of at least one nucleic acid. Also included are other major substitutions such as deletions that remove functional regions of the promoter and reduce the level of OTUB1 expression. In one embodiment, the mutation may be introduced at the position of at least one amino acid that forms the catalytic triad as defined above. Thus, for example, said mutation may be at least one, and preferably all of the following substitutions:

D to E at position 68 SEQ ID NO: 1

C to S at position 71 of SEQ ID NO: 1 and/or

H to R at position 267 of SEQ ID NO: 1.

In one embodiment of the invention, a mutation may be introduced into the OTUB1 promoter and at least one mutation is introduced into the OTUB1 gene.

In one embodiment of the invention, the mutation is introduced by mutagenesis or targeted genome editing. Those. in one embodiment, the present invention relates to a method and to a plant that was obtained by the methods of genetic engineering described above and does not include natural varieties.

Targeted genome modification or targeted genome editing is a genomic engineering technique that uses targeted DNA double-strand breaks (DSBs) to promote genome editing through homologous recombination (HR) mediated recombination events. To achieve efficient genome editing through the introduction of site-specific DNA DSBs, four main classes of specially designed DNA-binding proteins can be used: meganucleases derived from microbial transposable genetic elements; ZF nucleases based on eukaryotic transcription factors; transcription activator-like effectors (TALE) from Xanthomonas bacteria; and the RNA-regulated RNA-DNA endonuclease cas9 derived from the bacterial adaptive immune system CRISPR type II (clustered regularly interspersed short palindromic repeats). Meganuclease, ZF and TALE proteins recognize specific DNA sequences through protein-DNA interactions. Although meganucleases integrate nucleases and DNA-binding domains, however, the ZF and TALE proteins consist of separate modules targeting nucleotides 3 or 1 (nt) of DNA, respectively. ZF and TALE can be assembled in the desired combination and attached to the Fokl nuclease domain to direct nucleolytic activity to specific genomic loci.

Upon delivery to host cells via the type III bacterial secretion system, TAL effectors enter the nucleus, bind to effector-specific sequences in host gene promoters, and activate transcription. Their targeting specificity is determined by the central domain of the tandem of 33-35 amino acid repeats. They are followed by one truncated repeat of 20 amino acids. Most of the natural TAL effectors evaluated are located between 12 and 27 full-length repeats.

These repeats differ from each other only by two adjacent amino acids, i.e. two residues with variable repeats (RVD). The RVD that determines which one nucleotide the TAL effector will recognize is one RVD corresponding to one nucleotide, and each of the four most common RVDs that preferentially associates with one of the four bases. Natural recognition sites are evenly spaced after T, which is necessary to communicate TAL effector activity. TAL effectors can be attached to the catalytic domain of the Fokl nuclease to form the TAL effector nuclease (TALEN), allowing the introduction of targeted DNA double-strand breaks (DSBs) in vivo for genome editing. The use of this technology in genome editing is well documented in the literature, for example, US Pat. As described herein, the Golden Gate method uses type IIS restriction endonucleases that cleave sites outside the recognition sites to form unique 4 bp overhangs. Cloning is carried out by hydrolysis and ligation in the same reaction mixture, since correct assembly eliminates the site of racial

- 15 043050 enzyme cognition. Assembly of a custom-designed TALEN or TAL effector construction involves two steps: (i) assembling the repeat modules to produce intermediate arrays of 1-10 repeats, and (ii) attaching these intermediate arrays to the backbone to produce the final construct. Accordingly, using known techniques, a TAL effector can be constructed that will target the OTUB1 gene or promoter sequence described herein.

Another genome editing technique that can be applied in accordance with various aspects of the present invention is CRISPR. The use of this technology in genome editing is well documented in the literature, for example in US Pat. No. 8,697,359 and the documents cited therein. In short, CRISPR is a microbial nuclease system involved in defense against invasive phages and plasmids. CRISPR loci in microbial hosts contain a combination of CRISPR-associated genes (Cas) as well as non-coding RNA elements capable of programming the specificity of CRISPR-mediated nucleic acid cleavage (sssRNA). Three types (IIII) of CRISPR systems have been identified for a wide variety of host bacteria. One of the key features of each CRISPR locus is the presence of an array of repeating sequences (direct repeats) interspersed with short fragments of non-repeating sequences (spacers). The non-coding CRISPR array is transcribed and cleaved in direct repeats to form short crRNAs containing separate spacer sequences that direct Cas nucleases to the target site (protospacer). Type II CRISPR is one of the most well-characterized systems and performs the desired DNA double-strand break in four successive steps. First, two non-coding RNAs, array pre-cr-RNA and tracr-RNA, are transcribed from the CRISPR locus. Second, tracr-RNA hybridizes with regions of pre-cr-RNA repeats and mediates the processing of pre-cr-RNA into mature cr-RNAs containing individual spacer sequences. Third, the mature cr-RNA:tracr-RNA complex directs Cas9 to the target DNA via Watson-Crick base pairing between a spacer on the cr-RNA and a proto-spacer on the target DNA next to the adjacent proto-spacer motif (PAM), which is an additional condition. target recognition. Finally, Cas9 mediates target DNA cleavage to form a double-strand break in the protospacer.

One of the main advantages of the CRISPR-Cas9 system, compared to conventional gene targeting and other programmable endonucleases, is the ease of communicating multiplexity, where many genes can be mutated at the same time simply by using many ssrRNAs, each targeting a different gene. In addition, if two ssrRNAs are used to flank a genomic region, then the intermediate region can be deleted or inverted (Wiles et al., 2015).

Thus, Cas9 is a key protein of the type II CRISPR-Cas system and a large monomeric DNA nuclease that targets a DNA target sequence adjacent to the PAM sequence motif (adjacent protospacer motif) through a complex of two non-coding RNAs: CRISPR RNA (cr- RNA) and trans-activating cr-RNA (tracr-RNA). The Cas9 protein contains two nuclease domains homologous to RuvC and HNH nucleases. The HNH nuclease domain cleaves the complementary DNA strand, while the RuvC-like domain cleaves the non-complementary strand, introducing a blunt cut fragment into the target DNA. Heterologous expression of Cas9 together with ssrRNA allows the introduction of site-specific double-strand breaks (DSBs) into the genomic DNA of living cells originating from various organisms. For use in eukaryotic organisms, codon-optimized variants of Cas9 have been used, which are naturally derived from the bacterium Streptococcus pyogenes.

Single-stranded guide RNA (ssrRNA) is the second component of the CRISPR/Cas system, which forms a complex with the Cas9 nuclease. sssrRNA is a synthetic RNA chimera obtained by adding cr-RNA to tracr-RNA. The ssrRNA guide sequence is located at the 5' end and provides specificity for the target DNA. Thus, by modifying the guide sequence, ssrRNAs with different target specificities can be obtained. The canonical length of the guide sequence is 20 bp. In plants, ssrRNAs are expressed under the control of plant RNA polymerase III promoters such as U6 and U3. Accordingly, ssrRNA molecules targeting the OTUB1 gene or promoter sequence described herein can be constructed using known techniques. In one embodiment, the method includes using any of the nucleic acid constructs or ssrRNA molecules described herein.

Cas9 expression plasmids for use in the methods of the invention can be constructed as described in the literature.

Alternatively, more conventional mutagenesis techniques can be used to introduce at least one mutation in the OTUB1 gene or OTUB1 promoter sequence. These methods include physical and chemical mutagenesis. The person skilled in the art may be aware of other methods that can be used to obtain such mutants, and these methods of mutagenesis and modification of polynucleotides are well known to specialists. See, for example, Kunkel (1985) Proc.

- 16 043050

Natl. Acad. sci. USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol. 154:367-382; US Patent No. 4873192; Walker and Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and papers cited therein.

In one embodiment of the invention, insertion mutagenesis is used, such as by T-DNA mutagenesis (which allows T-DNA fragments from the Agrobacterium tumefaciens T-plasmid to be inserted into DNA that will result in a mutation causing loss of gene function or gain of gene function). ); site-directed nucleases (SDN) or transposons as a mutagen. Insertional mutagenesis is an alternative means of disrupting gene function and is based on the introduction of foreign DNA into a gene of interest (see Krysan et al, The Plant Cell, Vol. 11, 2283-2290, December 1999). Accordingly, in one embodiment of the invention, T-DNA is used as an insertion mutagen to terminate the expression of the OTUB1 gene or OTUB1 promoter. T-DNA not only disrupts the expression of the gene into which it is inserted, but also acts as a marker for the subsequent identification of the mutation. Because the sequence of the inserted element is known, the inserted gene can be isolated using various cloning or PCR-based strategies. Insertion of a T-DNA fragment of the order of 5-25 kb. usually results in a malfunction of the gene. When sufficiently large populations of T-DNA-transformed lines are obtained, there are sufficiently high chances of obtaining a transgenic plant carrying a T-DNA insert in any gene of interest. Transformation of spores by T-DNA is achieved using an Agrobacterium-mediated method, which involves treating plant cells and tissues with a suspension of Agrobacterium cells.

The details of this method are well known to those skilled in the art. In short, transformation of a plant using Agrobacterium results in the integration of a sequence called T-DNA into the nuclear genome, which is carried out on a bacterial plasmid. The application of T-DNA transformation provides stable single inserts. Subsequent analysis of the resulting transformed lines for the presence of a mutant is simple and each individual inserted line can be quickly characterized by direct sequencing and analysis of the DNA flanking the insert. The gene expression in the mutant is compared with the expression of the OTUB1 nucleic acid sequence in the wild type plant, and a phenotypic analysis is performed.

In another embodiment of the invention, the mutagenesis is physical mutagenesis, for example, using ultraviolet radiation, x-rays, gamma rays, fast or thermal neutrons or protons. The target population can then be screened to identify an OTUB1 mutant with reduced expression or activity.

In another embodiment of various aspects of the present invention, the method comprises mutagenesis of a plant population with a mutagen. The mutagen can be fast neutron irradiation or a chemical mutagen, for example, selected from the following non-limiting list: ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), N-ethyl-N-nitrosourea (ENU), triethylmelanin (1'EM), Nmethyl-N- nitrosourea (MNU), procarbazine, chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide monomer, melphalan, mustard gas nitrogen analogue, vincristine, dimethylnitrosamine, N-methyl-N'-nitro-nitrosoguanidine (MNNG), nitrosoguanidine, 2-aminopurine, 7.12 - dimethylbenz(a)anthracene (DMBA), ethylene oxide, hexamethylphosphoramide, bisulfan, diepoxyalkanes (diepoxyoctane (DEO), diepoxybutane (BEB), etc.), 2-methoxy-6-chloro-9[3-(ethyl- 2chloroethyl)aminopropylamino]acridine (ICR-170) or formaldehyde. And in this case, the target population can then be screened to identify the OTUB1 gene or promoter mutant.

In another embodiment of the invention, the method used to create and analyze mutations is the induction of local lesions in genomes by targeting (TILLING), described by Henikoff et al., 2004. In this method, seeds are subjected to mutagenesis with a chemical mutagen, for example, EMS. The resulting M1 plants are self-pollinating and the generation of M2 plants is used to obtain DNA samples for mutation screening. DNA samples are pooled and arrayed on microtiter plates and then subjected to gene-specific PNR. PCR amplification products can be screened for mutations in the target gene OTUB1 using any method that allows the identification of heteroduplexes between wild-type genes and mutant genes. Examples include, but are not limited to, denaturing high pressure liquid chromatography (dHPLC), constant denaturing capillary electrophoresis (CDCE), temperature gradient capillary electrophoresis (CTEG), or fragmentation by chemical digestion. Preferably, the PCR amplification products are incubated with an endonuclease that preferentially cleaves heteroduplex mismatches between wild-type and mutant sequences. The digests are subjected to electrophoresis on an automated gel sequencing machine and the gel images are analyzed using a standard commercially available image processing program. Any primer specific for the OTUB1 nucleic acid sequence can be used to amplify the OTUB1 nucleic acid sequence in the pooled DNA sample. Preferably, the primer is designed to amplify regions of the OTUB1 gene where appropriate mu

- 17 043050 mutations are likely to occur in particular in regions of the OTUB1 gene that are highly conserved and/or confer activity as described herein. To facilitate the detection of PCR products on the gel, the PCR primer can be labeled with any standard labeling method. In an alternative embodiment of the invention, the method used to generate and analyze mutations is EcoTILLING. EcoTILLING is a molecular method similar to TILLING, except that its purpose is to detect the natural variability of a given population compared to induced mutations. The EcoTILLING method was first described in Comai et al., 2004.

Thus, rapid high-throughput screening procedures allow the analysis of amplification products to identify a mutation conferring a reduction or inactivation of OTUB1 gene expression compared to the corresponding non-mutagenic wild-type plant. Once a mutation in a gene of interest has been identified, the seeds of an M2 plant carrying that mutation are cultured to produce adult M3 plants and screened for phenotypic traits associated with the OTUB1 target gene. Thus, loss and decrease in function in mutants with increased seed size compared to control can be identified.

Plants obtained or produced in this manner and carrying a functional mutation at an endogenous OTUB1 gene or promoter locus are also within the scope of the present invention.

In an alternative embodiment of the invention, the expression of the OTUB1 gene can be reduced at the level of transcription or translation. For example, expression of an OTUB1 nucleic acid sequence or an OTUB1 promoter as defined herein can be reduced or turned off using a number of gene silencing techniques known to those skilled in the art, such as, but not limited to, the use of short interfering nucleic acids. (kink) against OTUB1. The term gene silencing is a general term and means the suppression of gene expression through sequence-specific interactions that are mediated by RNA molecules. The degree of reduction can be adjusted to result in complete elimination of the production of the encoded gene product, but usually the elimination of expression is partial, ie. expression is preserved to some extent. Therefore, this term should not be construed as complete expression silencing.

In one embodiment, siNAs may include short interfering RNA (siRNA), double-stranded RNA (dsRNA), miRNA (miRNA), antagonists, and short hairpin RNA (shRNA) capable of mediating RNA interference.

Inhibition of expression and/or activity can be measured by determining the presence and/or amount of the OTUB1 transcript using methods well known to those skilled in the art (eg Northern blotting, RT-PCR, etc.).

Transgenes can be used to suppress endogenous plant genes. This was first discovered when chalcone synthase transgenes in petunia caused suppression of endogenous chalcone synthase genes, and this was observed by easily visible changes in pigmentation. Subsequently, many, if not all, plant genes have been described that can be silenced by transgenes. Gene silencing requires sequence similarity between the transgene and the gene that becomes silent. Such sequence homology may refer to promoter regions or coding regions of a silent target gene. If coding regions are present, then a transgene capable of causing gene silencing can be constructed together with a promoter that would initiate transcription of the coding RNA sequence in sense or antisense orientation. It is likely that different types of gene silencing are carried out by different mechanisms, which are not yet well understood. Various examples are transcriptional or post-transcriptional gene silencing, and both can be used in accordance with the methods of the invention.

The mechanisms of gene silencing and their application in genetic engineering, which were first discovered in plants in the early 1990s and later demonstrated in Caenorhabditis elegans, have been extensively described in the literature.

RNA-mediated gene suppression or RNA silencing according to the methods of the invention includes co-suppression wherein overexpression of the sense RNA or target mRNA, i.e. sense RNA or OTUB1 mRNA leads to a decrease in the level of expression of the genes in question. The RNA of the transgene and the homologous endogenous gene are suppressed together. Other methods used in the methods of the invention include the use of antisense RNA to reduce levels of endogenous target gene transcripts in a plant. In this method, RNA silencing does not affect the transcription of the gene locus, but only causes sequence-specific degradation of the target mRNA. The antisense nucleic acid sequence contains a nucleotide sequence that is complementary to the sense nucleic acid sequence encoding an OTUB1 protein or protein portion, i.e. complementary to the coding strand of the double-stranded cDNA molecule or complementary to the mRNA sequence of the transcript. The antisense nucleic acid sequence is preferably complementary to the endogenous OTUB1 gene, under

- 18 043050 Silencing. Complementarity may be present in the coding region and/or in the non-coding region of a gene. The term coding region means the region of a nucleotide sequence containing codons that are translated into amino acid residues. The term non-coding region refers to 5' and 3' sequences that flank the coding region and are transcribed but not translated into amino acids (also called 5' and 3' untranslated regions).

Antisense nucleic acid sequences can be designed according to the Watson and Crick base pairing rules. The antisense nucleic acid sequence may be complementary to the full length OTUB1 nucleic acid sequence as defined herein, but it may also be an oligonucleotide that is antisense for only a portion of the nucleic acid sequence (including the 5' and 3' UTRs of the mRNA). For example, the antisense oligonucleotide sequence may be complementary to the region surrounding the translation initiation site of the mRNA transcript encoding the polypeptide. The length of a suitable antisense oligonucleotide sequence is known to those skilled in the art and may be approximately 50, 45, 40, 35, 30, 25, 20, 15, or 10 nucleotides in length or less. The antisense nucleic acid sequence of the invention can be constructed by chemical synthesis and enzymatic ligation reactions using known methods. For example, an antisense nucleic acid sequence (e.g., an antisense oligonucleotide sequence) can be chemically synthesized using natural nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or increase the physical stability of the duplex formed between the antisense and sense nucleic acid sequences, for example, phosphorothioate derivatives and nucleotides substituted with acridine can be used. Examples of modified nucleotides that can be used to generate antisense nucleic acid sequences are well known to those skilled in the art. An antisense nucleic acid sequence can be produced biologically using an expression vector in which the nucleic acid sequence has been subcloned in an antisense orientation (i.e., RNA transcribed from an inserted nucleic acid will be in an antisense orientation with respect to the nucleic acid of interest - targets). Preferably, the production of antisense nucleic acid sequences in plants is carried out using a stably integrated nucleic acid construct comprising a promoter, an operably linked antisense oligonucleotide, and a terminator.

Nucleic acid molecules used for silencing in the methods of the invention hybridize or bind to mRNA transcripts and/or are inserted into the genomic DNA encoding the polypeptide, resulting in the inhibition of protein expression, for example, by inhibiting transcription and/or translation. Hybridization can be accomplished by standard nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid sequence that binds to DNA duplexes, through specific interactions in the main groove of the double helix. Antisense nucleic acid sequences can be introduced into a plant by transformation or direct injection into a specific tissue site. Alternatively, antisense nucleic acid sequences can be modified for selected target cells and then administered systemically. Thus, for example, for systemic administration, antisense nucleic acid sequences can be modified so that they specifically bind to receptors or antigens expressed on the surface of the selected cell, for example, by linking the antisense nucleic acid sequence to peptides or antibodies that bind to receptors or cell surface antigens. Antisense nucleic acid sequences can also be delivered to cells using vectors.

RNA interference (RNAi) is another post-transcriptional gene silencing phenomenon that can be applied in accordance with the methods of the invention. This interference is induced by double-stranded RNA, in which mRNA homologous to dsRNA undergoes specific degradation. This refers to the process of sequence-specific post-transcriptional gene silencing mediated by short interfering RNAs (siRNAs). The RNAi process begins when the DICER enzyme interacts with dsRNA and cuts it into fragments called short interfering RNAs (siRNAs). This enzyme belongs to the RNase III family of nucleases. The complex of proteins that combines these RNAs is preserved and serves as a code to search for and destroy any RNAs in the cell with the appropriate sequence, such as a target mRNA.

Artificial and/or natural microRNAs (miRNAs) can be used to knock out gene expression and/or mRNA translation. MicroRNAs (miRNAs) are typically single-stranded short RNAs that are typically 19-24 nucleotides in length. Most miRNA races

- 19 043050 shadows have perfect or almost perfect complementarity with their target sequences. However, there are natural targets with up to five mismatches. They are processed from longer non-coding RNAs with characteristic reverse fold structures via specific double-stranded RNases of the Dicer family. After processing, they are incorporated into the RNA-induced silencing complex (RISC) by binding to its main component, the Argonaute protein. miRNAs serve as components of RISC specificity as they base pair with target nucleic acids, mainly mRNAs in the cytoplasm. Subsequent regulatory events include cleavage and destruction of the target mRNA and/or inhibition of translation. Thus, the effects of miRNA overexpression are often observed when mRNA levels of target genes are reduced. Artificial microRNA (miRNA) technology has been applied to Arabidopsis thaliana and other plants to effectively silence target genes of interest. The principles for constructing miRNAs have been summarized and included in a software package available on the Web site (http://wmd.weigelworld.org).

Thus, in accordance with various aspects of the present invention, a plant can be transformed to introduce RNAi, shRNA, snRNA, dsRNA, siRNA, siRNA, takiRNA, siRNA, or co-suppression molecules that have been engineered to target expression of the OTUB1 nucleic acid sequence and selectively reducing or inhibiting gene expression or the stability of its transcript. Preferably, the RNAi, snRNA, dsRNA, shRNA, siRNA, siRNA, siRNA, ta-siRNA, or co-suppression molecule used in accordance with various aspects of the present invention comprises a fragment of at least 17 nucleotides, and preferably 22-26 nucleotides, and may be derived from the information provided in any of SEQ ID NOs: 1-12. Methods for constructing effective siRNAs are known to those skilled in the art. Briefly, a short fragment of the target gene sequence (for example, 19-40 nucleotides in length) was chosen as the target sequence of the siRNA according to the invention. A short sequence fragment of a target gene is an mRNA fragment of the target gene. In preferred embodiments of the invention, the criteria for selecting a sequence fragment from a target gene mRNA as a candidate for a siRNA molecule are: 1) a target gene mRNA sequence that has at least 50-100 nucleotides from the 5' or 3' end of the mRNA molecules, 2) a target gene mRNA sequence that has a G/C content ranging from 30 to 70%, and most preferably about 50%, 3) a target gene mRNA sequence that does not contain repeating sequences (e.g., AAA, CCC, GGG, TTT, AAAA, CCCC, GGGG, TTTT), 4) the mRNA sequence of the target gene, which is available in the mRNA, 5) the mRNA sequence of the target gene, which is unique to the target gene, 6) the exclusion of regions within 75 start codon bases. An mRNA sequence fragment of a target gene may meet one or more of the criteria listed above. The selected gene is entered as a nucleotide sequence into the prediction program, taking into account all the above variables used to design optimal oligonucleotides. This program scans any mRNA nucleotide sequence for regions susceptible to siRNA targeting. The result of this analysis is the evaluation of possible siRNA oligonucleotides. The highest scores are used to design double-stranded RNA oligonucleotides, which are usually prepared by chemical synthesis. Degenerate siRNA sequences can be used to target homologous regions other than the siRNA that is complementary to the target mRNA. siRNA according to the invention can be synthesized by any known method. The RNA is preferably chemically synthesized using suitably protected ribonucleoside phosphoramidites and a standard DNA/RNA synthesizer. In addition, siRNAs can be obtained from commercial suppliers involved in the synthesis of RNA oligonucleotides.

The siRNA molecules according to aspects of the present invention may be double stranded. In one embodiment, the double-stranded siRNA molecules contain blunt ends. In another embodiment, the double-stranded siRNA molecules contain overhangs (eg, 1-5 nucleotide overhangs, and preferably 2 nucleotide overhangs). In some embodiments, the siRNA is short hairpin RNA (shRNA); and the two strands of the siRNA molecule can be connected via a linker region (eg, a nucleotide linker or a non-nucleotide linker). The siRNAs of the invention may contain one or more modified nucleotides and/or non-phosphodiester linkages. Chemical modifications, well known to those skilled in the art, can increase the stability, availability, and/or uptake of the siRNA by the cell. The person skilled in the art may be aware of other types of chemical modification that can be included in the RNA molecules.

In one embodiment, the recombinant DNA constructs described in US Pat. No. 6,635,805, which is incorporated herein by reference, can be used.

The RNA molecule for silencing is introduced into the plant by standard methods, for example, by means of

- 20 043050 transformation mediated by the vector and Agrobacterium. Then, stably transformed plants are obtained and the expression of the OTUB1 gene is analyzed in comparison with the expression in the wild-type control plant.

Silencing the OTUB1 nucleic acid sequence or reducing its expression levels can also be achieved by virus-induced gene silencing.

Thus, in one embodiment, the plant expresses a nucleic acid construct comprising an RNAi, shRNA, snRNA, dsRNA, siRNA, siRNA, transactivating (ta) siRNA, siRNA, or cosuppression molecule that targets the OTUB1 nucleic acid sequence as described in of the present application, and which reduce the level of expression of the endogenous OTUB1 nucleic acid sequence. A gene is targeted if, for example, an RNAi, shRNA, snRNA, dsRNA, siRNA, siRNA, ta-siRNA, siRNA, or cosuppression molecule selectively reduces or inhibits the expression of the gene relative to that of a control plant. Alternatively, the RNAi, shRNA, snRNA, dsRNA, siRNA, siRNA, ta-siRNA, siRNA, or cosuppression molecule targets the OTUB1 nucleic acid sequence if the RNAi, shRNA, snRNA, dsRNA, siRNA, siRNA, ta-siRNA, siRNA, or cosuppression molecule cosuppressions hybridize under stringent conditions with the gene transcript. In one example, a plant expresses an RNAi-containing nucleic acid construct, wherein the RNAi sequence contains, or consists of, SEQ ID NO: 210 or a functional variant thereof as defined herein. The present invention also relates to the use of this RNAi-containing nucleic acid construct, wherein the RNAi sequence contains, or consists of, SEQ ID NO: 210 or a functional variant thereof, to reduce or abolish (and preferably reduce) the expression of OTUB1 and therefore increase grain harvest as described above.

Another way to achieve gene silencing is to target nucleic acid sequences that are complementary to the regulatory region of the OTUB1 gene (eg, promoter and/or enhancers) to form triple helical structures that prevent transcription of the gene in target cells. Other methods, such as the use of antibodies directed against the endogenous polypeptide to inhibit its function in planta, or the interference of a signal transduction pathway in which the polypeptide is involved, may be used, and these methods should be well known to the person skilled in the art. In particular, it is contemplated that artificial molecules can be used to inhibit the biological function of the target polypeptide or to interfere with a signal transduction pathway in which the target polypeptide is involved.

In one embodiment, the suppressor nucleic acids may be antisense suppressors of the expression of OTUB1 polypeptides. When using antisense sequences to inhibit gene expression, the nucleotide sequence is placed under the control of a promoter in reverse orientation so that transcription results in RNA complementary to the normal mRNA transcribed from the sense strand of the target gene.

The antisense suppressor nucleic acid may comprise an antisense sequence of at least 10 nucleotides derived from the target nucleotide sequence. It may be preferable that there be complete identity between the sequence used to inhibit expression of the target sequence and the target sequence itself, although overall sequence complementarity or similarity is not essential. One or more nucleotides may differ in the sequence used, derived from the target gene. Thus, the sequence used to inhibit the expression of a gene in accordance with the present invention may be a wild-type sequence (eg, a gene) selected from existing sequences or a variant of such a sequence.

The sequence optionally includes an open reading frame or is optionally specific for an RNA that can be translated. It may be preferable that the homology of the respective antisense and sense RNA molecules be sufficient for hybridization. Inhibition of gene expression can be observed even in the presence of approximately 5%, 10%, 15%, or 20% or more mismatches between the sequence used and the target gene. To achieve the desired effect, the homology must be sufficient to inhibit gene expression.

Suppressor nucleic acids can be operably linked to tissue-specific or inducible promoters. For example, coat and seed specific promoters can be used to specifically inhibit the OTUB1 nucleic acid in developing ovules and seeds to increase the final seed size.

A nucleic acid that represses expression of an OTUB1 polypeptide as described herein can be operably linked to a heterologous regulatory sequence such as a promoter, e.g., a constitutive promoter, an inducible promoter, a tissue-specific promoter, or a developmental stage-specific promoter. The construct or vector can be introduced into plant cells and expressed as described in this application. Plant cells, content

- 21 043050 such vectors are also within the scope of the present invention.

In another aspect, the present invention relates to a silencing construct obtained or obtained by the method described herein, and to a plant cell containing such a construct.

Thus, aspects of the present invention include methods of targeted mutagenesis, and in particular genome editing, and in a preferred embodiment of the invention, variants based only on growing plants by traditional breeding methods are excluded.

In yet another embodiment of the invention, the method may include reducing and/or abolishing OTUB1 activity. In one example, this method may include reducing the ability of OTUB1 to interact with UBC13 by reducing and/or abolishing its inhibition as described in this application and/or reducing the ability of OTUB1 to deubiquitinate SPL14, which leads to the accumulation of SPL14.

In another aspect, the present invention relates to a plant obtained or obtained by the method described here.

In another aspect, the present invention relates to a method of increasing the level of cell proliferation in the shell of the spikelet of a plant, preferably along the length of the grain and/or reducing the number of cells along the width of the grain, which will lead to a decrease in the length of the cells and to increase the width of the cells, where this method includes reducing or abolishing the expression of at least one nucleic acid encoding an OTUB1 polypeptide and/or reducing the activity of an OTUB1 polypeptide in said plant using any of the methods described herein. As used herein, the terms increase, improvement, or enhancement are synonymous. In one embodiment of the invention, cell proliferation is increased by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40 or 50% compared to the control plant.

Genetically altered or modified plants and methods for producing such plants.

In another aspect, the present invention relates to a genetically modified plant, part or cell thereof, characterized in that the plant does not express OTUB1, has reduced levels of OTUB1 expression, does not express a functional OTUB1 protein, or expresses an OTUB1 protein with reduced function and/or activity. For example, the plant is a down-function (knockdown) or loss-of-function (knockout) mutant, wherein the function of the OTUB1 nucleic acid sequence is reduced or absent compared to a control wild-type plant. Preferably, the plant has been knocked down, but not knocked out, which means that the plant has reduced levels of OTUB1 expression or expresses an OTUB1 protein with reduced function and/or activity. For this purpose, a mutation is introduced into the OTUB1 gene sequence or into the corresponding promoter sequence, which leads to disruption of gene transcription. Therefore, it is preferred that said plant contain at least one mutation in the promoter and/or in the OTUB1 gene. In one embodiment of the invention, the plant may contain a mutation in the promoter and in the OTUB1 gene.

In another aspect, the present invention relates to a plant, part or cell thereof, characterized by an increased seed yield compared to a wild-type plant or a control plant, where preferably the plant contains at least one mutation in the OTUB1 gene and/or promoter. Preferably, said increase in seed yield includes an increase in the number of grains, the number of grains per panicle, grain weight, grain width, grain thickness, thousand grain weight, and/or a decrease in grain length.

The plant can be obtained by introducing a mutation, preferably a deletion, insertion or substitution in the OTUB1 gene and/or promoter sequence by any of the methods described above. Preferably, said mutation is introduced into at least one plant cell and the plant is regenerated from at least one mutated plant cell.

Alternatively, the plant or plant cell may contain a nucleic acid construct expressing an RNAi molecule targeted to the OTUB1 gene described herein. In one example, the RNAi sequence contains, or consists of, SEQ ID NO: 210, or a variant thereof, as defined in this application. In one embodiment of the invention, said construct is stably incorporated into the plant genome. These methods also include gene targeting using vectors that target the gene of interest and allow integration of the transgene at a specific site. The targeting construct is designed to recombine with the target gene, which is achieved by including sequences of the gene itself in the construct. Recombination then occurs in the region of this sequence in the gene, which leads to the insertion of a foreign sequence and to disruption of the gene. If the sequence is interrupted, the altered gene will be translated into a non-functional protein, if it is translated at all.

In another aspect, the present invention relates to a method for producing a genetically modified plant as described herein. In one embodiment of the invention, the method comprises introducing at least one mutation in the OTUB1 gene and/or the OTUB1 promoter, preferably in at least one plant cell using any of the mutagenesis methods described herein.

- 22 043050

Preferably, said method further comprises regenerating a plant from a mutated plant cell.

The method may further include selecting one or more mutated plants preferably for further propagation. Preferably, said selected plants contain at least one mutation in the OTUB1 gene and/or promoter sequence. Preferably, these plants are characterized by the absence or reduced level of expression of OTUB1 and/or a reduced level of activity of the OTUB1 polypeptide. The levels of expression and/or activity of OTUB1 can be determined by any standard method known to those skilled in the art. In one embodiment of the invention, the level of deubiquitinase activity of OTUB1 can be determined. The reduction is as described in this application.

The selected plants can be propagated in various ways, for example by clonal propagation or classical breeding methods. Thus, for example, the first generation (or T1) of a transformed plant can self-pollinate, and selected homozygous second generation (or T2) transformants and T2 plants can then be further propagated by classical breeding methods. The resulting transformed plants can take various forms. For example, they may be chimeras of transformed cells and non-transformed cells; clonal transformants (eg, all cells transformed to contain an expression cassette); grafts of transformed and non-transformed tissues (eg, in plants, a transformed root shoot is grafted onto a non-transformed cutting).

In another aspect, the present invention relates to a plant obtained or obtainable by the methods described above.

In accordance with the present invention, a genetically modified plant or mutant plant is a plant that has been genetically modified from a natural wild-type (WT) plant. In one embodiment, the mutant plant is a plant that has been modified from a natural wild-type (WT) plant using a mutagenesis method, such as any of the mutagenesis methods described herein. In one embodiment of the invention, the mutagenesis method is targeted genome modification or genome editing. In one embodiment, the plant genome has been modified from wild-type sequences by mutagenesis. Such plants have the modified phenotype described above, such as increased seed yield. Thus, in this example, the increased seed yield is due to the presence of a modified plant genome, such as a mutated endogenous OTUB1 gene sequence or OTUB1 promoter. In one embodiment of the invention, an endogenous promoter or gene sequence is specifically targeted by targeted genome modification, and the presence of the mutated gene or promoter sequence is independent of the presence of transgenes expressed in the plant. In other words, a genetically modified plant can be described as a plant that does not contain the transgene.

A plant according to various aspects of the invention, including transgenic plants, methods and uses described herein, may be a monocot or dicot plant. The preferred plant is an agricultural crop. The term crop means any plant that is grown commercially for human or animal consumption or use. In a preferred embodiment of the invention, the plant is a cereal crop. In another embodiment, the plant is Arabidopsis.

In the most preferred embodiment of the invention, the plant is selected from rice, wheat, corn, barley, cabbage such as bok choy, soybeans and sorghum. In one example, the wheat is wild spelt. In another example, the plant is rice, preferably japonica or indica. In another embodiment, the plant contains a dep-1 mutant allele, or a functional variant or homologue thereof. Preferably, the plant contains or expresses (endogenously) a nucleic acid sequence comprising or consisting of SEQ ID NO: 156 or 158 which encodes a polypeptide as defined in SEQ ID NO: 157 or 159.

As used herein, the term plant encompasses whole plants, their ancestors and progeny, and parts of plants, including seeds, fruits, shoots, stems, leaves, roots (including tubers), flowers, tissues, and organs, where each of the aforementioned parts includes the nucleic acid construct described herein. . The term plant also encompasses plant cells, suspension cultures, callus tissue, embryos, meristems, gametophytes, sporophytes, pollen, and microspores, in which case each of the aforementioned parts includes the nucleic acid construct described here.

The present invention also relates to harvested plant parts according to the invention described in this application, including, but not limited to, seeds, leaves, fruits, flowers, stems, roots, rhizomes, tubers and bulbs. Aspects of the present invention also relate to products obtained, and preferably directly obtained, from the harvested part of such

- 23 043050 plants, such as, for example, dry granules or powders, oils, fats and fatty acids, starch or proteins. Another product that can be obtained from harvested plant parts according to the invention is biodiesel. The present invention also relates to food products and food supplements containing the plant according to the invention or parts thereof. In one embodiment of the invention, the food may be animal feed. In another aspect, the present invention relates to a product obtained from the plant described here or part thereof.

In the most preferred embodiment of the invention, the plant part or harvested product is a seed or grain. Therefore, in another aspect, the present invention relates to seeds obtained from the genetically modified plant described here. In an alternative embodiment of the invention, the plant part is the pollen, seedling, or progeny of the genetically modified plant described herein. Accordingly, in another aspect, the present invention relates to pollen, seedling or progeny of the genetically modified plant described here.

The control plant used here in accordance with all aspects of the present invention is a plant that has not been modified by the methods of the invention. Accordingly, in one embodiment, the control plant does not have reduced OTUB1 nucleic acid expression and/or reduced OTUB1 polypeptide activity. In an alternative embodiment of the invention, the plant has been genetically modified as described above. In one embodiment of the invention, the control plant is a wild-type plant. The control plant is usually a plant of the same species, and preferably a plant having the same genetic background as the modified plant.

Genome editing constructs for use in targeted genome modification methods described herein.

crRNA or CRISPR RNA means an RNA sequence that contains a protospacer element and additional nucleotides complementary to tracrRNA.

tracr-RNA (transactivating RNA) means an RNA sequence that hybridizes with srRNA and binds to a CRISPR enzyme such as Cas9 and thereby activates a nuclease complex to introduce double-strand breaks at specific sites in the genomic sequence of at least one nucleic acid sequence or the OTUB1 promoter.

A protospacer element means a portion of a crRNA (or ssrRNA) complementary to the target DNA genomic sequence, which is typically about 20 nucleotides in length. This element may also be known as a spacer or targeting sequence.

sssrRNA (single-stranded guide RNA) means the combination of tracr-RNA and cr-RNA in one RNA molecule, preferably also including a linker loop (which links tracr-RNA and cr-RNA into one molecule). ssrRNA may also be referred to as rRNA, and in this context, these terms are synonymous. ssrRNAs or rRNAs provide targeting specificity and have the ability to scaffold and bind to Cas nuclease. rRNA can mean a double RNA molecule containing a crRNA molecule and a tracrRNA molecule.

TAL effector (Transcription activator-like effector (TAL)) or TALE refers to a protein sequence that can bind to a target DNA genomic sequence (sequence in the OTUB1 gene or promoter sequence) and that can be attached to a cleavage domain of an endonuclease such as Fokl to form effector nucleases TAL or TALEN or meganuclease to create megaTAL. The TALE protein consists of a central domain that is responsible for DNA binding; a nuclear localization signal and a domain that activates transcription of the target gene. The DNA binding domain is made up of monomers, and each monomer can bind to one nucleotide in the target nucleotide sequence. The monomers are tandem repeats of 33-35 amino acids, of which the two amino acids present at positions 12 and 13 are highly variable (two repeat variable residues, RVD). It is these RVDs that are responsible for the recognition of one specific nucleotide. HD targets cytosine; NI targets adenine; NG targets thymine and NN targets guanine (although NN can also bind to adenine with lower specificity).

In another aspect, the present invention relates to a nucleic acid construct, wherein the nucleic acid construct comprises a nucleic acid sequence that encodes at least one DNA binding domain. In one embodiment, the DNA binding domain may bind to a sequence in the OTUB1 gene and/or promoter. Preferably said sequence is selected from SEQ ID NOs: 28, 34, 38, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93 , 96, 99, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142, and 146. In this example, SEQ ID NOs: 28 (rice), 34 (rice), 38 (rice ), 102 (wheat), 106 (wheat), 110 (barley), 114 (barley), 118 (corn), 122 (maize), 126 (sorghum), 130 (sorghum), 134 (soybean), 138 (soybean ), 142 (cabbage) and 146 (cabbage) are target sequences in the OTUB1 gene, a SEQ ID NOs: 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78 , 81, 84, 87, 90, 93, 96 and 99 are target sequences in the OTUB1 promoter, and

- 24 043050 preferably in a rice promoter. In one embodiment, the nucleic acid construct contains one or more DNA binding domains, and therefore, such a construct can bind to one or more, and preferably at least two or three sequences in the OTUB1 gene, where these sequences are selected from:

(i) SEQ ID NOs: 28 (rice), 34 (rice) and 38 (rice);

(ii) SEQ ID NO: 102 (wheat) and 106 (wheat);

(iii) SEQ ID NO: 110 (barley) and 114 (barley);

(iv) SEQ ID NO: 118 (corn) and 122 (corn);

(v) SEQ ID NO: 126 (sorghum) and 130 (sorghum);

(vi) SEQ ID NO: 134 (soy) and 138 (soy); or (vii) SEQ ID NOs: 142 (cabbage) and 146 (cabbage).

In an alternative or additional embodiment, the nucleic acid construct contains one or more DNA binding domains, where one or more DNA binding domains can bind to at least one sequence selected from SEQ ID NO: 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96 and 99.

In another embodiment of the invention, said construct also contains a nucleic acid encoding an SSN, such as a Fokl or Cas protein.

In one embodiment of the invention, the nucleic acid construct encodes at least one protospacer element, wherein the sequence of the protospacer element is selected from SEQ ID NOs: 29, 35, 39, 43, 46, 49, 52, 55, 58, 61, 64, 67, or variants thereof In one example, the nucleic acid construct may contain one, two, or three protospacer sequences, where the protospacer sequence is selected from:

(i) SEQ ID NOs: 29 (rice), 35 (rice) and 39 (rice);

(ii) SEQ ID NO: 103 (wheat) and 107 (wheat);

(iii) SEQ ID NO: 111 (barley) and 115 (barley);

(iv) SEQ ID NO: 119 (corn) and 123 (corn);

(v) SEQ ID NO: 125 (sorghum) and 131 (sorghum);

(vi) SEQ ID NO: 135 (soy) and 139 (soy); or (vii) SEQ ID NOs: 143 (cabbage) and 147 (cabbage).

In another embodiment, the nucleic acid construct contains a crRNA coding sequence. As defined above, the crRNA sequence may contain protospacer elements as defined above, and preferably additional nucleotides complementary to the tracrRNA. The corresponding sequence of additional nucleotides will be known to those skilled in the art as these sequences are determined by the choice of the Cas protein.

In another embodiment, the nucleic acid construct also contains a tracr RNA sequence. And in this case, the corresponding tracr-RNA sequence will be known to those skilled in the art, since this sequence is determined by the choice of the Cas protein. However, in one of the embodiments of the invention, the specified sequence contains, or consists of it, the sequence defined in SEQ ID NO: 30, or its variant.

In another embodiment of the invention, the nucleic acid construct contains at least one nucleic acid sequence that encodes sssrRNA (or rRNA). Again, as already discussed, the ssrRNA usually contains a crRNA or protospacer sequence and a tracrRNA sequence, and preferably a linker loop sequence. In a preferred embodiment of the invention, the nucleic acid construct comprises at least one nucleic acid sequence that encodes an sssrRNA sequence as defined in any of SEQ ID NOs: 31, 36, 40, 44, 47, 50, 53, 56, 59, 62, 65 68 71 74 77 80 83 86 89 92 95 98 101 104 108 112 116 120 124 128 132 136 140 144 and 148 or her version.

In another embodiment, the nucleic acid construct comprises or consists of a sequence selected from SEQ ID NOs: 33, 37, 41, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145 and 149.

In another embodiment, the nucleic acid construct may also comprise at least one nucleic acid sequence encoding an endoribonuclease cleavage site. Preferably the endoribonuclease is Csy4 (also known as Cas6f). If a nucleic acid construct contains multiple ssrRNA nucleic acid sequences, then the construct may include the same number of endoribonuclease cleavage sites. In another embodiment, the cleavage site is located at the 5' end of the ssrRNA nucleic acid sequence. Accordingly, each ssrRNA nucleic acid sequence is flanked by an endoribonuclease cleavage site.

The term variant means a nucleotide sequence where the nucleotides are substantially identical to one of the above sequences. The variant can be obtained by modifying

- 25 043050 cations, such as insertion, substitution or deletion of one or more nucleotides. In a preferred embodiment of the invention, the variant is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to any of the above sequences. In one embodiment of the invention, the sequence identity is at least 90%. In another embodiment of the invention, the sequence identity is 100%. Sequence identity can be determined using any sequence alignment program known to those skilled in the art.

The present invention also relates to a nucleic acid construct comprising a nucleic acid sequence operably linked to a suitable plant promoter. A suitable plant promoter may be a constitutive or strong promoter, or may be a tissue-specific promoter. In one embodiment, suitable plant promoters are selected from, but not limited to, Cestrum yellow leaf curl virus (CmYLCV) promoter or millet ubiquitin 1 (PvllbM) promoter, wheat CaMV35S RNA polymerase III U6 (TaU6), wheat U6 promoters or ubiquitin maize (eg Ubi1). Alternatively, expression can be specifically targeted to specific wheat seed tissues via gene expression control sequences. In one embodiment, the promoter is selected from the U3 promoter (SEQ ID NO: 163), the U6a promoter (SEQ ID NO: 164), the U6b promoter (SEQ ID NO: 165), the dicot U3b promoter (SEQ ID NO: 166) and the U6-1 promoter in dicot plants (SEQ ID NO: 167).

The nucleic acid construct of the invention may also further comprise a nucleic acid sequence that encodes a CRISPR enzyme. CRISPR enzyme refers to a DNA endonuclease which is under the control of RNA and which can associate with the CRISPR system. In particular, such an enzyme binds to the tracr-RNA sequence. In one embodiment, the CRIPSR enzyme is a Cas protein (CRISPR-associated protein), preferably Cas9 or Cpf1, and more preferably Cas9. In a particular embodiment, Cas9 is codon-optimized Cas9 (optimized for the plant in which it is expressed). In one example, Cas9 has the sequence described in SEQ ID NO: 150, or a functional variant or homologue thereof. In another embodiment, the CRISPR enzyme is a protein from the X class 2 candidate protein family, such as C2c1, C2C2, and/or C2c3. In one embodiment, the Cas protein is from Streptococcus pyogenes. In an alternative embodiment, the Cas protein may be derived from any of Staphylococcus aureus, Neisseria meningitides, Streptococcus thermophiies, or Treponema denticoia.

The term functional variant as used herein, when referring to Cas9, means a sequence variant of the Cas9 gene, or a portion of a gene sequence that retains the biological function of the full-length unmodified sequence, such as acting as a DNA endonuclease, or recognizing and/or binding to DNA. A functional variant also includes a variant of the gene of interest that has sequence modifications that do not affect function, such as non-conservative residues. The term also encompasses a variant that is substantially identical to the wild-type sequences provided herein, i.e. it has only some modifications in the sequence, for example, in non-conservative residues, and is biologically active. In one embodiment of the invention, the overall identity of the functional variant of SEQ ID NO: 150 and the amino acid sequence shown in SEQ ID NO: 150 is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% . In another embodiment, the Cas9 protein has been modified to increase activity.

Suitable homologs or orthologs can be identified by sequence comparison and identification of conserved domains. The function of a homologue or orthologue can be identified as described in this application, and thus, a person skilled in the art can confirm the function if it is expressed in a plant.

In another embodiment, the Cas9 protein has been modified to increase activity. Thus, for example, in one embodiment of the invention, the Cas9 protein may contain the amino acid substitution D10A, and this nickase only cleaves a DNA strand that is complementary to and recognized by rRNA. In an alternative embodiment of the invention, the Cas9 protein may alternatively or additionally contain the amino acid substitution H840A, and this nickase only cleaves a DNA strand that does not interact with ssRNA. In this embodiment, Cas9 can be used in conjunction with an ssrRNA pair (i.e., two molecules) (or with a construct expressing such a pair) and as a result it can cleave the target region on the opposite side of the DNA, with a possible increase in specificity in 100-1500 times. In another variant

- 26 043050 embodiment of the invention, the Cas9 protein may contain a substitution D1135E. The Cas9 protein may also be a VQR variant. Alternatively, the Cas9 protein may contain a mutation in both nuclease domains, and therefore, HNH and RuvCOIike are catalytically inactive. Rather, instead of cleaving the target strand, this catalytically inactive Cas protein can be used to prevent the transcriptional elongation process, which would result in the loss of function of incompletely translated proteins if they are co-expressed with the ssrRNA molecule. An example of a catalytically inactive protein is inactivated Cas9 (dCas9) induced by a point mutation in the RuvC and/or HNH nuclease domains (Komor et al., 2016 and Nishida et al., 2016).

In another embodiment, a Cas protein, such as Cas9, can also be coupled to a repression effector, such as a histone/DNA methylating enzyme or cytidine deaminase (Komor et al. 2016), to effect site-directed mutagenesis. In the latter case, the enzyme cytidine deaminase does not induce dsDNA breaks, but mediates the conversion of cytidine to uridine, and thereby induces the substitution of C for T (or G for A). These methods may be particularly suitable for targeting glutamine and proline residues in gliadins and for destroying toxic epitopes while maintaining the functional properties of gliadin.

In another embodiment, the nucleic acid construct contains an endoribonuclease. Preferably, the endoribonuclease is Csy4 (also known as Cas6f), and more preferably codon-optimized csy4, such as defined in SEQ ID NO: 168. In one embodiment, if the nucleic acid construct contains the Cas protein, then the nucleic acid construct may contain sequences for expression of an endoribonuclease such as Csy4 expressed as a 5' fusion of P2A (used as a self-cleaving peptide) with a Cas protein such as Cas9, for example as defined in SEQ ID NO: 169.

In one embodiment, the Cas protein, endoribonuclease, and/or endoribonuclease-Cas fusion sequence may be operably linked to a suitable plant promoter. Suitable plant promoters have already been described above, but in one embodiment, they may be the maize ubiquitin 1 promoter.

Suitable methods for producing nucleic acid systems and CRISPR vectors are known and, for example, are described in Molecular Plant (Ma et al., 2015, Molecular Plant, DOI:10.1016/j.molp.2015.04.007), which is introduced herein by links.

In an alternative aspect of the invention, the nucleic acid construct comprises at least one nucleic acid sequence that encodes a TAL effector, wherein said effector targets an OTUB1 gene and/or promoter sequence selected from SEQ ID NOs: 28, 34, 38, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142 and 146. Techniques for constructing a TAL effector are well known in the art and are performed depending on the target sequence. Examples of suitable methods are described in Sanjana et al., and Cermak T et al., which are incorporated herein by reference. Preferably, said nucleic acid construct contains two nucleic acid sequences encoding a TAL effector to form a TALEN pair. In another embodiment of the invention, the nucleic acid construct also contains a sequence-specific nuclease (SSN). Preferably, such an SSN is an endonuclease such as Fokl. In another embodiment of the invention, TALEN is constructed by Golden Gate cloning in a single plasmid construct or nucleic acid construct.

In another aspect, the present invention provides an ssrRNA molecule, wherein the ssrRNA molecule comprises a crRNA sequence and a tracrRNA sequence, and wherein the crRNA sequence can bind to at least one sequence selected from SEQ ID NOs: 28, 34, 38, 42, 45, 48, 51, 54, 57, 60, 63, 66, 69, 72, 75, 78, 81, 84, 87, 90, 93, 96, 99, 102, 106, 110, 114, 118, 122, 126, 130, 134, 138, 142 and 146 or its variant. In one embodiment, the nucleic acid sequence of the ssrRNA molecule is defined in any one of SEQ ID NOs: 31, 36, 40, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95, 98, 101, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144, and 148, or variants thereof. In other words, the ssrRNA RNA sequence is encoded by a nucleic acid sequence selected from SEQ ID NOs: 31, 36, 40, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83 , 86, 89, 92, 95, 98, 101, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144 and 148. In only one example, the RNA sequence of one ssrRNA according to the invention is defined in SEQ ID NO: 32 or its variant. The term variant is defined in this application. In one embodiment, the sssrRNA molecule may contain at least one chemical modification, for example, a modification that increases the stability and/or binding affinity of the target sequence or crRNA sequence to the tracrRNA sequence. Such modifications are well known to those skilled in the art and include, for example, but not limited to, the modifications described in Rahdar et al., 2015, which is incorporated herein by reference. In this example, the crRNA may contain a phosphorothioate backbone modification such as 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me) and Ssteric ethyl (cET) substitutions.

In another aspect, the present invention relates to an isolated nucleic acid sequence that encodes a protospacer element (as defined in any of SEQ ID NOs: 29, 35, 39, 43, 46, 49, 52, 55, 58, 61, 64, 67, 70, 73, 76, 82, 85, 88, 91, 94, 97, 100, 103, 107, 111, 115, 119, 123, 127, 131, 135, 139, 143, and 147) or ssrRNA (described in any of SEQ ID NOs: 31, 36, 40, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83, 86, 89, 92, 95 , 98, 101, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144 and 148).

In another aspect, the present invention relates to a plant or part thereof, or at least one isolated plant cell transfected with at least one nucleic acid construct described herein. Cas9 and ssrRNA may be combined or may be in separate expression vectors (or nucleic acid constructs, and these terms are synonymous). In other words, in one embodiment of the invention, the isolated plant cell is transfected with a single nucleic acid construct comprising sssrRNA and Cas9, as detailed above. In an alternative embodiment of the invention, the isolated plant cell is transfected with two nucleic acid constructs, i.e. a first nucleic acid construct comprising at least one ssrRNA as defined above, and a second nucleic acid construct comprising Cas9 or a functional variant or homologue thereof. The second nucleic acid construct may be transferred before, during, or after the transfer of the first nucleic acid construct. An advantage of a separate second construct containing a cas protein is that a nucleic acid construct encoding at least one sssRNA can pair with any type of cas protein described here, and therefore the protein is not limited to the function of a single Cas (as might be be the case when both cas and ssrRNA are encoded by the same nucleic acid construct).

In one embodiment, a nucleic acid construct comprising a cas protein is first transfected and stably integrated into the genome, followed by a second transfection with a nucleic acid construct comprising at least one sssRNA nucleic acid. In an alternative embodiment of the invention, the plant or part thereof, or at least one isolated plant cell, is transfected with mRNA encoding the cas protein and co-transfected with at least one nucleic acid construct as defined herein.

The Cas9 expression vectors used in the present invention can be constructed as described in the literature. In one example, the expression vector contains a nucleic acid sequence as defined in SEQ ID NO: 150, or a functional variant or homologue thereof, wherein said nucleic acid sequence is operably linked to a suitable promoter. Examples of suitable promoters are the actin promoter, CaMV35S, wheat U6 or maize ubiquitin (eg Ubi1).

In an alternative aspect, the present invention relates to an isolated plant cell transfected with at least one nucleic acid construct or ssrRNA molecule described herein.

In another aspect, the present invention relates to a genetically modified or edited plant containing the transfected cell described here. In one embodiment of the invention, the nucleic acid construct or constructs may be integrated in a stable form. In an alternative embodiment of the invention, the nucleic acid construct or constructs are not integrated (ie, they are transiently expressed). Accordingly, in a preferred embodiment of the invention, the genetically modified plant does not contain any nucleic acids encoding sssrRNA and/or Cas protein. In other words, this plant does not contain the transgene.

As used herein, the terms introduction, transfection, or transformation encompass the transfer of an exogenous polynucleotide into a host cell, regardless of the method of such transfer. Plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, can be transformed with the genetic construct of the invention and a whole plant regenerated from this tissue. The particular tissue selected will vary depending on the clonal propagation systems available and most suitable for transformation into particular species. Representative target tissues are leaf discs, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, meristem tissue present (eg, apical meristem, axillary buds, and root meristems), and induced meristem tissue (eg, cotyledon and hypocotyl meristems). The resulting transformed plant cell can then be used to regenerate the transformed plant in a manner known to those skilled in the art. The transfer of foreign genes into the plant genome is called transformation. Plant transformation of many species is now a routine technique. Any of several transformation methods known to those skilled in the art can be used to introduce a nucleic acid construct or ssrRNA molecule of interest into a suitable ancestor cell. The methods described for the transformation and regeneration of plants from plant tissues or cells can be applied to transient or stable transformation.

- 28 043050

Transformation methods include the use of liposomes, electroporation, the use of chemicals that increase free DNA uptake, injection of DNA directly into the plant (microinjection), gene firing (or the use of bioballistic particle delivery systems (bioballistics)) as described in the examples, lipofection, transformation with using viruses or pollen and microculturing. Methods may be selected from methods using calcium/polyethylene glycol for protoplasts; transfection of genes mediated by ultrasound; optical or laser transfection; transfections using silicon carbide fibers; protoplast electroporation; microinjection into plant material; bombardment with particles coated with DNA or RNA; infection with (non-integrating) viruses, and the like. Transgenic plants can also be obtained using Agrobacterium tumefaciens mediated transformation, including but not limited to the Agrobacterium flower dip method/vacuum infiltration method, as described in Clough & Bent (1998), which is incorporated herein by reference.

Accordingly, in one embodiment of the invention, at least one nucleic acid construct or ssrRNA molecule described in this application can be introduced into at least one plant cell by any of the methods described above. In an alternative embodiment of the invention, any nucleic acid construct described herein can be first transcribed to form a pre-assembled Cas9-sssrRNA ribonucleoprotein and then delivered to at least one plant cell by any of the methods described above, such as lipofection, electroporation, or microinjection.

In order to select transformed plants, the plant material resulting from the transformation process is usually, but not necessarily, subjected to selection under selective conditions so that the transformed plants can be distinguished from non-transformed plants. Thus, for example, seeds obtained in the above manner may be sown and, after an initial period of growth, subjected to suitable selection by spraying. Another possibility is to cultivate the seeds after sterilization, if necessary, on agar plates using a suitable selection agent, so that plants can only grow from the transformed seeds. As described in the examples, a suitable marker may be the phosphinothricin or PPT resistance bar gene. Alternatively, transformed plants are screened for the presence of a selectable marker such as, but not limited to, GFP, GUS (β-glucuronidase). Other examples may be well known to the person skilled in the art. Alternatively, no selection is made and the seeds obtained in the above manner are sown and cultivated, and then the expression levels of OTUB1 or protein are determined at the appropriate time period by standard methods described in the literature. This alternative method, which avoids the introduction of transgenes, is preferred for producing transgene-free plants.

After DNA transfer and regeneration, putative transformed plants can also be evaluated, for example, by PCR to detect the presence of the gene of interest, copy number and/or genomic organization. Alternatively or additionally, levels of integration and expression of the newly introduced DNA can be monitored by Southern, Northern/or Western blot analysis, and these methods are well known to those skilled in the art.

The resulting transformed plants can be propagated in various ways, for example by clonal propagation or classical breeding methods. Thus, for example, the first generation (or T1) of a transformed plant can self-pollinate, and selected homozygous second generation (or T2) transformants and T2 plants can then be further propagated by classical breeding methods.

In another related aspect, the present invention also relates to a method for obtaining a genetically modified plant, described in this application, where this method includes:

a. selection of a plant part;

b. transfection of at least one cell of the plant part according to paragraph (a) with at least one nucleic acid construct described herein, or at least one ssrRNA molecule described herein, by the transfection or transformation methods described above;

c. regenerating at least one plant derived from the transfected cell or transfected cells;

d. selection of one or more plants obtained in accordance with paragraph (c) in which silencing or a reduced level of expression of OTUB1 is observed.

In another embodiment of the invention, the method also includes the step of screening the genetically modified plant for SSN (preferably CRISPR)-induced mutations in the OTUB1 gene or promoter sequence. In one embodiment, the method includes obtaining a DNA sample from a transformed plant and amplifying the DNA to detect a mutation in at least one OTUB1 gene or promoter sequence.

In another embodiment of the invention, the methods include obtaining stable T2 plants that are preferably homozygous for the mutation (i.e., for a mutation in at least

- 29 043050 one sequence of the OTUB1 gene or promoter).

Plants that have a mutation in at least one OTUB1 gene or promoter sequence can also be crossed with another plant also containing at least one mutation in at least one OTUB1 gene and/or promoter sequence to produce plants having additional mutations in the OTUB1 gene or promoter sequence. Combinations will be apparent to those skilled in the art. Accordingly, this method can be used to produce T2 plants with mutations in all or most of the homologues compared to the number of homologue mutations in a single T1 plant transformed as described above.

The plant obtained or obtainable by the methods described above is also within the scope of the present invention.

A genetically modified plant according to the invention can also be obtained by transferring any sequences according to the invention by crossing, for example, using the pollen of the genetically modified plant described here to pollinate a wild-type plant or control plant, or to pollinate the gynoecium of plants described here with another pollen that does not contain mutations in at least one of the sequences of the OTUB 1 gene or promoter. Methods for producing plants according to the invention are not limited solely to the methods described in this paragraph, for example, genetic transformation of germ cells from an ear of wheat can be carried out as mentioned above, but without subsequent regeneration of the plant.

Method for screening plants for natural phenotypes of increased grain yield.

In another aspect, the present invention relates to a method for screening a plant population and identifying and/or selecting a plant that will have a reduced expression of OTUB1 and/or an increased grain yield phenotype, and preferably an increase in the number of grains, the number of grains per panicle, grain mass, grain width , grain thickness, thousand-kernel weight, and/or reduction in grain length (compared to a control or wild-type plant), wherein said method comprises detection in the plant or plant germplasm of at least one polymorphism (preferably a polymorphism with a low level of expression of OTUB1 ) in the OTUB1 gene promoter or in the OTUB 1 gene. Preferably, said screening comprises determining the presence of at least one polymorphism, wherein said polymorphism is at least one insertion and/or at least one deletion and/or substitution.

In one particular embodiment of the invention, said polymorphism may include at least one of the following substitutions:

C to T at position 1135 SEQ ID NO: 2 or 5;

G to C at position 1462 SEQ ID NO: 2 or 5; and/or

G to C at position 1798 SEQ ID NO: 2 or 5.

In another additional or alternative embodiment of the invention, said polymorphism is the insertion of one amino acid, and preferably T, at position 2234 of SEQ ID NO: 2 or 5.

In another additional or alternative embodiment of the invention, said polymorphism may be a G to A substitution at position 1824 of SEQ ID NO: 155.

As a result, the plants described above will have an increased grain yield phenotype as described above.

Suitable tests for evaluating the presence of polymorphisms are well known to those skilled in the art and include, but are not limited to, isozyme electrophoresis, restriction fragment length polymorphism (RFLP), randomly amplified polymorphic DNA (RAPD), polymerase chain reaction using random selected primers (AR-PCR), DNA amplification by fingerprinting (DAF), sequence-characterized amplified regions (SCAR), amplified fragment length polymorphism (AFLP), simple repeat sequence (SSR, also called microsatellites), and a single nucleotide polymorphism (SNP) test. In one embodiment of the invention, allele-specific genotyping by competitive binding PCR (KASP) is used.

In one embodiment of the invention, the method includes:

a) obtaining a nucleic acid sample from the plant, and

b) performing nucleic acid amplification of one or more alleles of the OTUB1 promoter using one or more primer pairs.

In another embodiment of the invention, the method may also include introgression of a chromosomal region containing at least one of these polymorphisms that expresses OTUB1 at a low level, or a chromosomal region containing a repetitive sequence with a deletion as described above, in a second plant or plant germplasm to obtain a plant or germplasm of an introgressive plant. Preferably, OTUB1 expression in

- 30 043050 said second plant was reduced or eliminated (compared to a control plant or a wild type plant), and more preferably, said second plant gave a greater grain yield, and preferably gave an increase in at least the number of grains, the number of grains per panicle, grain weight, grain width, grain thickness, thousand grain weight, and/or reduction in grain length.

In one embodiment, the plant may endogenously express SEQ ID NO: 2 or 4 and the levels of OTUB1 nucleic acid and/or OTUB1 protein activity have been reduced or further reduced by any of the methods described herein.

Accordingly, in another aspect, the present invention relates to a method for increasing yield, and preferably seed or grain yield, of a plant, said method comprising:

A. screening the plant population for at least one plant with at least one of the polymorphisms described above;

b. further reducing or abolishing the expression of at least one OTUB1 nucleic acid and/or reducing the activity of an OTUB1 polypeptide in said plant by introducing at least one mutation in the nucleic acid sequence encoding OTUB1 or at least one mutation in the OTUB1 promoter described herein, as described in this application or using the RNA interference described here.

Further reduction means a decrease in the expression level of OTUB1 to a level lower than that of a plant with at least one of the above described OTUB1 polymorphisms. The term reduction means a decrease in the levels of expression and/or activity of OTUB1 by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% compared to the level in the control plant.

UBC13.

The inventors of the present invention have also unexpectedly found that increasing the expression level of the ubiquitin E2 binding protein, UBC13, produces a phenotype similar to that observed in plants carrying the npt1 allele, such as an increase in the number of grains, a thousand grain mass, a stem diameter, and a decrease in the number shoots. Accordingly, overexpression of UBC13 will also result in increased grain yield. Therefore, in another aspect, the present invention relates to a method of modifying, and preferably increasing the levels of at least one transcription factor like a protein that binds to the SQUAMOSA promoter (SBP domain), which method includes increasing the level of expression or activity of UBC13 described here or reduction or abolition of the expression or activity of OTUB1 described here. In one embodiment, the transcription factor for the SBP domain is SPL14.

The terms increase, improvement, or enhancement are used interchangeably herein. In one embodiment of the invention, the grain length increases by at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40 or 50% compared to the control plant. Preferably, such an increase is at least 5-50%.

Accordingly, in another aspect, the present invention relates to a nucleic acid construct comprising a nucleic acid sequence encoding a polypeptide defined in SEQ ID NO: 161 or a functional variant or homologue thereof, wherein said sequence is operably linked to a regulatory sequence, wherein said preferred regulatory sequence is is a tissue-specific or constitutive promoter. In another embodiment, the nucleic acid construct comprises a nucleic acid sequence as defined in SEQ ID NO: 160 (cDNA) or 162 (genomic) or a functional variant or homologue thereof. The functional variant or homologue is as defined above.

As used herein, operably linked refers to a functional link between a promoter sequence and a gene of interest, and therefore, the promoter sequence is capable of initiating transcription of the gene of interest.

A plant promoter includes regulatory elements that mediate the expression of a coding sequence segment in plant cells. Accordingly, a plant promoter need not be derived from a plant, i. e. it can come from viruses or microorganisms, such as viruses that attack plant cells. A plant promoter can also be derived from a plant cell, for example from a plant that has been transformed with a nucleic acid sequence expressed by the method of the invention and described herein. This also applies to other plant regulatory signals such as plant terminators. Promoters upstream of the nucleotide sequences used in the methods of the invention may be modified by one or more nucleotide substitutions, insertions and/or deletions without affecting the functionality or activity of either the promoters or the open reading frame (ORF) or 3' regulatory region. , such as terminators or other 3'regulatory regions located at a distance from the ORF. The activity of promoters can also be increased by modifying their sequence, or by completely replacing them with more active promoters and even promoters derived from heterologous organisms. For expression in

- 31 043050 plants, the nucleic acid molecule must be, as described above, functionally attached to a suitable promoter or contain a suitable promoter that expresses the gene at the right time and with the desired spatial expression pattern. As used herein, operably linked refers to a functional link between a promoter sequence and a gene of interest such that the promoter sequence is capable of initiating transcription of the gene of interest.

In one embodiment of the invention, the promoter is a constitutive promoter. A constitutive promoter means a promoter that is transcriptionally active in most, but not necessarily all, phases of growth and development, and under most environmental conditions, in at least one cell, tissue, or organ. Examples of constitutive promoters include, but are not limited to, actin, HMGP, CaMV19S, GOS2, rice cyclophilin, maize histone H3, alfalfa histone H3, 34S FMV, Rubisco small subunit, OCS, SAD1, SAD2, NOS, V-ATPase, superpromoter, G-box proteins and synthetic promoters.

In another aspect, the present invention relates to a vector containing the above-described nucleic acid sequence.

In another aspect, the present invention relates to a host cell containing a nucleic acid construct. The host cell may be a bacterial cell such as Agrobacterium tumefaciens or an isolated plant cell.

The present invention also relates to a culture medium or a kit containing a culture medium and an isolated host cell, as described below.

In another embodiment, the present invention relates to a transgenic plant expressing the nucleic acid construct described above. In one embodiment of the invention, said nucleic acid construct is stably incorporated into the plant genome.

The nucleic acid sequence is introduced into said plant in a manner referred to as transformation, as described above.

The resulting transformed plants can be propagated in various ways, for example by clonal propagation or by classical breeding methods. Thus, for example, the first generation (or T1) of a transformed plant can self-pollinate, and selected homozygous second generation (or T2) transformants and T2 plants can then be further propagated by classical breeding methods. The resulting transformed plants can take various forms. Thus, for example, they may be chimeras of transformed cells and N-transformed cells; clonal transformants (eg, all cells transformed to contain an expression cassette); grafts of transformed and non-transformed tissues (eg, in plants, a transformed root shoot is grafted onto a non-transformed cutting).

A suitable plant is defined above.

In another aspect, the present invention relates to the use of a nucleic acid construct as described herein to increase grain yield, and preferably increase the number of grains and/or thousand grains.

In another aspect, the present invention relates to a method for increasing the yield of grain, and preferably increasing the number of grains and/or the weight of a thousand grains, which method includes the introduction of the nucleic acid construct described herein into the specified plant and its expression in the specified plant.

In another aspect, the present invention relates to a method for producing a plant with an increased grain yield, and preferably an increased number of grains and/or an increased mass of a thousand grains, where this method includes the introduction of the nucleic acid construct described herein into the specified plant and its expression in the specified plant.

This increase is considered compared to a control plant or a wild type plant.

Although the above disclosure is a general description of the subject matter of the invention, which is within the scope of the present invention, including methods, as well as the best way to carry out, develop and use this invention, however, the following examples are provided for a better understanding of the method of carrying out the invention and for more complete it. descriptions. However, one skilled in the art will appreciate that the specific details provided in these examples should not be construed as limiting the invention, the scope of which is to be set forth in the claims and in the equivalents appended to this disclosure. Various other aspects and embodiments of the present invention will be apparent to those skilled in the art from the disclosure of the present invention.

The compound union and/or used here should be considered as a specific disclosure of each of the two indicated features or components, taken together or separately from each other. Thus, for example, A and/or B should be taken as a specific disclosure of each of (i) A; (ii) B and (iii) A and B, just as if each of them were separately defined in this description.

- 32 043050

Unless it conflicts with the context of the invention, the description and definition of the above features is not limited to any particular aspect or embodiment of the invention and is equally applicable to all aspects and embodiments of the invention described herein.

The application described above and all serial numbers of documents and sequences cited therein at the time of their consideration (documents cited in the application), and all documents cited or mentioned in the documents cited here, and all documents cited or mentioned here (documents cited here), and all documents cited or referenced in the documents cited herein, together with any manufacturer's instructions, description, product specification, and listing of any products mentioned herein, or products mentioned in any document incorporated herein by reference, are incorporated herein by reference, and may be used for the practical implementation of the invention. More specifically, all cited documents are introduced by reference, as if each individual document were specifically and separately included in the present description by reference.

The present invention is described in the following non-limiting examples.

Example 1

Achieving an increase in grain yield has long been the focus of cereal breeding programs. The authors have shown that the qNPT1 rice yield quantification locus, which acts by defining a new plant type (NPT) architecture characterized by fewer barren shoots, stronger stems, and larger panicles, encodes a deubiquitinizing enzyme homologous to human OTUB1. Inhibition of OsOTUB1 enhances meristem activity, which leads to a decrease in the number of shoots per plant, an increase in the number of grains per panicle, an increase in grain mass, and, as a result, an increase in grain yield. OsOTUB1 interacts with OsUBC13 and with the SBP domain transcription factor OsSPL14 and restricts Lys63-bound ubiquitin in OsSPL14 to regulate proteasome-dependent destruction. Conversely, loss of OsOTUB1 function results in high levels of OsSPL14 accumulating, which in turn regulates NPT architecture and increases grain yield. Pyramidizing the high yield alleles nptl and dep1-1 provides a new strategy to increase the potential yield of rice, which may exceed current levels.

A set of 670 recombinant inbred lines (RIL) was developed by crossing rice cv. japonica Chunjiang06 and a selected NPT line (IR66167-27-5-1-6), and RIL52 was selected from this set based on its NPT phenotype. , responsible for the increase in the number of grains, the decrease in the number of shoots, and the thickening of the stem (Fig. 1a-1e). Subsequent QTL analysis identified the qNPT1 locus (new type 1 plants) as pleiotropically responsible for all three traits (Fig. 1f). Positional cloning of qNPT1 was performed using BC ₂ F ₂ and BC2F3 progeny from a backcross between RIL52 and indica Zhefu802 (recurrent parent). The candidate region was narrowed down to a ˜4.1 kb segment flanked by P139 and P143 markers that contain the promoter and part of the gene coding sequence at LOC_Os08g42540 (Fig. 1g). This gene was predicted to encode a deubiquitinizing enzyme homologous to human OTUB1 (ubiquitin aldehyde binding protein containing ovarian tumor domain 1) (FIG. 5), i.e. a protein associated with regulation of p53 stability and repair of DNA damage ^9-12 . Based on this, LOC_Os08g42540 is hereinafter referred to as OsOTUB1. Sequence comparison revealed five nucleotide variants in which the alleles responsible for NPT differed from those of the standard phenotype (Fig. 1h), three of which had a single nucleotide polymorphism (SNP), one had an insertion-deletion polymorphism (Indel) in the intron sequence, and one had a SNP in the promoter region.

Then, the authors obtained a near-isogenic line (NIL) ZH11-npt1, which contains a segment of ~240 kb, including the npt1 allele from IR66167-27-5-1-6 in the initial culture of rice japonica Zhonghua11 (ZH11) ( Fig. 6). Quantitative RT-PCR analysis showed that a peak of OsOTUB1 transcript excess was found in the shoot meristem and young panicles (Fig. 2a). Histochemical analyzes for GUS targeting the OsOTUB1 promoter showed that this gene is highly expressed in vascular tissue, as well as in root cap cells and resting central cells (QC) (Fig. 7). The OsOTUB1 transcript excess in ZH11-npt1 was lower than in ZH11 (Fig. 2a). The knockout phenotype of OsOTUB1 generated using CRISPR/Cas9 ¹³ was similar to the ZH11-npt1 phenotype, resulting in a decrease in shoot number, an increase in kernel number, an increase in grain mass and a subsequent increase in grain yield (Fig. 6). In transgenic plants carrying the pOsOTUB1::OsOTUB1-GFP construct, the GFP signal was detected both in the nucleus and in the cytoplasm of root sheath and leaf sheath cells (Fig. 8). Based on a projected database annotated with descriptions of rice plants, the OsOTUB1 gene was predicted to generate two alternatively spliced transcripts (Fig. 1g). The phenotype of the transgenic ZH11-npt1 expressing OsOTUB1.1 cDNA induced by its native promoter was similar to that of ZH11, while the OsOTUB1.2 overexpressing phenotype was similar to that of ZH11-npt1 (Fig. 6). In addition, transgenic

- 33 043050 ZH11 plants overexpressing OsOTUB1.1 were dwarfed, had fewer grains, and had leaf necrosis (FIG. 9). These results indicate that the loss of function of OsOTUB1 is associated with an ideotypical architecture.

Haplotype analysis showed that the npt1 allele was not used by breeders of elite indica cultivars and japonica cultivars of temperate latitudes (Fig. 1h). Since the high-yielding allele dep1-1 has been extensively used by Chinese breeders ^14,15 it was interesting to evaluate the effect of combining npt1 and dep1-1. NILs for allelic combinations of the qNPT1 and qDEP1 loci were generated in the elite rice cultivar japonica Wuyunjing7 (which contains the NPT1 and dep1-1 alleles, hereinafter referred to as WYJ7-NPT1-dep1-1). WYJ7-npt1-dep1-1 and WYJ7-NPT1-dep1-1 plants did not differ from each other in head panicle formation time and plant height (Figs. 2b, 2e and 2f), while WYJ7-npt1-dep1-1 plants produced fewer shoots and had more heavier grains than WYJ7-NPT1-dep1-1 (Fig. 2g-2j). The shoot tip meristems produced by WYJ7-npt1-dep1-1 plants were larger than those of WYJ7NPT1-dep1-1 (Fig. 2k), indicating that npt1 and dep1-1 act synergistically to increase meristem activity ⁵ . WYJ7-npt1-dep1-1 plants also showed more vascular bundles, thicker stalk, and thicker parenchyma and sclerenchyma cell walls (Fig. 2l-2n). Importantly, for three consecutive seasons, the total grain yield of WYJ7-npt1-dep1-1 plants was 10.4% greater than that of WYJ7-NPT1-dep1-1 plants (Fig. 2o). Therefore, pyramiding of the npt1 and dep1-1 alleles is an effective means of increasing the grain yield of rice.

Given the predicted deubiquitinase activity of OsOTUB1, the authors investigated its ability to cleave linear Lys48- and Lys63-bound tetra-ubiquitin. Consistent with the behavior of the human homologue, it exhibited high cleavage activity when Lys48-linked tetraubiquitin was present ^16,17 but unlike OTUB1, it also exhibited a moderate level of activity if it was present in Lys63-linked forms (Fig. 10 ). ZH11-npt1 plants expressing OTUB1 or its orthologues derived from mouse, maize or barley and regulated by the OsOTUB1 promoter showed a phenotype not dissimilar to that of ZH11 or transgenic ZH11-npt1 plants expressing OsOTUB1.1 (Fig. 11), allowing suggest that OTUB1 orthologs from all these species are functionally interchangeable. OTUB1 is known to interact with UBC13 resulting in inhibition of double-strand break-induced chromatin ubiquitination ^9,10 and the same in vitro and in vivo interactions have been established between OsOTUB1 and OsUBC13 (Fig. 12). OsUBC13 overexpression produced a phenotype similar to that of plants carrying the npt1 allele, at least in terms of grain number, shoot number, and stem diameter (FIG. 12). Conversely, constitutive RNAi expression directed to OsUBC13 produced a phenotype resembling that of plants in which the OsOTUB 1.1 cDNA was overexpressed (FIG. 9 and FIG. 12).

Yeast screening for the presence of two targeting proteins interacting with OsOTUB1 revealed interactions of 72 candidates that included a rice homologue consisting of a transcription factor like the SQUAMOSA promoter-binding protein (SBP domain) OsSPL14, which is known to regulate plant architecture in in relation to the reduction in the number of shoots, the thickening of the stem and the increase in the number of grains ^18.19 . Bimolecular fluorescence (BiFC) complementarity assays and co-immunoprecipitation assays showed that the OsSPL14-OsOTUB1 interaction clearly occurs inplanta (FIGS. 3a, 3b). Deletion analysis showed that a conserved SBP domain is both necessary and sufficient for in vitro and in vivo interactions (Fig. 3a and Fig. 13). Further analyzes of BiFC showed that OsOTUB1 was able to interact with the full array of rice SPL transcription factors (Fig. 14), consistent with data indicating that excess transcripts of OsSPL7, OsSPL13, OsSPL14, and OsSPL16 correlate with one or more of the traits: reduction in shoot number , increasing the number of grains or increasing the mass of the grain ^18-23 . The presence of the npt1 or OsSPL14 ^WFP allele associated with a high level of the OsSPL14 transcript ¹⁹ was shown to generate the NPT architecture, and the phenotype of ZH11-npt1 plants in which OsSPL14 was silencing by RNAi was similar to that of ZH11 plants (Fig. 3c). -3h). Comparative transcriptomic analysis based on RNA sequencing of ZH11, ZH11-npt1 and ZH11-OsSPL14 ^WFP showed that transcript levels of 453 common target genes were higher in ZH11-npt1 and in ZH11-OsSPL14 ^WFP than in ZH11 (Fig. 15). and this result was confirmed with the help of col. RT-PCR ^22-24 Conversely, the number of target genes assessed was significantly reduced in the case of OsSPL14 with ZH11-npt1 silencing (FIG. 15). Thus, OsOTUB1 and OsSPL14 act antagonistically and regulate plant architecture through the regulation of common target genes.

EMSA analyzes indicated that the OsOTUB1-OsSPL14 interaction is unlikely to affect the binding affinity of OsSPL14 to its GTAC targeting motifs (FIG. 16). There was no difference in the amount of OsSPL14 transcript between ZH11 and ZH11-npt1 (Fig. 3e), but accumulation of OsSPL14 was much higher in the latter genotype (Fig. 4a). In the case of treatment with the proteasome inhibitor MG132, accumulation of OsSPL14 was clearly higher in ZH11 (Fig. 4b). These results suggest that OsOTUB1, in contrast to OTUB1, which regulates the stability of p53 and SMAD proteins ^11,12

- 34 043050 promotes the degradation of OsSPL14. If lysates obtained from young ZH11 panicles were exposed to GST-OsSPL14, the GST-OsSPL14 titer decreased over time, and in the case of treatment with MG132, the opposite effect was observed (Fig. 4c). The stable accumulation of GST-OsSPL14 was greater when lysates were obtained from ZH11-npt1 panicles but not from ZH11, while the degradation of GSTOsSPL14 was accelerated in lysates formed from ZH11-npt1 panicles in the presence of His-OsOTUB1 (Fig. 4c). Western blot analysis showed that it was possible to detect the presence of polyubiquitinated forms of the Myc-OsSPL14 immunoprecipitate isolated from young panicles using an antibody that recognized total ubiquitin or Lys48-polyubiquitin or Lys63polyubiquitin conjugates (Fig. 4d). This suggests that OsSPL14 is modulated by Lys48- and Lys63-related ubiquitination ²⁵ .

This assay was also performed to investigate the ubiquitination of OsSPL14 mediated by the endogenous E3 ligase. In the presence of wild type ubiquitin, MG132 treatment of rice protoplasts expressing Flag-OsSPL14 resulted in increased accumulation of polyubiquitinized FlagOsSPL14; however, in the presence of K48R ubiquitins, MG132 treatment did not result in any significant accumulation of polyubiquitinized Flag-OsSPL14 (Fig. 4e). This observation is consistent with the notion that Lys48-bound ubiquitin OsSPL14 is required for proteasome-mediated degradation. In the presence of Myc-OsOTUB1.1, the titer of polyubiquitinized Flag-OsSPL14 clearly decreased in the case of K48R or K63O mutations, but remained unchanged in the presence of K48O or K63R mutations (Fig. 4f). In addition, Myc-OsOTUB1 stimulated the degradation of Flag-OsSPL14 only in the presence of wild-type ubiquitin, K48O- or K63R-linked ubiquitin (Fig. 4f), indicating that OsSPL14 stabilization correlates with Lys48-linked ubiquitinization. Overall, these results suggest that OsOTUB1-mediated inhibition of Lys48-related ubiquitination of OsSPL14 is required for its proteasome-dependent degradation.

SBP-domain transcription factors targeting miR156 play an important role in the regulation of stem cell function and flowering in plants ^26-28 . The non-canonical OsOTUB1-mediated regulation of SBP-domain transcription factors provides a new basis for studying the fate of meristem cells, inflorescence architecture, and flower development. The results obtained by the authors shed light on the molecular basis of the ideatypic approach in rice breeding programs, and manipulation of the OsOTUB1-OsSPL14 module also represents a potential strategy to facilitate the breeding of new rice varieties with higher grain yields.

Plant materials and cultivation conditions. A set of 670 RIL populations was developed by crossing the Chinese temperate rice cultivar japonica Chunjiang06 and breeding NPT IR66167-27-5-1-6. The rice samples used for sequence diversity analysis have been described in other publications ^14,21 . NIL plants carrying either npt1, ZH11-OsSPL14 ^WFP19,29 or allelic combinations of the qNPT1 and qDEP1 loci were bred by crossing RIL52 seven times with Zhonghua11 or Wuyunjing7. Plants grown under irrigated conditions were planted 20 cm apart and grown during the standard growing season at three experimental stations: one in Lingshui (Hainan Province), one in Hefei (Anhui Province) and one in Beijing.

transgenic constructs. OsOTUB1.1 and OsOTUB1.2 encoding sequences, their UTRs (5': from transcription initiation site to -2.8 kb; 3': 1.5 kb downstream of termination site), was amplified from ZH11 genomic DNA (gDNA) and introduced into the pCAMBIA2300 (CAMBIA) vector to give pOsOTUB1::OsOTUB1.1 and pOsOTUB1::OsOTUB1.2. Full-length human OTUB1 cDNA (and its mouse, barley, and maize orthologues) was amplified from the corresponding template cDNA and then subcloned into the pActin::nos ¹⁴ vector, while the OsOTUB1.1 cDNA and its 5'-UTR were inserted into the p35S::GFP vector -nos ²¹ to get pOsOTUB1::OsOTUB1.1-GFP-nos construct. To obtain p35S::MycOsSPL14, OsSPL14 cDNA was amplified from the ZH11 cDNA template and cloned into p35S::Myc-nos. The rRNA constructs required for CRISPR/Cas9 knockout of OsOTUB1 were obtained as described elsewhere ¹³ . A 300 bp OsSPL14 cDNA fragment and a 300 bp OsUBC13 cDNA fragment were amplified from the ZH11 cDNA and used to construct the pActin::RNAi-OsSPL14 and pActin::RNAiOsUBC13 transgenes, as described elsewhere ¹⁴ . Transgenic rice plants were obtained by Agrobacterium-mediated transformation as previously described ⁵ . The corresponding primer sequences are shown in FIG. 17.

Quantitative real-time PCR analysis (q.RT-PCR). Total RNA was extracted from plant tissues using the TRIzol reagent (Invitrogen) and treated with RNase-free DNase I (Invitrogen) according to the manufacturer's protocol. The resulting RNA was subjected to reverse transcription using a cDNA synthesis kit (TRANSGEN). Subsequent co-RTPCR was performed as described elsewhere ²¹ , including three independent RNA preparations as biological duplicates. Rice Actinl was used as a reference. The corresponding primer sequences are shown in FIG. 17.

Yeast two-hybrid system assays. Yeast two-hybrid system assays were performed as described elsewhere ^14,21 . Full-length OsOTUB1.1 cDNA and C-terminal frag

- 35 043050 ment OsOTUBl was amplified from ZH11 cDNA and inserted into pGBKT7 (Takara Bio Inc.), while full-length OsUBC13 cDNA and the C-terminal fragment of OsSPL14 were inserted into pGADT7 (Takara Bio Inc.). Each of these plasmids was confirmed by sequencing followed by transformation into yeast strain AH109. Necessary β-galactosidase assays were performed according to the manufacturer's protocol (Takara Bio Inc.). Cells carrying either empty pGBKT7 or empty pGADT7 were used as negative controls. The entire OsOTUB1 sequence or C-terminal fragment was used as bait to screen a cDNA library prepared from pol·y(A)-containing RNA isolated from young rice panicles (<0.2 cm long). Experimental screening and plasmid isolation procedures were performed according to the manufacturer's protocol (Takara Bio Inc.). The corresponding primer sequences are shown in FIG. 17.

BiFC analyses. Full-length cDNAs of OsOTUB1.1, OsUBC13, OsSPL1-OsSPL13, and OsSPL15OsSPL19, together with their deletion and non-deletion variants of OsSPL14, were amplified from the ZH11 cDNA, and the amplicons were inserted into the vectors pSY-735-35S-cYFP-HA or pSY-736-35S- nYFP-EE ³⁰ to create a series of hybrid designs. Two vectors used to test protein-protein interactions (eg, nYFP-OsSPL14 and cYFP-OsOTUB1) were co-transfected into rice protoplasts. After incubation in the dark for 14 hours, the YFP signal was assessed and photographs taken under a confocal microscope (Zeiss LSM710) as described elsewhere ³¹ . Each BiFC assay was repeated at least three times. The corresponding primer sequences are shown in Figure 17.

Inhibition in vitro. The recombinant fusion protein GST-OsOTUB1 was immobilized on spheres with glutathione-sepharose and incubated with His-OsUBC13 for 30 minutes at 4°C. Glutathione Sepharose spheres were washed three times and eluted with elution buffer (50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0). The supernatant was subjected to immunoblot analysis using anti-His and anti-GST antibodies (Santa Cruz).

Coimmunoprecipitation and Western blotting. Myc-OsSPL14 was extracted from young panicles (<0.2 cm long) of transgenic ZH11 plants bearing pActin::Myc-OsSPL14 using a buffer consisting of 50 mM HEPES (pH 7.5), 150 mM KCl, 1 mM EDTA , 0.5% Triton-X 100, 1 mM DTT, and proteinase inhibitor cocktail (Roche LifeScience, Basel, Switzerland). Then, agarose-conjugated anti-Myc antibodies (Sigma-Aldrich) were added and the reaction mixture was left at least 4 hours at 4°C, and then washed ~5-6 times with TBS-T buffer and eluted 2x with loading buffer. Immunoprecipitates and lysates were subjected to SDS-PAGE and the separated proteins were transferred to a nitrocellulose membrane (GE Healthcare). Myc-OsSPL14 fusion proteins were detected by membrane probing with an anti-Myc antibody (Santa Cruz), and its polyubiquitinated forms were detected by probing with antibodies that recognize common ubiquitin conjugates; antibodies that specifically recognize Lys48-polyubiquitin conjugates; or antibodies that specifically recognize Lys63-polyubiquitin conjugates (Abcam).

OsSPL14 degradation analysis. Lysates obtained from young panicles (<0.2 cm long) harvested from ZH11 and ZH11-npt1 plants were incubated with the respective recombinant GST-OsSPL14 fusion protein in the presence or absence of the recombinant His-OsOTUB1 fusion protein. Protein was extracted from lysates that were untreated or treated with 50 μM MG132 for predetermined time periods and subjected to SDS-PAGE and anti-GST Western blotting (Santa Cruz). As a loading control, HSP90 excess was determined by probing with an anti-HSP90 antibody (BGI). Lysis buffer contains 25 mM Tris-HCl (pH 7.5), 10 mM NaCl, 10 mM MgCl ₂ , 4 mM PMSF, 5 mM DTT and 10 mM ATP as described elsewhere ³² .

Cleavage assays for linear K48- and K63-linked tetra-ubiquitin. An aliquot of ~1 µg of recombinant GST-OsOTUB1.1, GST-OsOTUB1.2 or OTUB1 was added to 20 µl of 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 0.5 mM dithiothreitol containing 2.5 µg of linear K48- and K63-linked tetraubiquitin (Boston Biochem) and left for 1 hour at 37°C. The reaction products were analyzed by western blot analysis based on an anti-ubiquitin antibody (Abcam) as described elsewhere ¹⁶ .

In vitro ubiquitination assay. Rice protoplasts derived from young ZH11npt1 panicles (<0.2 cm long) were transfected with plasmids pUC19-35S-Flag-OsSPL14-RBS (33) and pUC19-35SHA-Ubiq-RBS (HA-labeled ubiquitin (wild-type), K48R (with K48 replaced with arginine), K63R (with K63 replaced with arginine), K48O (ubiquitin with K48 only, with other lysine residues replaced with arginine) or K63O (ubiquitin with K63 only with other lysine residues replaced with arginine) in the presence of or in the absence of plasmid pUC19-35S-Myc-OsOTUB1-RBS After 15 hours, protoplasts were lysed in extraction buffer [50 mM Tris-HCl (pH 7.4), 150 mM KCl, 1 mM EDTA, 0.5% Triton- x 100, 1 mM DTT] containing a cocktail of proteinase inhibitors (Roche Life Science).The resulting lysates were treated with agarose-conjugated anti-Flag antibodies (Sigma-Aldrich) for at least 4 hours at 4°C, and then 5- Washed 6 times in extraction buffer and eluted with 3xFlag peptide (Sigma-Aldrich) Immunoprecipitates were separated by SDS-PAGE and

- 36 043050 was transferred to a nitrocellulose membrane (GE Healthcare) which was used for Western blot analysis using anti-HA and anti-Flag conjugate antibodies (Sigma-Aldrich).

Example 2

Results.

The wtg1-1 mutant produces wide, thick, short, and heavy grains.

To determine the grain size of rice, the authors mutagenized japonica Zhonghuajing (ZHJ) with γ-irradiation, and isolated a mutant with a wide and thick grain 1 (wtg1-1) in M2 populations. The wtg1-1 grains were wider than the ZHJ grains (Fig. 18a, b, e). The grain width ZHJ was 3.41 mm, while the grain width wtg1-1 was 3.84 mm (Fig. 18e). Conversely, the grain length of wtg1-1 was shorter than that of ZHJ (Fig. 18a, b, d). The ZHJ grain length was 7.47 mm, while the wtg1-1 grain length was 6.89 mm. The wtg1-1 grains were clearly thicker than the ZHJ grains (Fig. 18c). The average grain thicknesses of ZHJ and wtg1-1 were 2.27 mm and 2.66 mm, respectively (FIG. 18f). Importantly, the wtg1-1 grains were significantly heavier than the ZHJ grains (Fig. 18g). The weight of 1000 grains of ZHJ and wtg1-1 was 25.56 g and 29.09 g, respectively. These results showed that WTG1 affects grain width, grain thickness and length, and grain mass.

The wtg1-1 mutant increases the number of grains per panicle.

Mature wtg1-1 plants were slightly shorter than wild-type plants (Fig. 19a, b, f). The wtg1-1 plants had broader leaves compared to the ZHJ plants, while the wtg1-1 leaf length was similar to that of the ZHJ leaves (Fig. 19c, d, g, h). The panicles of wtg1-1 were short, thick and dense compared to those of the wild-type plant, indicating an erect panicle phenotype (Fig. 19e, i). The length of the wtg1-1 panicle axis was shorter compared to the wild-type panicle axis (Fig. 19j), indicating that WTG1 influences the panicle size. Since the panicle branches determine the structure and shape of the panicle, the authors studied the primary and secondary panicle branches of the wild type and wtg1-1. Wtg1-1 panicles had more primary and secondary branches than ZHJ (Fig. 19k, l). The authors estimated the number of grains per panicle in ZHJ and wtg1-1. As shown in FIG. 2M, the number of grains per panicle in wtg1-11 was higher than in ZHJ. Thus, these data showed that an increased number of primary and secondary panicle branches in wtg1-1 leads to an increase in the number of grains per panicle.

Identification of the WTG1 gene.

The authors attempted to identify the wtg1-1 mutation using the MutMap method (Abe et al., 2012), which was used to clone rice genes. The authors crossed wtg1-1 with ZHJ and obtained the F2 population. In the F2 population, offspring segregation has shown that one recessive mutation determines wtg1-1 phenotypes. The authors extracted DNA from fifty F2 plants that showed wide and thick grain phenotypes, and the same amount of DNA was mixed for whole genome sequencing. The authors also sequenced ZHJ as a control. 6.2 Gbp and 5.4 Gbp short reads were obtained for ZHJ and pooled F2 plants, respectively. The authors detected 1399 SNPs and 157 INDELs between the combined F2 and ZHJ. We then calculated the SNP/INDEL ratio in pooled F2 plants. Whereas all mutant plants in the F ₂ population must have a causal SNP/INDEL, the SNP/INDEL ratio for this causal mutation in the pooled F ₂ plants must be 1. Among them, only INDELs show a SNP/INDEL ratio of 1. These INDELs contain a 4 bp deletion in the gene. (LOC_Os08g42540) (Figs. 20a, 23). The authors also confirmed this deletion in wtg1-1 by displaying the dCAPS1 marker (Fig. 20b). Thus, these analyzes suggest that LOC_Os08g42540 may represent the WTG1 gene.

To confirm that WTG1 is the LOC_Os08g42540 gene, a genetic complementarity test was performed by the authors. Genomic fragment containing 2337 p. 5' flanking sequence, gene LOC_Os08g42540 and 1706 bp. The 3' flanking sequence (gWTG1) was transformed into the wtg1-1 mutant. The gWTG1 construct complemented the phenotypes of the wtg1-1 mutant (Fig. 20f-k). The grain width, grain thickness, and grain length of transgenic plants gWTG1;wtg1-1 were comparable with those of ZHJ. Therefore, this genomic complementarity experiment confirmed that the WTG1 gene is LOC_Os08g42540.

WTG1 encodes an otubain-like protease with deubiquitinizing activity.

The WTG1 gene encodes an unknown protein with the predicted otubain domain (Fig. 20d). Homologues of WTG1 have been found in Chlamydomonas reinhardtii, Physcomitrella patents, Selaginella moellendorffii, and other plant and animal species (Fig. 24). Homologues in plants of the grass family (eg, wheat, Brachypodium, corn, and sorghum) have high amino acid sequence identity with WTG1 (FIG. 24). WTG1 also shares similarities with human otubain 1/2 (OTUB1/2) proteins (Fig. 25), which are involved in multiple physiological and developmental processes (Nakada et al., 2010, Herhaus et al., 2013). In wtg1-1, a 4 bp deletion observed at the junction of the exonintron of the fourth intron (Fig. 20a). The wtg1-1 mutation results in a change in WTG1 splicing, which in turn results in the premature appearance of a stop codon (Fig. 20c). The protein encoded by the wtg1-1 allele lacks half of the predicted otubain domain and also contains completely

- 37 043050 related peptide (FIG. 20e), suggesting that wtg1-1 is a loss of function allele.

In animals, otubain proteins are known to have deubiquitinizing activity because they contain histidine, cysteine, and aspartate residues in the conserved catalytic domain of cysteine proteases (Fig. 25) (Balakirev et al., 2003). WTG1 resembles human otubain proteins and has an otubain-like domain (Fig. 20d, 25), suggesting that WTG1 may be an otubain-like protease. Therefore, the authors carefully examined the amino acid sequence of WTG1 and found that WTG1 contains conserved aspartate (D68), cysteine (C71) and histidine (H267) residues in the predicted otubain domain, which define the ubiquitin protease catalytic triad in animals (Fig. 20d, 25) (Balakirev et al., 2003). The authors then determined whether WTG1 had deubiquitinizing activity. The authors expressed WTG1, WTG1 ^wtg1 ' ¹ encoded by the wtg1-1 allele and WTG1 ^d68e ' ^C71S ' ^h267R , with mutations in conserved amino acids, as proteins labeled with MBP (maltose binding protein) (MBP-WTG1, MBP-WTG1 ^{wtg1- 1} and WTG1 ^d68e ' ^C71S ' ^h267R ), and UBQ10 (hexameric polyubiquitin) as His-tagged protein (His-UBQ10). Deubiquitination assays showed that MBP-WTG1 was able to cleave His-UBQ10, while MBP-WTG1 ^wtg1 ' ¹ , MBP-WTG1 ^{d68E;C71S;H267R} and MBP did not cleave His-UBQ10 (Fig. 20l). These results showed that WTG1 has deubiquitinizing activity, WTG1 ^wtg1 ' ¹ does not have deubiquitinizing activity, and conserved aspartate (D68), cysteine (C71), and histidine (H267) residues play an important role in communicating the deubiquitinizing activity of WTG1. Thus, WTG1 is an otubain-like protease with deubiquitinizing activity.

Expression and subcellular localization of WTG1.

The authors evaluated the expression of WTG1 using quantitative real-time RT-PCR analysis. As shown in FIG. 21a, the WTG1 gene is expressed in leaves, in developing panicles, and in roots. The authors obtained transgenic plants with the WTG1:GUS promoter (proWTG1:GUS) and studied the patterns of WTG1 expression in various tissues. We observed GUS staining in leaves and roots of young proWTG1:GUS seedlings (Fig. 21b). GUS staining in developing panicles was also detectable (Fig. 21d). GUS activity in younger panicles was stronger than in older panicles (Fig. 21d), consistent with the function of WTG1 influencing the number of panicle branches. During the development of the spikelet coat, WTG1 expression started at the tips and then spread throughout the spikelet coat and finally disappeared at later stages of development. Therefore, the WTG1 expression pattern supports its functions in the development of the panicle and spikelet rind.

Then the authors obtained transgenic pro35S:GFP-WTG1 plants and investigated the subcellular localization of WTG1. As shown in Figures 5e-g, the GFP signal in pro35S:GFP-WTG1 roots was predominantly found in nuclei. The authors also tried to find out whether GFP-WTG1 can be localized exclusively in the nucleus. The authors obtained cytoplasmic and nuclear protein fractions from pro35S:GFP-WTG1 transgenic plants. Surprisingly, GFP-WTG1 fusion proteins were found to be present in both the nuclear fraction and the cytoplasmic fraction, although the GFP signal in pro35S:GFP-WTG1 plants was clearly not observed in the cytoplasm (Fig. 21h). Thus, these data indicate that WTG1 is localized both in the nucleus and in the cytoplasm of rice.

Overexpression of WTG1 produces a narrow, thin, and long grain due to the presence of narrow and long cells in the spikelet rind.

To further reveal the functions of WTG1 that regulate grain size and shape, the authors obtained the proActin:WTG1 construct, which was then transformed into a wild-type plant (ZHJ). As shown in FIG. 22g, proActin:WTG1 transgenic lines had higher levels of WTG1 expression than wild-type (ZHJ) plants. The authors then examined the phenotypes of grain size and shape in proActin:WTG1 transgenic lines. As shown in FIG. 22a-f, the proActin:WTG1 transgenic lines produced narrow, thin and long grain compared to ZHJ. WTG1 expression levels in proActin:WTG1 transgenic lines were associated with grain size and shape phenotypes (Fig. 22d-g). These data also showed that WTG1 affects grain size and shape.

Considering that the proActin:WTG1 transgenic lines had a narrow and long grain, the authors tried to find out whether WTG could affect cell reproduction. The authors examined the size of outer epidermal cells in the ZHJ and proActin:WTG1 spikelets. The outer epidermis of the spikelet rind of proActin:WTG1 contained narrow and long cells compared to the spikelet rind of ZHJ (Fig. 26a,b). In contrast, the outer epidermis of the spikelet rind of proActin:WTG1 had the same number of epidermal cells in grain width and length as the number of epidermal cells in the spikelet rind of wild-type plants (Fig. 26c, d). These results also showed that WTG1 determines grain size and shape by influencing the size and shape of cells in the spikelet rind.

Discussion.

Grain size and shape are critical to the yield and appearance of grain in crops. The width, thickness and length of the grain together determine the size and shape of the grain of rice. However, the molecular mechanisms underlying the formation of rice grain size and shape are

- 38 043050 are not yet known exactly. In this study, the authors isolated a mutant with a wide and thick grain (wtg1-1), which produced a thick, wide, short and heavy grain compared to the wild-type plant. WTG1 encodes an otubain-like protease with deubiquitinizing activity. Overexpression of WTG1 results in a narrow, thin, and long grain. Thus, the results obtained by the authors identify otubain-like protease as an important factor influencing the size and shape of rice grains, suggesting that it can increase grain yield and improve grain size and shape.

The wtg1-1 mutant produced a thick, wide and short grain (FIGS. 18a-f), indicating that WTG1 acts as a factor influencing rice grain size and shape. Wtg1-1 grains were heavier than ZHJ grains (Fig. 18g), indicating that WTG1 plays a key role in determining grain mass. The wtg1-1 mutant showed short, thick and dense panicles compared to the wild-type plant (Fig. 19e), suggesting that WTG1 function influences the size and shape of the panicle. The wtg1-1 mutation resulted in an increase in the number of grains per panicle as a result of an increase in both primary and secondary panicle branches (Fig. 19k, l, m). Previous studies have shown that an increase in the number of grains per panicle usually causes a decrease in grain size and mass (Huang et al., 2009). Conversely, some rice mutants have been described to produce larger grains as well as more grains per panicle (Li et al., 2011a, Hu et al., 2015). The results obtained by the authors showed that the wtg1-1 mutant produces a heavy grain and a greater number of grains per panicle.

The WTG1 gene encodes an otubain-like protease. WTG1 homologues have been found in various plant and animal species. Homologues of WTG1 in humans are members of the deubiquitinizing enzyme (DUB) family of proteases that bind to the ovarian tumor domain (OTU). OTUB1 is involved in DNA damage repair and transformation of growth factor-b (TGFb) signal transduction pathways (Nakada et al., 2010, Herhaus et al., 2013). OTUB1 has deubiquitinizing activity and functions to remove bound ubiquitin chains or molecules from their targets. The OTU domain of OTUB1 contains three conserved amino acids (D88/C91/H265) (Balakirev et al., 2003). Similarly, WTG1 contains the predicted catalytic triad (D68/C71/H267) in the predicted otubain domain, suggesting that WTG1 may have deubiquitinizing activity. Accordingly, the biochemical data obtained by the authors showed that WTG1 can cleave polyubiquitins, and therefore, WTG1 is a functional deubiquitinizing enzyme. Conversely, mutations in the predicted catalytic triad (WTG1 ^D68E > ^C71S > ^H267R ) disrupted the deubiquitinizing activity of WTG1 (Fig. 20l), indicating that these conserved amino acids are critical for deubiquitinizing activity. In addition, the protein encoded by the wtg1-1 allele (WTG1 ^wtg1-1 ) did not have any deubiquitinizing activity, indicating that wtg1-1 is a loss of function allele. It is possible that WTG1 can remove ubiquitin chains from its targets and prevent destruction of its targets.

The wtg1-1 mutant produced wide, thick, and short grains, while WTG1 overexpression produced narrow, thin, and long grains. In addition, wtg1-1 grains were significantly heavier than wild-type grains, and the wtg1-1 mutant showed an increased number of grains per panicle, suggesting that it may increase grain yield. Thus, it should be tested whether WTG1 and its homologues in crops (eg corn and wheat) can be used to increase grain yield and improve grain size and shape in the future. It is known that the characteristics of grain size and shape were chosen by rice growers during the process of rice domestication. The authors found that these rice varieties contained many SNPs in the region of the WTG1 gene (http://ricevarmap.ncpgr.cn).

Materials and methods.

Plant materials and growing conditions.

Grain of japonica Zhonghuajing (ZHJ) cultivar was irradiated with γ-rays, and a wide and thick grain mutant 1 (wtg1-1) was identified from this M2 population. Rice plants were grown in irrigated fields at a density of 20 cm x 20 cm. A total of 48 rice seedlings were transferred from the seedling nursery to an irrigated paddy rice plot (1.92 m ² ). Rice plants were cultivated in the irrigated fields of Lingshui (110°03'E, 18°51N, altitude 10 m, Hainan, China) from December 2015 to April 2016 and in Guangzhou (119°95'E, 30 °07N, altitude 12 m, Zhejiang Province, China) from July to November 2016, respectively. The soil in Lingshui is sandy loam soil, while the soil in Guangzhou is clay soil. During the growing season, temperatures ranged from 9 to 32°C in Lingshui, and from 12 to 39°C in Guangzhou (http://data.cma.cn/site/index.html). During the rice growing cycle, nitrogen, phosphorus and potash fertilizers were applied (120 kg per hectare for each).

Morphological and cellular analysis.

ZHJ and wtg1-1 plants grown in irrigated rice fields were dug up and planted in photographic pots. A MICROTEK Scan Marker i560 (MICROTEK, Shanghai, China) was used to scan mature grains. A WSEEN rice testing system (WSeen, Zhejiang, China) was used to automatically measure grain width and length. The grain thickness was measured using

- 39 043050 digital caliper (JIANYE TOOLS, Zhejinag, China). Grains from 30 main panicles were used to measure grain weight. A total of 1000 dry seeds were weighed on an electronic analytical balance. The grain weight was evaluated in triplicate.

Identification of WTG1.

To clone the WTG1 gene, the authors crossed wtg1-1 with ZHJ and received a population of F ₂ . From the F2 population, we selected 50 plants that had wtg1-1 phenotypes and pooled their DNA in equal proportions for whole genome resequencing on a NextSeq 500 (Illumina, America). MutMap was performed as described in a previous study (Abe et al., 2012) and the SNP/INDEL ratio was calculated according to a previous report (Fang et al., 2016). Of these, only one INDEL gave the ratio SNP/INDEL=1. This INDEL contained a 4 bp deletion in the gene that was present at the exon-intron junction of the fourth intron LOC_Os08g42540. The dCAPS1 marker was also expressed based on this 4 bp deletion. Thus, LOC_Os08g42540 is a candidate gene for WTG1.

Constructions and transformation of plants.

Primers C99-WTG1-GF and C99-WTG1-GR were used to amplify the 2337 bp WTG1 genomic sequence. 5' flanking sequence, WTG1 gene and 1706 bp. 3' flanking sequence. The genomic sequence was then inserted into the PMDC99 vector using the GBclonart Seamless Clone Kit (GB2001-48, Genebank Biosciences) to obtain the gWTG1 construct. Primers 003-CDSWTG1-F and 003-CDSWTG1-R were used to amplify the CDS of the WTG1 gene. The CDS was inserted into the pIPKb003 vector using the ACTIN promoter using the GBclonart Seamless Clone Kit (GB2001-48, Genebank Biosciences) to obtain the proACTIN:WTG1 plasmid. Primers C43-CDSWTG1-F and C43CDSWTG1-R were used to amplify the CDS of the WTG1 gene. The CDS was inserted into the PMDC43 vector using the GBclonart Seamless Clone Kit (GB2001-48, Genebank Biosciences) to obtain the pro35S:GFP-WTG1 plasmid. Primers proWTG1-F and proWTG1-R were used to amplify 3798 bp. 5' flanking sequence of WTG1. The promoter sequence was then inserted into the pMDC164 vector using the GBclonart Seamless Clone Kit (GB2001-48, Genebank Biosciences) to obtain the proWTG1:GUS plasmid. Plasmids gWTG1, proACTIN:WTG1, pro35S:GFP-WTG1 and proWTG1:GUS were introduced into Agrobacterium tumefaciens GV3101, respectively. GWTG1 was transferred to wtg1-1 and proACTIN:WTG1, pro35S:GFPWTG1 and proWTG1:GUS were transferred to ZHJ as previously described (Hiei et al., 1994).

GUS staining and subcellular localization of WTG1.

GUS staining of various tissues (proWTG1:GUS) was performed as previously described (Xia et al., 2013, Fang et al., 2016). Roots of pro35S:GFP-WTG1 transgenic lines were used to observe GFP fluorescence. Zeiss LSM 710 confocal microscopy was used to observe GFP fluorescence. Root cell nuclei were labeled with 4',6-diamidino-2-phenylindole (DAPI) (1 μg/ml).

RNA isolation, reverse transcription and quantitative real-time RT-PCR.

Young panicles of ZHJ and wtg1-1 and seedlings of transgenic lines ZHJ and proACTIN:WTG1 were used for total RNA isolation using an RNA extraction kit (Tiangen, China). RNA (2 μg) was subjected to reverse transcription to obtain complementary DNA using the FastQuant RT kit (Tiangen, China) according to the user manual. Real-time quantitative RT-PCR was performed as previously described (Wang et al., 2016). Three copies were tested for each sample. The list of primers is given in the additional table. 1.

Deubiquitination tests.

WTG1 and wtg1-1 coding sequences were amplified from cDNA transcribed from total panicle RNA using the MBP-WTG1F/R and MBP-WTG1-F/MBP-wtg1-R primers and cloned into the pMAL-C2 vector using the kit. GBclonart Seamless Clone (GB2001-48, Genebank Biosciences) to construct the MBP-WTG1 and MBPWTG1 ^wtg1-1 plasmids, respectively. To construct MBP-WTG1 ^d68E:C71S ' ^h267R , primers WTG1-WTG1MutF and MBP-WTG1-MutR1 (with two mutation sites) were used to amplify the first part of wtgi ^d68E:C71S ' ^h267R , and primers MBP-WTG1-MutF1 (with two mutation sites) and MBP-WTG1-MutR (with one mutation site) were used to amplify the second part of wtgi ^{d68E:C71S:H267R} These two products were then mixed as templates and primers MBP-WTG1-MutF and MBP-WTG1-MutR were used to amplification of the complete wtgi ^d68E:C71S ' ^h267R sequence and finally the wtgi ^{d68E;C71S;H267R} sequence was cloned into the pMAL-C2 vector using the GBclonart Seamless cloning kit (GB2001-48, Genebank Biosciences) to construct the MBPWIG plasmid '' ··· Plasmid His-UBQ10 was constructed as described in previous studies (Xu et al., 2016). Plasmids MBP-WTG1, MBP-WTG1 ^wtg1-1 and MBP-WTG1 ^{d68E;C71S;H267R} were transferred into Escherichia coli BL21. Induction, isolation and purification of MBP-WTG1, MBP-WTG1 ^{wtg1 1} and MBPD68E;C71S;H267R proteins

WTG1 ’ ’ was carried out in accordance with previous studies (Xia et al., 2013). 15 µl

His-UBQ10 was incubated with 2 µl of purified MBP, MBP-WTG1, MBP-WTG1 ^wtg1-1 and MBP

- 40 043050

Wtgi ^{d68E;C71S;H267R} _{in 100 µl} reaction buffer (50 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM DTT) at 30° C. for 20 minutes, respectively. The cleaved ubiquitin products, MBP-WTG1, MBPWTG1 ^wtg1-1 and MBP-WTG1 ^{d68E;C71S;H267R} were analyzed by SDS-PAGE. Anti-His and anti-MBP antibodies were used to detect cleaved ubiquitin and MPB-labeled proteins, respectively.

Protein extraction and Western blot analysis.

Leaves of pro35S:GFP-WTG1 transgenic plants were used to prepare cytoplasmic and nuclear protein fractions according to the previously described method (Alvarez-Venegas and Avramova, 2005). Anti-GFP (Beyotime), anti-Bip (Abcam) and anti-H4 (active motif) antibodies were used to detect GFP-WTG1, Bip and histone H4, respectively.

Example 3

The RNAi strategy was used to obtain transgenic plants with OsOTUB1 knockdown. The authors inserted two identical segments of the OsOTUB1 coding region head-to-head into the pUCCRNAi intermediate vector, which contained the potato GA20 oxidase intron. Using pUCCRNAiOsOTUB1, the RNA interference structure was introduced into the pCambia-actin-2300 binary plant vector to generate the pActin::OsOTUB1-RNAi construct. The OsOTUBI-RNAi construct was then transferred to rice using an Agrobacterium tumefaciens mediated transformation system. Transgenic plants were selected in half strength Murashige and Skoog (MS) medium containing 50 mg/l G418, and G418 resistant plants were transplanted into soil and grown in the field. To identify transgenic plants homozygous for T2 with OsOTUB1 knockdown, a count was performed. RT-PCR. As shown in FIG. 28a, OsOTUB1 RNAi silencing showed a ZH11-npt1-like phenotype. In addition, as shown in FIG. 28b, plants expressing RNAi had an increased number of primary branches per panicle, an increased number of secondary branches per panicle, and an increased number of grains per panicle and, most importantly, gave an increase in the total grain yield per plant.

Sequence list

Arabidopsis thaliana (AtOTUB1), soybean (GmOTUB1), corn (ZmOTUB1), sorghum (SbOTUB1), barley (HvOTUB1), wild spelled wheat (TuOTUB1) rice (OsOTUB1)

SEQ ID NO: 1: OsOTUB1 polypeptide

MGGDYYHSCCGDPDPDLRAPEGPKLPYVGDKEPLSTLAAEFQSGSPILQEKIKLL

GEQYDALRRTRGDGNCFYRSFMFSYLEHILETQDKAEVERILKKIEQCKKTLADLGYIEF TFEDFF SIFIDQLES VLQGHES SIGAEELLERTRDQMVSD YVVMFFRF VTSGEIQRRAEFF EPFISGLTNSTVVQFCKASVEPMGEESDHVHIIALSDALGVPIRVMYLDRSSCDAGNISVN HHDFSPEANSSDGAAAA EKPYITLLYRPGHYDILYPK

SEQ ID NO: 2: OsOTUB1 nucleic acid (cDNA from Zhefu802) atgggcggggactactaccactcgtgctgcggcgaccccgaccccgacctccgcgcgcccgaggggcccaagctgccgtac gtcggggacaaggaacctctctccactttagccgctgagtttcagtctggcagccccattttacaggagaaaaataaag ttgcttggtgaacag tatgatgctttaagaaggacacgaggagatggaaactgcttttatcgaagcttttgttttcctacttggaacatatcctagagacacaagacaa tattcattgatcagctggaaagtgttctgcagggacatgaatcctccataggggccgaagagcttctagaaagaaccagggatcagatgg cttccgtggagccgatgggcgaggaaagtgaccatgtccacataattgccctatcagatgcgttgggt gtgccaatccgtgtgtgtacctagacagaagctcatgtgatgctggaaatataagtgtgaaccaccatgatttcagccctgaggccaattcat cggacggtgctgctgctgctgagaaaccttacattactttgctc taccgtcctggtcactacgacattctctcccgaagtga

- 41 043050

SEQ ID NO: 3: OsOTUBl nucleic acid (genomic from Zhefu802) atgggcggggactactaccactcgtgctgcggcgaccccgaccccgacctccgcgcgcccgaggggcccaagctgccgtac gtcggggacaaggtgagatgttgacgcctctctctctttctgtctctctcgctcgctttgactcatctg cgctttgactcatctgcggtcgatag atttgttcatgtggtagaaatgggtctgaatcgtggtaagacgcccagtgttgccatgccagtatccgctagttgtgccagcaggtgaggcga tagatcagtcctgttagtctagttggatgctgattgttggtcatcattactgttgtattggtgctccatttctatgtgaattgacattt taaggcgtcta tacaagcagtggggactagaatttggataacaagtaacaatttccccttattgcgtcatcttaaaggataagaggagttatcagatgctggattt caggcgctattgttgtgagttgtaactgttgtacttgattttttagcttcgttggtgaactgatgtttggaggttcatgcagaagtcag aaccatggggaattagaatttggatgaacagacagcaattacctttcgtgttctcttctaggaaaagaagggtttatctgttatcatttgctggat gtgctccttacttaatatttatgcatggaataa tagagatggccaaacaagtttgctccatagcgtattatgattctaggataaaagtggtgtcatc ctttaatcgtcacacctaggacaataaatgtgaaggacgaacttgttgatgcagaataatagcctatgctagtcaattcagcacagaaactgag gttaaatgtgtcccaaaagtttcagttaaggttcacacactaggatttacac gaacagaacaaatctgcaaatatagtccatatgagaaggtggag agctacatacacacaatttcatatgaaatggaaaatgatgttggcactaaacttggatttaggtcaagtagttagatatttgatgcctcaaattcat tttgttctgttaattgcaagcaccttttcacaatggaggacactaatgcattgctatgattatctctgtgttt gtacagttattttaggctatcgtatga ctttcttctgctttcatttgtgttcattattgatatcttttgaaccttgcaggaacctctctccactttagccgctgagtttcagtctggcagccccattt tacaggagaaaataaaggtccatatggatttggcagttataatgtaatagacatattttggttgatctatcgtatcaatggatggt gcttcaattt gttcttataatttcttgcttggtctgcagttgcttggtgaacagtatgatgctttaagaaggacacgaggagatggaaactgcttttatcgaagctt tatgttttcctacttggtattatttttggtctgtttccatacaaactttgactattttataagctgatgatcttatcatttgcttctaggaacatatcctaga ga cacaagacaaagctgaggttgagcgcattctaaaaaaaattgagcagtgcaagaagactcttgcagatcttggatacattgagttcacctt tgaagatttcttctctgtaagtctttattgttactttgtgtggtcctccttacttatcctgttcaattgctgttttgcaacttatgccagatgtattccctct gaatagtatga agatctgtccgattattttcatgtatgcttgtttgcatttcctttttagatgttcctggaataatttttgtatgagctagttataatgaga gcttgtgcattttcctgtcatgcaacaaattaaatactagtgtctaatccttgtgcattgttaataactttgaaaatgattagccttgaagattggtcc attatatatatgttcacttgtttct tagttaggatcactcaccagtcacccttctgaagttcataatgtatcacttataagtaagtaagctagcaaaacaaaa tttggactgtttgtagccacccagaacccaaatagatggatttcacattattttctactggctttgggagttatttgatcgatgctagtacaacgttg aaatttgggtagttgagatgcgtttttcacaaaggactcctttattgg tgcttgatctacaactggtgttttacttttttacaaaaaatgtaatctcct gtattaacaacaaagcaatttttgtaattgtt tataaagtatggcatgtccatcataaatgttttccttctgtagtgaatctattttgttttcctgtatccttagggccgaagagcttctagaaagaacca gggatcagatggtttctgattatggtttgtacatccagatatgtgtagtatgcctttctctgctttgctctcattatttaactatg tcttctttagttgtca tgttctttaggtttgtcacctctggtgaaatccaaaggagggctgagttcttcgaaccattcatctctggcttgacaaattcgactgtggttcaggt tagctccatacttccattttatgaggggttgtacagtcgttgggggaggtattatgaggtacaatacctccaggtaccgggagttaccacacataa gca taaaatgtgtggtgcctctccaaggaccacaagaaatctcttcattatatttgtaatgcacagagcagagagtacagacaaatagacctg cactctgcattttcattaagtatttagatgtgagattattctatgttttatctctcttgttagtattttttgctctgttttataatggaagttcattttcttggg aactgtcattcacaaaaacaatgag ttatcgtaccctgccatttagtagggaatttggtggtaaaaaaccattaactttttcttcaattttgtgccttct gcacaaggtgggatagggcatatattgtggaacaaaagagtgcacaatgactaattatttagtatgcatcacactggagtatgatatactagtg gaaaggttatggcaaaataccatgatagtagcttgatagattagcaggtccgta agtatttttccaatgataatgttttattcattaaactgtagca ggataaaatctacttatgcaccttttttcatgagtagcaaacaatgcattctctggtttgaaaaacttgttcaagttgcagtgtgttattccaatccg tgtttgtgtgacaagcaattgctggagttactgatcctgagttcaattcaatttgcagttctgcaaggcttccgtggagccgatgggcgaggaa gtgaaccaccatgatttcagccctgaggccaattcatcggacggtgctgctgctgctgagaaaccttacattactttgctctaccgtcct ggtcactacgacattctctacccgaagtga

SEQ ID NO: 4: OsOTUB1 nucleic acid (cDNA from IR66167-27-5-1-6) atgggcggggactactaccactcgtgctgcggcgaccccgaccccgacctccgcgcgcccgaggggcccaagctgccgtac gtcggggacaaggaacctctctccactttagccgctgagtttcagtctggcagccccat tttacaggagaaaataaagttgcttggtgaacag tatgatgctttaagaaggacacgaggagatggaaactgcttttatcgaagcttttgttttcctacttggaacatatcctagagacacaagacaa agctgaggttgagcgcattctaaaaaaaattgagcagtgcaagaagactcttgcagatcttggatacattgagttcaccttt gaagatttcttctc tatattcattgatcagctggaaagtgttctgcagggacatgaatcctccataggggccgaagagcttctagaaagaaccagggatcagatgg tttctgattatgttgtcatgttctttaggttgtcacctctggtgaaatccaaaggagggccgagttcttcgaaccattcatctctggcttgacaaatt cgactgt ggttcagttctgcaaggcttccgtggagccgatgggcgaggaaagtgaccatgtccacataattgccctatcagatgcgttgggt gtgccaatccgtgtgatgtacctagacagaagctcatgtgatgctggaaaatataagtgtgaaccaccatgatttcagccctgaggccaattcat cggacggtgctgctgctgctgagaa accttacattactttgctctaccgtcctggtcactacgacattctctctaccgaagtga

- 42 043050

SEQ ID NO: 5: OsOTUBl nucleic acid (genomic from IR66167-27-5-1-6) atgggcggggactactaccactcgtgctgcggcgaccccgaccccgacctccgcgcgcccgaggggcccaagctgccgtac gtcggggacaaggtgagatgttgacgcctctctctctctctctgtctctctcgct cgctttgactcatctgcgctttgactcatctgcggtcgataga tttgttcatgtggtagaaatgggtctgaatcgtggtaagacgcccagtgttgccatgccagtatccgctagttgtgccagcaggtgaggcgat agatcagtcctgttagtctagttggatgctgattgttgtcatcattactgttgtattggtgctcca tttctatgtgaattgacattttaaggcgtctat acaagcagtggggactagaatttggataacaagtaacaatttccccttattgcgtcatcttaaaggataagaggagttatcagatgctggatttt ctcctttatttttagtcgtggtgcctggaataatggagattggctgaacaagttcatatcagttgtgtccattttcatcctctggatggt cagtcctta agtattgtcaggcgctattgttgtgagttgtaactgttgtacttgattttttagcttcgttggtgaactgatgtttggaggttcatgcagaagtcaga accatgggaattagaatttggatgaacagacagcaattacctttcgtgttctcttctaggaaaagaagggtttatctgttcatttgctggatgt gctccttact taatattttgcatggaataatagatggccaaacaagtttgctccatagcgtattatgattctaggataaaagtggtgtcatcct ggttcacactaggatttacacgaacagaacaaatctgcaaatatagtccatatgagaaggtggaga gctacatacacacaatttcatatgaaatggaaaatgatgttggcactaaacttggatttaggtcaagtagttagatatttgatgcctcaaatttattt tgttctgttaattgcaagcaccttttcacaatggaggacactaatgcattgctat gattatctctgtgtttgtacagttattttaggctatcgtatgact ttcttctgctttcatttgtgttcattattgatatcttttgaaccttgcaggaacctctctccactttagccgctgagtttcagtctggcagccccatttta caggagaaaataaaggtccatatggatttggcagttataatgtaatagacatattttggttgatctat cgtatcaatggatggtgcttcaatttgt tcttataatttcttgcttggtctccagttgcttggtgaacagtatgatgctttaagaaggacacgaggagatggaaactgcttttatcgaagctttat gttttcctacttggtattatttttggtctgtttccatacaaactttgactattttataagctgatgatcttatcatttgcttctag gaacatatcctagaga cacaagacaaagctgaggttgagcgcattctaaaaaaaattgagcagtgcaagaagactcttgcagatcttggatacattgagttcacctttg aagatttcttctctgtaagtctttattgttactttgtgtggtcctccttacttatcctgttcaatttctgttttgcaacttatgccagatgtattccctct ga atagtatgaagatctgtccgattattttcatgtatgcttgtttgcatttcctttttagatgttcctggaataatttttgtatgagctagttataatgagagc ttgtgcattttcctgtcatgcaacaaattaaatactagtgtctaatccttgtgcattgttaataactttgaaaatgattagccttgaagattggtccatt atatatgttc acttgtttcttagttaggatcactcaccagtcacccttctgaagttcataatgtatcacttataagtaagctagcaaaacaaaattt tattggtgcttgatctacaactggtgttttacttttttacaaaaaaatgtaatctccttg cagtgcactcaaattattgcaacctccttccttatgttcccaccctcattattttcagatattcattgatcagctggaaagtgttctgcagggacatg aatcctccatagggtaaatatcctagagttatttgtatccttaatgcatatgaccaataatcat gtattaacaacaaagcatttttgtaattgttta taaagtatggcatgtccatcataaatgttttccttctgtagtgaatctattttgttttcctgtatccttagggccgaagagcttctagaaagaaccag ggatcagatggttctgattatggtttgtacatccagatatgtgtagtatgcctttctctgctttgctctcattatttaactat gtcttctttagttgtcat gttctttaggtttgtcacctctggtgaaatccaaaggagggccgagttcttcgaaccattcatctctggcttgacaaattcgactgtggttcaggt cctctccaaggaccacaagaaatctcttcattatttgtaatgcacagagcagagagtacagacaaatagacctg cactctgcattttcattaagtatttagatgtgagattattctatgttttatctctcttgttagtattttttgctctgttttataatggaagttcattttcttggg aactgtcattcacaaaacaatgagttatcgtaccctgccatttag tagggaatttggtggtaaaaaaccattaactttttcttcaattttgtgccttct gcaaaggtgggatagggcatatattgtggaacaaaagagtgcacaatgactaattatttagtatgcatcacactggagtatgatatactagtg gaaaggttatggcaaaataccatgatagtagcttgatagattagcaggtccgtaagtatttttccaatgataatg ttttattcattaaactgtagca ggataaaatctacttatgcacctttttttcatgagtagcaaacaatgcattctctggtttgaaaaacttgttcaagttgcagtgtgttattccaatccg tgtttgtgtgacaagcaattgctggagttactgatcctgagttcaattcaatttgcagttctgcaaggcttccgtggagccgat gggcgaggaa agtgaccatgtccacataattgccctatcagatgcgttgggtgtgccaatccgtgtgatgtacctagacagaagctcatgtgatgctggaaata taagtgtgaaccaccatgatttcagccctgaggccaattcatcggacggtgctgctgctgctgagaaaccttacattactttgctctaccgtcct gg tcactacgacattctctacccgaagtga

- 43 043050

SEQ ID NO: 6: Zhefu802 OsOTUBl promoter sequence aaggcc aaaataattttcgatgctggttggtgctaccaagtcccacgtatgatacttaagaatcaatcatgaatattacaaatcaagtcaaactacgttatgt attgaactcttataattactgcaacatatcacactggaatttcctatggtaattcctcgccagccttatcctacccatcccttgcagtatattaagag catcaacaacaaacatgattca agacaacttttattaacactgaacaacataaattgggaacaaaaacaaaccacttggaggcatgattaggat aatcggtattaaagaactggacatcacaattcacaactagatgttgaaataatacctgtctctctctttggctcatggcaggtgtcagtgaaatata ctgatgctccaagagagctggaagcaccgtttccacgtaatcaaaatgtccttttc gtttgctgcaatcaaccttaaagggctcttttgatgctat ctcttcaggcatgtcctttacaacttccacataacctctggtttgctcaatgaagtaatcaacatcgaaaacgtctgcaaatccactgacagaga ataccaataagtgatgaactaccttttgaacagaaataaactgcataactacaagtagcacagtcgttcatcttgtagagtgattct catacctag attcattccagtaggcagcaacctcaaacttgggcagaaccattgttgcgttgagaaggcgcgcaaccgcaattccatcacatagctgaaga aacattcggaagaataattacaaccaggagtaacataataacatagccagttgaaatcacattcgccttgcaatgtgaaaattttcataaataat ctgaaaatttagttatgccactatata tcatgcaacctgcctccacgacattttaatcatggagtagaagataaaacatatgatccccctcattga ccctactatcttactacttgtgcatggccgaacgatctaacagcgaaatccagaaagccaacactcatttgatcccactaacaacggaagaga gaaacgctagccgagatcgcttaacgtacatcgcgtcgcagctggttgagcccgcc gtagcagtcgatccggatgtacccattcctcctcg acggagctgcagaagaagaggaggttcaaaaccgcaatcaccaccacagtctcaagcagagatgtccactacccggatccttaaaccca aaccacaaatcacggcgaggtctcacccggcattgccgcccgccaccacccgcacgaccgccactccgccaccgccgctgcgcccat atgacccgcgaccccgacgccgacggcgactcctccctaaagaccaaaagcgagtaagcgagatccgtaagcttctctggaacaatctcga gcatcagctgcaagaggtgaggctgggccgcgtacctggaggtgggaagagtgaagaagaaaggcggagaggagggtggagag gaggaagtagagcgcgggggcgaggaagatgaccgg taggaggatgcggacgcggctgcgcgcccaccacgccgccggcgacgc cgacgacgacatcgcctcgccgcgagaagcactggatctgatcggccgccgcctccacgccggagtggagagcgtatataagctcgtca gaatgtgggcccgtggctatgtgggcccaccatgtcatcgacgcttatcaagatcga gcggtggcgtgaggaaaccggtaggggtgggg gggctaaccaatcggaaacgcgtaataactcacccgcggttcactttctccttatgacacgtgggcccatctctcctggacccacctgtcagt tacccttacggcctccactctgaggatctaaacgtaaaaacgaatttatcggagggcttatccgcgaggggaaaa aaacgcgcacttatttct cgccttcgccgagatctcggaagaagaagaacacgcaccgcggggagaggggggagaagcggaaagctccaccgaatcgaagccccc tcccggaggaggcggtgatgggggaggcggcggcggcagccgcggcccccgagccggtcgtcgaggggggag gaggggggggggaggggttgaatcctaaccctagcggcggtgggggaggaggtggtggagggtgctcggactccgtgtcggtcgagc tctcg

SEQ ID NO: 7: TuOTUB1 cDNA sequence

Atgggcggggactactaccacgcctgctgcggcgacccggaccccgaccacaagcccgagggaccccaggtgccgtacat cggtaacaaggaacctctctccgccttagcagcagagttccagtctggcagccccattttacaggagaaaataaagttgcttggtgaacaata tgatgctttaaggcgaacacgaggagatggaaactgcttt tatcgaagctttatgttctcctacctggaacatatcctagagacacaagataga gctgaggttgagcgcatcctaaaaaacattgaacagtgcaagatgacactttcaggtcttggatacattgaattcacttttgaagacttcttctct atgttcattgaggagctgcaaaatgttctgcagggacacgaaacttctattgggcctgaagaacttctaga aagaaccagggatcaaacgac ttctgattatgttgtcatgttctttaggttgttacctggtgaaattcaaaggagggctgagttctttgaaccatttatttctggcttgacaaattcg accgtggctcagttttgcaagtcttctgtggagccaatgggcgaggaaagcgaccatgtgcacattattgctctctgtcagatgcg g

SEQ ID NO: 8: HvOTUB1 cDNA sequence

Atgggcggggactactaccacgcctgctgcggcgaccccgaccccgaccccaagcccgagggaccccaggtgccgtacat cggtaacaaggaacctctctccgccttagcagcagagttccagtctggcagccccattttacaggagaaaataaagttgcttggtgaacaata tgatgctttaagacggacacgaggagatggaaactgcttttat cgaagctttatgttctcctacctggaacatatccttgagacacaagacaga gctgaggttgagcgcatcctaaaaaacattgaacaatgcaagaagaacactttcaggtcttggatacattgagttcacttttgaggacttcttctct atgttcattgaggagctgcaaaatgttctgcagggacacggaacttctattgggcctgaagaacttctagaaaga accagggatcagacgac ttctgattatgttgtcatgttctttagatttgttacctctggtgaaattcaaaggagggctgagttctttgaaccatttatttctggcttgacaaattcg accgtggttcagttttgcaagtcttctgtggagccaatgggcgaggaaagtgaccatgtgcacattattgctctgtcagatgcgttaggg gtgc caatccgcgtgatgtacctagaccgaagctcttgtgacacaggcaatctaagtgtgaaccaccatgatttcatccctgcagccaattcctctga aggtgatgctgcaatgggattaaatcctgctgatgagaaaccttacattactctgctctaccggcctggtcactatgacattctctacccgaagt ga

- 44 043050

SEQ ID NO: 9: ZmOTUB1 cDNA sequence atgggcgacgttccacaggcgccgcacgctgcgggaggtggagaagagtgggcggggccggaccctaaccctagcccga gcctcggcggctgctcggaccccgtgtcggtggagctctccatgggcggggactactaccgcgcctgctgcggcgagcccgatcccgac atccccgaggggcccaagctgccgtgcgttggggacaaggaacctctctcctctttagcagctgagtttcagtctctggcagccccattttacaa gagaaaattaagttgcttggcgagcaatatggtgctttaagacgtacacgtggagatggaaactgctttttatcgaagctttatgttttcctacctg gaacacatcctagagacacaagaca aagctgaggctgatcgcatcatggtaaaaattgaggaatgcaagaaaacactcctctctcttggata tattgagttcacttttgaggacttcttttcgatattcattgaactgctggaaagtgttctgcagggacatgaaactcctatagggtttgtcacttctg gtgaaattcaaaggaggtctgacttctttgaaccgttcatatctggctt gacaaattcaaccgtggttcagttctgcaaggcttctgtggaaccta tgggtgaggaaagtgaccatgtgcacataattgccctatcagatgcactaggcgtaccaatccgtgttatgtacctagaccgaagctcgtgtg acactggcaacctgagcgtgaatcaccacgatttcatcccgtcggccaatgattcgg agggtgatgcggccacgacacctgctcctgccac agagaaaccgtacatcactttgctctaccgtcctggccactacgatattctctacccaaagtga

SEQ ID NO: 10: SbOTUB1 cDNA sequence atgggcgacgtgccccaggcgccgcacgccgcggaaggaggaggaggaggactggaggagggggcggtgcccgaccct aaccctagcccgagcctgagcctcggcggctgctcggaccccgtgtcgctggagctctccatgggcggggactactaccgcgcctgct g cggcgaccccgaccccgacatccccgaggggcccaagctgccgtgcgttggggaaaaggaacctctctcctctttagcagccgagtttca gtctggcagccccattttacaagaagaaaattaagttgcttggcgaacaatatggtgctttaagacggacacgtggagatggaaactgcttttat cgaagctttatgttctcctacttggaac acatcctagagacacaagacaaagctgaggctgatcgcatcatggtaaaaattgaggattgcaag aagacgctcctgtctcttggatatattgagttcacttttgaggatttctttgcgatattcattgatatgctggaaagtgttctgcagggacatgaaac tcctatagggtttgtcacttctggtgaaattcaaaggaggtctgacttct ttgaaccattcatatctggcttgacaaattcaactgtggttcagttct gcaaggcttctgtggaacctatgggtgaggaaagtgaccatgttcacataattgccctatcggatgcactaggtgtacctatccgtgttatgta cctagaccgaagctcgtgtgatactggcaatctgagtgtgaatcaccatgatttcatcccttc gtccaatgcttctgagggtgatgctgcgatg acatctactcctgacgctgagaaaccttacatcactttgctctaccgtcctggtcactatgatattctctacccaaagtga

SEQ ID NO: 11: cDNA sequence AtOTUBl atgcagaatcagattgatatggtgaaggatgaagcggaagtagctgcatcgatttcagcaattaagggtgaagaatggggaaatt gttcatcagtggaagatcaaccatcttttcaagaagaagaagctgctaaagttccttatgttggtgataaggaacctgtctagtttagctgca gagtatcaat cagggagtcccattttgctggagaagattaagatactggacagtcaatatatcggaatccggcgaacaagaggagatggaa attgcttcttccgaagttttatgttctcttaccttgagcatatattggaatcacaagatcgtgctgaagtcgatcgtatgttgagaaa tgtagaaagactctgcaaaacttaggttatacagat tttacatttgaggacttctttgcgttgttccttgagcaactagatgacattctccaaggaa ctgaagagtctataagctacgatgagctggttaacagaagtagagatcagtcagtctcagattacattgtaatgttctttaggtttgttactgctg gtgatatacgaacgcgtgccgattttttcgagcctttttataacaggcttatcaaatgcaac agtggatcagttttgcaagtcctcggtcgaacca atgggggaagaggtgaccatattcacataactgctttgtcggacgcacttggtgttgcaatccgtgttgtgtatcttgaccgtagctcatgtga tagtgggggcgtcactgtgaatcatcatgactttgttcctgtgggcattaccaatgagaaagatgaagaagcttctgctcca tttataaccttgct gtatcgtccaggccattacgatatcctctaccccaagccatcttgtaaggtatcagacaatgtggggaaa

SEQ ID NO: 12: GmOTUB1 cDNA sequence

Atgcagagtaaagaagctgttgtggaagatggggaaataaagagtgtgactgctgtagggtctgaaattgatgggtggaccaat tttggggacgatgacataatgcagcagcagtatacaattcagtctgaagaggctaagaaagttccatctgtgggcgacaaggaaccactga gtagcttagctgctgaatataaatcaggcagtcc tatcttgctggagaaaataaaggtgcttgatgagcaatacgctgccattcgtcgtactcg aggagatggaaactgcttctttcgaagctttatgttttcatatcttgagcatgttatgaaatgtcaagaccaagcagaagttgatcgtatccaagc caatgttgaaaaaagtagaaaagcactgcagaccttgggttatgcag acttgacttttgaagatttttttgcgttattccttgagcagctggaatct gttattcaagggaaagagacttccataagtcatgaagagcttgttcttagaagccgagatcagtcagtatctgattatgtcgttatgttcttcagat ttgttacctctgccgcaatacaaaagcgcacagaattttttgaaccattcatactaggcttaacta atacaacggtcgagcagttttgcaaatcat ctgttgaaccaatgggtgaagagagcgaccatgtgcacattactgccctttcagatgcattgggcattccagtccgtgttgtgtaccttgaccg cagctcaagtgatactggtggtgtcagtgtaaatcatcatgatttcatgccagtggctggtgatctcccaaatgctagttgca gctctgaaaag aacattcctttcatcacactactatatcgtcctggtcactatgacatcctctatccaaaatga

- 45 043050

SEQ ID NO: 13: Chinese cabbage cDNA sequence

Atgcagaatcagaatgatacggtgaaggatgatgcggagctcgctgcttccatctcggctgaacaatggggatgctgttcagtg gaggaaccatcttttcaagatgatgaagctgctaaagttccttatgttggtgataaggagcctatgtctagtttagctgcagagtaccaagcag ggagccccattttgcttgagaagataaaggt actggacagtcaatatgttgcaatcaggcgaacaagaggagatggaaactgcttcttccga agttttatgttctcttaccttgagcatattttggaatcacaagatggtgctgaagttgaccgtatcaagctcaatgttgaaaaatgtagaaagaatc tgcagaacttaggctacacagatttcacatttgaggacttctttgcgttgttccttgag caactagatgacatcctccaaggaggcgaagagtct ataagctatgatgagctggttaacagaagtagagatcagtctgtttccgactacattgtgatgttcttcaggtttgttactgctggtgaaataaaa gatgggggaag agagtgaccatattcacataacagctttgtcggacgcgcttggtgttgcaatccgggttgtgtatcttgaccgtagctcatgtgatactggaggt ggtgtcactgtgaaccatcacgactttgttcccgttggcagtggcactaatgagaaagaagaagcttcttctgctgctcccttttaacattgctc ta tcgtccaggccattacgatatcctctaccccaaggtattggagaatgtggaaaaatga

SEQ ID NO: 14: amino acid sequence of TuOTUB1

MGGDYYHACCGDPDPDHKPEGPQVPYIGNKEPLSALAAEFQSGSPILQEKIKLLG EQYDALRRTRGDGNCFYRSFMFSYLEHILETQDRAEVERILKNIEQCKMTLSGLGYIEFT FEDFFSMFIEELQNVLQGHETSIGPEELLERTRDQTTSDYVVMFFVTSGEIQRRAEFFE PFISGLTNST VAQFCKS S VE PMGEESDHVHIIALSDALGVPIRVMYLDRS SCDTGNL S VN HHDFIPAANSSEGDAAMGLNPAEEKPYITLLYRPGHYDILYPK

SEQ ID NO: 15: Amino acid sequence of HvOTUB1

SVE PMGEESDHVHIIALSDALGVPIRVMYLDRS SCDTGNL SVN HHDFIPAANSSEGDAAMGLNPADEKPYITLLYRPGHYDILYPK

SEQ ID NO: 16: Amino acid sequence of ZmOTUB1

MGDVPQAPHAAGGGEEWAGPDPNPSPSLGCSDPVSVELSMGGDYYRACCGEP DPDIPEGPKLPCVGDKEPLSSLAAEFQSGSPILQEKIKLLGEQYGALRRTRGDGNCFYRSF MFSYLEHILETQDKAEADRIMVKIEECKKTLLSLGYIEFTFEDFFSIFIELLESVLQGHETPI GFVTSGEIQRRSDFFEPFISGLTNSTV VQFCKASVEPMGEESDHVHIIALSDALGVPIRVM YLDRSSCDTGNLSVNHHDFIPSANDSEGDAATTPAPATEKPYITLLYRPGHYDILYPK SEQ ID NO: 17: SbOTUB1 amino acid sequence

MGDVPQAPHAAEGGGGGLEEGAVPDPNPSPSLGGCSDPVSLELSMGGDYYR ACCGDPDPDIPEGPKLPCVGEKEPLSSLAAEFQSGSPILQEKIKLLGEQYGALRRTRGDG NCFYRSFMFSYLEHILETQDKAEADRIMVKIEDCKKTLLSLGYIEFTFEDFFAIFIDMLESV LQGHETPIGFVTSGEIQRRSDFFEPFISGLT NSTVVQFCKASVEPMGEESDHVHIIALSDAL GVPIRVMYLDRSSCDTGNLSVNHHDFIPSSNASEGDAAMTSTPDAEKPYITLLYRPGHY DILYPK

SEQ ID NO: 18: Amino acid sequence of AtOTUB1

MQNQIDMVKDEAEVAASISAIKGEEWGNCSSVEDQPSFQEEEAAKVPYVGDKEP LSSLAAEYQSGSPILLEKIKILDSQYIGIRRTRGDGNCFFRSFMFSYLEHILESQDRAEVDRI KVNVEKCRKTLQNLGYTDFTFEDFFALFLEQLDDILQGTEESISYDELVNRSRDQSVSDY IVMFFRFVTAGDIRTRADFFEP FITGLSNATVDQFCKSSVEPMGEESDHIHITALSDALGV AIRVVYLDRSSCDSGGVTVNHHDFVPVGITNEKDEEASAPFITLLYRPGHYDILYPKPSC KVSDNVGK

SEQ ID NO: 19: Amino acid sequence of GmOTUB1

MQSKEAVVEDGEIKSVTAVGSEIDGWTNFGDDDIMQQQYTIQSEEAKKVPSVGD KEPLSSLAAEYKSGSPILLEKIKVLDEQYAAIRRTRGDGNCFFRSFMFSYLEHVMKCQDQ AEVDRIQANVEKSRKALQTLGYADLTFEDFFALFLEQLESVIQGKETSISHEELVLRSRD QSVSDYVVMFFRFVTSAAI QKRTEFFEPFILGLTNTTVEQFCKSSVEPMGEESDHVHITAL SDALGIPVRVVYLDRS S SDTGGVS VNHHDFMP VAGDLPNASC S SEKNIPFITLLYRPGHY DILYPK

- 46 043050

SEQ ID NO: 20: amino acid sequence of Chinese cabbage rdqsvsdy ivmffrfvtageiktraeffepfitglsnttvdqfcktsvepmgeesdhihitalsdalgvairvvyldrsscdtgggvtvnhhdfvpvgsg tnekeeassaapfitllyrpghydilypkvlenvek

SEQ ID NO: 21: TuOTUB1 promoter sequence:

cagta aa gcggattatacctgtgaaaagtattcttggactgatgacaatcattagaacagaaccca gcacaaatttatcagtaggtcatcatatcacaaagcagacaacaaatcaagattaggtaagttgatgaagtggttggagttagtaaagaagtc atgtaaccaacaaatttagggcacgtgaaagtcttgcattgcatgaactgacatcttagttaaacaaagttat cactcaagaatatagcgtatcg gtgtatttattccaattatgtgagctcaagatgattgcaaagggaaagggggaagggctataaagctatgaagataacttttatgagagtgta cagttttggatagtaacgcagcatgccaaattcgaaggttgatagtcagaatcgtgattctgaatcggatcggggccctaatggtagggtcgc atatcg cagaatcttcactacgaaaatcacagaaacatagattttaccaaacagaatcatagaatcatgagttagttggattgtcaagtcgtag aatcgtacaacagaatcgcgattctgactactacttttaacatattcactcctaatttgcactgagaaaaaaaagataaatacagggaaataatttgc atggttacatcacacatctaaacaaagacaaatacgacaa atacatatgattttctacactgcttgatgatactatgtctcatgtatgccaacttaa gaatgcagtcaatgacaccacgaatctgcttcatacaaggaacacctactactactgcaatatatatttttcgaaaaggattactgcaatctatctacttt aagttacaggtagatacgtgccctcgaaagatataagaattttagcactggagaacttcatcgccagccttattactcc ctccaatccgaatt aattgatgcagcctctatacaatgacattatgctaagatcggagggagtattctatatccatccttcacattatgctaagatcctcagcagcagat acaactcaagacctttgatgtgaacaatgaacaaaaatagcttggaaaataataaatggacagtatgactaagataattggtattatgaaaatgg aaagtagatttca ggtatcaactagataactcggcaatacctatcttttctttggctcatggcaggtgtcagagaaatgtatcgatgttctaaaag agctggaagaactgtttctacataatcaaaatgtcctttccgttttcggcaatcgaccttaaatggctctttcaacgatatctcctcgggcatatcc ttcaccacttccacataacctctagtttgctcaatga agtgatcaacatcaaacacgtctgcaaaaccactgacaaagcattctgataagcacat gattagttttgaacagaagtgcactacatagaaacaacagtagaagccttcgtttcaccccaagtgactccagtacctagactcattccagtat gcagcaacctcaaacttgggcagaaccattgtcgcgttcagaagacgtgcaaccgcaattccat cgcacagctgcagaacaattgcgcca gaggccagaataattacaaccgaggaacttgtaacatagccaatcgacaacgcacaccactgcacaacattctatacaatgctacctatttcc taaagctaaatcatatgatcccactattgactccacgagcctatacttgtcacttgtgcattcagtacatggccagatgatccaagtttaacagta catacata agaaaaggaagtcttactccattacaaggaggataaaccaagtttaacatacatcgcgccgcagctggttgaggccgccgtag caatctatcctgatgtagccattcctcgtcgacggagctgctgaaggagcggggacagaaaccaatcatcaccacaacccaagccaat cctaaactctgaccgaaaaccacgaagcacacaaggcctcaccgggcattg ccgtctgccaccactcgcacggccgccactccgccaaa cgccgctgcgcccatatgagccgcgaccccgacggcgaatgctccctgcgggaaaatcccaatgcgggggatctgtaacttccaggaag agtccagaagaacctggtatggcaggtcggggcggggcgcgtacctggaggccggtagggcgaagaaggatggggagaggagc gcgg ggaggcgaggaagaagaagagggcggcggcgaggaagagggccgggagaaggaggcgtatgtggtggggggccgcgcctgttcg gggtcccacatgtaaataagggaccacctgtcatggatggttatccagatcgagaggtggcgtgtggg aatggagcagggtggtggtccagcaggaacagcgttaacttcctgaaaatggaacgcgggcccgctgcgtcagccccacatgtcagggt cacgaagtaccatgaaggtggatgacgcgaagggcttatctt caaaacgacacgcacataccctttgtttcgcctgcggcgagatctccg actgccacaaaagcgaaggtctcctccgcccgaatcaaagccgtcgtctcgaannnnnnnnnnnnnnnnnnnnnnnnnnnnn nnnnnnnnnnnnnnnnnnnnntccggccccgaggacgcggcc

- 47 043050

SEQ ID NO: 22: HvOTUBl promoter sequence:

aatacagaaagtacgaaactcaggtaaccacgaggaagttgcatcacgtctcctcaggtttaagtcattgacttgggagtcctcg gtgttcacacaaggtcaagatatctactggcttttcccccttcagagacagcaaagaaagcacgcccaccactgtcctttctaacatgtgattta taggttgtccaatgctgtcaacactgca acaaagcgatggccgctggtatgtgcagcatgtttgaaatcaaaatgcttgcatcagtatatatga aaatgtaacgatgagaagaggtatacactggggagtatgattagtgaatgaagggaggttattcttccatccgctgcttgcttgagctgccaa acggaaggaatgagcagcggaataccaagtgtcatgtacaccttcc ttacagttaaagatcgcatgctcaacaccaatttgtttaagcttgctt gcttgccacgacgtaaaatatgaaaataaccactagtcacatagtccctccattcttaaacattccaaccgtccgaaaatacttgtcatatgaatt catatatctagatgtaatttagttctagatatccattttttatatatttgtgcgataagtaatttcaggcgg agggagtataagttttttaaagatttca ctagggactacatacggatgtatatagacatactttagagtatagatacatccattttgctttgtatgcagtctgtagtggaatctctaaaaaagac tgggtgattgatgcacactccactcgttggcgcagaatgcatcatgcattgcatttggggtttctagttaagcataactctcattga cttgttaacaattgagatggacagttgttggaatcgattctccttctagaagcaaaattgaccagggcaacaagaaccaaacaacttcctagcc aaagaaatgaataagcagctaagtatgtaaacatt gcgatgcacacaataagaatccggttatggaacccaactctacaacaaactatgtaaa tgtggtgtatttcccagtgatggatcagattaatcacaggccattacaccacctgacagtagatcagtagctagtttctgagcaatgcaaaattc aaaaattatacatatatccccccaaaaatttcctgcttgttagttatctaagaacaaagtt ttcaacctatctatacccctcaaatgttgagtttgg gcctgggcatatttccagttgcaaaaggaaatgccagaatgattcgctaggtagtaagtgtgcgtgtacatgtctgagctactatctgaactca ccgaaaacagtatccagaagaggccatgatgcattgttgtatgagtgataattgctctctgaatcaagaggatgtgcagct tgcgactctgatg aagaacggtttctctgaagggcattacattatgcccactgcagtagcaactcagccacaatgcgatctattagtcgctaccgataagaaggt atataatcctctcgatgagcatcccacatggccttagctagttggtctcctgccagtccagcagccgtgtagtttgcgaaggc cctcctactcaagaccaatgtcttctgtagcccttgcattgttctcattgcagttaccatcttatccatatttccaaagaacgtggcaacataagca tcgctgttgactgaaacatagtagtcgagagcagct tttgtgttgccatgcatcctctcaaaatcctcatgagtcaggagagacgatttagtata catatttttgtagatcgaagtgaagccctcgagttccattagcccatccccagcagctaaataaatgttgtctctgtaggaatacctagtgcttg gaggataaatgctgtttcacttggtgttagagggcactttccacggttcctccacagatgag cagcatcaccaatcaatacattcctgtcctccc cacgtgcagcttcaatcgcatccagtgatttggaagaaagggcactgtattcacatcgactgtaggcaaccatatctggttcaaatctaaggtg aagtgataagaaaggttttggtattgcttggagaagcttcatagctttggcttctaccttcttgttcaactggagtgcattgtagcaacct tggcag taacaagctttcgcatgtgatggatatctgagaaaatacaggcatatacatcagcatggggaaaaggcagatccaaggtgaataattacaac agacattgttacatcgcaaagaactaactacagagtaacattaactaagtttacacaactagttatct taaaggctttattctccgcaaatccgcatcttgcagattatacctgtgaaaagtcttcttggactgatgacaatcattagaacagaaaccggcac aaatttatcagtaggtcatcgtatcgcaaagcagacaacaaatcaagattaggtaagtt gaagtggttggagttagtaaagaattcatgtaacc aacaaatttaaggcacgtgaaagtcttgcattgtatgaaactatgaattgacatcttagttaaacaaagttatcacgcgatagtatagtgtatcag tgtatttattctaattatgcatagtttttaaggagtcgaggcgttttaaggcgttgaggggggggct

SEQ ID NO: 24: ZmOTUB1 promoter sequence:

tcaatacatttggaaagtacaaatgaacatcacatcaaacatgtacaaacattagaacacaactcaaaagatgtagtattaaataaaggagaat gaggtcaagttataaaatgaactaatcctaattaggagttgattactctataagtgtttggaacatatctcaaaaaggtaaagtagtaatttaaac aagaaagagattaaatcataagatggacttatctcatt taggaatttgattattctttaagtgttatctataattacataacctaattctattcatttcatt gagacctaaaagttaaaccaaaatgaattcatgtaacacatattattaatctcacaagattaaatatttttaactcctagggctttcatttttatttat ttcattaaatgaataaatcaacatatacattaaacttatactctaggtgtaaaattaaagtgcacagaccatagatga acttaatttatttggacag agaataatattatgaacattttacaatttgaaccactaaatttggagttcatatgaaaaagatatgaaataaacaagttttgaagttgaaatatgaa attagggctaaatttgtgattaaataaaagtacagggggctatccaaaagaaaccaggggcctacgcgctaaaacagaggacgtcgggttg

- 48 043050 atttcctaaaagccgagggtctcttaagtaaaacttacacgtgaagggggtacggggtaacctcggccatcagatcagagatcaacgacaa ggattagatcgcgtgggcgcgggcgcgtggacgcgactaacaggcggggacggggcgtcagcgagtcagggcgggctgaccagccg ggcccaggggcaggggcgcaggtgaggggggagaagggagagcggccggatctagatcggagcgctgcgattagatccgcaataatg aaacctaaaccgcacgatctcggacgaacgcccgagatctatcgactgggggcggacacaaacgcggtggcgccgctcggtcccgcgc cgacagtgagcggtggcgaagct ctctgttcgcgcgggcagagcgaggagagggcgaggggtttggctgagggcgcaagtgagc gaggggaggtgggcgagcagggcgcggggctcaaaagggacgcaggcgcggggacgtggccggagaacgcgcggacgtgggcg cgtccaccgcgggggatcgtgggcgggaggttggggatgaccgaca ggtgggctcggtgggacagagagagagagagagcggg cgcggagggaaaggaacgacgtcgacaactcggtcccacagagcagcgagagagggggagagggcgcgctggagagacagac taactgggccagattggct ttttctatttccagggaatttctatttgctttttttatttattctctagggttttcaattcaaattcaaatctagtttcaaattcaaaccaaatcaaacatgt gcaacaattcaagaatatttaggctcaatatgatgcaacatttcatgactcatatgttttgggcaaaataaaataaataatccctcactaattaaac taac cctaatcaaaagaggaagagggggagaaactagagagagaaactagagtgagatagagaagagtaacacctgaatttggat tagattcttaatagattattattactagttggttgctcatactttgctacggttattatatactc catccgttccataatataaggcgtaaccatttttgttcttgtccccacaatataagacatgctctctctatgcatacgtacattaatgcagtgatat agagaaaattaaatatatttcttggtatttgaaccagaggtggttacgccttatatactaggacagagggagtat catataaataggtatttagat atttattcaaatgtaactattgtttatttctaaacactaaggtatgtgtggtctggtggttagctccaaatttctgaagcaggggggcgtgggctcga gtagttgccctgcattttttgcgcggtgtgatgggtgggcctagagtgtcagaaggtgaagctgtgatatcatcatgtgcccacattagat tctt aatagattagtatagattattaattattactaggtatgtgtccgcttgttgccacgaggctgagaagcgtgggggaggggcgcctaagctacaaa tttgtgggctggacgtggggagagtgaagccgtggtaacactaggtgcccacattaggttcttaatagatagtagatagatagattgtt tagcaccaggtacctacattagattcttaatagatagcaggggagtagaaatatatatatagaacttatatat atgccgaagctttgttttatacattattcttaaccgtttatttttaaataacatatatttttaatagcacgaaatagatgcaaatatttttatggccttaaa caatttaaatagattttattttaatttttaggacttaattatacgactttttgaacgctataag taaacggtaaataacaagggcttatcc

SEQ ID NO: 25: SbOTUBl promoter sequence:

tattcaacctgagagcactgtagcagccttgacagtatgaagcttttgcatatgaggggtttgaaaacacacaaacatatatcaatttggcgc tcattgccacaatcgtgaatcatagaatagctaaccacgcaaatctgtaggtacatacata agctggaaggactaaactactcgttaccttattatcttatttcactcaacctacatctttagaagatagtaggaaactaaaagattaggtaaattaa agtggttgctggttcgcaaaaaagtcatataaccaacaaataaaggatacccaaattctcataatata tagacaggcatcttcaccatcaaaatt catagtgtgtacgctctaaccaattaaggtcaggttcgtttactgcaaagggacaaaaagaattgataaatgcagatcactacagcttttcaca gtaagttctaagttgtaaataaaggaaatccagcaaactaaaattttaaaggtttacaaaaatatgctacactaaatagttgttagaagtag gaag gttatttcaaacaatgaaatcaacaaaacaccttatccatgtaaagtagcaattaccaactgcttggtcacatcagatgctaacatctaaag cataaatgactaaataaatactgatttatacttcatcacggcttagttgtagtactttcacatatactagtcactttagtcaggaacagtaatgataaa actattatacttcataaactattttcccttc ggtaggaataggaaatatgaacttcttataaactataatatagtacaaccaagatgccaatcttgta gtcgtacaatattttattgctaactgatttattatcaccccttccaacatactaatacaatgagtcaatgaatatagaataacatattctattagatgtc atgcaaaacccactgtaacttgtgaaacgaatcacttgtaagcatgagtaagagcattagtatattatgg aaatggatggaaaatgcaaagga tctatgagatatacaagcagtacctatctcttctttcatggcaggtgtcagtgagatgtattgatgctccaagagagctggaagtacacttt caacataatcaaaatgtcctttccgtttactgcagtcaaccttaaacggctctttggatgctatctccacaggcaagtccttcataacttccaca ta

- 49 043050 tcctctagtctgctcaatgaagtaatcaacatcgaaaacatctgcaaagccactgacagaacaatccaataagaactgtaagcatatttca aacagaactgaactatagaacttcaatcatcatacctagactcattccaataggcggcaacctcaaacttgggcagaatcatcgttgcgttcag aagacgtgcaaccccaattccg tcacatagctgcagaaccatcacaatagtatcatttagtggcccctccagaactcataagcacaagatatg aagttatgaacaaaagaaagcacaattgctttatggaacatcaagatcttctgactgaaagttcagcactctcagtctcaggctcagagagtca ctactccatgcagtgctgctacgatactgacaaccagagcatgtttcggctaaa atcaatcatttgatctctacaggaaggaggaaactctaac cgaggtctcttaacgtacatcacgtcgcagctggttgagaccaccgtagcagtctattcggatgtacccgttcctcctggacggagctgcaca cacagcgcaggcgtcaatcatcagtacgtccaagtccagtatccaaattccacccccaaacagcgaggccttaccttgc actggtgccgtc cgccaccacccgcacgggcgccactccaccaaccgccgctgcgcccatatgagccgcgagcccgaaggaggcagctctctctgctacaaa agggcggcggcgacggcggcgacaggaggaagaagaggacgggcgcgaggaagagggacggtaggaggaacc ggaggcggtgctgcgcgcaccacgccgccgccggtgacatggccggcggcgactcaccgtcgccggagccgccgggtgaggcgcct ggatctgatcggccggtggcggcgtgtgatgaggaggagaa agaaatcgaatcccctagcacactgcgcacaccggagccgcaccag ggccttgtttagttccaattttttttttcaaaatagaattagtagcacattcgtttgtatttgacaaattgttcaatcatgaactaactaggcttaaaa gattcgtctcgttaattccgactaaactgtataattagttttttttttcgtttatatttaatacttcatgta tgcgtctaaagatttgatgtgacggagaat ctgaaaaa gate

SEQ ID NO: 26: GmOTUBl promoter sequence:

tctgttaataatcaataatttgataattaattaatttttttatcatacaagttaataatgactacacatctttatcaaaattgaaagaagattga aatctccttaaatcaacttaactgattgtacaagttaaattaggatgcataaattaattaattacataattaaaattaatttctcaatatatttttgtctgaa tttcatttctcatagtatgttcacttgaaaaatatga taaatgaaactaaaataaaataaaaaatataataaataatgtgattagaaagaaagaa aaagacaaaagaagaagaaaaagtaaatatatacatttttacatatttaggtcaaatgactttttttttaatttgatcttttattttctaaaaaaattattt taatctttttgtttttaaattgattcgaaatagtccttctgttaaaagcaa atttaacaccattaataatataaagataacccgccacaaaaacataa tttcttttctgaaatatctccatactataattaattaatacacatcaacaatttttaactcctactataatatttacaaagtttgaaaaaatacacaaaaacat gttctaacctccatgaacctagaacatccccaacccataaccaaaaacaattgcaacaattcttaaaattggtttagggctt ctaaattgcatatc tttacaagcaaaacccaaaaacaatctttgattttgattcaaaaacttgagagaatttacaaatcttgtattcaaaaccaaataaaaagttgcaca acaatctccctctagtgctccttccttcgttgtgtcactactactactgctacaatgattatgagtttgaatgctattatcatcattgttgtcgtcg cattgtt cacttctgttgtcacaaattattttaatgatgtttccaacgacgaatgaaatccatcatattagaaaaaagtaaacaaacgagcacaaatatcata gatctaaaattaatctacaaattaaaaaaccaaacacatatattggtatcagtgagtgtttaaaaaaaatactataagtgaaacccattgtgactc tgatgg tttcgatgatgacaaacataaactattcaacgaaagtcacaaaatactaatagtagggactaatttgaatcattttaaataaaaaaataa tattatgaatcttttaaaagagtgaaactgaagaccttgtggttagaattatataaattttcaagggcataatttta catttacaaggtttaaaactttcaaaatgaacgaaattatttttttgaaatgaaacattgtttcaatctaaatattttatcatcgatgttttaaaatgta gattctattaaaaaatatcatctcaatattatcctagggtgtatt ggattaagatttcaaaggattttaaaagacttttttgattaaaaagtcttgt ggtattcaatcaagactttttaaaaatataaacaaatcttgtggtattcaattaagacaataaagattttttttcagggcaacaaaatctgttggtat tcaattgagatttcttaccacttttaaaatgtcttttggtattcaaaagtaaatagattttga tggattcttttgtggaatggattttgagagactttttag ttagaaatacacataaaatactcctccaacaatctcacccaaacccttgagacttttcataatcttccaaagactttttctctcctgccgaacaag acacaaaccactaccaggtttattctcttacaacttttcaatcaattttacctactttttaatgacaatcgatagtcaatattgttcatactact atatgtata ttacgcaaatcaaaagaaaagggtgctgtatatgcttcaagatttcgtattcatattgttttcaacgtccaggcaatgaatatgcatcaaaagaat cat gtgcccttaagacacttgttagtgtattatgaacccattgttcaaacacaagagcaggaacgagaagatgctaatatatggaggactaata aacattataatttatttatcatctttttcattcacttttgtaacttccgttattttttgtttaaaatgtat taatctttcaaaatcttaaaaatccataaagtactttgaaatcttaaaaatctgtgttagaaatccattaaaatcttgggttaagaatcctgattctaa aaagtcttttaaaaaaaatcttttaaaatcccacaaa gec

- 50 043050

SEQ ID NO: 27: BnOTUBl promoter sequence:

tgtttaaatacatgtggtcactttcactatgtggattgtggaccctctcgtttattctaaagatggctttgtggaatttttatagatacaaa aagtgggtaacatggaaatgtgcaacatcatattatcatcaccatctaatcatatgactgaaagtgaattgagtatatggcttttaggattggcca aagtaagcatgtaagattatgagccaat ctgcaatgagttttttttttcttctatttcttttttgttcttctctctaattttcttttcactttaggcatgct aaaaatcatattaattaagaaaaaattagtctgaaaatttgttagcttattcctaaatc gttataagaaaaattggttccatcccaaataggtttatatttgttttaatataacgatggcatttgagttctacatggatgtcacttgtctaagacaat tttcttatcttttttttgattaaatagagtatcccagacctatggaaatatcaaactaat cttccaaaaagaagtatagctcacgagttagatcatt tgcgggtatccaaataaagttcatactctatagtatagttccatatcttcgtgaccacacatatttagtgtcgtgggattgctttaaaccgaattgct taactaaccggaattcgcctagatcactcatttgttttttcttactccctccgtttttattgtaagtagttttagcaaaa aatgtttgtttcacaatataa gtagttttcatatttcaatgcacattttctctttattgaatattgtgtaaccaatcaaataatgctagcttttttataataggttgaatttattaattaaataa taaaattctatttccatcaaactgaattaaaattgttgacaaaattcattgtcatctattcttgtcatttacaaatttgactctacacctaatatgc tttcccatgaatatcatatatgtgtcacttgatcaaatgatgggctaatcacatgctttacaatactcatgattgcaagccctgcaat aaaccattcgttatcgttttcttaattttattttgtggctttttcatatggctattccaatgctttt atgattattagcatgaacttgagcatgttctttatcccccaaagaaagtaaactacagatacatcgtatgatattattgtgtattattaattattatgta cgatcgattttacaacatatatagaagagctgatgagtgttactgacgtaaaatagtaattaata catatatcacttaactatatatgcataaagtc aagtttggttttgggttaaaaacgtacatcaaaccaaacataatagttcagtttatcttagtttaggtttgattatggtccagtagatcgtaatccaa ggtcactatttttgttgacaatggttttttttctggttaaattttgattttttttttgtttttaatttgcaagtccccgcatacactaa aagataaagctcgat atatttgaattaaacgggaatataatagcatagattcaatcatggacgtattatcattctctagtgactttttccacgctttcattaaaaaaagaaca cttatctacatactgataacaactaacatagtataattaaggtcgttaagagtttagaaaaaaaatgcaaaaagaaagaataatagttgatgttcg tgtagattttt ggttactttcttgaacgagcaaggatcaaaatcatcttcggaagaggagtatcaacctcacaagtcacaagaataataagctac tttacttgtagcagagtcaaatgaattcacgcgatcagcaataatcttatttttaacaaaca gaaatagtggacatccatagatgatataaaccttaatttttgtttcaaattcaccacgaaacccttacattcgaaataagttataagctatcatatgt tagttcattaccacaggtccacaacatctgtgcttcatcagctggtatcagaaccatattaag tcttaaatattaatatatgaatcatcatgtgatta ttgttgtgcgtttatgttgctagtccaaattccgatttggtcgtttactaacattcgcaaaagaggcccaaatttcggcccattctactaacatgat gtcgttccattattagcgaagccacatgcacgtcgttttaatttcccataaacgcttcgtctttctttattgta cattggaaaaaagccaaacgtctg ttaccttttctgcccttccaatcaaattggtgtaaatcttttggtttcctctttttttcagaaatttattttccctaaagttttgttttctttgtaaaaacattgca gagtgcccccaaaccg

CRISPR Sequence Information

Rice

Example I; OTUB1 gene

SEQ ID NO: 28: OsOTUB1 CRISPR target sequence tcagctggaaagtgttctgcagg

SEQ ID NO: 29: OsOTUB1 CRISPR protospacer sequence tcagctggaaagtgttctgc

SEQ ID NO: 30: Nucleotide sequence of tracr-RNA

GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT GAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTT

SEQ ID NO: 31: Full-length ssrRNA nucleotide sequence Os tcagctggaaagtgttctgcGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTT

SEQ ID NO: 32: ssrRNA OTUB1 Os

GUUUUAGAGCUAGAAAUAGCAAGUUAAAUAAGGCUAGUCCGUUAUCAAC UUGAAAAAGUGGCACCGAGUCGGUGC

- 51 043050

SEQ ID NO: 33: Full length OTUB1 CRISPR nucleic acid construct

TGGAATCGGCAGCAAAGGATTTTTTCCTGTAGTTTTCCCACAACCATTTTTTA CCATCCGAATGATAGGATAGGAAAAATATCCAAGTGAACAGTATTCCTATAAAATT CCCGTAAAAAGCCTGCAATCCGAATGAGCCCTGAAGTCTGAACTAGCCGGTCACCT GTACAGGCTATCGAGATGCCATACAAGAGACGGTAGTAGGAACTAGGAAGACGATG GTTGATTC GTCAGGCGAAATCGTCGTCCTGCAGTCGCATCTATGGGCCTGGACGGAA TAGGGGAAAAAGTTGGCCGGATAGGAGGAAAGGCCCAGGTGCTTACGTGCGAGG TAGGCCTGGGCTCTCAGCACTTCGATTCGTTGGCACCGGGGTAGGATGCAATAGAG AGCAACGTTTAGTACCACCTCGCTTAGCTAGAGCAAACTGGACTGCCTTATATGCGC Example II; OTUB1 gene

SEQ ID NO: 34: OsOTUB1 CRISPR target sequence gactactaccactcgtgctgcgg

SEQ ID NO: 35: protospacer sequence OsOTUB1 gactactaccactcgtgctg

SEQ ID NO: 36: OsOTUB1 ssrRNA nucleic acid sequence gactactaccactcgtgctgGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTT

SEQ ID NO: 37: Monocot plant OsOTUB1 U3-ssrRNA cluster nucleic acid construct

ACACTG GGTACGTTGGAAACCACGTGTGATGTGAAGGAGTAAGATAAACTGTAGGA GAAAAGCATTTCGTAGTGGGCCATGAAGCCTTTCAGGACATGTATTGCAGTATGGGC cgtgctgGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT TGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTT (Lower letters show the protospacer sequence that binds to the target sequence. The highlighted sequence is the ssrRNA coding sequence).

Example III; OTUB1 gene

SEQ ID NO: 38: OsOTUB1 CRISPR target sequence ccaccatgatttcagccctgagg

SEQ ID NO: 39: OsOTUB1 protospacer sequence ccaccatgatttcagccctg

SEQ ID NO: 40: OsOTUB1 ssrRNA nucleic acid sequence ccaccatgatttcagccctgGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTC

CGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCT

SEQ ID NO: 41: Monocot plant OsOTUB1 U6b-ssrRNA cluster nucleic acid construct

TGGAATCGGCAGCAAAGGATGCAGAACGAACTAAGCCGGACAAAAAAAAA AGGAGCACATATACAAACCGGTTTTATTCATGAATGGTCACGATGGATGATGGGGC TCAGACTTGAGCTACGAGGCCGCAGGCGAGAGAAGCCTAGTGTGCTCTCTGCTTGTT GGGCGCTGCGGGGCCGTTGGATGGGGATCGGTGGGTCGCGGGAGCGTTGAGGGA GACAGGTTTAGTACCACCTCGCCTACCGAACAATGAAGAACCCACCTTATAACCCCG CGAGTCGGTGCTTTTTTT CAAGAGCT (lowercase letters show the protospacer sequence that binds to after

- 52 043050 target range. The isolated sequence is the ssrRNA encoding sequence).

Example IV; OTUB1 promoter

SEQ ID NO: 42: target sequence agcggcggtgggggaggaggtgg

SEQ ID NO: 43; protospacer sequence agcggcggtgggggaggagg

SEQ ID NO: 44: full-length ssrRNA sequence (protospacer sequence highlighted (same as the following sequences)

TGGAATCGGCAGCAAAGGATTTTTTCCTGTAGTTTTCCCACAACCATTTTTTA CCATCCGAATGATAGGATAGGAAAAATATCCAAGTGAACAGTATTCCTATAAAATT CCCGTAAAAAGCCTGCAATCCGAATGAGCCCTGAAGTCTGAACTAGCCGGTCACCT GTACAGGCTATCGAGATGCCATACAAGAGACGGTAGTAGGAACTAGGAAGACGATG GTTGATTC GTCAGGCGAAATCGTCGTCCTGCAGTCGCATCTATGGGCCTGGACGGAA TAGGGGAAAAAGTTGGCCGGATAGGAGGAAAGGCCCAGGTGCTTACGTGCGAGG TAGGCCTGGGCTCTCAGCACTTCGATTCGTTGGCACCGGGGTAGGATGCAATAGAG AGCAACGTTTAGTACCACCTCGCTTAGCTAGAGCAAACTGGACTGCCTTATATGCGC GGGTGCTGGCTTGGCTGCCGagcggcggtgggggaggaggGTTTTAGAGCTAGAAATAGCAAG TTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT TTTCAAGAGCTT

Example V; OTUB1 promoter

SEQ ID NO: 45: target sequence

Gggaggaggggggggggggg

SEQ ID NO: 46 protospacer sequence gggaggaggagggggggggg

SEQ ID NO: 47: full-length ssrRNA sequence

TGGAATCGGCAGCAAAGGATTTTTTCCTGTAGTTTTCCCACAACCATTTTTTA CCATCCGAATGATAGGATAGGAAAAATATCCAAGTGAACAGTATTCCTATAAAATT CCCGTAAAAAGCCTGCAATCCGAATGAGCCCTGAAGTCTGAACTAGCCGGTCACCT GTACAGGCTATCGAGATGCCATACAAGAGACGGTAGTAGGAACTAGGAAGACGATG GTTGATTC GTCAGGCGAAATCGTCGTCCTGCAGTCGCATCTATGGGCCTGGACGGAA TAGGGGAAAAAGTTGGCCGGATAGGAGGAAAGGCCCAGGTGCTTACGTGCGAGG TAGGCCTGGGCTCTCAGCACTTCGATTCGTTGGCACCGGGGTAGGATGCAATAGAG AGCAACGTTTAGTACCACCTCGCTTAGCTAGAGCAAACTGGACTGCCTTATATGCGC GGGTGCTGGCTTGGCTGCCGgggaggaggaggggggggggGTTTTAGAGCTAGAAATAGCAA GTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTT TTTTCAAGAGCTT

Example VI; OTUB1 promoter

SEQ ID NO: 48: target sequence

Ggggggaggaggagggggggggg

SEQ ID NO: 49 protospacer sequence

Ggggggaggagggggggggg

SEQ ID NO: 50: full-length ssrRNA sequence

TGGAATCGGCAGCAAAGGATTTTTTCCTGTAGTTTTCCCACAACCATTTTTTA CCATCCGAATGATAGGATAGGAAAAATATCCAAGTGAACAGTATTCCTATAAAATT CCCGTAAAAAGCCTGCAATCCGAATGAGCCCTGAAGTCTGAACTAGCCGGTCACCT GTACAGGCTATCGAGATGCCATACAAGAGACGGTAGTAGGAACTAGGAAGACGATG GTTGATTC GTCAGGCGAAATCGTCGTCCTGCAGTCGCATCTATGGGCCTGGACGGAA TAGGGGAAAAAGTTGGCCGGATAGGAGGAAAGGCCCAGGTGCTTACGTGCGAGG TAGGCCTGGGCTCTCAGCACTTCGATTCGTTGGCACCGGGGTAGGATGCAATAGAG AGCAACGTTTAGTACCACCTCGCTTAGCTAGAGCAAACTGGACTGCCTTATATGCGC GGGTGCTGGCTTGGCTGCCGggggggaggaggagggggggGTTTTAGAGCTAGAAATAGCAA GTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTT TTTTCAAGAGCTT

- 53 043050

Example VII; OTUB1 promoter

SEQ ID NO: 51: target sequence tcgaggggggaggaggagggggg

SEQ ID NO: 52; protospacer sequence tcgaggggggaggaggaggg

SEQ ID NO: 53: full-length ssrRNA sequence

TGGAATCGGCAGCAAAGGATTTTTTCCTGTAGTTTTCCCACAACCATTTTTTA CCATCCGAATGATAGGATAGGAAAAATATCCAAGTGAACAGTATTCCTATAAAATT CCCGTAAAAAGCCTGCAATCCGAATGAGCCCTGAAGTCTGAACTAGCCGGTCACCT GTACAGGCTATCGAGATGCCATACAAGAGACGGTAGTAGGAACTAGGAAGACGATG GTTGATTC GTCAGGCGAAATCGTCGTCCTGCAGTCGCATCTATGGGCCTGGACGGAA TAGGGGAAAAAGTTGGCCGGATAGGAGGAAAGGCCCAGGTGCTTACGTGCGAGG TAGGCCTGGGCTCTCAGCACTTCGATTCGTTGGCACCGGGGTAGGATGCAATAGAG AGCAACGTTTAGTACCACCTCGCTTAGCTAGAGCAAACTGGACTGCCTTATATGCGC GGGTGCTGGCTTGGCTGCCGtcgaggggggaggaggagggGTTTTAGAGCTAGAAATAGCAAG TTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT TTTCAAGAGCTT

Example VIII; OTUB1 promoter

SEQ ID NO: 54: target sequence

Gtcgtcgagggggggaggaggagg

SEQ ID NO: 55; protospacer sequence gtcgtcgaggggggaggagg

SEQ ID NO: 56: full-length ssrRNA sequence

TGGAATCGGCAGCAAAGGATTTTTTCCTGTAGTTTTCCCACAACCATTTTTTA CCATCCGAATGATAGGATAGGAAAAATATCCAAGTGAACAGTATTCCTATAAAATT CCCGTAAAAAGCCTGCAATCCGAATGAGCCCTGAAGTCTGAACTAGCCGGTCACCT GTACAGGCTATCGAGATGCCATACAAGAGACGGTAGTAGGAACTAGGAAGACGATG GTTGATTC GTCAGGCGAAATCGTCGTCCTGCAGTCGCATCTATGGGCCTGGACGGAA TAGGGGAAAAAGTTGGCCGGATAGGAGGAAAGGCCCAGGTGCTTACGTGCGAGG TAGGCCTGGGCTCTCAGCACTTCGATTCGTTGGCACCGGGGTAGGATGCAATAGAG AGCAACGTTTAGTACCACCTCGCTTAGCTAGAGCAAACTGGACTGCCTTATATGCGC GGGTGCTGGCTTGGCTGCCGgtcgtcgaggggggaggaggGTTTTAGAGCTAGAAATAGCAAG TTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTT TTTCAAGAGCTT

Example IX; OTUB1 promoter

SEQ ID NO: 57: Target sequence ggcagccgcggcccccgagccgg

SEQ ID NO: 58; protospacer sequence ggcagccgcggcccccgagc

SEQ ID NO: 59: full-length ssrRNA sequence

TGGAATCGGCAGCAAAGGATTTTTTCCTGTAGTTTTCCCACAACCATTTTTTA CCATCCGAATGATAGGATAGGAAAAATATCCAAGTGAACAGTATTCCTATAAAATT CCCGTAAAAAGCCTGCAATCCGAATGAGCCCTGAAGTCTGAACTAGCCGGTCACCT GTACAGGCTATCGAGATGCCATACAAGAGACGGTAGTAGGAACTAGGAAGACGATG GTTGATTC GTCAGGCGAAATCGTCGTCCTGCAGTCGCATCTATGGGCCTGGACGGAA TAGGGGAAAAAGTTGGCCGGATAGGAGGAAAGGCCCAGGTGCTTACGTGCGAGG TAGGCCTGGGCTCTCAGCACTTCGATTCGTTGGCACCGGGGTAGGATGCAATAGAG AGCAACGTTTAGTACCACCTCGCTTAGCTAGAGCAAACTGGACTGCCTTATATGCGC GGGTGCTGGCTTGGCTGCCGggcagccgcggcccccgagcGTTTTAGAGCTAGAAATAGCAAG TTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT TTTCAAGAGCTT

Example X; OTUB1 promoter

SEQ ID NO: 60: Target sequence tcccggaggaggcggtgatgggg

- 54 043050

SEQ ID NO: 61; protospacer sequence tcccggaggaggcggtgatg

SEQ ID NO: 62: Full-length ssrRNA sequence

TGGAATCGGCAGCAAAGGATTTTTTCCTGTAGTTTTCCCACAACCATTTTTTA CCATCCGAATGATAGGATAGGAAAAATATCCAAGTGAACAGTATTCCTATAAAATT CCCGTAAAAAGCCTGCAATCCGAATGAGCCCTGAAGTCTGAACTAGCCGGTCACCT GTACAGGCTATCGAGATGCCATACAAGAGACGGTAGTAGGAACTAGGAAGACGATG GTTGATTC GTCAGGCGAAATCGTCGTCCTGCAGTCGCATCTATGGGCCTGGACGGAA TAGGGGAAAAAGTTGGCCGGATAGGAGGAAAGGCCCAGGTGCTTACGTGCGAGG TAGGCCTGGGCTCTCAGCACTTCGATTCGTTGGCACCGGGGTAGGATGCAATAGAG AGCAACGTTTAGTACCACCTCGCTTAGCTAGAGCAAACTGGACTGCCTTATATGCGC GGGTGCTGGCTTGGCTGCCGtcccggaggaggcggtgatgGTTTTAGAGCTAGAAATAGCAAG TTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTT TTTCAAGAGCTT

Example XI; OTUB1 promoter

SEQ ID NO: 63: target sequence cgccgggaagcgcaaggcggcgg

SEQ ID NO: 64; protospacer sequence cgcccggaagcgcaaggcgg

SEQ ID NO: 65: full-length ssrRNA sequence

TGGAATCGGCAGCAAAGGATTTTTTCCTGTAGTTTTCCCACAACCATTTTTTA CCATCCGAATGATAGGATAGGAAAAATATCCAAGTGAACAGTATTCCTATAAAATT CCCGTAAAAAGCCTGCAATCCGAATGAGCCCTGAAGTCTGAACTAGCCGGTCACCT GTACAGGCTATCGAGATGCCATACAAGAGACGGTAGTAGGAACTAGGAAGACGATG GTTGATTC GTCAGGCGAAATCGTCGTCCTGCAGTCGCATCTATGGGCCTGGACGGAA TAGGGGAAAAAGTTGGCCGGATAGGAGGAAAGGCCCAGGTGCTTACGTGCGAGG TAGGCCTGGGCTCTCAGCACTTCGATTCGTTGGCACCGGGGTAGGATGCAATAGAG AGCAACGTTTAGTACCACCTCGCTTAGCTAGAGCAAACTGGACTGCCTTATATGCGC GGGTGCTGGCTTGGCTGCCGcgccgggaagcgcaaggcggGTTTTAGAGCTAGAAATAGCAAG TTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTT TTTCAAGAGCTT

Example XII; OTUB1 promoter

SEQ ID NO: 66: Target sequence gaagagaagaacacgcaccgcgg

SEQ ID NO: 67; protospacer sequence gaagagaagaacacgcaccg

SEQ ID NO: 68: full-length ssrRNA sequence

TGGAATCGGCAGCAAAGGATTTTTTCCTGTAGTTTTCCCACAACCATTTTTTA CCATCCGAATGATAGGATAGGAAAAATATCCAAGTGAACAGTATTCCTATAAAATT CCCGTAAAAAGCCTGCAATCCGAATGAGCCCTGAAGTCTGAACTAGCCGGTCACCT GTACAGGCTATCGAGATGCCATACAAGAGACGGTAGTAGGAACTAGGAAGACGATG GTTGATTC GTCAGGCGAAATCGTCGTCCTGCAGTCGCATCTATGGGCCTGGACGGAA TAGGGGAAAAAGTTGGCCGGATAGGAGGAAAGGCCCAGGTGCTTACGTGCGAGG TAGGCCTGGGCTCTCAGCACTTCGATTCGTTGGCACCGGGGTAGGATGCAATAGAG AGCAACGTTTAGTACCACCTCGCTTAGCTAGAGCAAACTGGACTGCCTTATATGCGC GGGTGCTGGCTTGGCTGCCGgaagagaagaacacgcaccgGTTTTAGAGCTAGAAATAGCAAG TTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT TTTCAAGAGCTT

Example XIII; OTUB1 promoter

SEQ ID NO: 69: target sequence cccttacggcctccactctgagg

SEQ ID NO: 70; protospacer sequence cccttacggcctccactctg

- 55 043050

SEQ ID NO: 71: full-length ssrRNA sequence

TGGAATCGGCAGCAAAGGATTTTTTCCTGTAGTTTTCCCACAACCATTTTTTA CCATCCGAATGATAGGATAGGAAAAATATCCAAGTGAACAGTATTCCTATAAAATT CCCGTAAAAAGCCTGCAATCCGAATGAGCCCTGAAGTCTGAACTAGCCGGTCACCT GTACAGGCTATCGAGATGCCATACAAGAGACGGTAGTAGGAACTAGGAAGACGATG GTTGATTC GTCAGGCGAAATCGTCGTCCTGCAGTCGCATCTATGGGCCTGGACGGAA TAGGGGAAAAAGTTGGCCGGATAGGAGGAAAGGCCCAGGTGCTTACGTGCGAGG TAGGCCTGGGCTCTCAGCACTTCGATTCGTTGGCACCGGGGTAGGATGCAATAGAG AGCAACGTTTAGTACCACCTCGCTTAGCTAGAGCAAACTGGACTGCCTTATATGCGC GGGTGCTGGCTTGGCTGCCGcccttacggcctccactctgGTTTTAGAGCTAGAAATAGCAAGTT AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT TCAAGAGCTT

Example XIV; OTUB1 promoter

SEQ ID NO: 72: target sequence cactttctccttatgacacgtgg

SEQ ID NO: 73; protospacer sequence cactttctccttatgacacg

SEQ ID NO: 74: full-length ssrRNA sequence

TGGAATCGGCAGCAAAGGATTTTTTCCTGTAGTTTTCCCACAACCATTTTTTA CCATCCGAATGATAGGATAGGAAAAATATCCAAGTGAACAGTATTCCTATAAAATT CCCGTAAAAAGCCTGCAATCCGAATGAGCCCTGAAGTCTGAACTAGCCGGTCACCT GTACAGGCTATCGAGATGCCATACAAGAGACGGTAGTAGGAACTAGGAAGACGATG GTTGATTC GTCAGGCGAAATCGTCGTCCTGCAGTCGCATCTATGGGCCTGGACGGAA TAGGGGAAAAAGTTGGCCGGATAGGAGGAAAGGCCCAGGTGCTTACGTGCGAGG TAGGCCTGGGCTCTCAGCACTTCGATTCGTTGGCACCGGGGTAGGATGCAATAGAG AGCAACGTTTAGTACCACCTCGCTTAGCTAGAGCAAACTGGACTGCCTTATATGCGC GGGTGCTGGCTTGGCTGCCGcactttctccttatgacacgGTTTTAGAGCTAGAAATAGCAAGTT AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT TCAAGAGCTT

Example XV; OTUB1 promoter

SEQ ID NO: 75: Target sequence tatataagctcgtcagaatgtgg

SEQ ID NO: 76; protospacer sequence tatataagctcgtcagaatg

SEQ ID NO: 77: full-length ssrRNA sequence

TGGAATCGGCAGCAAAGGATTTTTTCCTGTAGTTTTCCCACAACCATTTTTTA CCATCCGAATGATAGGATAGGAAAAATATCCAAGTGAACAGTATTCCTATAAAATT CCCGTAAAAAGCCTGCAATCCGAATGAGCCCTGAAGTCTGAACTAGCCGGTCACCT GTACAGGCTATCGAGATGCCATACAAGAGACGGTAGTAGGAACTAGGAAGACGATG GTTGATTC GTCAGGCGAAATCGTCGTCCTGCAGTCGCATCTATGGGCCTGGACGGAA TAGGGGAAAAAGTTGGCCGGATAGGAGGAAAGGCCCAGGTGCTTACGTGCGAGG TAGGCCTGGGCTCTCAGCACTTCGATTCGTTGGCACCGGGGTAGGATGCAATAGAG AGCAACGTTTAGTACCACCTCGCTTAGCTAGAGCAAACTGGACTGCCTTATATGCGC GGGTGCTGGCTTGGCTGCCGtatataagctcgtcagaatgGTTTTAGAGCTAGAAATAGCAAGTT AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT

TCAAGAGCTT

Example XVI; OTUB1 promoter

SEQ ID NO: 78: target sequence cgagaagcactggatctgatcgg

SEQ ID NO: 79; protospacer sequence cgagaagcactggatctgat

- 56 043050

SEQ ID NO: 80: full-length ssrRNA sequence

TGGAATCGGCAGCAAAGGATTTTTTCCTGTAGTTTTCCCACAACCATTTTTTA CCATCCGAATGATAGGATAGGAAAAATATCCAAGTGAACAGTATTCCTATAAAATT CCCGTAAAAAGCCTGCAATCCGAATGAGCCCTGAAGTCTGAACTAGCCGGTCACCT GTACAGGCTATCGAGATGCCATACAAGAGACGGTAGTAGGAACTAGGAAGACGATG GTTGATTC GTCAGGCGAAATCGTCGTCCTGCAGTCGCATCTATGGGCCTGGACGGAA TAGGGGAAAAAGTTGGCCGGATAGGAGGAAAGGCCCAGGTGCTTACGTGCGAGG TAGGCCTGGGCTCTCAGCACTTCGATTCGTTGGCACCGGGGTAGGATGCAATAGAG AGCAACGTTTAGTACCACCTCGCTTAGCTAGAGCAAACTGGACTGCCTTATATGCGC GGGTGCTGGCTTGGCTGCCGcgagaagcactggatctgatGTTTTAGAGCTAGAAATAGCAAGT TAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTT TTCAAGAGCTT

Example XVII; OTUB1 promoter

SEQ ID NO: 81: target sequence gagaggaggaagtagagcgcggg

SEQ ID NO: 82; protospacer sequence gagaggaggaagtagagcgc

SEQ ID NO: 83: full-length ssrRNA sequence

TGGAATCGGCAGCAAAGGATTTTTTCCTGTAGTTTTCCCACAACCATTTTTTA CCATCCGAATGATAGGATAGGAAAAATATCCAAGTGAACAGTATTCCTATAAAATT CCCGTAAAAAGCCTGCAATCCGAATGAGCCCTGAAGTCTGAACTAGCCGGTCACCT GTACAGGCTATCGAGATGCCATACAAGAGACGGTAGTAGGAACTAGGAAGACGATG GTTGATTC GTCAGGCGAAATCGTCGTCCTGCAGTCGCATCTATGGGCCTGGACGGAA TAGGGGAAAAAGTTGGCCGGATAGGAGGAAAGGCCCAGGTGCTTACGTGCGAGG TAGGCCTGGGCTCTCAGCACTTCGATTCGTTGGCACCGGGGTAGGATGCAATAGAG AGCAACGTTTAGTACCACCTCGCTTAGCTAGAGCAAACTGGACTGCCTTATATGCGC GGGTGCTGGCTTGGCTGCCGgaggaggaagtagagcgcGTTTTAGAGCTAGAAATAGCAAG TTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT TTTCAAGAGCTT

Example XVIII; OTUB1 promoter

SEQ ID NO: 84: Target sequence taaacccaaaccacaaatcacgg

SEQ ID NO: 85; protospacer sequence taaacccaaaccacaaatca

SEQ ID NO: 86: full-length ssrRNA sequence

TGGAATCGGCAGCAAAGGATTTTTTCCTGTAGTTTTCCCACAACCATTTTTTA CCATCCGAATGATAGGATAGGAAAAATATCCAAGTGAACAGTATTCCTATAAAATT CCCGTAAAAAGCCTGCAATCCGAATGAGCCCTGAAGTCTGAACTAGCCGGTCACCT GTACAGGCTATCGAGATGCCATACAAGAGACGGTAGTAGGAACTAGGAAGACGATG GTTGATTC GTCAGGCGAAATCGTCGTCCTGCAGTCGCATCTATGGGCCTGGACGGAA TAGGGGAAAAAGTTGGCCGGATAGGAGGAAAGGCCCAGGTGCTTACGTGCGAGG TAGGCCTGGGCTCTCAGCACTTCGATTCGTTGGCACCGGGGTAGGATGCAATAGAG AGCAACGTTTAGTACCACCTCGCTTAGCTAGAGCAAACTGGACTGCCTTATATGCGC GGGTGCTGGCTTGGCTGCCGtaaacccaaaccacaaatcaGTTTTAGAGCTAGAAATAGCAAGT TAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTT TTCAAGAGCTT

Example XIX; OTUB1 promoter

SEQ ID NO: 87: target sequence atgtacccattcctcctcgacgg

SEQ ID NO: 88; protospacer sequence atgtacccattcctcctcga

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SEQ ID NO: 89: full-length ssrRNA sequence

TGGAATCGGCAGCAAAGGATTTTTTCCTGTAGTTTTCCCACAACCATTTTTTA CCATCCGAATGATAGGATAGGAAAAATATCCAAGTGAACAGTATTCCTATAAAATT CCCGTAAAAAGCCTGCAATCCGAATGAGCCCTGAAGTCTGAACTAGCCGGTCACCT GTACAGGCTATCGAGATGCCATACAAGGAACGGTAGTAGGAACTAGGAAGACGATG GTTGATTC GTCAGGCGAAATCGTCGTCCTGCAGTCGCATCTATGGGCCTGGACGGAA TAGGGGAAAAAGTTGGCCGGATAGGAGGAAAGGCCCAGGTGCTTACGTGCGAGG TAGGCCTGGGCTCTCAGCACTTCGATTCGTTGGCACCGGGGTAGGATGCAATAGAG AGCAACGTTTAGTACCACCTCGCTTAGCTAGAGCAAACTGGACTGCCTTATATGCGC GGGTGCTGGCTTGGCTGCCGtatgtacccattcctcctcgaGTTTTAGAGCTAGAAATAGCAAGTT AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT TCAAGAGCTT

Example XX; OTUB1 promoter

SEQ ID NO: 90: Target sequence aacgtacatcgcgtcgcagctgg

SEQ ID NO: 91; protospacer sequence aacgtacatcgcgtcgcagc

SEQ ID NO: 92: full-length ssrRNA sequence

TGGAATCGGCAGCAAAGGATTTTTTCCTGTAGTTTTCCCACAACCATTTTTTA CCATCCGAATGATAGGATAGGAAAAATATCCAAGTGAACAGTATTCCTATAAAATT CCCGTAAAAAGCCTGCAATCCGAATGAGCCCTGAAGTCTGAACTAGCCGGTCACCT GTACAGGCTATCGAGATGCCATACAAGAGACGGTAGTAGGAACTAGGAAGACGATG GTTGATTC GTCAGGCGAAAATCGTCGTCCTGCAGTCGCATCTATGGGCCTGGACGGAA

TAGGGGAAAAAAGTTGGCCGGATAGGAGGGAAAGGCCCAGGTGCTTACGTGCGAGG TAGGCCTGGGCTCTCAGCACTTCGATTCGTTGGCACCGGGGTAGGATGCAATAGAG AGCAACGTTTAGTACCACCTCGCTTAGCTAGAGCAAACTGGACTGCCTTATATGCGC GGGTGCTGGCTTGGCTGCCGaacgtacatcgcgtcgcagc GTTTTAGAGCTAGAAATAGCAAGT TAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTT TTCAAGAGCTT

Example XXI; OTUB1 promoter

SEQ ID NO: 93: target sequence actatcttactacttgtgcatgg

SEQ ID NO: 94; protospacer sequence actatcttactacttgtgca

SEQ ID NO: 95: full-length ssrRNA sequence

TGGAATCGGCAGCAAAGGATTTTTTCCTGTAGTTTTCCCACAACCATTTTTTA CCATCCGAATGATAGGATAGGAAAAATATCCAAGTGAACAGTATTCCTATAAAATT CCCGTAAAAAGCCTGCAATCCGAATGAGCCCTGAAGTCTGAACTAGCCGGTCACCT GTACAGGCTATCGAGATGCCATACAAGAGACGGTAGTAGGAACTAGGAAGACGATG GTTGATTC GTCAGGCGAAATCGTCGTCCTGCAGTCGCATCTATGGGCCTGGACGGAA TAGGGGAAAAAGTTGGCCGGATAGGAGGAAAGGCCCAGGTGCTTACGTGCGAGG TAGGCCTGGGCTCTCAGCACTTCGATTCGTTGGCACCGGGGTAGGATGCAATAGAG AGCAACGTTTAGTACCACCTCGCTTAGCTAGAGCAAACTGGACTGCCTTATATGCGC GGGTGCTGGCTTGGCTGCCGactatcttactacttgtgcaGTTTTAGAGCTAGAAATAGCAAGTT AAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT TCAAGAGCTT

Example XXII; OTUB1 promoter

SEQ ID NO: 96 target sequence tcggaagaataattacaaccagg

SEQ ID NO: 97; protospacer sequence tcggaagaataattacaacc

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SEQ ID NO: 98: full-length ssrRNA sequence

TGGAATCGGCAGCAAAGGATTTTTTCCTGTAGTTTTCCCACAACCATTTTTTA CCATCCGAATGATAGGATAGGAAAAATATCCAAGTGAACAGTATTCCTATAAAATT CCCGTAAAAAGCCTGCAATCCGAATGAGCCCTGAAGTCTGAACTAGCCGGTCACCT GTACAGGCTATCGAGATGCCATACAAGGAACGGTAGTAGGAACTAGGAAGACGATG GTTGATTC GTCAGGCGAAATCGTCGTCCTGCAGTCGCATCTATGGGCCTGGACGGAA TAGGGGAAAAAGTTGGCCGGATAGGAGGAAAGGCCCAGGTGCTTACGTGCGAGG TAGGCCTGGGCTCTCAGCACTTCGATTCGTTGGCACCGGGGTAGGATGCAATAGAG AGCAACGTTTAGTACCACCTCGCTTAGCTAGAGCAAACTGGACTGCCTTATATGCGC GGGTGCTGGCTTGGCTGCCGtcggaagaataattacaaccGTTTTAGAGCTAGAAATAGCAAGT TAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTT TTCAAGAGCTT

Example XXIII; OTUB1 promoter

SEQ ID NO: 99: target sequence gtaggcagcaacctcaaacttgg

SEQ ID NO: 100; protospacer sequence gtaggcagcaacctcaaact

SEQ ID NO: 101: full-length ssrRNA sequence

TGGAATCGGCAGCAAAGGATTTTTTCCTGTAGTTTTCCCACAACCATTTTTTA CCATCCGAATGATAGGATAGGAAAAATATCCAAGTGAACAGTATTCCTATAAAATT CCCGTAAAAAGCCTGCAATCCGAATGAGCCCTGAAGTCTGAACTAGCCGGTCACCT GTACAGGCTATCGAGATGCCATACAAGGAACGGTAGTAGGAACTAGGAAGACGATG GTTGATTC GTCAGGCGAAATCGTCGTCCTGCAGTCGCATCTATGGGCCTGGACGGAA TAGGGGAAAAAGTTGGCCGGATAGGAGGAAAGGCCCAGGTGCTTACGTGCGAGG TAGGCCTGGGCTCTCAGCACTTCGATTCGTTGGCACCGGGGTAGGATGCAATAGAG AGCAACGTTTAGTACCACCTCGCTTAGCTAGAGCAAACTGGACTGCCTTATATGCGC GGGTGCTGGCTTGGCTGCCGgtaggcagcaacctcaaactGTTTTAGAGCTAGAAATAGCAAGT TAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTT TTCAAGAGCTT

Spelled wild wheat

Example I; OTUB1 gene

SEQ ID NO: 102: TuOTUB1 CRISPR target sequence ttaaggcgaacacgaggagatgg

SEQ ID NO: 103: protospacer sequence; TuOTUB1 (double target) ttaaggcgaacacgaggaga

SEQ ID NO: 104: TuOTUB1 ssrRNA nucleic acid ttaaggcgaacacgaggagaGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGT

CCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTT

SEQ ID NO: 105 TuOTUB1; construction of nucleic acid cluster U6a-ssrRNA of a monocot plant

TGGAATCGGCAGCAAAGGATTTTTTCCTGTAGTTTTCCCACAACCATTTTTTA CCATCCGAATGATAGGATAGGAAAAATATCCAAGTGAACAGTATTCCTATAAAATT CCCGTAAAAAGCCTGCAATCCGAATGAGCCCTGAAGTCTGAACTAGCCGGTCACCT GTACAGGCTATCGAGATGCCATACAAGAGACGGTAGTAGGAACTAGGAAGACGATG GTTGATTC GTCAGGCGAAATCGTCGTCCTGCAGTCGCATCTATGGGCCTGGACGGAA TAGGGGAAAAAGTTGGCCGGATAGGAGGAAAGGCCCAGGTGCTTACGTGCGAGG TAGGCCTGGGCTCTCAGCACTTCGATTCGTTGGCACCGGGGTAGGATGCAATAGAG AGCAACGTTTAGTACCACCTCGCTTAGCTAGAGCAAACTGGACTGCCTTATATGCGC GGGTGCTGGCTTGGCTGCCGttaaggcgaacacgaggagaGTTTTAGAGCTAGAAATAGCAAG TTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT TTTCAAGAGCTT (lowercase letters show the protospacer sequence that binds to the target sequence. The highlighted sequence is the ssrRNA coding sequence).

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Example II; OTUB1 gene

SEQ ID NO: 106: TuOTUB1 CRISPR target sequence attgctctgtcagatgcgttagg

SEQ ID NO: 107: protospacer sequence; TuOTUB1 (double target) attgctctgtcagatgcgtt

SEQ ID NO: 108: TuOTUB1 ssrRNA nucleic acid

AttgctctgtcagatgcgttGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTC

CGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTT

SEQ ID NO: 109: TuOTUB1; construction of nucleic acid cluster of U3-ssrRNA of a monocot plant

ACACTG GGTACGTTGGAAACCACGTGTGATGTGAAGGAGTAAGATAAACTGTAGGA GAAAAGCATTTCGTAGTGGGCCATGAAGCCTTTCAGGACATGTATTGCAGTATGGGC ca gatgcgttGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT GAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTT (lowercase letters show the protospacer sequence that binds to the target sequence. The highlighted sequence is the ssrRNA coding sequence).

Barley

Example I; OTUB1 gene

SEQ ID NO: 110: HvOTUB1 CRISPR target sequence ttaagacggacacgaggagatgg

SEQ ID NO: 111: Protospacer Hv OTUB1 ttaagacggacacgaggaga

SEQ ID NO: 112: ssrRNA nucleic acid Hv OTUB1 ttaagacggacacgaggagaGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGT CCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTT

SEQ ID NO: 113: Monocot plant Hv OTUB1 U6a-ssrRNA cluster nucleic acid construct

TGGAATCGGCAGCAAAGGATTTTTTCCTGTAGTTTTCCCACAACCATTTTTTA CCATCCGAATGATAGGATAGGAAAAATATCCAAGTGAACAGTATTCCTATAAAATT CCCGTAAAAAGCCTGCAATCCGAATGAGCCCTGAAGTCTGAACTAGCCGGTCACCT GTACAGGCTATCGAGATGCCATACAAGGAACGGTAGTAGGAACTAGGAAGACGATG

GTTGATTCGTCAGGCGAAATCGTCGTCCTGCAGTCGCATCTATGGGCCTGGACGGAA TAGGGAAAAAAGTTGGCCGGATAGGAGGGAAAGGCCCAGGTGCTTACGTGCGAGG TAGGCCTGGGCTCTCAGCACTTCGATTCGTTGGCACCGGGGTAGGATGCAATAGAG AGCACGTTTAGTACCACCTCGCTTAGCTAGAGCAAACTGGACTGCC TTATATGCGC GGGTGCTGGCTTGGCTGCCGttaagacggacacacgaggagaGTTTTAGAGCTAGAAATAGCAAG TTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTT TTTCAAGAGCTT (lowercase letters show the protospacer sequence that binds to the target sequence. The highlighted sequence is the ssrRNA coding sequence).

Example II; OTUB1 gene

SEQ ID NO: 114: HvOTUB1 CRISPR target sequence Attgctctgtcagatgcgttagg

SEQ ID NO: 115: protospacer Hv OTUB1 attgctctgtcagatgcgtt

SEQ ID NO: 116: ssrRNA nucleic acid Hv OTUB1 attgctctgtcagatgcgttGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCC GTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTT

- 60 043050

SEQ ID NO: 117: Nucleic acid construct of Hv OTUB1 u3-ssrRNA cluster of monocot plant

ACACTG GGTACGTTGGAAACCACGTGTGATGTGAAGGAGTAAGATAAACTGTAGGA GAAAAGCATTTCGTAGTGGGCCATGAAGCCTTTCAGGACATGTATTGCAGTATGGGC ca gatgcgttGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT GAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTT (lowercase letters show the protospacer sequence that binds to the target sequence. The highlighted sequence is the ssrRNA coding sequence).

Corn

Example I; OTUB1 gene

SEQ ID NO: 118: ZmOTUB1 CRISPR target sequence aagctttatgttttcctacctgg

SEQ ID NO: 119: ZmOTUB1 protospacer sequence aagctttatgttttcctacc

SEQ ID NO: 120: ZmOTUB1 ssrRNA nucleic acid sequence aagctttatgttttcctaccGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCC GTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTT

SEQ ID NO: 121: Monocot Plant U6a-ssrRNA Cluster Nucleic Acid Construct of ZmOTUB1

TGGAATCGGCAGCAAAGGATTTTTTCCTGTAGTTTTCCCACAACCATTTTTTA CCATCCGAATGATAGGATAGGAAAAATATCCAAGTGAACAGTATTCCTATAAAATT CCCGTAAAAAGCCTGCAATCCGAATGAGCCCTGAAGTCTGAACTAGCCGGTCACCT GTACAGGCTATCGAGATGCCATACAAGAGACGGTAGTAGGAACTAGGAAGACGATG GTTGATTC GTCAGGCGAAATCGTCGTCCTGCAGTCGCATCTATGGGCCTGGACGGAA TAGGGGAAAAAGTTGGCCGGATAGGAGGAAAGGCCCAGGTGCTTACGTGCGAGG TAGGCCTGGGCTCTCAGCACTTCGATTCGTTGGCACCGGGGTAGGATGCAATAGAG AGCAACGTTTAGTACCACCTCGCTTAGCTAGAGCAAACTGGACTGCCTTATATGCGC GGGTGCTGGCTTGGCTGCCGaagctttatgttttcctaccGTTTTAGAGCTAGAAATAGCAAGTTA AAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT CAAGAGCTT (lowercase letters show the protospacer sequence that binds to the target sequence. The highlighted sequence is the ssrRNA coding sequence).

Example II; OTUB1 gene

SEQ ID NO: 122: ZmOTUB1 CRISPR target sequence attgccctatcagatgcactagg

SEQ ID NO: 123: ZmOTUB1 protospacer sequence attgccctatcagatgcact

SEQ ID NO: 124: ssrRNA nucleic acid sequence of ZmOTUB1 attgccctatcagatgcactGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTT

- 61 043050

SEQ ID NO: 125: Monocot plant ZmOTUBl US-ssrRNA cassette nucleic acid construct

ACACTG GGTACGTTGGAAACCACGTGTGATGTGAAGGAGTAAGATAAACTGTAGGA GAAAAGCATTTCGTAGTGGGCCATGAAGCCTTTCAGGACATGTATTGCAGTATGGGC agatgcactGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACT TGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTT (lowercase letters show the protospacer sequence that binds to the target sequence. The highlighted sequence is the ssrRNA coding sequence). Sorghum

Example I; OTUB1 gene SEQ ID NO: 126: SbOTUBl CRISPR target sequence aagctttatgttctcctacttgg

SEQ ID NO: 127: SbOTUBl protospacer sequence aagctttatgttctcctact

SEQ ID NO: 128: SbOTUB1 ssrRNA nucleic acid sequence aagctttatgttctcctactGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCC GTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTT

SEQ ID NO: 129: Monocot plant U6a-ssrRNA cluster nucleic acid construct of ZmOTUB1

TGGAATCGGCAGCAAAGGATTTTTTCCTGTAGTTTTCCCACAACCATTTTTTA CCATCCGAATGATAGGATAGGAAAAATATCCAAGTGAACAGTATTCCTATAAAATT CCCGTAAAAAGCCTGCAATCCGAATGAGCCCTGAAGTCTGAACTAGCCGGTCACCT GTACAGGCTATCGAGATGCCATACAAGAGACGGTAGTAGGAACTAGGAAGACGATG GTTGATTC GTCAGGCGAAATCGTCGTCCTGCAGTCGCATCTATGGGCCTGGACGGAA TAGGGGAAAAAGTTGGCCGGATAGGAGGAAAGGCCCAGGTGCTTACGTGCGAGG TAGGCCTGGGCTCTCAGCACTTCGATTCGTTGGCACCGGGGTAGGATGCAATAGAG AGCAACGTTTAGTACCACCTCGCTTAGCTAGAGCAAACTGGACTGCCTTATATGCGC GGGTGCTGGCTTGGCTGCCGaagctttatgttctcctactGTTTTAGAGCTAGAAATAGCAAGTTA AAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT CAAGAGCTT (lowercase letters show the protospacer sequence that binds to the target sequence. The highlighted sequence is the ssrRNA coding sequence).

Example II; OTUB1 gene

SEQ ID NO: 130: SbOTUB1 CRISPR target sequence tgttcacataattgccctatcgg

SEQ ID NO: 131: SbOTUB1 protospacer sequence tgttcacataattgccctat

SEQ ID NO: 132: ssrRNA nucleic acid sequence of SbOTUB1 tgttcacataattgccctatGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCC GTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTT

SEQ ID NO: 133: Monocot plant US-ssrRNA cassette SbOTUB1 nucleic acid construct

TGGAATCGGCAGCAAAGGACGCGTTGACATTGTAGGACTATATTGCTCTAAT AAAGGAAGGAATCTTTAAACATACGAACAGATCACTTAAAGTTTCTTCTGAAGCAAC TTAAAGTTATCAGGCATGCATGGATCTTGGAGGAATCAGATGTGTCAGTCAGGGACC ATAGCACAAGACAGGCGTCTTCTACTGTGCTACCAGCAAATGCTGGAAGCCGGGA

- 62 043050

ACACTGGGTACGTTGGAAACCACGTGTGTGATGTGAAGGAGTAAGATAAACTGTAGGA GAAAAGCATTTCGTAGTGGGCCATGAAGCCTTTCAGGACATGTATTGCAGTATGGGC cacataat tgccctatGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTT GAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCTT (Lower letters show the protospacer sequence that binds to the target sequence. The highlighted sequence is the ssrRNA coding sequence).

Soya

Example I; OTUB1 gene

SEQ ID NO: 134: GmOTUB1 CRISPR target sequence attcgtcgtactcgaggagatgg

SEQ ID NO: 135: GmOTUB1 protospacer sequence attcgtcgtactcgaggaga

SEQ ID NO: 136: ssrRNA nucleic acid sequence of GmOTUB1 attcgtcgtactcgaggagaGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTC CGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCT

SEQ ID NO: 137: Monocot plant GmOTUB1 U3b-ssrRNA cluster nucleic acid construct

TGGAATCGGCAGCAAAGGATTTACTTTAAATTTTTTCTTATGCAGCCTGTGAT GGATAACTGAATCAAACAAATGGCGTCTGGGTTTAAGAAGATCTGTTTTGGCTATGT CATGCCCAGTACCCATGTATGCGTTTCATATAAGCTCCTAATTTCTCCC ACATCGCTCAAATCTAAACAAATCTTGTTGTATATATAACACTGAGGGAGCAACATT GGTCAattcggtcgtactcgaggagaGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGT CCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCT (lowercase letters sssrRNA is shown as the protospacer sequence that binds to the target sequence.

Example II; OTUB1 gene

SEQ ID NO: 138: GmOTUB1 CRISPR target sequence tactgccctttcagatgcattgg

SEQ ID NO: 139: GmOTUB1 protospacer sequence tactgccctttcagatgcat

SEQ ID NO: 140: ssrRNA nucleic acid sequence of GmOTUB1 tactgccctttcagatgcatGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTC

CGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCT

SEQ ID NO: 141: U6-1-sssrRNA cluster nucleic acid construct

GmOTUB 1 monocot plant

TGGAATCGGCAGCAAAGGAAGAAATCTCAAAATTCCGGCAGAACAATTTTGA ATCTCGATCCGTAGAAACGAGACGGTCATTGTTTTAGTTCCACCACGATTATATTTG AAATTTACGTGAGTGTGTGAGACTTGCATAAGAAAATAAAATCTTTAGTTGGGA AAAATTCAATAATATAAATGGGCTTGAGAAGGAAGCGAGGGATAGGCCTTTTTCT AAAATAGGCCCAT TTAAGCTATTAACAATCTTCAAAAGTACCACAGCGCTTAGGTAA AGAAAGCAGCTGAGTTTATATGGTTAGAGACGAAGTAGTGATTGtactgccctttcagatgc atGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA AAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCT a sequence that binds to a target sequence (the isolated sequence is the ssrRNA encoding sequence).

Cabbage

Example I; OTUB1 gene

SEQ ID NO: 142: BnOTUB1 CRISPR target sequence atcaggcgaacaagaggagatgg

- 63 043050

SEQ ID NO: 143: protospacer sequence BnOTUBl atcaggcgaacaagaggaga

SEQ ID NO: 144: Nucleic acid sequence of ssrRNA BnOTUB1 atcaggcgaacaagaggagaGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGT

CCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCT

SEQ ID NO: 145: Monocot plant U3b-ssrRNA cluster nucleic acid construct BnOTUB1

TGGAATCGGCAGCAAAGGATTTACTTTAAATTTTTTCTTATGCAGCCTGTGAT GGATAACTGAATCAAACAAATGGCGTCTGGGTTTAAGAAGATCTGTTTTGGCTATGT CATGCCCAGTACCCATGTATGCGTTTCATAAGCTCCTAATTTCTCCC ACATCGCTCAAATCTAAACAAATCTTGTTGTATATATAACACTGAGGGAGCAACATT GGTCAatcaggcgaacaagaggagaGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAG TCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCT a protospacer sequence that binds to a target sequence (the isolated sequence is the ssrRNA encoding sequence).

Example II; OTUB1 gene

SEQ ID NO: 146: BnOTUB1 CRISPR target sequence tattcacataacagctttgtcgg

SEQ ID NO: 147: protospacer sequence BnOTUB1 tattcacataacagctttgt

SEQ ID NO: 148: Nucleic acid sequence of ssrRNA BnOTUB1 tattcacataacagctttgtGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCC

GTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCT

SEQ ID NO: 149: Monocot Plant U6-1-ssrRNA BnOTUB1 Cluster Nucleic Acid Construct

TGGAATCGGCAGCAAAGGAAGAAATCTCAAAATTCCGGCAGAACAATTTTGA ATCTCGATCCGTAGAAACGAGACGGTCATTGTTTTAGTTCCACCACGATTATATTTG AAATTTACGTGAGTGTGTGAGACTTGCATAAGAAAATAAAATCTTTAGTTGGGA AAAATTCAATAATATAAATGGGCTTGAGAAGGAAGCGAGGGATAGGCCTTTTTCT AAAATAGGCCCAT TTAAGCTATTAACAATCTTCAAAAGTACCACAGCGCTTAGGTAA AGAAAGCAGCTGAGTTTATATGGTTAGAGACGAAGTAGTGATTGtattcacataacagcttt gtGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAA AAAGTGGCACCGAGTCGGTGCTTTTTTTCAAGAGCT , which binds to the target sequence.The isolated sequence is the ssrRNA encoding sequence).

- 64 043050

SEQ ID NO: 150; Cas9 atggctcctaagaagaagcggaaggttggtattcacggggtgcctgcggctgacaagaagtactccatcggcctcgacatcgg caccaacagcgtcggctgggcggtgatcaccgacgagtacaaggtcccgtccaagaagttcaaggtcctgggcaacaccgaccgccact ccatcaagaagaacctcatcggcgccctcctcttcg actccggcgagacggcggaggcgacccgcctcaagcgcaccgcccgccgccg ctacacccgccgcaagaaccgcatctgctacctccaggagatcttctctccaacgagatggcgaaggtcgacgactccttcttccaccgcctc gaggagtccttcctcgtggaggaggacaagaagcacgagcgccaccccatcttcggcaacatc gtcgacgaggtcgcctaccacgagaa gtaccccactatctaccaccttcgtaagaagcttgttgactctactgataaggctgatcttcgtctcatctaccttgctctcgctcacatgatcaag ttccgtggtcacttccttatcgagggtgaccttaaccctgataactccgacgtggaccaagctcttcatccagctcgtccagacctaca accagc tcttcgaggagaaccctatcaacgcttccggtgtcgacgctaaggcgatcctttccgctaggctctccaagtccaggcgtctcgagaacctc atcgcccagctccctggtgagaagaagaacggtcttttcggtaacctcatcgctctctccctcggtctgacccctaacttcaagtccaacttcg acctcgctgaggacg ctaagcttcagctctccaaggatacctacgacgatgatctcgacaacctcctcgctcagattggagatcagtacgct ctcctcaaggctcttgttcgtcagcagctccccgagaagtacaaggagat acggctctatccctc accagatccaccttggtgagcttcacgccatccttcgtaggcaggaggacttctaccctttcctcaaggacaaccgtgagaagatcgagaag atccttactttccgtattccttactacgttggtcctcttgctcgtggtaactcccgtttcgcttggatgactaggaagtccgaggagactatcaccc cttggaacttc gaggaggttgttgacaagggtgcttccgcccagtccttcatcgagcgcatgaccaacttcgacaagaacctccccaacgag aaggtcctccccaagcactccctcctctacgagtacttcacggtctacaacgagctcaccaaggtcaagtacgtcaccgagggtatgcgca agcctgccttcctctccggcgagcagaagaaggctatc gttgacctcctcttcaagaccaaccgcaaggtcaccgtcaagcagctcaagga ggactacttcaagaagatcgagtgcttcgactccgtcgagatcagcggcgttgaggaccgttcaacgcttctctcggtacctaccacgatct cctcaagatcatcaaggacaaggacttcctcgacaacgaggagaacgaggacatcctcgaggacat cgtcctcactcttactctcttcgagg atagggagatgatcgaggagaggctcaagacttacgctcatctcttcgatgacaaggttatgaagcagctcaagcgtcgccgttacaccggt tggggtaggctctcccgcaagctcatcaacggtatcagggataagcagagcggcaagactatcctcgacttcctcaagtctgatggtttcgc taacaggaacttcatgcagctcatccacgatgactctctcttaccttcaaggaggatattcagaaggctcaggtgtccggtcagggcgactctct cgtcatcgagatggctcgtgagaaccagactacccagaagggtcagaagaactcgagggagcgcat gaagaggattgaggagggtatcaaggagcttggttctcagatccttaaggagcaccctgtcgagaacacccagctccagaacgagaagct ctacctactactacctccagaacggtagggatatgtacgttgaccaggagctcgacatcaacaggctttct gactacgacgtcgaccacattgt tcctcagtctttccttaaggatgactccatcgacaacaaggtcctcacgaggtccgacaagaacaggggtaagtcggacaacgtcccttccg aggaggttgtcaagaagaatgaagaactactggaggcagcttctcaacgctaagctcattacccagaggaagttcgacaacctcacgaagg ctg agaggggtggcctttccgagcttgacaaggctggtttcatcaagaggcagcttgttgagacgaggcagattaccaagcacgttgctca gatcctcgattctaggatgaacaccaagtacgacgagaacgacaagctcatccgcgaggtcaaggtgatcaccctcaagtccaagctcgtc tccgacttccgcaaggacttccagttctacaaggt ccgcgagatcaacaactaccaccacgctcacgatgcttaccttaacgctgtcgttggt accgctcttatcaagaagtaccctaagcttgagtccgagttcgtctacggtgactacaaggtctacgacgttcgtaagatgatcgccaagtcc gagcaggagatcggcaaggccaccgccaagtacttcttctactccaacatcatgaacttct tcaagaccgagatcaccctcgccaacggcg agatccgcaagcgccctcttatcgagacgaacggtgagactggtgagatcgtttgggacaagggtcgcgacttcgctactgttcgcaaggt cctttctatgcctcaggttaacatcgtcaagaagaccgaggtccagaccggtggcttctccaaggagtctatccttccaaagaagaaact cgga caagctcatcgctaggaagaaggattgggaccctaagaagtacggtggtttcgactcccctactgtcgcctactccgtcctcgtggtcgcca aggtggagaagggtaagtcgaagaagctcaagtccgtcaaggagctcctcggcatcaccatcatggagcgctcctccttcgagaagaacc cgatcgacttcctcgaggccaaggg ctacaaggaggtcaagaaggacctcatcatcaagctccccaagtactctctcttttcgagctcgagaa cggtcgtaagaggatgctggcttccgctggtgagctccagaagggtaacgagcttgctcttccttccaagtacgtgaacttcctctacctcgc ctcccactacgagaagctcaagggttcccctgaggataacgagcagaagcagctcttcg tggagcagcacaagcactacctcgacgagat catcgagcagatctccgagttctccaagcgcgtcatcctcgctgacgctaacctcgacaaggtcctctccgcctacaacaagcaccgcgac aagcccatccgcgagcaggccgagaacatcatccacctcttcacgctcacgaacctcggcgccctgctgctttcaagtacttc gacacca ccatcgacaggaagcgttacacgtccaccaaggaggttctcgacgctactctcatccaccagtccatcaccggtctttacgagactcgtatc gacctttcccagcttggtggtgataagcgtcctgctgccaccaaaaaggccggacaggctaagaaaaagaagtag

- 65 043050

SEQ ID NO: 151: Cys4-P2A-TaCas9 nucleic acid sequence

5’ATGGACCACTACCTCGACATCAGGCTCAGGCCAGACCCAGAGTTCCCACCA GCCCAGCTCATGTCCGTCCTCTTCGGCAAGCTCCACCAGGCCCTCGTGGCCCAGGGC GGCGACAGGATCGGCGTGTCCTTCCCAGACCTCGACGAGTCCAGGTCCAGGCTCGG CGAGAGGCTCCGCATCCACGCCTCCGCCGACGACCTCAGGGCCCTCCTCGCCAG GCC GTGGCTGGAGGGCCTCAGGGACCACCTCCAGTTCGGCGAGCCAGCCGTGGTGCCAC ACCCAACCCCATACAGGCAAGTGTCCAGGGTGCAAGCCAAGTCCAACCCAGAGG CTCAGGAGGAGCTCATGAGGAGGCACGACCTCTCCGAGGAAGAGGCCAGGAAGGGCATCCCAGACACCGTGGCCAGGGCCCTCGACCTCCCATTCGTGACCCTCAG GTCCC AGTCCACCGGCCAGCACTTCCGCCTCTTCATCAGGCACGGCCCACTCCAGGTGACCG CCGAGGAGGGCGGCTTTACCTGCTACGGCCTCTCCAAGGGCGGCTTCGTGCCGTGGT TCGGCTCCGGCGCCACCAACTTCTCCCTCCTCAAGCAAGCCGGCGACGTGGAGG AGAACCCAGGCCCAATGGACAAGAAGTACTCGATCGGCCTCGACATCGGGA CGAACTC AGTTGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCTCTAAGAAGTTCAAGGTCCT GGGGAACACCGACCGCCA TTCCA TCAAGAAGAACCTCA TCGGCGCTCTCCTGTTCGACAG CGGGAGACCGCTGAGGCTACGAGGCTCAAGAGAACCGCTAGGCGCCGGTACACGAGA AGGAAGAACAGGATCTGCTACCTCCAAGAGATTTTC TCCAACGAGATGGCCAAGGTTGAC GATTCATTCTTCCACCGCCTGGAGGAGTCTTTCCTCGTGGAGGAGGATAAGAAGCACGAG

- 66 043050

CGGCA TCCCA TCTTCGGCAACA TCGTGGACGAGGTTGCCTACCACGAGAAGTACCCTACG ATCTACCATCTGCGGAAGAAGCTCGTGGACTCCACCGATAAGGCGGACCTCAGACTGATC TACCTCGCTCTGGCCCACATGATCAAGTTCCGCGGCCATTTCCTGATCGAGGGGGATCTC AACCCAGACAAACGCGATGTTGACAAGCTGTTCATCCAACTCGTG CAGACCTACAACCAAC TCTTCGAGGAGAACCCGA TCAACGCCTCTGGCGTGGACGCGAAGGCTA TCCTGTCCGCG AGGCTCTCGAAGTCCAGGAGGCTGGAGAACCTGATCGCTCAGCTCCCAGGCGAGAAGAA GAACGGCCTGTTCGGGAACCTCATCGTCTCAGCCTGGGGCTCACCCGAACTTCAAGTC GAACTTCGATCTCGCTGAGGACGCCAAGCTGCA ACTCTCCAAGGACACCTACGACGATGA CCTCGA TAACCTCCTGGCCCAGA TCGGCGATCAA TACGCGGACCTGTTCCTCGCTGCCAA GAACCTGTCGGACGCCATCCTCCTGTCAGATATCCTCCGCGTGAACACCGAGATCACGAA GGCTCCACTCTCTGCCTCCATGATCAAGCGCTACGACGAGCACCATCAGGATCTGACCCT CCTGAAGGCGCTGGTCCGCCA ACAGCTCCCGGAGAAGTACAAGGAGA TTTTCTTCGA TCA GTCGAAGAACGGCTACGCTGGTACATCGACGGCGGGGCCTCACAAGAGGAGTTCTACA AGTTCATCAAGCCAATCCTGGAGAAGATGGACGGCACGGAGGAGCTCCTGGTGAAGCTC AACAGGGAGGACCTCCTGCGGAAGCAGAGAACCTTCGA TAACGGCAGCA TCCCCCACCA AA TCCATCTCGGGGAGCTGCACGCCATCCTGAGAAGGCAAGAGGACTTCTACCCTTTCCT CAAGGATAACCGGGAGAAGATCGAGAAGATCCTGACCTTCAGAATCCCATACTACGTCGG CCCTCTCGCGCGGGGGAACTCAAGATTCGCTTGGATGACCCGCAAGTCTGAGGAGACCA AGTCGTTCATC GAGAGGATGACCAACTTCGACAAGAACCTGCCCAACGAAGGTGCTCCCTAAGCACTCG CTCCTGTACGAGTACTTCACCGTCTACAACGAGCTCACGAAGGTGAAGTACGTCACCGAG GGCATGCGCAAGCCAGCGTTCCTGTCCGGGGAGCAGAAGAAGGCTATCGTGGACCTCCT GTTCAAGACCAACCGGAAGGTCACGGTTAAGCAACTCAAGGA GGACTACTTCAAGAAGAT CGAGTGCTTCGATTCGGTCGAGATCAGCGGCGTTGAGGACCGCTTCAACGCCAGCCTCG GGACCTACCACGATCTCCTGAAGATCATCAAGGATAAGGACTTCCTGGACAACGAGGAGA ACGAGGATATCCTGGAGGACATCGTGCTGACCCTCACGCTGTTCGAGGACAGGGAGATG A TCGAGGAGCGCCTGAAGACGTACGCCCA TCTCT TCGA TGACAAGGTCA TGAAGCAACTC AAGCGCCGGAGA TACACCGGCTGGGGGAGGCTGTCCCGCAAGCTCA TCAACGGCA GGGGACTCGCTGCACGAGCATATCGCGAACCTCGCTGGCTCC CCCGCGATCAAGAAGGGCATCCTCCAGACCGTGAAGGTTGTGGACGAGCTCGTGAAGGT CATGGCCGGCACAAGCCTGAGAACATCGTCATCGAGATGGCCAGAGAGAACCAAACCA ACCCAACTGCAGAACGA GAAGCTCTACCTGTACTACCTCCAGAACGGCAGGGATATGTACGTGGACCAAGAGCTGGA TATCAACCGCCTCAGCGATTACGACGTCGATCATATCGTTCCCCAGTCTTTCCTGAAGGAT GACTCCATCGACAACAAGGTCCTCACCAGGTCGGACAAGAACCGCGGCAAGTCAGATAAC GTTCCATCTGAGGAGGTCGTTAAGAAGATGAAGA ACTACTGGAGGCAGCTCCTGAACGCC AAGCTGATCACGCAAAGGAAGTTCGACAACCTCACCAAGGCTGAGAGAGGCGGGCTCTC

- 67 043050

AGAGCTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTCGAGACCAGACAAATCACGA AGCACGTTGCGCAAATCCTCGACTCTCGGATGAACACGAAGTACGATGAGAACGACAAGC TGA TCAGGGAGGTTAAGGTGA TCACCCTGAAGTCTAAGCTCGTCTCCGACTTCAGGAAGG A TTTCCAGTTTCTACAAGGTTCGCGAGA TCAACAACTACCAC CA TGCCCA TGACGCTTACCT CAACGCTGTGGTCGGCACCGCTCTGATCAAGAAGTACCCAAAGCTGGAGTCCGAGTTCGT GTACGGGGACTACAAGGTTTACGA TGTGCGCAAGATGA TCGCCAAGTCGGAGCAAGAGA T CGGCAAGGCTACCGCCAAGTACTTCTTCTACTCAAACATCATGAACTTCTTCAAGACCGAG ATCACGCTGGCCAACGGCGATC CGGAAGAGACCGCTCATCGAGACCAACGGCGAGAC GGGGGAGA TCGTGTGGGACAAGGGCAGGGA TTTCGCGACCGTCCGCAAGGTTCTCTCCA GAAGTACGGCGGGTTCGACAGCCCTACCGTGGCCTACTCGGTCCTGGTTGTGGCGAA GGTTGAGAAGGGCAAGTCCAAGAAGCTCAAGAGCGTGAAGGAGCTCCTGGGGATCACCA TCA TGGAGAGGTCCAGCTTCGAGAAGAACCCAA TCGACTTCCTGGAGGCCAAGGGCTACA AGGAGGTGAAGAAGGACCTGATCATCAAGCTCCCGAAGTACT CTCTCTTCGAGCTGGAGA ACGGCAGGAAGGAATGCTGGCTTCCGCTGGCGAGCTCCAGAAGGGGAACGAGCTCGC GCTGCCAAGCAAGTACGTGAACTTCCTCTACCTGGCTTCCCACTACGAGAAGCTCAAGGG CAGCCCGGAGGACAACGAGCAAAAGCAGCTGTTCGTCGAGCAGCACAAGCATTACCTCG ACGAGATCATCGAGCAAATCTCCGAGTTCAGCAAGCGCG TGATCCTCGCCGACGCGAACC TGGA TAAGTCCTCTCCGCCTACAACAAGCACCGGGACAAGCCCA TCAGAGAGCAAGCG GAGAACA ACCGGCCTGTACGAGACGAGGATCGACCTCTCACAACTCGGC GGGGATAAGAGACCCGCAGCAACCAAGAAGGCAGGGCAAGCAAAGAAGAAGAAGTGA 3’

SEQ ID NO: 152; Otubain-like domain

PYVGDKEPLSTLAAEFQSGSPILQEKIKLLGEQYDALRRTRGDGNCFYRSFMFSY LEHILETQDKAEVERILKKIEQCKKTLADLGYIEFTFEDFFSIFIDQLESVLQGHESSIGAEE LLERTRDQMVSDYVVMFFRFVTSGEIQRRAEFFEPFISGLTNSTVVQFCKASVEPMGEES DHVHIIALSDALGVPIRVMYLD RSSCDAGNISVNHHDFSPEANSSDGAAAAAEKPYITLLY RPGHYDILYP

SEQ ID NO: 153; Mutated OsOTUB1

ATGGGCGGGGACTACTACCACTCGTGCTGCGGCGACCCCGACCCCGACCTCC GCGCGCCCGAGGGGCCCCAAGCTGCCGTACGTCGGGGACAAGGAACCTCTCTCCACT CTTTATTGTTTCCTACTTGGAACATATCCTAGACACAAGACAAAGCTGAGGT TGAGCGCATTCTAAAAAAAATTGAGCGTGCAAGAAGACTCTTGCAGATCTTGGAT ACATTGAGTTCACCTTTGAAGATTTCTTCTCTGTCTTTATTGTTACTTTGTGTGGCCCT CCTTACTTATCCTGTTCAATTGCTGTTTTGCAACTTATGCCAGATGTATTCCCTCTGA ATAG TATGAAGATCTGTCCGATTATTTTCATGTATGCTTGTTTGCATTTCCTTTTAG

- 68 043050

ATGTTCCTGGAATAATTTTTGTATGAGCTAGTTATAATGAGAGCTTGTGTGCATTTTCCT GTCATGCAACAAATTAAATACTAGTGTCTAATCCTTGTGCATTGTTAATAACTTTGA AAATGATTAGCCTTGAAGATTGGTCCATTATATATATGTTCACTTGTTTCTTAGTTAG GATCACTCACCAGTCACCCTTCTGAAGTTCATAATGTATCACTTATAAGTAAGCTAG CAAAACAAAATTTG GACTGTTTGTAGCCACCCAGAACCCAAATAGATGGATTTCAC ATTATTTTCTACTGGCTTTGGGAGTTATTTGATCGATGCTAGTACAACGTTGAAATTT GGGTAGTTGAGATGCATTTTTCACAAAGGACTCCTTTATTGGTGCTTGATCTACAAC TGGTGTTTTACTTTTTCAAAAAAATGTAATCTCCTTGCAGTGCACTCAAATTATTG CAACCTCCTTCCTTA TGTTCCCACCCTCATTATTTTCAGATATTCATTGATCAGCTGG AAAGTGTTCTGCAGGGACATGAATCCTCCATAGGGGCCGAAGAGCTTCTAGAAAGA ACCAGGGATCAGATGGTTTCTGATTATGTTGTCATGTTCTTTAGGTTTGTCACCTCTG GTGAAATCCAAAGGAGGCCGAGTTCTTCGAACCATTCATCTCTGGCTTGACAAATT CGACTGTGGTTCAG TTCTGCAAGGCTTCCGTGGAGCCGATGGGCGAGGAAAGTGAC CATGTCCACATAATTGCCCTATCAGATGCGTTGGGTGTGCCAATCCGTGTGTGATGTAC CTAGACAGAAGCTCATGTGATGCTGGAAATATAAGTGTGAACCACCATGATTTCAG CCCTGAGGCCAATTCATCGGACGGTGCTGCTGCTGCTGAGAAACCTTACATTACTTT GCCTCTACCGTCCTGGTC ACTACGACATTCTCTACCCGAAGTGA

SEQ ID NO: 154; mutation wtg OsOTUBl

MS

SEQ ID NO: 155: 2.5 kb NPT1 promoter sequence. from IR66167-27-5-1-6 ataattttcgatgctggttggtgctaccaagtcccacgtatgatacttaagaatcaatcatgaatattacaaatcaagtcaaactacgttatgt attgaactcttataattactgcaacatatcacactggaatttcctatggtaattcctcgccagccttatcctacccatcccttgcagtatattaagag catcaacaacaaacatgattcaagacaacttttattaac actgaacaacataaattgggaacaaaaacaaaccacttggaggcatgattaggat aatcggtattaaagaactggacatcacaattcacaactagatgttgaaataatacctgtctcttctttggctcatggcaggtgtcagtgaaatata ctgatgctccaagagagctggaagcaccgtttccacgtaatcaaaatgtccttttcgtttgctgca atcaaccttaaagggctcttttgatgctat ctcttcaggcatgtccttcacaacttccacataacctctggttgctcaatgaagtaatcaacatcgaaaacgtctgcaaatccactgacagaga ataccaataagtgatgaactaccttttgaacagaaataaactgcataactacaagtagcacagtcgttcatcttgtagagtgattctcatacctag attcat tccagtaggcagcaacctcaaacttgggcagaaccattgttgcgttgagaaggcgcgcaaccgcaattccatcacatagctgaaga aacattcggaagaataattaattacaaccaggagtaacataataacatagccagttgaaatcacattcgccttgcaatgtgaaaattttcataaataat ctgaaaatttagttatgccactatatatcatgcaacctg cctccacgacattttaatcatggagtagaagataaaacatatgatccccctcattga ccctactatcttactacttgtgcatggccgaacgatctaacagcgaaatccagaaagccaacactcatttgatcccactaacaacggaagaga gaaacgctagccgagatcgcttaacgtacatcgcgtcgcagctggttgagcccgccgtagcagtcga tccggatgtacccattcctcctcg acggagctgcagaagaagaggaggttcaaaaccgcaatcaccaccacagtctcaagcagagatgtccactacccggatccttaaaccca aaccacaaatcacggcgaggtctcacccggcattgccgcccgccaccacccgcacgaccgccactccgccacccgccgctgcgcccat atgacccgcga ccccgacgccgacggcgactcctccctaaagaccaaaagcgagtaagcgagatccgtaagcttctggaacaatctcga acgcggctgcgcgcccaccacgccgccggcgacgc cgacgacgacatcgcctcgccgcgagaagcactggatctgatcggccgccgcctccacgccggagtggagagcatatataagctcgtca gaatgtgggcccgtggctatgtgggcccaccatgtcatcgacgcttatcaagatcgagcggtggcgtga ggaaaccggtaggggtgggg gggctaaccaatcggaaacgcgtaataactcacccgcggttcactttctccttatgacacgtgggcccatctctcctggacccacctgtcagt tacccttacggcctccactctgaggatctaaacgtaaaaacgaatttatcggagggcttatccgcgaggggaaaaaaacgcgcactta tttct cgccttcgccgagatctcggaagaagaagaacaccgcaccgcggggagaggggagagaagcggaaagctccaccgaatcgaagccccc tgatgggggaggcggcggcggcagccgcggcccccgagccggtcgtcgaggggggag gaggggggggggaggggttgaatcctaaccctagcggcggtgggggaggaggtggtggagggtgctcggactccgtgtcggtcgagc tctcg

- 69 043050

SEQ ID NO: 156: DEP1 cDNA sequence:

atgggggaggaggcggtggtgatggaggcgccgaggcccaagtcgccgccgaggtacccggacctgtgcggccggcggc ggatgcagctggaggtgcagatcctgagccgcgagatcacgttcctcaaggatgagcttcacttccttgaaggagctcagccgtttctcgtt ctggatgcattaaagagataaat gagtttgttggtacaaaacatgacccactaataccaacaaagaaggaggcacagatcttgccgtcttt ttcggtggatcggatcaaaattgtgtatctgcatttcatgtctttgctactgttgcaagtgctcacccaagtgcaaaagaccaaggtgcctcaatt gttcttgcagctcatgctgcgacgagccatgctgta agccaaactgcagtgcgtgctgcgctgggtcatgctgtagtccagactgctgctcat gctgtaaacctaactgcagttgctgcaagaccccttcttgctgcaaaccgaactgctcgtgctcctgtccaagctgcagctcatgctgcgata catcgtgctgcaaaccgagctgcacctgcttcaacatcttttcatgctt caaatccctgtacagctgcttcaagatcccttcatgcttcaagtccc agtgcaactgctctagccccaattgctgcacttgcacccttccaagctgtagctgcaagggctgtgcctgtccaagctgtggatgcaacggct gtggctgtccaagctgcggatgcaacggttgtggctgtccaagctgcggttgcaacggctgtggcc ttccaagctgcggttgcaacggctg cggctcgtgctcttgcgcccaatgcaaacccgattgtggctcgtgctctaccaattgctgtagctgcaagccaagctgcaacggctgctgcg gcgagcagtgctgccgctgcgcggactgcttctcctgctcgtgccctcgttgctccagctgcttcaacatcttcaa atgctcctgcgctggctg ctgctcgagcctgtgcaagtgcccctgcacgacgcagtgcttcagctgccagtcgtcatgctgcaagcggcagccttcgtgctgcaagtgc cagtcgtcttgctgcgaggggcagccttcctgctgcgagggacactgctgcagcctcccgaaaccgtcg tgccctgaatgttcctgtgggt gtgtctggtcttgcaagaattgtacagagggttgtcgatgcccacggtgtcgtaacccatgctgtctcagtggttgcttatgttga

SEQ ID NO: 157: DEP1 protein:

mgeeavvmeaprpkspprypdlcgrrrmqlevqilsreitflkdelhflegaqpvsrsgcikeinefvgtkhdpliptkrrrhr scrlfrwigsklcicisclcycckcspkckrprclncscssccdepcckpncsaccagsccspdccscckpncsccktpscckpncscsc pscssccdtscckpsctcfnifsc fkslyscfkipscfksqcncsspncctctlpscsckgcacpscgcngcgcpscgcngcgcpscgcn gcglpscgcngcgscscaqckpdcgscstnccsckpscngccgeqccrcadcfscscprcsscfnifkcscagccsslckcpcttqcfs cqsscckrqpscckcqss ccegqpscceghccslpkpscpecscgcvwscknctegcrcprcmpcclsgclc

SEQ ID NO: 158: cDNA sequence of dep1:

ATGGGGGAGGAGGCGGTGGTGATGGAGGCGCCGAGGCCCAAGTCGCCGCCG AGGTACCCGGACCTGTGCGGCCGGCGGCGGATGCAGCTGGAGTGCAGATCCTGAG CCGCGAGATCACGTTCCTCAAGGATGAGCTTCACTTCCTTGAAGGAGCTCAGCCCGT TTTCCGTTCTGGATGCATTAAAGAGATAAATGAGTTTGTTGGTACAAAAACAT GACCC ACTAATACCAACAAAGAGAAGGAGGCACAGATCTTGCCGTCTTTTTCGGTGGATCG GATCAAAATTGTGTATCTGCATTTCATGTCTTTGCTACTGTTGCAAGTGCTCACCCAA

GTGCAAAAGACCAAGGTGCCTCAATTGTTTCTTGCAGCTCATGCTGCGACGAGCCATG CTGTAAGCCAAACTGCAGTGCGTGTGCTGCGCTGGGTCATGCTGCAGTCCAGACTGCTG CCTGCTTCAACATCTAG

SEQ ID NO: 159: dep1 protein:

mgeeavvmeaprpkspprypdlcgrrrmqlevqilsreitflkdelhflegaqpvsrsgcikeinefvgtkhdpliptkrrrhr scrlfrwigsklcicisclcycckcspkckrprclncscssccdepcckpncsaccagsccspdccscckpncsccktpscckpncscsc pscssccdtscckpsctcfnif

SEQ ID NO: 160: OsUBC13 cDNA atggccaacagcaacctcccccggcgaatcatcaaggaacgcagcgactcctcagcgagccagcgccgggaatcagcgc gtctccgtcggaggagaacatgcgctacttcaacgtcatgatccttggcccggcacagtccccctatgaaggtggagtttttaagcttgaact cttt ttacctgaggaatatcctatggctgctccaaaggttaggttcctgaccaaaatataccaccccaacattgacaagcttggtaggatatgcc agccaatgaagcagaagctgttgaaacagcaaaggagtggactcgcctgtatgccagc ggtgcataa

SEQ ID NO: 161: OsUBC13 protein:

Mansnlprriiketqrllsepapgisaspseenmryfnvmilgpaqspyeggvfklelflpeeypmaapkvrfltkiyhpni dklgricldilkdkwspalqirtvllsiqallsapnpddplsdniakhwkaneaeavetakewtrlyasga

- 70 043050

SEQ ID NO: 162: gDNA OsUBC13 atggccaacagcaacctcccccggcgaatcatcaaggtcgcgcctccgatctgattgcctgggcgtagatctcccccgcctct aacccgattccccccttttcttttctttttttttttttttccctttctgtgctgatttttgttttgctgtctgtgtgttcgcgtct ggtttggatctgcgcag gagacgcagcgactcctcagcgagccaggtgcgggcggtttttttttttttttgtatcgcggattttggcgatggtgttgtgtttttccgtggttt gggttcgtgttgttctgatggttttgggttggtttgtttgtgcagcgccgggaatcagcgcgtctccgtcgg aggagaacatgcgctacttcaa cgtcatgatccttggcccggcacagtccccctatgaaggtacggcacccgatggatacgttatttgttgtagagggtgttacggctcccgatg gatatgttatttgttgtagagggtggtcgattagggcgtttctaggtataccgcagaattgggtgattctgtataaacccaattactagaagtgt ta attatgtgtcagtcatggacgggcacgcatttcgtaagcctgcttttgctgattaggctggttcttttgcacttaccagttttagtttctcgggcgg cgggcacttgcatgttctatgtgtctcatattccctttctagtatgaagttagtaatgctaaaacactagtgacttttagcaataatgttctaaactcg gattatatacg tgacttataagtgcttttctttttgtgttaacttgtttgatgctttgtgcggcaaaggttgcccttgattttccataaccaaattgatc gggttggatatataatgggtgttaacagttagtagctttagatggtttattctattatcatttggcttcctgatattgttctttctatcaggtggag tttttaagcttgaactctttttacctgaggaatatcctatggctgctccaaaggtaacaagacatagataaactttggttatgttttatgatattatg gggactgtataccttcaagctaattatgtcgta tttagaattgcgtgtggttatatactcatattaaaagtcaggacatttctgcattcattgacttgt gttgtgaagcggagcattaatttggtgacattggactcatgcatataaccaaatacaggaaagtagctgaaacatttaatttgtttgaaccattct ttggcaaaaaaaaatttttattcttatagcctaatttttagttacccatatttaatc gtgtcgctctggaaccatcgttggttttatatgttatatgtgat gtcttgttggtaatagttttgttacatttgcttgatttccttttagggataatgcaaaccctgcctatttgagatgtaatttgcaatgagatcctgtgcc aaggaccaaggttgtaagatacttttggtcctgtgggaacttttagctgtgtcctgag catcagcacgccattgcttctcttgaccgtctttttg cttcaagttcttaaaggtgttcttaacatcacattgttcgtttggtgttcagccaaattataccttgattagttatttttttagtctgtttggcttgacaat gtcatagattggaagggtgaagaaatattactcatatgttatacaaccataccggattattgtctacttcagtctccctaag tgtaaagctatataa ttcattattgtctacctcagtctccattttctttgttgcgtatgcaggttaggttcctgaccaaaatataccaccccaacattgacaaggtttgcag caaatggagcccagcccttcagattcggacagttcttttgaggtctctcccccgttgcctgcacatgtgtattagctttttatatcctgcacaaatt ccaacaaaaactcttgagcattgtcaactattggaagttaggggtatcactttgctatacttaccttgtgacaacactctttttttcag tatccaggcactcctaagtgcaccaaaccctgatgatcctctctctgataacattgcaaagcactggaaagccaatgaagcagaagctgttga aacaggtaaattgaagctagcaatccagttaac agtgtctcccctcacactctgattttattggttgcctgataagcgatggtctggcatttgcag caaaggagtggactcgcctgtatgccagcggtgcataa

SEQ ID NO: 163: U3 promoter sequence:

AAAGGAAGGAATCTTTAAACATACGAACAGATCACTTAAAGTTCTTCTGAAG CAACTTAAAGTTATCAGGCATGCATGGATCTTGGAGGAATCAGATGTGTCAGTCAGG AGGAGAAAAGCATTTCGTAGTGGGCCATGAAGCCTTTCAGGACATGTATTGCAGTA TGGGCCGGCCCATTACGCAATTGGACGACAACAAAGACTAGTATTAGTACCACCTC GGCTATCCACATAGATCAAAGCTGGTTTAAAAGAGTTGTGCAGATGATCCGTGGCA SEQ ID NO: 164: U6a promoter sequence:

TTTTTTCCTGTAGTTTTCCCACAACCATTTTTACCATCCGAATGATAGGATAG GAAAAATATCCAAGTGAACAGTATTCCTATAAAATTCCCGTAAAAAGCCTGCAATC CGAATGAGCCCTGAAGTCTGAACTAGCCGGTCACCTGTACAGGCTATCGATGCC ATACAAGAGACGGTAGTAGGAACTAGGAAGACGATGGTTGATTCGTCAGGCGAAAT CG TCGTCCTGCAGTCGCATCTATGGGCCTGGACGGAATAGGGGAAAAAGTTGGCCG GATAGGAGGAAAGGCCCAGGTGCTTACGTGCGAGGTAGGCCTGGGCTCTCAGCAC TTCGATTCGTTGGCACCGGGGTAGGATGCAATAGAGAGCAACGTTTAGTACCACCTC GCTAGCTAGAGCAAACTGGACTGCCTTATATGCGCGGGTGCTGGCTTG GCTGCCG SEQ ID NO: 165: U6b promoter sequence:

TGCAAGAACGAACTAAGCCGGACAAAAAAAAAAGGAGCACATATACAAACC GGTTTTATTCATGAATGGTCACGATGGATGATGGGCTCAGACTTGAGCTACGAGGC CGCAGGCGAGAGAAGCCTAGTGTGTGCTCTCTGCTTGTTTGGGCCGTAACGGAGGATA CGGCCGACGAGCGTGTACTACCGCGCGGGATGCCGCTGGGCGCTGCGGGGCCGTT GGA TGGGGATCGGTGGGTCGCGGGAGCGTTGAGGGGAGACAGGTTTAGTACCACCT CGCCTACCGAACAATGAAGAACCCACCTTATAACCCCGCGCGCTGCCGCTTGTGTTG

- 71 043050

SEQ ID NO: 166: U3b in a dicot plant

CCCATGTA TGCGTTTCATATAAGCTCCTAATTTCTCCCACATCGCTCAAATCTAAACA AATCTTGTTGTATATATAACACTGAGGGAGCAACATTGGTCA

SEQ ID NO: 167: U6-1 in a dicot plant

AGAAATCTCAAAATTCCGGCAGAACAATTTTGAATCTCGATCCGTAGAAACG AGACGGTCATTGTTTTAGTTCCACCACGATTATATTTGAAATTTACGTGAGTGTGAG TTCAAAAGTACCACAGCGCTTAGGTAAAGAAAGCAGCTGAGTTTAT ATATGGTTAGAGACGAAGTAGTGATTG

SEQ ID NO: 168; nucleic acid sequence Cys4-P2A-TaCas9

5'ATGGACCACTACCTCGACATCAGGCTCAGGCCAGACCCAGAGTTCCCACCA GCCCAGCTCATGTCCGTCCTCTTCGGCAAGCTCCACCAGGCCCTCGTGGCCCAGGGC GGCGACAGGATCGGCGTGTCCTTCCCAGACCTCGACGAGTCCAGGTCCAGGCTCGG CGAGAGGCTCCGCATCCACGCCTCCGCCGACGACCTCAGGGCCCTCCTCGCCAG GCC GTGGCTGGAGGGCCTCAGGGACCACCTCCAGTTCGGCGAGCCAGCCGTGGTGCCAC ACCCAACCCCATACAGGCAAGTGTCCAGGGTGCAAGCCAAGTCCAACCCAGAGG CTCAGGAGGAGCTCATGAGGAGGCACGACCTCTCCGAGGAAGAGGCCAGGAAGGGCATCCCAGACACCGTGGCCAGGGCCCTCGACCTCCCATTCGTGACCCTCAG GTCCC AGTCCACCGGCCAGCACTTCCGCCTCTTCATCAGGCACGGCCCACTCCAGGTGACCG CCGAGGAGGGCGGCTTTACCTGCTACGGCCTCTCCAAGGGCGGCTTCGTGCCGTGGT TCGGCTCCGGCGCCACCAACTTCTCCCTCCTCAAGCAAGCCGGCGACGTGGAGG AGAACCCAGGCCCAATGGACAAGAAGTACTCGATCGGCCTCGACATCGGGA CGAACTC AGTTGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCTCTAAGAAGTTCAAGGTCCT GGGGAACACCGACCGCCA TTCCA TCAAGAAGAACCTCA TCGGCGCTCTCCTGTTCGACAG CGGGAGACCGCTGAGGCTACGAGGCTCAAGAGAACCGCTAGGCGCCGGTACACGAGA AGGAAGAACAGGATCTGCTACCTCCAAGAGATTTTC TCCAACGAGATGGCCAAGGTTGAC GATTCATTCTTCCACCGCCTGGAGGAGTCTTTCCTCGTGTGGAGGAGGATAAGAAGCACGAG GATCAAGTTCCGCGGCCATTTCCTGATCGAGGGGGATCTC AACCCAGACAACAGCGATGTTGACAAGCTGTTCATCCAACTCGTGCAGACCTACAACCAAC TCTTCGAGGAGAACCCGA TCAACGCCTCTGGCGTGGACGCGAAGGCTA TCCTGTCCGCG AGGCTCTCGAAGTCCAGGAGGCTGGAGAACCTGATCGCTCAGCTCCAGGCGAGAAGAA GAACGGCCT GTTCGGGAACCTCATCGCTCTCAGCCTGGGGCTCACCCCCGAACTTCAAGTC GAACTTCGATCTCGCTGAGGACGCCAAGCTGCAACTCTCCAAGGACACCTACGACGATGA CCTCGA TAACCTCCTGGCCCAGA TCGGCGATCAA TACGCGGACCTGTTCCTCGCTGCCAA GAACCGTGTCGGACGCCATCCTCCTGTCAGATATCCTCCGCGTGAACACCGAGATCAC GAA GGCTCCACTCTCTGCCTCCATGATCAAGCGCTACGACGAGCACCATCAGGATCTGACCCT CCTGAAGGCGCTGGTCCGCCAACAGCTCCCGGAGAAGTACAAGGAGA TTTTCTTCGA TCA GTCGAAGAACGGCTACGCTGGGTACATCGACGGCGGGGCCTCACAAGAGGAGTTCTACA AGTTCATCAAGCCAATCCTGGAGAAGATGGACGGCACGGAG GAGCTCCTGGTGAAGCTC AACAGGGAGGACCTCCTGCGGAAGCAGAGAACCTTCGA TAACGGCAGCA TCCCCCACCA AATCCATCTCGGGGAGCTGCACGCCATCCTGAGAAGGCAAGAGGACTTCTACCCTTTCCT CAAGGATAACCGGGAGAAGATCGAGAAGATCCTGACCTTCAGAATCCCATACTACGTCGG

- 72 043050

CCCTCTCGCGCGGGGGAACTCAAGATTCGCTTGGATGACCCGCAAGTCTGAGGAGACCA TCACGCCGTGGAACTTCGAGGAGGTGGTGGACAAGGGCGCTAGCGCTCAGTCGTTCATC GAGAGGATGACCAACTTCGACAAGAACCTGCCCAACGAGAAGGTGCTCCCTAAGCACTCG CTCCTGTACGAGTACTTCACCGTCTACAACGAGCTCACGAAGGTGAAG TACGTCACCGAG GGCATGCGCAAGCCAGCGTTCCTGTCCGGGGAGCAGAAGAAGGCTATCGTGGACCTCCT GTTCAAGACCAACCGGAAGGTCACGGTTAAGCAACTCAAGGAGGACTACTTCAAGAAGAT CGAGTGCTTCGATTCGGTCGAGATCAGCGGCGTTGAGGACCGCTTCAACGCCAGCCTCG TCCTGGACAACGAGGAGA ACGAGGATA TCCTGGAGGACA TCGTGCTGACCCTCACGCTGTTCGAGGACAGGGAGA TG A TCGAGGAGCGCCTGAAGACGTACGCCCA TCTCTTCGA TGACAAGGTCA TGAAGCAACTC AAGCGCCGGAGA TACACCGGCTGGGGGAGGCTGTCCCGCAAGCTCA TCAACGGCA TCCG GGACAAGCAGTCCGG GAAGACCATCCTCGACTTCCTCAAGAGCGATGGCTTCGCCAACA GGAACTTCATGCAACTGATCCACGATGACAGCCTCACCTTCAAGGAGGATATCCAAAAGG CTCAAGTGAGCGGCCAGGGGGACTCGCTGCACGAGCATATCGCGAACCTCGCTGGCTCC CCCGCGATCAAGAAGGGCATCCTCCAGACCGTGAAGGTTGTGGACGAGCTCGTGAAGGT CATG GGCCGGCACAAGCCTGAGAACATCGTCATCGAGATGGCCAGAGAGAACCAAACCA CGCAGAAGGGCAAAAGAACTCTAGGGAGCGCATGAAGCGCATCGAGGAGGGCATCAAG GAGCTGGGGTCCCAAATCCTCAAGGAGCACCCAGTGGAGAACACCCAACTGCAGAACGA GAAGTCTCTACCTGTACTACCTCCAGAACGGCAGGGATATGTACGTGGACCAAGAGC TGGA TATCAACCGCCTCAGCGATTACGACGTCGATCATATCGTTCCCCAGTCTTTCCTGAAGGAT GACTCCATCGACAACAAGGTCCTCACCAGGTCGGACAAGAACCGCGGCAAGTCAGATAAC GTTCCATCTGAGGAGGTCGTTAAGAAGATGAAGAACTACTGGAGGCAGCTCCTGAACGCC AAGCTGATCACGCAAAGGAAGTTCGACAACCTCACCAAGGCTGAGAG GCGGGCTCTC AGAGCTGGACAAGGCCGGCTTCATCAAGCGGCAGCTGGTCGAGACCAGACAAATCACGA AGCACGTTGCGCAAA TCCTCGACTCTCGGA TGAACACGAAGTACGA TGAGAACGACAAGC TGA TCAGGGAGGTTAAGGTGA TCACCCTGAAGTCTAAGCTCGTCTCCGACTTCAGGAAGG A TTTCCAGTTCTA CAAGGTTCGCGAGA TCAACAACTACCA CCA TGCCCA TGA CGCTTA CCT CAACGCTGTGGTCGGCACCGCTCTGATCAAGAAGTACCCAAAGCTGGAGTCCGAGTTCGT GT A CGGGGA STA CAA GGTTTA CGA TGTGCGCAA GA TGA TCGCCAA GTCGGA GCAA GA GA T CGGCAAGGCTACCGCCAAGTACTTCTTCTACTCAAACATCATGAACTTCTTCAAGACCGAG ATCACGCTGGCCAACGGCGAGATCCGGAAGAG ACCGCTCATCGAGACCAACGGCGAGAC GGGGGAGATCGTGTGGGACAAGGGCAGGGATTTCGCGACCGTCCGCAAGGTTCTCTCCA TGCCCCAGGTGAACATCGTCAAGAAGACCGAGGTCCAAACGGGCGGGTTCTCAAAGGAG TCTA TCTGCCTAAGCGGAACAGCGACAAGCTCA TCGCCAGAAAGAAGGACTGGGACCCA AAGAAGTACG TCA TGGAGAGTCCAGCTTCGAGAAGAACCCAA TCGACTTCCTGGAGGCCAAGGGCTACA AGGAGTGAAGAAGGACCTGATCATCAAGCTCCCGAAGTACTCTCTCTTC GAGCTGGAGA ACGGCAGGAAGAATGCTGGCTTCCGCTGGCGAGCTCCAGAAGGGGAACGAGCTCGC GCTGCCAAGCAAGTACGTGAACTTCCTCTACCTGGCTTCCCACTACGAGAAGCTCAAGGG

CAGCCCGGAGGACAACGAGCAAAAGCAGCTGTTCGTCGAGCAGCACAAGCATTACCTCG ACGAGATCATCGAGCAAATCTCCGAGTTCAGCAAGCGCGTGATCCTCGCCGACGCGAACC TGCTTTCAAGTACT TCGACACCACGATCGATCGGAAGAGATACACCTCCACGAAGGAGGTCCTGGACGCGACC CTCATCCACCAGTCGATCACCGGCCTGTACGAGACGAGGATCGACCTCTCAACTCGGC GGGGATAAGAGACCCGCAGCAACCAAGAAGGCAGGGCAAGCAAAGAAGAAGAAGTGA 3’

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SEQ ID NO: 169: Cys 4 endoribonuclease nucleic acid sequence

5'ATGGACCACTACCTCGACATCAGGCTCAGGCCAGACCCAGAGTTCCCACCA

GCCCAGCTCATGTCCGTCCTCTTCGGCAAGCTCCACCAGGCCCTCGTGGCCCAGGGC GGCGACAGGATCGGCGTGTCCTTCCCAGACCCTCGACGAGTCCAGGTCCAGGCTCGG TGCCAC ACCCAACCCCATACAGGCAAGTGTCCAGGGTGCAAGCCAAGTCCAACCCAGAGG CTCAGGAGGAGCTCATGAGGAGGCACGACCTCTCCGAGGAAGAGGCCAGGAAGG GCATCCCAGACACCGTGGCCAGGGCCCTCGACCTCCCATTCGTGACCCTCAGGTCCC TGACCG CCGAGGAGGGCGGCTTTACCTGCTACGGCCTCTCCAAGGGCGGCTTCGTGCCGTGGT TC 3’

Table I

Primers used to construct ssrRNA

Number Direct primer reverse primer 1 Agcggcggtgggggaggagggttttagagct agaaat (SEQ ID NO: 170) Cctcctcccccaccgccgctcggcagccaagccagca (SEQ ID NO: 171) 2 Gggaggaggagggggggggggttttagagc tagaaat (SEQ ID NO: 172) Cccccccccctcctcctccccggcagccaagccagca (SEQ ID NO: 173) 3 Ggggggaggaggaggggggggttttagagc tagaaat (SEQ ID NO: 174) Ccccccctcctcctccccccggcagccaagccagca (SEQ ID NO: 175) 4 Tcgaggggggaggaggaggggttttagagct agaaat (SEQ ID NO: 176) Ccctcctcctcccccctcgacggcagccaagccagca (SEQ ID NO: 177) 5 Gtcgtcgaggggggagggggttttagagcta gaaat (SEQ ID NO: 178) Cctcctcccccctcgacgaccggcagccaagccagca (SEQ ID NO: 179) 6 Ggcagccgcggccccgagcgttttagagcta gaaat (SEQ ID NO: 180) Gctcggggggccgcggctgcccggcagccaagccag ca (SEQ ID NO: 181) 7 Tcccggaggaggcggtgatggttttagagcta gaaat (SEQ ID NO: 182) C atcaccgcctcctccgggacggcagc caagcc age a (SEQ W NO: 183) 8 Cgccgggaagcgcaaggcgggttttagagct Ccgccttgcgcttccggggcgcggcagccaagccagc

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agaat (SEQ ID NO: 184) a (SEQ ID NO: 185) 9 Gaagaagaagaacacgcaccggttttagagcta gaaat (SEQ ID NO: 186) Cggtgcgtgttcttctcttccggcagccaagccagca (SEQ ID NO: 187) 10 cccttacggcctccactctggtttttagagctaga aat (SEQ ID NO: 188) Cagagtggaggccgtaagggcggcagccaagccag ca (SEQ ID NO: 189) eleven C actttctc cttatgacacggttttagagctagaa at (SEQ ID NO: 190) Cgtgtcataaggagaaagtgcggcagccaagccagc a (SEQ ID NO: 191) 12 Tatataagctcgtcagaatggttttagagctaga aat (SEQ ID NO: 192) Cattctgacgagcttatatacggcagccaagccagca (SEQ ID NO: 193) 13 Cgagaagcactggatctgatgttttagagctag aaat (SEQ ID NO: 194) Atcagatccagtgcttctcgcggcagccaagccagca (SEQ ID NO: 195) 14 Gagaggaggaagtagagcgcgttttagagcta gaaat (SEQ ID NO: 196) Gcgctctacttcctcctctccggcagccaagccagca (SEQ ID NO: 197) 15 Taaacccaaaccacaaatcagttttagagctag aaat (SEQ ID NO: 198) Tgatttgtggtttgggtttacggcagccaagccagca (SEQ ID NO: 199) 16 Atgtacccattcctcctcgagttttagagctaga aat (SEQ ID NO: 200) Tcgaggaggaatgggtacatcggcagccaagccagc a (SEQ ID NO: 201) 17 Aacgtacatcgcgtcgcagcgttttagagctag aaat (SEQ ID NO: 202) Gctgcgacgcgatgtacgttcggcagccaagccagca (SEQ ID NO: 203) 18 Actatcttactacttgtgcagttttagagctagaa at (SEQ ID NO: 204) Tgcacaagtagtaagatagtcggcagccaagccagca (SEQ ID NO: 205) 19 Tcggaagaataattacaaccgttttagagctag aaat (SEQ ID NO: 206) Ggttgtaattattcttccgacggcagccaagccagca (SEQ ID NO: 207) 20 Gtaggcagcaacctcaaactgttttagagctag aaat (SEQ ID NO: 208) Agtttgaggttgctgcctaccggcagccaagccagca (SEQ ID NO: 209)

SEQ ID NO: 210: RNAi nucleic acid sequence

G ATTTCAGCCCTGAGGCCAATTCATCGGACGGTGCTGCTGCTGCTGAGAAACCTTAC ATTACTTTGCTCTACCGTCCTGGTCACTACG

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Bibliography

1. Abe, A., Kosugi, S., Yoshida, K., Natsume, S., Takagi, H., Kanzaki, H., Matsuura, H., Mitsuoka, C, Tamiru, M., Innan, H. , Cano, L., Kamoun, S. and Terauchi, R. (2012) Genome sequencing reveals agronomically important loci in rice using MutMap. Nat Biotechnol, 30, 174-178.

2. Alvarez-Venegas, R. and Avramova, Z. (2005) Methylation patterns of histone H3 Lys 4, Lys 9 and Lys 27 in transcriptionally active and inactive Arabidopsis genes and in atxl mutants. Nucleic Acids Res, 33, 5199-5207.

3. Balakirev, M.Y., Tcherniuk, S.O., Jaquinod, M. and Chroboczek, J. (2003) Otubains: a new family of cysteine proteases in the ubiquitin pathway. EMBO Rep. 4, 517-522.

4. Che, R., Tong, H., Shi, B., Liu, Y., Fang, S., Liu, D., Xiao, Y., Hu, B., Liu, L., Wang, H ., Zhao, M. and Chu, C. (2016) Control of grain size and rice yield by GL2-mediated brassinosteroid responses. Nat Plants 2

5. Dong, H., Dumenil, J., Lu, F.H., Na, L., Vanhaeren, H., Naumann, C, Klecker, M., Prior, R., Smith, C, McKenzie, N., Saalbach , G., Chen, L., Xia, T., Gonzalez, N., Seguela, M., Inze, D., Dissmeyer, N., Li, Y. and Bevan, M.W. (2017) Ubiquitylation activates a peptidase that promotes cleavage and destabilization of its activating E3 ligases and diverse growth regulatory proteins to limit cell proliferation in Arabidopsis. Genes Dev, 31, 197-208.

6. Du, L., Li, N., Chen, L., Xu, Y., Li, Y., Zhang, Y. and Li, C. (2014) The ubiquitin receptor DAI regulates seed and organ size by modulating the stability of the ubiquitin-specific protease UBP15/SOD2 in Arabidopsis. Plant Cell, 26, 665-677.

7. Duan, P., Ni, S., Wang, J., Zhang, B., Xu, R., Wang, Y., Chen, H., Zhu, X. and Li, Y. (2016) Regulation of OsGRF4 by OsmiR396 controls grain size and yield in rice. Nat Plants, 2.

8. Duan, P., Rao, Y., Zeng, D., Yang, Y., Xu, R., Zhang, B., Dong, G., Qian, Q. and Li, Y. (2014) SMALL GRAIN 1 , which encodes a mitogen-activated protein kinase kinase 4, influences grain size in rice. Plant J, 77, 547-557.

9. Duan, P., Xu, J., Zeng, D., Zhang, B., Geng, M., Zhang, G., Huang, K., Huang, L., Xu, R., Ge, S ., Qian, Q. and Li, Y. (2017) Natural Variation in the Promoter of GSE5 Contributes to Grain Size Diversity in Rice. Mol Plant, 10, 685-694.

10. Fan, C, Xing, Y., Mao, H., Lu, T., Han, B., Xu, C, Li, X. and Zhang, Q. (2006) GS3, a major QTL for grain length and weight and minor QTL for grain width and thickness in rice, encodes a putative transmembrane protein. Theor AppI Genet, 112, 1164-1171.

11. Fang, N., Xu, R., Huang, L, Zhang, B., Duan, P., Li, N., Luo, Y. and Li, Y. (2016) SMALL GRAIN 11 Controls Grain Size, Grain Number and Grain Yield in Rice. Rice (N Y), 9, 64.

12. Herhaus, L., Al-Salihi, M., Macartney, T., Weidlich, S. and Sapkota, G.P. (2013) OTUB1 enhances TGFbeta signaling by inhibiting the ubiquitylation and degradation of active SMAD2/3. Nat Commun, 4, 2519.

13. Hisanaga, T., Kawade, K. and Tsukaya, H. (2015) Compensation: a key to clarifying the organ-level regulation of lateral organ size in plants. J Exp Bot, 66, 1055-1063.

14. Hu J., Wang, Y., Fang, Y., Zeng, L., Xu, J., Yu, H., Shi, Z., Pan, J., Zhang, D., Kang, S. , Zhu, L., Dong, G., Guo, L., Zeng, D., Zhang, G., Xie, L., Xiong, G., Li, J. and Qian,

- 76 043050

Q. (2015) A Rare Allele of GS2 Enhances Grain Size and Grain Yield in Rice. Mol Plant, 8, 14551465.

15. Hu Z., He, H., Zhang, S., Sun, F., Xin, X., Wang, W., Qian, X., Yang, J. and Luo, X. (2012) A Kelch Motif-Containing Serine/Threonine Protein Phosphatase Determines the Large Grain QTL Trait in Rice. J Integr Plant Biol, 54, 979-990.

16. Huang, X., Qian, Q., Liu, Z., Sun, H., He, S., Luo, D., Xia, G., Chu, C, Li, J. and Fu, X. (2009) Natural variation at the DEP1 locus enhances grain yield in rice. Nat Genet, 41, 494-497.

Li M., Tang, D., Wang, K., Wu, X., Lu, L., Yu, H., Gu, M., Yan, C. and Cheng, Z. (2011a) Mutations in the F -box gene LARGER PANICLE improve the panicle architecture and enhance the grain yield in rice. Plant Biotechnol J, 9, 1002-1013.

18. Li N. and Li, Y. (2014) Ubiquitin-mediated control of seed size in plants. Front Plant Sci, 5, 332.

19. Li N. and Li, Y. (2016) Signaling pathways of seed size control in plants. Curr Opin Plant Biol, 33, 23-32.

20. Li S., Gao, F., Xie, K., Zeng, X., Cao, Y., Zeng, J., He, Z., Ren, Y., Li, W., Deng, Q. , Wang, S., Zheng, A., Zhu, J., Liu, H., Wang, L. and Li, P. (2016) The OsmiR396cOsGRF4-OsGIFl regulatory module determines grain size and yield in Rice. Plant Biotechnol J, 14, 2134-2146.

21. Li, Y. Fan, C, Xing, Y., Jiang, Y., Luo, L., Sun, L, Shao, D., Xu, C, Li, X., Xiao, J., He, Y. and Zhang, Q. (2011b) Natural variation in GS5 plays an important role in regulating grain size and yield in rice. Nat Genet, 43, 1266-1269.

22. Li Y., Zheng, L., Corke, F., Smith, C. and Bevan, M.W. (2008) Control of final seed and organ size by the DAI gene family in Arabidopsis thaliana. Genes Dev, 22, 1331-1336.

23. Liu, S., Hua, L., Dong, S., Chen, H., Zhu, X., Jiang, J., Zhang, F., Li, Y., Fang, X. and Chen, F (2015) OsMAPK6, a mitogen-activated protein kinase, influences rice grain size and biomass production. Plant J, 84, 672-681.

Mao, H., Sun, S., Yao, J., Wang, C, Yu, S., Xu, C, Li, X. and Zhang, Q. (2010) Linking differential domain functions of the GS3 protein to natural variation of grain size in rice. Proc Natl Acad. sci. USA, 107, 19579-19584.

25. Nakada, S., Tai, L, Panier, S., Al-Hakim, A., Iemura, S., Juang, Y.C., O'Donnell, L., Kumakubo, A., Munro, M., Sicheri , F., Gingras, A.C., Natsume, T., Suda, T. and Durocher, D. (2010) Non-canonical inhibition of DNA damage-dependent ubiquitination by OTUB1. Nature, 466, 941-946.

Nijman, S.M., Luna-Vargas, M.P., Velds, A., Brummelkamp, T.R., Dirac, A.M., Sixma, T.K. and Bernards, R. (2005) A genomic and functional inventory of deubiquitinating enzymes. Cell, 123, 773-786.

27. Qi, P., Lin, Y.S., Song, X.J., Shen, J.B., Huang, W., Shan, J.X., Zhu, M.Z., Jiang, L., Gao, J. P. and Lin, H.X. (2012) The novel quantitative trait locus GL3.1 controls rice grain size and yield by regulating Cyclin-Tl ;3. Cell Res, 22, 1666-1680.

- 77 043050

28. Shomura, A., Izawa, T., Ebana, K., Ebitani, T., Kanegae, H., Konishi, S. and Yano, M. (2008) Deletion in a gene associated with grain size increased yields during rice domestication. Nat Genet, 40, 1023-1028.

29. Si L., Chen, J., Huang, X., Gong, H., Luo, J., Hou, Q., Zhou, T., Lu, T., Zhu, J., Shangguan, Y. , Chen, E., Gong, C, Zhao, Q., Jing, Y., Zhao, Y., Li, Y., Cui, L., Fan, D., Lu, Y., Weng, Q., Wang, Y., Zhan, Q., Liu, K., Wei, X., An, K., An, G. and Han, B. (2016) OsSPL13 controls grain size in cultivated rice. Nat Genet, 48, 447-456.

Song, X.J., Huang, W., Shi, M., Zhu, M.Z. and Lin, H.X. (2007) A QTL for rice grain width and weight encodes a previously unknown RING-type E3 ubiquitin ligase. Nat Genet, 39, 623-630.

31. Sun, P., Zhang, W., Wang, Y., He, Q., Shu, F., Liu, H., Wang, J., Yuan, L. and Deng, H. (2016) OsGRF4 controls grain shape, panicle length and seed shattering in rice. J Integr Plant Biol, 58, 836-847.

32. Wang, S., Li, S., Liu, Q., Wu, K., Zhang, J., Wang, Y., Chen, X., Zhang, Y., Gao, C, Wang, F. , Huang, H. and Fu, X. (2015a) The OsSPL16-GW7 regulatory module determines grain shape and simultaneously improves rice yield and grain quality. Nat Genet, 47, 949-954.

33. Wang, S., Wu, K., Yuan, Q., Liu, X., Liu, Z., Lin, X., Zeng, R., Zhu, H., Dong, G., Qian, Q ., Zhang, G. and Fu, X. (2012) Control of grain size, shape and quality by OsSPU 6 in rice. Nat Genet, 44, 950-954.

34. Wang, Y., Xiong, G., Hu, J., Jiang, L., Yu, H., Xu, J., Fang, Y., Zeng, L., Xu, E., Ye, W ., Meng, X., Liu, R., Chen, H., Jing, Y., Zhu, X., Li, J. and Qian, Q. (2015b) Copy number variation at the GL7 locus contributes to grain size diversity in rice. Nat Genet, 47, 944948.

Wang, Z., Li, N., Jiang, S., Gonzalez, N., Huang, X., Wang, Y., Inze, D. and Li, Y. (2016) SCF(SAP) controls organ size by targeting PPD proteins for degradation in Arabidopsis thaliana. Nat Commun, 7, 1 1 192.

36. Weng, J., Gu, S., Wan, X., Gao, H., Guo, T., Su, N., Lei, C, Zhang, X., Cheng, Z., Guo, X. , Wang, J., Jiang, L., Zhai, H. and Wan, J. (2008) Isolation and initial characterization of GW5, a major QTL associated with rice grain width and weight. Cell Res, 18, 1 199-1209.

Wu, Y., Fu, Y., Zhao, S., Gu, P., Zhu, Z., Sun, C. and Tan, L. (2016) CLUSTERED PRIMARY BRANCH 1, a new allele of DWARF 1 1 , controls panicle architecture and seed size in rice. Plant Biotechnol J, 14, 377-386.

38. Xia, T., Li, N., Dumenil, J., Li, J., Kamenski, A., Bevan, M.W., Gao, F. and Li, Y. (2013) The Ubiquitin Receptor DAI Interacts with the E3 Ubiquitin Ligase DA2 to Regulate Seed and Organ Size in Arabidopsis. Plant Cell, 25, 3347-3359.

Xu, Y., Jin, W., Li, N., Zhang, W., Liu, C, Li, C. and Li, Y. (2016) UBIQUITINSPECIFIC PROTEASE14 Interacts with ULTRAVIOLET-B INSENSITIVE4 to Regulate Endoreduplication and Cell and Organ Growth in Arabidopsis. Plant Cell, 28, 1200-1214.

40. Yamamuro, C, Ihara, Y., Wu, X., Noguchi, T., Fujioka, S., Takatsuto, S., Ashikari, M., Kitano, H. and Matsuoka, M. (2000) Loss of function of a rice brassinosteroid insensitivel

- 78 043050 homolog prevents internode elongation and bending of the lamina joint. Plant Cell, 12, 1591-1606.

Zhang, X., Wang, J., Huang, J., Lan, H., Wang, C, Yin, C, Wu, Y., Tang, H., Qian, Q., Li, J. and Zhang, H. (2012) Rare allele of OsPPKU associated with grain length causes extralarge grain and a significant yield increase in rice. Proc Natl Acad. sci. USA, 109, 21534-21539.

Zhou, Y., Miao, J., Gu, H., Peng, X., Leburu, M., Yuan, F., Gao, Y., Tao, Y., Zhu, J., Gong, Z., Yi, C, Gu, M., Yang, Z. and Liang, G. (2015) Natural Variations in SLG7 Regulate Grain Shape in Rice. Genetics, 201, 1591-1599.

43. Zhao, M. et al. Regulation of OsmiR156h through alternative polyadenylation improves grain yield in rice. PLOS One 10, e0126154 (2015).

Bracha-Drori, K. et al. Detection of protein-protein interactions in plants using bimolecular fluorescence complementation. Plant J 40, 419-427 (2004).

Wang, F. et al. Biochemical insights on degradation of Arabidopsis DELLA proteins gained from a cell-free assay system. Plant Cell 21,2378-2390 (2009).

Liu, Z. et al. BIK1 interacts with PEPRs to mediate ethylene-induced immunity.

Proc.Natl.Acad.Sci.USA 110, 6205-6210 (2013).

Cermak, T. et al. Efficient design and assembly of custom TALEN and other TAL effector-based constructs for DNA targeting. Nucleic Acids Res. 39 (201 1 ).

Neville E Sanjana,, Le Cong, Yang Zhou, Margarei M Cunniff, Guoping Feng & Feng

Zhang A transcription activator-like effector toolbox for genome engineering, Nature Protocols

7, 171-192 (2012).

Wiles, M.V., Qin, W., Cheng, A.W., Wang, H. CRISPR-Cas9-mediated genome editing and guide RNA design. Mamm Genome 26(9-10), 501-510 (2015).

Henikoff, S., Jill, B.J., Comai, L. TILLING. Traditional Mutagenesis Meets Functional

Genomics. Perspectives on Translational Biology. 135(2), 630-636 (2004).

Comai, L., Young, K., Reynolds, S.H., Greene, E.A., Codomo, C.A., Enns, L.C., Johnson, J.E., Burtner, C, Odden A.R., Henikoff, S. Efficient discovery of DNA polymorphisms in natural populations by Ecotilling. Plant J 37(5), 778-786 (2004).

Komor, A.C., Kim, Y.B., Packer, M.S., Zuris, J.A., Liu, D.R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533(7603), 420424 (2016).

Nishida, K., Arazoe, T., Yachie, N., Banno, S., Kakimoto, M., Tabata, M., Mochizuki, M., Miyabe, A., Araki, M., Hara, K.Y., Shimatani , Z., Kondo, A. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems. Science 353(6305), aaf8729 (2016).

Clough, S.J., Bent A.F. Floral dip: a simplified method for Agrobacterium-mediate transformation of Arabidopsis thaliana. Plant J. 16(6), 735-743 (1998).

Claims (24)

1. A method for increasing the grain yield of a plant, comprising reducing the expression level of at least one nucleic acid encoding an otubain-like protease (OTUB1) and/or reducing the activity of OTUB1, where the nucleic acid encodes OTUB1 defined in SEQ ID NO: 1, or a functional variant or homologue thereof, wherein the functional variant shares at least 60% sequence identity with SEQ ID NO: 3. 2. The method of claim 1, wherein the method comprises introducing at least one mutation in at least one nucleic acid sequence encoding an OTUB1 polypeptide and/or an OTUB1 polypeptide promoter. 3. The method according to claim 1 or 2, wherein the nucleic acid encoding the OTUB1 polypeptide comprises a nucleic acid sequence as defined in SEQ ID NO: 2, 3, 4, or 5, or a functional homologue or variant thereof, and wherein the nucleic acid sequence, encoding the OTUB1 promoter contains SEQ ID NO: 6 or a functional variant or homologue thereof, where the functional variant has at least 60% sequence identity with SEQ ID NO: 2, 3, 4, 5 or 6. 4. The method of any one of claims 1 to 3, wherein the method comprises using RNA interference to reduce the expression of at least one OTUB1 nucleic acid. 5. The method according to claims 1 to 4, wherein the plant is selected from rice, wheat, corn, sorghum, barley, soybeans and cabbage - 79 043050 you. 6. A genetically modified plant or part thereof, wherein said plant contains at least one mutation in at least one nucleic acid sequence encoding an OTUB1 polypeptide and/or an OTUB1 promoter, or where the plant contains an RNAi construct that reduces the expression of an OTUB1 polypeptide, where the nucleic acid sequence encoding the OTUB1 polypeptide contains SEQ ID NO: 1 or its functional variant or homologue, and where the nucleic acid sequence encoding the OTUB1 promoter contains SEQ ID NO: 6 or its functional variant or homologue; where the functional variant has at least 60% identity with SEQ ID NO: 1 or 6. 7. A genetically modified plant cell, wherein said plant contains at least one mutation in at least one nucleic acid sequence encoding an OTUB1 polypeptide and/or an OTUB1 promoter, or where the plant contains an RNA interference construct that reduces the expression of an OTUB1 polypeptide, wherein the nucleic acid sequence an acid encoding an OTUB1 polypeptide contains SEQ ID NO: 1 or a functional variant or homologue thereof, and wherein the nucleic acid sequence encoding the OTUB1 promoter contains SEQ ID NO: 6 or a functional variant or homologue thereof; where the functional variant has at least 60% identity with SEQ ID NO: 1 or 6 8. A genetically modified plant according to claim 6, or a plant cell according to claim 7, where the plant is selected from rice, wheat, corn, sorghum, barley, soybeans and cabbage. 9. A plant part according to any one of claims 6 to 8, wherein said plant part is a grain or seed. 10. A method for producing a plant with increased grain yield, comprising introducing at least one mutation in at least one nucleic acid sequence encoding an OTUB1 polypeptide and/or an OTUB1 polypeptide promoter, where the mutation reduces the expression or activity of OTUB1, where the nucleic acid sequence encoding OTUB1 polypeptide contains SEQ ID NO: 1 or its functional homologue or variant, and where the nucleic acid sequence encoding the OTUB1 promoter contains SEQ ID NO: 6 or its functional variant or homologue; where the functional variant has at least 60% sequence identity with SEQ ID NO: 1 or 6. 11. The method according to claim 10, where the nucleic acid sequence encoding the OTUB1 polypeptide contains SEQ ID NO: 2, 3, 4 or 5 or its functional homologue or variant, and the functional homologue or variant has at least 60% sequence identity with SEQ ID NO: 2, 3, 4 or 5. 12. A method for producing a plant with an increased grain yield, comprising introducing into said plant and expressing in said plant an RNA interference construct that reduces the expression of an OTUB1 nucleic acid, wherein the nucleic acid encodes OTUB1 as defined in SEQ ID NO: 1, or a functional thereof. a variant or homolog wherein the functional variant shares at least 60% sequence identity with SEQ ID NO: 1. 13. The method of claim 12, wherein the method also includes assessing a decrease in the expression level of the OTUB1 nucleic acid and/or assessing a decrease in activity, and preferably deubiquitinase activity, of the OTUB1 polypeptide. 14. The method of claim 13, wherein the method also includes regenerating the plant and screening for increased grain yield. 15. The method according to claim 13 or 14, wherein the plant is selected from rice, wheat, corn, sorghum, barley, soy and cabbage. 16. A plant or plant part obtained or which can be obtained by the method according to any one of claims 12-15. 17. A plant cell obtained or which can be obtained by the method of any one of claims 1215. 18. A method for identifying and/or selecting a plant that will produce an increased grain yield compared to a wild-type plant or a control plant, comprising detecting in the plant or in the germplasm of the plant at least one polymorphism in the OTUB1 gene and/or in the OTUB1 promoter, and selecting said plant or progeny thereof, wherein the polymorphism results in reduced expression of OTUB1, wherein the OTUB1 gene comprises a nucleic acid sequence as defined in SEQ ID NO: 2, 3, 4, or 5, or a functional homologue or variant thereof, and wherein the nucleic acid sequence is coding for the OTUB1 promoter includes SEQ ID NO: 6 or a functional variant or homologue thereof; where the functional variant has at least 60% sequence identity with SEQ ID NO: 2, 3, 4, 5 or 6. 19. The method of claim 18, wherein the method also includes introgression of a chromosomal region containing at least one polymorphism in the OTUB1 gene and/or OTUB1 promoter into a second plant or plant germplasm to obtain an introgressed plant or plant germplasm. 20. A nucleic acid construct containing a nucleic acid sequence that - 80 043050 cleaving at least one single-stranded guide RNA (sssrRNA), wherein the ssrRNA contains, or consists of, a sequence selected from 31, 36, 40, 44, 47, 50, 53, 56, 59, 62, 65, 68 71 74 77 80 83 86 89 92 95 98 101 104 108 112 116 120 124 128 132 136 140 144 and 148 or her variant, the variant having at least 60% sequence identity with SEQ ID NOs: 31, 36, 40, 44, 47, 50, 53, 56, 59, 62, 65, 68, 71, 74, 77, 80, 83 , 86, 89, 92, 95, 98, 101, 104, 108, 112, 116, 120, 124, 128, 132, 136, 140, 144 and 148. 21. The nucleic acid construct of claim 20, wherein the nucleic acid construct also includes a nucleic acid sequence encoding a CRISPR enzyme. 22. The nucleic acid construct of claim 20, wherein the nucleic acid construct encodes a TAL effector, and wherein the nucleic acid construct also contains a sequence encoding an endonuclease or its DNA cleavage domain, wherein the endonuclease is Fokl. 23. A single-stranded guide (ssr) RNA molecule, wherein said ssrRNA contains or consists of SEQ ID NO: 32. 24. A genetically modified plant, wherein the nucleic acid according to any one of claims 20-22 is integrated in a stable form.