WO2023131616A1 - Means and methods to increase abiotic stress tolerance in plants - Google Patents
Means and methods to increase abiotic stress tolerance in plants Download PDFInfo
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
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Definitions
- the present invention belongs to the field of agricultural biology.
- the present invention relates to improved crops which have a gene disruption in a histonl like gene which results in crops being tolerant to abiotic stress.
- Abiotic stresses such as drought, high temperature, and salinity, affect plant growth and productivity. Furthermore, global climate change may increase the frequency and severity of abiotic stresses, suggesting that development of plants with improved stress tolerance is critical for future sustainable crop production. Accordingly, there is a need to generate crops which are more resistant to abiotic stress.
- LER and LED are anticorrelated in mild drought conditions in a selected panel of a corn B73xH99 recombinant inbred line (RIL) population.
- RIL recombinant inbred line
- H1L histone Hl variants
- FIG. 2 Heatmap of all histone genes. Heat map showing the Iog2 fold change (log2FC) of expression of all histone genes based on description from PLAZA (Van Bel M. et al 2018, Nucleic Acids Research, Volume 46, Issue DI, 4 January 2018, Pages D1190-D1196). Water deficit (WD)/well-watered (WW) values indicate ratios of gene expression under WD relative to WW conditions at four to seven days after leaf four appearance. Re-watering (RW)/WD and RW/RW values indicate ratios of gene expression under RW relative to WD and RW at five or seven days after leaf four appearance, respectively. Histones with an opposite expression pattern were marked with a green box. Heat maps were made using pheatmap (R package from CRAN). The number refers to the day after leaf 4 appearance.
- FIG. 3 Hl-LIKE protein with annotation of the linker histone domain (green) and the gRNAs used in the CRISPR constructs.
- Construct 1 contains HlL_gRNA_l and HlL_gRNA_2.
- Construct 2 contains HlL_gRNA_3 and HlL_gRNA_4.
- the protein length of H1L is 236 amino acids.
- hll homozygous hll mutant
- WT wildtype
- WD water deficit
- WW well-watered
- LER leaf elongation rate.
- Arabidopsis thaliana AT
- Glycine max Glyma
- Solanum lycopersicum Solyc
- Sorghum bicolor Sobic
- Hl variants Protein alignment of Hl variants in dicots and monocots.
- the linker domain of the Hl variants shows a high degree of conservation, whereas the N- and C-terminal domain is highly variable.
- Blast hits for AtHISTONE1.3 in Zea mays (extracted from NCBI). re 10: Blast hits for ZmHISTONEl-LIKE.
- Selected species Sorghum bicolor, Oryza sativa, Triticum aestivum, Arabidopsis thaliana, Solanum lycopersicum, Gossypium hirsutum, Nicotiana tabacum and Glycine max (extracted from NCBI).
- Blast hits for ZmHISTONEl Selected species: Sorghum bicolor, Oryza sativa, Triticum aestivum, Arabidopsis thaliana, Solanum lycopersicum, Gossypium hirsutum, Nicotiana tabacum and Glycine max (extracted from NCBI).
- red box linker histone domain
- blue box RKP(K/R)SAG motif
- yellow box (S/A)EE(K/R)K
- green box (A/V)RxKRA(R/K)(R/K) motif
- * identical or conserved in all sequences in the alignment
- : conserved substitutions
- . semi-conserved substitutions. Analysis was done using ClustalO.
- the invention provides plants which are tolerant to abiotic stress, particularly drought stress, more particularly mild drought stress.
- the plants of the invention do not suffer from a yield penalty when they are submitted to conditions of abiotic stress such as drought stress.
- the present invention provides plants which have a disruption in the genome of the histonllike (H1L) gene.
- the corn H1L polynucleotide sequence is depicted in SEQ ID NO: 21 and the corresponding encoded polypeptide sequence is depicted in SEQ ID NO: 1.
- the invention provides a plant having a gene disruption in a polynucleotide encoding for SEQ ID NO: 1 or having a gene disruption in a polynucleotide encoding a plant orthologous polypeptide sequence of SEQ ID NO: 1.
- a plant is a cultivated crop.
- a plant orthologous polypeptide sequence of SEQ ID NO: 1 comprises SEQ ID NO: 18, 19 and 20.
- plant orthologous polypeptide sequences of SEQ ID NO: 1 are depicted in SEQ ID NO: 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17.
- the invention provides a seed or a plant cell derived from a plant having a gene disruption in a polynucleotide encoding a histonllike protein.
- a method for increasing tolerance to abiotic stress in a plant comprising: disrupting the expression of a polynucleotide in the plant encoding a histonllike protein.
- plant yield generally refers to a measurable product from a plant, particularly a crop. Yield and yield increase (in comparison to a starting plant which does not have a gene disruption in a particular gene or wild-type plant) can be measured in a number of ways, and it is understood that a skilled person will be able to apply the correct meaning in view of the particular embodiments, the particular crop concerned and the specific purpose or application concerned.
- the terms “improved yield” or “increased yield” can be used interchangeable.
- the term “improved yield” or the term “increased yield” means any improvement in the yield of any measured plant product, such as grain, fruit, leaf, stem, root, or fiber.
- yield preservation refers to conditions wherein the yield of the plant is not reduced, for example under conditions of abiotic stress.
- the activity of a histonllike protein may be reduced or eliminated by disrupting the gene encoding the histonllike gene.
- the disruption inhibits expression or activity of histonllike protein compared to a corresponding control plant cell lacking the disruption.
- the endogenous histonllike gene comprises two or more endogenous histonllike genes.
- plants the endogenous histonllike gene comprises three or more endogenous histonllike genes.
- two or more endogenous histonllike genes or “three or more endogenous histonllike genes” refers to two or more or three or more homologs of histonllike but it is not excluded that two or more or three or more combinations of homologs of histonllike are disrupted (or their activity reduced).
- the disruption step comprises insertion of one or more transposons, where the one or more transposons are inserted into the endogenous histonllike gene.
- the disruption comprises one or more point mutations in the endogenous histonllike gene.
- the disruption can be a homozygous disruption in the histonllike gene.
- the disruption is a heterozygous disruption in the histonllike gene.
- there is more than one disruption which can include homozygous disruptions, heterozygous disruptions or a combination of homozygous disruptions and heterozygous disruptions.
- Detection of expression products is performed either qualitatively (by detecting presence or absence of one or more product of interest) or quantitatively (by monitoring the level of expression of one or more product of interest).
- the expression product is an RNA expression product.
- aspects of the invention optionally include monitoring an expression level of a nucleic acid, polypeptide as noted herein for detection of histonllike or measuring the amount of abiotic stress tolerance in a plant or in a population of plants.
- a polynucleotide (such as an antisense polynucleotide) is introduced into a plant that upon introduction or expression, inhibits the expression of a histonllike gene of the invention.
- an expression cassette capable of expressing a polynucleotide that inhibits the expression of a histonllike polypeptide is an expression cassette capable of producing an RNA molecule that inhibits the transcription and/or translation of a histonllike polypeptide of the invention.
- the "expression” or “production” of a protein or polypeptide from a DNA molecule refers to the transcription and translation of the coding sequence to produce the protein or polypeptide
- the "expression” or “production” of a protein or polypeptide from an RNA molecule refers to the translation of the RNA coding sequence to produce the protein or polypeptide.
- polynucleotide includes reference to a deoxyribopolynucleotide, ribopolynucleotide or analogs thereof that have the essential nature of a natural ribonucleotide in that they hybridize, under stringent hybridization conditions, to substantially the same nucleotide sequence as naturally occurring nucleotides and/or allow translation into the same amino acid(s) as the naturally occurring nucleotide(s).
- a polynucleotide can be full-length or a subsequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes reference to the specified sequence as well as the complementary sequence thereof.
- DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotides" as that term is intended herein.
- DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art.
- polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including inter alia, simple and complex cells.
- nucleic acid includes reference to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g. peptide nucleic acids).
- encoding or “encoded,” with respect to a specified nucleic acid, is meant comprising the information for transcription into an RNA and in some embodiments, translation into the specified protein.
- a nucleic acid encoding a protein may comprise non-translated sequences (e.g., introns) within translated regions of the nucleic acid, or may lack such intervening non-translated sequences (e.g., as in cDNA).
- the information by which a protein is encoded is specified by the use of codons. Typical ly, the amino acid sequence is encoded by the nucleic acid using the "universal" genetic code.
- ZFNs Zinc Finger Nucleases
- TALENS Transcription Activator-Like Effector Nucleases
- CRISPR/Cas Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated nuclease with an engineered crRNA/tracr RNA
- U.S. Patent Publication No. 20080182332 describes use of non-canonical zinc finger nucleases (ZFNs) for targeted modification of plant genomes and U.S. Patent Publication No. 20090205083 describes ZFN-mediated targeted modification of a plant EPSPs genomic locus.
- ZFNs non-canonical zinc finger nucleases
- zinc fingers defines regions of amino acid sequence within a DNA binding protein binding domain whose structure is stabilized through coordination of a zinc ion.
- a “zinc finger DNA binding protein” (or binding domain) is a protein, or a domain within a larger protein, that binds DNA in a sequence-specific manner through one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized through coordination of a zinc ion.
- the term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP.
- Zinc finger binding domains can be "engineered” to bind to a predetermined nucleotide sequence.
- Nonlimiting examples of methods for engineering zinc finger proteins are design and selection.
- a designed zinc finger protein is a protein not occurring in nature whose design/composition results principally from rational criteria.
- Rational criteria for design include application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP designs and binding data. See, for example, U.S. Pat. Nos. 6,140,081; 6,453,242; 6,534,261 and 6,794,136; see also WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496.
- a "TALE DNA binding domain” or "TALE” is a polypeptide comprising one or more TALE repeat domains/units. The repeat domains are involved in binding of the TALE to its cognate target DNA sequence.
- a single “repeat unit” (also referred to as a “repeat”) is typically 33-35 amino acids in length and exhibits at least some sequence homology with other TALE repeat sequences within a naturally occurring TALE protein. See, e.g., U.S. Patent Publication No. 20110301073, incorporated by reference herein in its entirety.
- CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
- Cas CRISPR Associated nuclease system.
- a "CRISPR DNA binding domain" is a short stranded RNA molecule that acting in concert with the CAS enzyme can selectively recognize, bind, and cleave genomic DNA.
- the CRISPR/Cas system can be engineered to create a double-stranded break (DSB) at a desired target in a genome, and repair of the DSB can be influenced by the use of repair inhibitors to cause an increase in error prone repair. See, e.g., Jinek et al (2012) Science 337, p. 816-821, Jinek et al, (2013), eLife 2:e00471, and David Segal, (2013) eLife 2:e00563).
- Zinc finger, CRISPR and TALE binding domains can be "engineered” to bind to a predetermined nucleotide sequence, for example via engineering (altering one or more amino acids) of the recognition helix region of a naturally occurring zinc finger.
- TALEs can be “engineered” to bind to a predetermined nucleotide sequence, for example by engineering of the amino acids involved in DNA binding (the repeat variable diresidue or RVD region). Therefore, engineered DNA binding proteins (zinc fingers or TALEs) are proteins that are non-naturally occurring.
- Non-limiting examples of methods for engineering DNA-binding proteins are design and selection.
- a designed DNA binding protein is a protein not occurring in nature whose design/composition results principally from rational criteria.
- Rational criteria for design include application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP and/or TALE designs and binding data. See, for example, U.S. Pat. Nos. 6,140,081; 6,453,242; and 6,534,261; see also WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496 and U.S. Publication Nos. 20110301073, 20110239315 and 20119145940.
- a "selected" zinc finger protein, CRISPR or TALE is a protein not found in nature whose production results primarily from an empirical process such as phage display, interaction trap or hybrid selection. See e.g., U.S. Pat. No. 5,789,538; U.S. Pat. No. 5,925,523; U.S. Pat. No. 6,007,988; U.S. Pat. No. 6,013,453; U.S. Pat. No. 6,200,759; WO 95/19431; WO 96/06166; WO 98/53057; WO 98/54311; WO 00/27878; WO 01/60970 WO 01/88197 and WO 02/099084 and U.S. Publication Nos. 20110301073, 20110239315 and
- the polynucleotide encodes a zinc finger protein that binds to a gene encoding a histonllike polypeptide, resulting in reduced expression of the gene.
- the zinc finger protein binds to a regulatory region of a histonllike gene.
- the zinc finger protein binds to a messenger RNA encoding a histonllike polypeptide and prevents its translation.
- the TALE protein binds to a regulatory region of a histonllike gene.
- the TALE protein binds to a messenger RNA encoding a histonllike polypeptide and prevents its translation.
- Additional methods for decreasing or eliminating the expression of endogenous genes in plants are also known in the art and can be similarly applied to the instant invention. These methods include other forms of mutagenesis, such as ethyl methanesulfonate-induced mutagenesis, deletion mutagenesis and fast neutron deletion mutagenesis used in a reverse genetics sense (with PCR) to identify plant lines in which the endogenous gene has been deleted. For examples of these methods see, Ohshima, et al, (1998) Virology 243:472-481; Okubara, et al, (1994) Genetics 137:867-874 and Quesada, et al, (2000) Genetics 154:421-436, each of which is herein incorporated by reference.
- mutagenesis such as ethyl methanesulfonate-induced mutagenesis, deletion mutagenesis and fast neutron deletion mutagenesis used in a reverse genetics sense (with PCR) to identify plant lines in which the endogen
- a fast and automatable method for screening for chemically induced mutations TILLING (Targeting Induced Local Lesions in Genomes), using denaturing HPLC or selective endonuclease digestion of selected PCR products is also applicable to the instant invention.
- TILLING Targeting Induced Local Lesions in Genomes
- Mutations that impact gene expression or that interfere with the function of the encoded protein are well known in the art. Insertional mutations in gene exons usually result in null-mutants. Mutations in conserved residues are particularly effective in inhibiting the activity of the encoded protein. conserveed residues of plant histonllike polypeptides suitable for mutagenesis with the goal to eliminate histonllike activity have been described.
- single stranded DNA can be used to downregulate the expression of histonllike genes.
- Methods for gene suppression using ssDNA are e.g. described in W02011/112570.
- protein interference as described in the patent application W02007071789 can be used to downregulate a gene product.
- the latter technology is a knock-down technology which in contrast to RNAi acts at the post-translational level (i.e. it works directly on the protein level by inducing a specific protein aggregation of a chosen target). Protein aggregation is essentially a misfolding event which occurs through the formation of intermolecular beta-sheets resulting in a functional knockout of a selected target.
- the invention encompasses still additional methods for reducing or eliminating the activity of the histonllike polypeptide.
- methods for altering or mutating a genomic nucleotide sequence in a plant include, but are not limited to, the use of RNA:DNA vectors, RNA:DNA mutational vectors, RNA:DNA repair vectors, mixed-duplex oligonucleotides, self-complementary RNA:DNA oligonucleotides and recombinogenic oligonucleotide bases.
- Such vectors and methods of use are known in the art. See, for example, US5565350; US5731181; US5756325; US5760012; US5795972 and US5871984, each of which are herein incorporated by reference.
- expression means the transcription of a specific gene or specific genes or specific genetic construct.
- expression in particular means the transcription of a gene or genes or genetic construct into structural RNA (rRNA, tRNA) or mRNA with or without subsequent translation of the latter into a protein. The process includes transcription of DNA and processing of the resulting mRNA product.
- introduction or “transformation” as referred to herein encompass the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer.
- Plant tissue capable of subsequent clonal propagation may be transformed with a genetic construct of the present invention and a whole plant regenerated there from.
- the particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed.
- tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, mega-gametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem).
- the polynucleotide may be transiently or stably introduced into a host cell and may be maintained non-integrated, for example, as a plasmid. Alternatively, it may be integrated into the host genome.
- the resulting transformed plant cell may then be used to regenerate a transformed plant in a manner known to persons skilled in the art.
- Transformation of plant species is now a fairly routine technique.
- any of several transformation methods may be used to introduce the gene of interest into a suitable ancestor cell.
- the methods described for the transformation and regeneration of plants from plant tissues or plant cells may be utilized for transient or for stable transformation. Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant, particle gun bombardment, transformation using viruses or pollen and microprojection. Methods may be selected from the calcium/polyethylene glycol method for protoplasts (Krens, F.A. et al., (1982) Nature 296, 72-74; Negrutiu I et al.
- Transgenic plants including transgenic crop plants, are preferably produced via Agrobacterium-mediated transformation.
- An advantageous transformation method is the transformation in planta.
- agrobacteria to act on plant seeds or to inoculate the plant meristem with agrobacteria. It has proved particularly expedient in accordance with the invention to allow a suspension of transformed agrobacteria to act on the intact plant or at least on the flower primordia. The plant is subsequently grown on until the seeds of the treated plant are obtained (Clough and Bent, Plant J. (1998) 16, 735-743).
- Methods for Agrobacterium-mediated transformation of rice include well known methods for rice transformation, such as those described in any of the following: European patent application EP1198985, Aldemita and Hodges (Planta 199: 612-617, 1996); Chan et al.
- nucleic acids or the construct to be expressed is preferably cloned into a vector, which is suitable for transforming Agrobacterium tumefaciens, for example pBinl9 (Bevan et al (1984) Nucl. Acids Res. 12-8711).
- Agrobacteria transformed by such a vector can then be used in known manner for the transformation of plants, such as plants used as a model, like Arabidopsis or crop plants such as, by way of example, tobacco plants, for example by immersing bruised leaves or chopped leaves in an agrobacterial solution and then culturing them in suitable media.
- plants used as a model like Arabidopsis or crop plants such as, by way of example, tobacco plants, for example by immersing bruised leaves or chopped leaves in an agrobacterial solution and then culturing them in suitable media.
- the transformation of plants by means of Agrobacterium tumefaciens is described, for example, by Hofgen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877 or is known inter alia from F.F. White, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1 , Engineering and Utilization, eds. S.D. Kung and R. Wu, Academic Press, 1993, pp. 15-
- the transformation of the chloroplast genome is generally achieved by a process which has been schematically displayed in Klaus et al., 2004 [Nature Biotechnology 22 (2), 225-229], Briefly the sequences to be transformed are cloned together with a selectable marker gene between flanking sequences homologous to the chloroplast genome. These homologous flanking sequences direct site specific integration into the plastome. Plastidal transformation has been described for many different plant species and an overview is given in Bock (2001) Transgenic plastids in basic research and plant biotechnology. J Mol Biol. 2001 Sep 21; 312 (3):425-38 or Maliga, P (2003) Progress towards commercialization of plastid transformation technology. Trends Biotechnol. 21, 20-28. Further biotechnological progress has recently been reported in form of marker free plastid transformants, which can be produced by a transient co-integrated maker gene (Klaus et al., 2004, Nature Biotechnology 22(2), 225-229).
- plant as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, leaves, roots (including tubers), flowers, and tissues and organs, wherein each of the aforementioned comprise the gene/nucleic acid of interest.
- plant also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the aforementioned comprises the gene/nucleic acid of interest.
- Plants that are particularly useful in the methods of the invention include in particular monocotyledonous and dicotyledonous plants including fodder or forage legumes, ornamental plants, food crops, trees or shrubs selected from the list comprising Acer spp., Actinidia spp., Abelmoschus spp., Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium spp., Amaranthus spp., Ammophila arenaria, Ananas comosus, Annona spp., Apium graveolens, Arachis spp, Artocarpus spp., Asparagus officinalis, Avena spp.
- Avena sativa e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida
- Averrhoa carambola e.g. Bambusa sp.
- Benincasa hispida Bertholletia excelsea
- Beta vulgaris Brassica spp.
- Brassica napus e.g. Brassica napus, Brassica rapa ssp.
- the plant cell according to the invention is non-propagating or cannot be regenerated into a plant.
- HISTONE1-LIKE H1L
- knock-out mutants were generated in the B104 inbred line using the CRISPR-Cas9 system.
- the polynucleotide sequence of the corn H1L gene is depicted in SEQ ID NO: 21 and this gene was knocked out.
- SEQ ID NO: 21 encodes for the polypeptide depicted in SEQ ID NO: 1.
- Two independent constructs were made, each containing two guide RNAs (gRNAs) that specifically target the linker histone domain of the H1L gene and have no off targets in other (linker histone) genes (see Figure 3).
- gRNAs guide RNAs
- HlL_gRNA_l AGCAGGAAGCCCAAGTCCGC (SEQ ID NO: 22)
- HlL_gRNA_2 GCCCAATTACCGCAAGGTGC (SEQ ID NO: 23)
- HlL_gRNA_3 CGATCCTGTCGCAGGACGGC (SEQ ID NO: 24)
- HlL_gRNA_4 GGCCAGCACCTTGCGGTAAT (SEQ ID NO: 25)
- hll plants tend to have larger leaves compared to their segregating WT siblings under drought stress. Even more, hll plants under drought have one to two leaves more compared to WT resulting in a lower yield penalty and a more vigorous growth under water deficit conditions. No significant differences were found between hll and WT seedlings under well-watered conditions.
- HISTONE1- LIKE subclade can be recognized as a distinct group of linker histones in monocots
- H1L is upregulated under drought and downregulated upon re-watering. Under drought, hll CRISPR mutants show a more vigorous growth compared to their WT siblings.
- Arabidopsis Arabidopsis (AtH1.3, AT2G18050 - polypeptide sequence depicted in SEQ ID NO: 3) and tomato (SIH1-S, Solyc02g084240.3)
- Hl variants that belong to the same subclade of H1L, have been shown to be induced upon drought stress (Rutowicz et al., 2015; Scippa et al., 2004).
- H1L has one close homolog, namely HISTONE1 (Hl) ( Figure 5).
- Hl and H1L are closely related, Hl is downregulated under drought and upregulated upon rewatering, suggesting an opposite role for Hl compared to H1L in response to drought (see Figure 6).
- Hl and H1L protein sequences of rice, wheat and maize in the Hl-subclade and the H1L- subclade were aligned, together with AtH1.3 (SEQ. ID NO: 3) (Arabidopsis) and SIH1-S (tomato) and other dicot orthologs in tobacco (Nitab), soya bean (Glyma) and cotton (Gohir) (see Figure 7).
- Linker histones typically have a tripartite structure which is composed of a conserved central globular domain flanked by a highly variable short N-terminal domain and a longer highly basic C-terminal domain.
- the N- and C-terminal domains are prone to post-translational modifications which can cause alterations in chromatin condensation, resulting in differential gene expression (Gibbs and Kriwacki, 2018).
- Hl variants can be divided into two groups according to the length of the N-terminal domain, the first group has a short N- terminal domain consisting of 8 to 9 AA, the second group has a longer N-domain ranging from 25 to 47 AA.
- Monocot proteins belonging to the Hl subclade have a basic N-terminal domain containing no acidic amino acid residues.
- dicot proteins and proteins belonging to the H1L subclade have a mix of acidic and basic AA residues, most of them being acidic.
- Hl Blasting the protein sequence of AtH1.3 at NCBI, yields Hl as a first hit in Zea mays (see Figure 9). If we blast the sequence of H1L and Hl in the species described above in the protein alignment (see figure 9), the ranking of the monocot proteins is identical to their divergence into H1L and Hl subclades. For example, blasting H1L gives Sobic.010G021300, TraesCS7A03G0150000 and 0s06g0130800 as best hits in Sorghum, wheat and rice, respectively (see Figure 10). For Hl, the best hits in Sorghum, wheat and rice are Sobic.006G186700, TraesCS2A03G0956900 and 0s04g0253000 (see Figure 11).
- Orthologous protein sequences of corn H1L of different monocotyledonous plants are depicted in SEQ I D NO: 2, 4, 5, 6, 7, 8 and 9. Alignment of monocot species belonging to the H1L subclade with the dicot proteins from tomato (Solyc02g084240.2), tobacco (Nitab4.5_0002880g0020, Nitab4.5_0005721g0020) and cotton (Gohir.AllG176800) that were found in the H1L blast, shows conserved substitutions in the RKP(K/R)SAG motif, in the N-terminal domain (see Figure 13).
- the RKP(K/R)SAG motif changes into a (K/R)KP(K/R)SA motif by conserved substitution of residues. This conserved motif is not found in cotton, Arabidopsis thaliana and Glycine max, nor in proteins belonging to the monocot Hl subclade.
- the (A/V)RxKRA(R/K)(R/K) motif shows a low degree of conservation whereas the (S/A)EE(K/R)K motif is conserved as a AGKKE motif by amino acid substitutions in tomato (Solyc02g084240.2), tobacco (Nitab4.5_0002880g0020, Nitab4.5_0005721g0020) and cotton (Gohir.AllG176800, Gohir.DllG184700).
- the AGKKE and the (S/A)EE(K/R)K motif were not found in monocot proteins belonging to the Hl subclade, nor in Arabidopsis thaliana, Glycine max and cotton (with the exception of Gohir.AllG176800, Gohir.DllG184700).
- Dicotyledonous orthologous protein sequences of corn H1L (SEQ ID NO: 1) are depicted in SEQ ID NO: 10-17.
- SEQ ID NO: 1 Zea mays polypeptide sequence of ZmHlL
- SEQ ID NO: 2 Zea mays polypeptide sequence of ZmHIL (orthologue of SEQ ID NO: 1)
- SEQ ID NO: 3 (Arabidopsis thaliana Hl.3 polypeptide, encoded from gene AT2G18050)
- SEQ ID NO: 4 (Sorghum bicolor, H1L orthologue of SEQ ID NO: 1, NCBI accession: OQU75744)
- SEQ ID NO: 5 (Oryza sativa, orthologue of SEQ ID NO: 1, NCBI accession: XP_015644353)
- SEQ ID NO: 6 (Triticum aestivum, orthologue of SEQ ID NO: 1, NCBI accession: KAF7093214)
- SEQ ID NO: 7 (Triticum aestivum, orthologue of SEQ ID NO: 1, NCBI accession: KAF7093216)
- SEQ ID NO: 8 (Triticum aestivum, orthologue of SEQ ID NO: 1, NCBI accession: KAF7045985)
- SEQ ID NO: 9 (Triticum aestivum, orthologue of SEQ ID NO: 1, NCBI accession: XP_044442749) MATVMAAAAPAMVGAGEEVKEAVAAPEKVEEVKEAVAAPEKVEEVKEAGAGEEVMEVAAGEAKEAGAGEEAME
- SEQ ID NO: 10 Solanum lycopersicum, orthologue of SEQ ID NO: 1, NCBI accession: NP_001234389
- SEQ ID NO: 11 Gossypium hirsutum, orthologue of SEQ ID NO: 1, NCBI accession: XP_040937395)
- SEQ ID NO: 12 (Nicotiana tabacum, orthologue of SEQ ID NO: 1, NCBI accession: AAN37904)
- SEQ ID NO: 13 (Glycine max, orthologue of SEQ ID NO: 1, NCBI accession: XP_003537627)
- SEQ ID NO: 14 (Glycine max, orthologue of SEQ ID NO: 1, NCBI accession: KAG5089416)
- SEQ ID NO: 15 (Glycine max, orthologue of SEQ ID NO: 1, NCBI accession: NP_001237870)
- SEQ ID NO: 17 (Glycine max, orthologue of SEQ ID NO: 1, NCBI accession: ACU15107)
- SEQ ID NO: 21 polynucleotide sequence encoding SEQ ID NO: 1
- Przewloka M. R., Wierzbicki, A. T., Slusarczyk, J., Kuras, M., Grasser, K. D., Stemmer, C., & Jerzmanowski, A. (2002).
- a specialized histone Hl variant is required for adaptive responses to complex abiotic stress and related DNA methylation in Arabidopsis. Plant Physiology, 169(3), 2080-2101.
- N-and C-terminal domains determine differential nucleosomal binding geometry and affinity of linker histone isotypes H10 and Hlc. Journal of Biological Chemistry, 287(15), 11778-11787.
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Abstract
The present invention relates to plants having a decreased expression of the Histon1like gene which result in an increased tolerance to abiotic stress, such as drought stress. The invention also provides methods which can be used to decrease the Histon1like gene expression.
Description
MEANS AND METHODS TO INCREASE ABIOTIC STRESS TOLERANCE IN PLANTS
Field of the invention
The present invention belongs to the field of agricultural biology. In particular the present invention relates to improved crops which have a gene disruption in a histonl like gene which results in crops being tolerant to abiotic stress.
Introduction to the invention
Abiotic stresses, such as drought, high temperature, and salinity, affect plant growth and productivity. Furthermore, global climate change may increase the frequency and severity of abiotic stresses, suggesting that development of plants with improved stress tolerance is critical for future sustainable crop production. Accordingly, there is a need to generate crops which are more resistant to abiotic stress. In the present invention we have studied the molecular mechanisms underlying the re-watering effects on LER and LED, by detailed analysis of the transcriptome (by application of the RNAseq technology) at different time points upon drought and re-watering in corn leaves. Two main determinants for maize leaf size are the maximal growth rate (leaf elongation rate; LER) and the duration of the growth period (leaf elongation duration; LED). We identified how changes in environmental conditions affect LER and LED by applying mild drought stress on corn. The mild drought stress lowers the maximal growth rate, but prolongs the duration of growth, suggesting that the latter compensates, at least in part, for the reduction in growth rate (see Figure 1). Furthermore, LER and LED are anticorrelated in mild drought conditions in a selected panel of a corn B73xH99 recombinant inbred line (RIL) population. The prolonged growth duration increased the possibility to resume growth upon alleviation of the drought. Depending on the growth phase of the leaf, upon re-watering, growth could be either fully restored by increasing the maximal growth rate or partially restored by prolonging the duration of growth. To investigate the molecular mechanisms underlying the re-watering effects on LER and LED, the transcriptome (analysed via RNAseq technology) of the most basal half centimeter (dividing cells) of the fourth leaf of RIL89 corn plants (Baute et al., 2015) was profiled at different time points upon drought and re-watering. From the RNAseq data, it became clear that growth recovery from mild drought conditions activates or represses specific gene sets depending on the timing of re-watering during leaf development. Maize plants re-watered at four days after leaf appearance (resulting in a restoration of the maximal LER) showed one day later an increase in histone gene expression (Hl; H2A; H2B; H3 and H4). The majority of these histone encoding genes were downregulated under drought, as was reported
previously in rice (Hu and Lai, 2015). However, remarkably we identified three histones which showed an opposite expression pattern (see Figure 2) including GRMZM2G069911 (sequence is depicted in SEQ ID NO: 21), HISTONE1-LIKE (abbreviated herein further as H1L), which encodes for the protein depicted in SEQ ID NO: 1. Also in Arabidopsis, tobacco, tomato and cotton, specialized histone Hl variants were previously identified which are induced by drought stress and ABA (Przewloka et al., 2002; Scippa et al., 2004; Trivedi et al., 2012; Rutowicz et al., 2015). However, the H1L identified in corn is not the predicted closest orthologue of the latter histone Hl variants (such as Histone 1.3 depicted in SEQ ID NO: 3), H1L is also about 70 amino acids longer than the reported histone sequences and H1L was only identified after careful analysis of the transcriptome changes during mild drought stress. Remarkably, we show that the knockout of this histon like gene (H1L) in corn leads to an enhanced tolerance to abiotic stress, such as drought stress.
Figure legends
Figure 1: Leaf elongation rate (LER) and duration (LED) of leaf 4 under drought and upon rewatering in the RIL89 recombinant inbred line. Depending on the time of rewatering, different compensation mechanisms are used by the plant to compensate for the growth penalty under drought conditions. To study the recovery mechanisms upon rewatering, plants were sampled at different time point for RNAseq analysis. Legend: WW = well-watered, WD = water deficit, R(e)W = rewatering, the number refers to the day after leaf 4 appearance.
Figure 2: Heatmap of all histone genes. Heat map showing the Iog2 fold change (log2FC) of expression of all histone genes based on description from PLAZA (Van Bel M. et al 2018, Nucleic Acids Research, Volume 46, Issue DI, 4 January 2018, Pages D1190-D1196). Water deficit (WD)/well-watered (WW) values indicate ratios of gene expression under WD relative to WW conditions at four to seven days after leaf four appearance. Re-watering (RW)/WD and RW/RW values indicate ratios of gene expression under RW relative to WD and RW at five or seven days after leaf four appearance, respectively. Histones with an opposite expression pattern were marked with a green box. Heat maps were made using pheatmap (R package from CRAN). The number refers to the day after leaf 4 appearance.
Figure 3: Hl-LIKE protein with annotation of the linker histone domain (green) and the gRNAs used in the CRISPR constructs. Construct 1 contains HlL_gRNA_l and HlL_gRNA_2. Construct 2 contains HlL_gRNA_3 and HlL_gRNA_4. The protein length of H1L is 236 amino acids.
re 4 hll CIRPSR mutants under mild drought conditions hll mutants suffer less from drought compared to their WT siblings, resulting in a more vigorous growth, higher biomass and a lower yield penalty. Legend: hll = homozygous hll mutant, WT = wildtype, WD = water deficit, WW = well-watered, LER = leaf elongation rate.
Phylogenetic tree of Hl variants. Phylogenetic tree was built on protein sequences from
Arabidopsis thaliana (AT), Glycine max (Glyma), Solanum lycopersicum (Solyc), Sorghum bicolor (Sobic),
Oryza sativa (Os) and Zea mays (Zm). According to the presence of Hl and H1L in maize, each subclade was named respectively.
Heatmap of Hl and H1L in maize (zoomed in from Figure 2). Legend: WD water deficit, WW
= well-watered, RW = rewatering, the number refers to the day after leaf 4 appearance.
Protein alignment of Hl variants in dicots and monocots. The linker domain of the Hl variants shows a high degree of conservation, whereas the N- and C-terminal domain is highly variable. re 8 Alignment of the N-terminal domain of Hl variants in dicots and monocots showing the polarity and charge of the amino acid residues. Residues are coloured according to polarity and charge: green = neutral, polar, black = neutral, nonpolar, red = acidic, polar, blue = basic, polar.
Blast hits for AtHISTONE1.3 in Zea mays (extracted from NCBI). re 10: Blast hits for ZmHISTONEl-LIKE. Selected species: Sorghum bicolor, Oryza sativa, Triticum aestivum, Arabidopsis thaliana, Solanum lycopersicum, Gossypium hirsutum, Nicotiana tabacum and Glycine max (extracted from NCBI).
Blast hits for ZmHISTONEl. Selected species: Sorghum bicolor, Oryza sativa, Triticum aestivum, Arabidopsis thaliana, Solanum lycopersicum, Gossypium hirsutum, Nicotiana tabacum and Glycine max (extracted from NCBI).
Conserved motifs in monocot proteins belonging the HISTONE1-LIKE subclade Legend: red box = linker histone domain, blue box = RKP(K/R)SAG motif, yellow box = (S/A)EE(K/R)K, green box = (A/V)RxKRA(R/K)(R/K) motif, * = identical or conserved in all sequences in the alignment, : = conserved substitutions, . = semi-conserved substitutions. Analysis was done using ClustalO.
Conserved motifs in selected dicot proteins and monocot proteins belonging the HISTONE1-
LIKE subclade. Legend: red box = linker histone domain, blue, green and yellow boxes represent motifs that are conserved between species: blue box = (K/R)KP(K/R)SA motif, yellow box = (S/A)(G/E)(K/E)(K/R)(K/E) motif, green box = (A/V)(R/G)xK(K/R)x(R/K)(R/K) motif, * = identical or
conserved in all sequences in the alignment, : = conserved substitutions, . = semi-conserved substitutions. In Nicotiana tabacum, the Nitab4.5_0002880g0020 protein is identical to Nitab4.5_0005721g0020. Analysis was done using ClustalO.
Detailed description of the invention
To facilitate the understanding of this invention a number of terms are defined below. Terms defined herein (unless otherwise specified) have meanings as commonly understood by a person of ordinary skill in the areas relevant to the present invention. As used in this specification and its appended claims, terms such as "a", "an" and "the" are not intended to refer to only a singular entity, but include the general class of which a specific example may be used for illustration, unless the context dictates otherwise. The terminology herein is used to describe specific embodiments of the invention, but their usage does not delimit the invention, except as outlined in the claims.
The invention provides plants which are tolerant to abiotic stress, particularly drought stress, more particularly mild drought stress. The plants of the invention do not suffer from a yield penalty when they are submitted to conditions of abiotic stress such as drought stress.
The present invention provides plants which have a disruption in the genome of the histonllike (H1L) gene. The corn H1L polynucleotide sequence is depicted in SEQ ID NO: 21 and the corresponding encoded polypeptide sequence is depicted in SEQ ID NO: 1.
Thus in a first embodiment the invention provides a plant having a gene disruption in a polynucleotide encoding for SEQ ID NO: 1 or having a gene disruption in a polynucleotide encoding a plant orthologous polypeptide sequence of SEQ ID NO: 1.
In a particular embodiment a plant is a cultivated crop.
In a particular embodiment a plant orthologous polypeptide sequence of SEQ ID NO: 1 comprises SEQ ID NO: 18, 19 and 20.
In yet another particular embodiment plant orthologous polypeptide sequences of SEQ ID NO: 1 are depicted in SEQ ID NO: 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17.
In another particular embodiment the invention provides a seed or a plant cell derived from a plant having a gene disruption in a polynucleotide encoding a histonllike protein.
A method for increasing tolerance to abiotic stress in a plant, the method comprising: disrupting the expression of a polynucleotide in the plant encoding a histonllike protein.
The term "plant yield" as used herein generally refers to a measurable product from a plant, particularly a crop. Yield and yield increase (in comparison to a starting plant which does not have a gene disruption in a particular gene or wild-type plant) can be measured in a number of ways, and it is understood that
a skilled person will be able to apply the correct meaning in view of the particular embodiments, the particular crop concerned and the specific purpose or application concerned. The terms "improved yield" or "increased yield" can be used interchangeable. As used herein, the term "improved yield" or the term "increased yield" means any improvement in the yield of any measured plant product, such as grain, fruit, leaf, stem, root, or fiber. The term "yield preservation" refers to conditions wherein the yield of the plant is not reduced, for example under conditions of abiotic stress.
Thus, the activity of a histonllike protein may be reduced or eliminated by disrupting the gene encoding the histonllike gene. The disruption inhibits expression or activity of histonllike protein compared to a corresponding control plant cell lacking the disruption. In one embodiment, the endogenous histonllike gene comprises two or more endogenous histonllike genes. Similarly, in another embodiment, in particular plants the endogenous histonllike gene comprises three or more endogenous histonllike genes. The wording "two or more endogenous histonllike genes" or "three or more endogenous histonllike genes" refers to two or more or three or more homologs of histonllike but it is not excluded that two or more or three or more combinations of homologs of histonllike are disrupted (or their activity reduced)..
In another embodiment, the disruption step comprises insertion of one or more transposons, where the one or more transposons are inserted into the endogenous histonllike gene. In yet another embodiment, the disruption comprises one or more point mutations in the endogenous histonllike gene. The disruption can be a homozygous disruption in the histonllike gene. Alternatively, the disruption is a heterozygous disruption in the histonllike gene. In certain embodiments, when more than one histonllike gene is involved, there is more than one disruption, which can include homozygous disruptions, heterozygous disruptions or a combination of homozygous disruptions and heterozygous disruptions.
Detection of expression products is performed either qualitatively (by detecting presence or absence of one or more product of interest) or quantitatively (by monitoring the level of expression of one or more product of interest). In one embodiment, the expression product is an RNA expression product. Aspects of the invention optionally include monitoring an expression level of a nucleic acid, polypeptide as noted herein for detection of histonllike or measuring the amount of abiotic stress tolerance in a plant or in a population of plants.
Thus, many methods may be used to reduce or eliminate the activity of a histonllike gene. More than one method may be used to reduce the activity of a single plant histonllike gene. In addition, combinations of methods may be employed to reduce or eliminate the activity of two or more different histonllike genes. Non-limiting examples of methods of reducing or eliminating the expression of a plant histonllike are given below.
In some embodiments of the present invention, a polynucleotide (such as an antisense polynucleotide) is introduced into a plant that upon introduction or expression, inhibits the expression of a histonllike gene of the invention. The term "expression" as used herein refers to the biosynthesis of a gene product, including the transcription and/or translation of said gene product. For example, for the purposes of the present invention, an expression cassette capable of expressing a polynucleotide that inhibits the expression of a histonllike polypeptide is an expression cassette capable of producing an RNA molecule that inhibits the transcription and/or translation of a histonllike polypeptide of the invention. The "expression" or "production" of a protein or polypeptide from a DNA molecule refers to the transcription and translation of the coding sequence to produce the protein or polypeptide, while the "expression" or "production" of a protein or polypeptide from an RNA molecule refers to the translation of the RNA coding sequence to produce the protein or polypeptide.
As used herein, "polynucleotide" includes reference to a deoxyribopolynucleotide, ribopolynucleotide or analogs thereof that have the essential nature of a natural ribonucleotide in that they hybridize, under stringent hybridization conditions, to substantially the same nucleotide sequence as naturally occurring nucleotides and/or allow translation into the same amino acid(s) as the naturally occurring nucleotide(s). A polynucleotide can be full-length or a subsequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes reference to the specified sequence as well as the complementary sequence thereof. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotides" as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are polynucleotides as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term polynucleotide as it is employed herein embraces such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including inter alia, simple and complex cells.
As used herein, "nucleic acid" includes reference to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues having the essential nature of natural nucleotides in that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g. peptide nucleic acids).
By "encoding" or "encoded," with respect to a specified nucleic acid, is meant comprising the information for transcription into an RNA and in some embodiments, translation into the specified protein. A nucleic acid encoding a protein may comprise non-translated sequences (e.g., introns) within translated regions of the nucleic acid, or may lack such intervening non-translated sequences (e.g., as in cDNA). The
information by which a protein is encoded is specified by the use of codons. Typical ly, the amino acid sequence is encoded by the nucleic acid using the "universal" genetic code.
Significant advances have been made in the last few years towards development of methods and compositions to target and cleave genomic DNA by site specific nucleases (e.g., Zinc Finger Nucleases (ZFNs), Meganucleases, Transcription Activator-Like Effector Nucleases (TALENS) and Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated nuclease (CRISPR/Cas) with an engineered crRNA/tracr RNA), to induce targeted mutagenesis, induce targeted deletions of cellular DNA sequences, and facilitate targeted recombination of an exogenous donor DNA polynucleotide within a predetermined genomic locus. See, for example, U.S. Patent Publication No. 20030232410; 20050208489; 20050026157; 20050064474; and 20060188987, and International Patent Publication No. WO 2007/014275, the disclosures of which are incorporated by reference in their entireties for all purposes. U.S. Patent Publication No. 20080182332 describes use of non-canonical zinc finger nucleases (ZFNs) for targeted modification of plant genomes and U.S. Patent Publication No. 20090205083 describes ZFN-mediated targeted modification of a plant EPSPs genomic locus. Current methods for targeted insertion of exogenous DNA typically involve co-transformation of plant tissue with a donor DNA polynucleotide containing at least one transgene and a site specific nuclease (e.g., ZFN) which is designed to bind and cleave a specific genomic locus of an actively transcribed coding sequence. This causes the donor DNA polynucleotide to stably insert within the cleaved genomic locus resulting in targeted gene addition at a specified genomic locus comprising an actively transcribed coding sequence.
As used herein the term "zinc fingers," defines regions of amino acid sequence within a DNA binding protein binding domain whose structure is stabilized through coordination of a zinc ion.
A "zinc finger DNA binding protein" (or binding domain) is a protein, or a domain within a larger protein, that binds DNA in a sequence-specific manner through one or more zinc fingers, which are regions of amino acid sequence within the binding domain whose structure is stabilized through coordination of a zinc ion. The term zinc finger DNA binding protein is often abbreviated as zinc finger protein or ZFP. Zinc finger binding domains can be "engineered" to bind to a predetermined nucleotide sequence. Nonlimiting examples of methods for engineering zinc finger proteins are design and selection. A designed zinc finger protein is a protein not occurring in nature whose design/composition results principally from rational criteria. Rational criteria for design include application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP designs and binding data. See, for example, U.S. Pat. Nos. 6,140,081; 6,453,242; 6,534,261 and 6,794,136; see also WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496.
A "TALE DNA binding domain" or "TALE" is a polypeptide comprising one or more TALE repeat domains/units. The repeat domains are involved in binding of the TALE to its cognate target DNA sequence. A single "repeat unit" (also referred to as a "repeat") is typically 33-35 amino acids in length and exhibits at least some sequence homology with other TALE repeat sequences within a naturally occurring TALE protein. See, e.g., U.S. Patent Publication No. 20110301073, incorporated by reference herein in its entirety.
The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR Associated) nuclease system. Briefly, a "CRISPR DNA binding domain" is a short stranded RNA molecule that acting in concert with the CAS enzyme can selectively recognize, bind, and cleave genomic DNA. The CRISPR/Cas system can be engineered to create a double-stranded break (DSB) at a desired target in a genome, and repair of the DSB can be influenced by the use of repair inhibitors to cause an increase in error prone repair. See, e.g., Jinek et al (2012) Science 337, p. 816-821, Jinek et al, (2013), eLife 2:e00471, and David Segal, (2013) eLife 2:e00563).
Zinc finger, CRISPR and TALE binding domains can be "engineered" to bind to a predetermined nucleotide sequence, for example via engineering (altering one or more amino acids) of the recognition helix region of a naturally occurring zinc finger. Similarly, TALEs can be "engineered" to bind to a predetermined nucleotide sequence, for example by engineering of the amino acids involved in DNA binding (the repeat variable diresidue or RVD region). Therefore, engineered DNA binding proteins (zinc fingers or TALEs) are proteins that are non-naturally occurring. Non-limiting examples of methods for engineering DNA-binding proteins are design and selection. A designed DNA binding protein is a protein not occurring in nature whose design/composition results principally from rational criteria. Rational criteria for design include application of substitution rules and computerized algorithms for processing information in a database storing information of existing ZFP and/or TALE designs and binding data. See, for example, U.S. Pat. Nos. 6,140,081; 6,453,242; and 6,534,261; see also WO 98/53058; WO 98/53059; WO 98/53060; WO 02/016536 and WO 03/016496 and U.S. Publication Nos. 20110301073, 20110239315 and 20119145940.
A "selected" zinc finger protein, CRISPR or TALE is a protein not found in nature whose production results primarily from an empirical process such as phage display, interaction trap or hybrid selection. See e.g., U.S. Pat. No. 5,789,538; U.S. Pat. No. 5,925,523; U.S. Pat. No. 6,007,988; U.S. Pat. No. 6,013,453; U.S. Pat. No. 6,200,759; WO 95/19431; WO 96/06166; WO 98/53057; WO 98/54311; WO 00/27878; WO 01/60970 WO 01/88197 and WO 02/099084 and U.S. Publication Nos. 20110301073, 20110239315 and
20119145940.
In one embodiment, the polynucleotide encodes a zinc finger protein that binds to a gene encoding a histonllike polypeptide, resulting in reduced expression of the gene. In particular embodiments, the zinc finger protein binds to a regulatory region of a histonllike gene. In other embodiments, the zinc finger protein binds to a messenger RNA encoding a histonllike polypeptide and prevents its translation. Methods of selecting sites for targeting by zinc finger proteins have been described, for example, in US6453242, and methods for using zinc finger proteins to inhibit the expression of genes in plants are described, for example, in US2003/0037355, each of which is herein incorporated by reference.
In another embodiment, the polynucleotide encoded a TALE protein that binds to a gene encoding a histonllike polypeptide, resulting in reduced expression of the gene. In particular embodiments, the TALE protein binds to a regulatory region of a histonllike gene. In other embodiments, the TALE protein binds to a messenger RNA encoding a histonllike polypeptide and prevents its translation. Methods of selecting sites for targeting by zinc finger proteins have been described in e.g. Moscou MJ, Bogdanove AJ (2009) (A simple cipher governs DNA recognition by TAL effectors. Science 326:1501) and Morbitzer R, Romer P, Boch J, Lahaye T (2010) (Regulation of selected genome loci using de novo-engineered transcription activator-like effector (TALE)-type transcription factors. Proc Natl Acad Sci USA 107:21617- 21622).
Additional methods for decreasing or eliminating the expression of endogenous genes in plants are also known in the art and can be similarly applied to the instant invention. These methods include other forms of mutagenesis, such as ethyl methanesulfonate-induced mutagenesis, deletion mutagenesis and fast neutron deletion mutagenesis used in a reverse genetics sense (with PCR) to identify plant lines in which the endogenous gene has been deleted. For examples of these methods see, Ohshima, et al, (1998) Virology 243:472-481; Okubara, et al, (1994) Genetics 137:867-874 and Quesada, et al, (2000) Genetics 154:421-436, each of which is herein incorporated by reference. In addition, a fast and automatable method for screening for chemically induced mutations, TILLING (Targeting Induced Local Lesions in Genomes), using denaturing HPLC or selective endonuclease digestion of selected PCR products is also applicable to the instant invention. See, McCallum, et al, (2000) Nat. Biotechnol 18:455- 457, herein incorporated by reference. Mutations that impact gene expression or that interfere with the function of the encoded protein are well known in the art. Insertional mutations in gene exons usually result in null-mutants. Mutations in conserved residues are particularly effective in inhibiting the activity of the encoded protein. Conserved residues of plant histonllike polypeptides suitable for mutagenesis with the goal to eliminate histonllike activity have been described.
Also single stranded DNA can be used to downregulate the expression of histonllike genes. Methods for gene suppression using ssDNA are e.g. described in W02011/112570.
In yet another embodiment protein interference as described in the patent application W02007071789 (means and methods for mediating protein interference) can be used to downregulate a gene product. The latter technology is a knock-down technology which in contrast to RNAi acts at the post-translational level (i.e. it works directly on the protein level by inducing a specific protein aggregation of a chosen target). Protein aggregation is essentially a misfolding event which occurs through the formation of intermolecular beta-sheets resulting in a functional knockout of a selected target. Through the use of a dedicated algorithm it is possible to accurately predict which amino acidic stretches in a chosen target protein sequence have the highest self-associating tendency (Fernandez-Escamilla A. M. et al (2004) Nat Biotechnol 22(10): 1302-6. By expressing these specific peptides in the cells the protein of interest can be specifically targeted by inducing its irreversible aggregation and thus its functional knock-out.
In yet another embodiment the invention encompasses still additional methods for reducing or eliminating the activity of the histonllike polypeptide. Examples of other methods for altering or mutating a genomic nucleotide sequence in a plant are known in the art and include, but are not limited to, the use of RNA:DNA vectors, RNA:DNA mutational vectors, RNA:DNA repair vectors, mixed-duplex oligonucleotides, self-complementary RNA:DNA oligonucleotides and recombinogenic oligonucleotide bases. Such vectors and methods of use are known in the art. See, for example, US5565350; US5731181; US5756325; US5760012; US5795972 and US5871984, each of which are herein incorporated by reference.
The term "expression" or "gene expression" means the transcription of a specific gene or specific genes or specific genetic construct. The term "expression" or "gene expression" in particular means the transcription of a gene or genes or genetic construct into structural RNA (rRNA, tRNA) or mRNA with or without subsequent translation of the latter into a protein. The process includes transcription of DNA and processing of the resulting mRNA product.
The term "introduction" or "transformation" as referred to herein encompass the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer. Plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a genetic construct of the present invention and a whole plant regenerated there from. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed. Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, mega-gametophytes, callus tissue, existing meristematic tissue (e.g., apical meristem, axillary buds, and root meristems), and induced meristem tissue (e.g., cotyledon meristem and hypocotyl meristem). The polynucleotide may be transiently or stably introduced into a
host cell and may be maintained non-integrated, for example, as a plasmid. Alternatively, it may be integrated into the host genome. The resulting transformed plant cell may then be used to regenerate a transformed plant in a manner known to persons skilled in the art.
The transfer of foreign genes into the genome of a plant is called transformation. Transformation of plant species is now a fairly routine technique. Advantageously, any of several transformation methods may be used to introduce the gene of interest into a suitable ancestor cell. The methods described for the transformation and regeneration of plants from plant tissues or plant cells may be utilized for transient or for stable transformation. Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant, particle gun bombardment, transformation using viruses or pollen and microprojection. Methods may be selected from the calcium/polyethylene glycol method for protoplasts (Krens, F.A. et al., (1982) Nature 296, 72-74; Negrutiu I et al. (1987) Plant Mol Biol 8: 363- 373); electroporation of protoplasts (Shillito R.D. et al. (1985) Bio/Technol 3, 1099-1 102); microinjection into plant material (Crossway A et al., (1986) Mol. Gen Genet 202: 179-185); DNA or RNA-coated particle bombardment (Klein TM et al., (1987) Nature 327: 70) infection with (non-integrative) viruses and the like. Transgenic plants, including transgenic crop plants, are preferably produced via Agrobacterium-mediated transformation. An advantageous transformation method is the transformation in planta. To this end, it is possible, for example, to allow the agrobacteria to act on plant seeds or to inoculate the plant meristem with agrobacteria. It has proved particularly expedient in accordance with the invention to allow a suspension of transformed agrobacteria to act on the intact plant or at least on the flower primordia. The plant is subsequently grown on until the seeds of the treated plant are obtained (Clough and Bent, Plant J. (1998) 16, 735-743). Methods for Agrobacterium-mediated transformation of rice include well known methods for rice transformation, such as those described in any of the following: European patent application EP1198985, Aldemita and Hodges (Planta 199: 612-617, 1996); Chan et al. (Plant Mol Biol 22 (3): 491 - 506, 1993), Hiei et al. (Plant J 6 (2): 271 -282, 1994), which disclosures are incorporated by reference herein as if fully set forth. In the case of corn transformation, the preferred method is as described in either Ishida et al. (Nat. Biotech. 14(6): 745-50, 1996) or Frame et al. (Plant Physiol 129(1): 13-22, 2002), which disclosures are incorporated by reference herein as if fully set forth. Said methods are further described by way of example in B. Jenes et al., Techniques for Gene Transfer, in: Transgenic Plants, Vol. 1 , Engineering and Utilization, eds. S.D. Kung and R. Wu, Academic Press (1993) 128-143 and in Potrykus Annu. Rev. Plant Physiol. Plant Mol. Biol. 42 (1991) 205-225). The nucleic acids or the construct to be expressed is preferably cloned into a vector, which is suitable for transforming Agrobacterium tumefaciens, for example pBinl9 (Bevan et al (1984) Nucl. Acids Res. 12-8711). Agrobacteria transformed
by such a vector can then be used in known manner for the transformation of plants, such as plants used as a model, like Arabidopsis or crop plants such as, by way of example, tobacco plants, for example by immersing bruised leaves or chopped leaves in an agrobacterial solution and then culturing them in suitable media. The transformation of plants by means of Agrobacterium tumefaciens is described, for example, by Hofgen and Willmitzer in Nucl. Acid Res. (1988) 16, 9877 or is known inter alia from F.F. White, Vectors for Gene Transfer in Higher Plants; in Transgenic Plants, Vol. 1 , Engineering and Utilization, eds. S.D. Kung and R. Wu, Academic Press, 1993, pp. 15-38.
In addition to the transformation of somatic cells, which then have to be regenerated into intact plants, it is also possible to transform the cells of plant meristems and in particular those cells which develop into gametes. In this case, the transformed gametes follow the natural plant development, giving rise to transgenic plants. Thus, for example, seeds of Arabidopsis are treated with agrobacteria and seeds are obtained from the developing plants of which a certain proportion is transformed and thus transgenic [Feldman, KA and Marks MD (1987). Mol Gen Genet 208:1 -9; Feldmann K (1992). In: C Koncz, N-H Chua and J Shell, eds, Methods in Arabidopsis Research. Word Scientific, Singapore, pp. 274-289], Alternative methods are based on the repeated removal of the inflorescences and incubation of the excision site in the center of the rosette with transformed agrobacteria, whereby transformed seeds can likewise be obtained at a later point in time (Chang (1994). Plant J. 5: 551 -558; Katavic (1994). Mol Gen Genet, 245: 363-370). However, an especially effective method is the vacuum infiltration method with its modifications such as the "floral dip" method. In the case of vacuum infiltration of Arabidopsis, intact plants under reduced pressure are treated with an agrobacterial suspension [Bechthold, N (1993). CR Acad Sci Paris Life Sci, 316: 1 194-1 199], while in the case of the "floral dip" method the developing floral tissue is incubated briefly with a surfactant-treated agrobacterial suspension [Clough, SJ and Bent AF (1998) The Plant J. 16, 735-743], A certain proportion of transgenic seeds are harvested in both cases, and these seeds can be distinguished from non-transgenic seeds by growing under the above-described selective conditions. In addition the stable transformation of plastids is of advantages because plastids are inherited maternally is most crops reducing or eliminating the risk of transgene flow through pollen. The transformation of the chloroplast genome is generally achieved by a process which has been schematically displayed in Klaus et al., 2004 [Nature Biotechnology 22 (2), 225-229], Briefly the sequences to be transformed are cloned together with a selectable marker gene between flanking sequences homologous to the chloroplast genome. These homologous flanking sequences direct site specific integration into the plastome. Plastidal transformation has been described for many different plant species and an overview is given in Bock (2001) Transgenic plastids in basic research and plant biotechnology. J Mol Biol. 2001 Sep 21; 312 (3):425-38 or Maliga, P (2003) Progress towards
commercialization of plastid transformation technology. Trends Biotechnol. 21, 20-28. Further biotechnological progress has recently been reported in form of marker free plastid transformants, which can be produced by a transient co-integrated maker gene (Klaus et al., 2004, Nature Biotechnology 22(2), 225-229).
The term "plant" as used herein encompasses whole plants, ancestors and progeny of the plants and plant parts, including seeds, shoots, stems, leaves, roots (including tubers), flowers, and tissues and organs, wherein each of the aforementioned comprise the gene/nucleic acid of interest. The term "plant" also encompasses plant cells, suspension cultures, callus tissue, embryos, meristematic regions, gametophytes, sporophytes, pollen and microspores, again wherein each of the aforementioned comprises the gene/nucleic acid of interest.
Plants that are particularly useful in the methods of the invention include in particular monocotyledonous and dicotyledonous plants including fodder or forage legumes, ornamental plants, food crops, trees or shrubs selected from the list comprising Acer spp., Actinidia spp., Abelmoschus spp., Agave sisalana, Agropyron spp., Agrostis stolonifera, Allium spp., Amaranthus spp., Ammophila arenaria, Ananas comosus, Annona spp., Apium graveolens, Arachis spp, Artocarpus spp., Asparagus officinalis, Avena spp. (e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida), Averrhoa carambola, Bambusa sp., Benincasa hispida, Bertholletia excelsea, Beta vulgaris, Brassica spp. (e.g. Brassica napus, Brassica rapa ssp. [canola, oilseed rape, turnip rape]), Cadaba farinosa, Camellia sinensis, Canna indica, Cannabis sativa, Capsicum spp., Carex elata, Carica papaya, Carissa macrocarpa, Carya spp., Carthamus tinctorius, Castanea spp., Ceiba pentandra, Cichorium endivia, Cinnamomum spp., Citrullus lanatus, Citrus spp., Cocos spp., Coffea spp., Colocasia esculenta, Cola spp., Corchorus sp., Coriandrum sativum, Corylus spp., Crataegus spp., Crocus sativus, Cucurbita spp., Cucumis spp., Cynara spp., Daucus carota, Desmodium spp., Dimocarpus longan, Dioscorea spp., Diospyros spp., Echinochloa spp., Elaeis (e.g. Elaeis guineensis, Elaeis oleifera), Eleusine coracana, Eragrostis tef, Erianthus sp., Eriobotrya japonica, Eucalyptus sp., Eugenia uniflora, Fagopyrum spp., Fagus spp., Festuca arundinacea, Ficus carica, Fortunella spp., Fragaria spp., Ginkgo biloba, Glycine spp. (e.g. Glycine max, Soja hispida or Soja max), Gossypium hirsutum, Helianthus spp. (e.g. Helianthus annuus), Hemerocallis fulva, Hibiscus spp., Hordeum spp. (e.g. Hordeum vulgare), Ipomoea batatas, Juglans spp., Lactuca sativa, Lathyrus spp., Lens culinaris, Linum usitatissimum, Litchi chinensis, Lotus spp., Luffa acutangula, Lupinus spp., Luzula sylvatica, Lycopersicon spp. (e.g. Lycopersicon esculentum, Lycopersicon lycopersicum, Lycopersicon pyriforme), Macrotyloma spp., Malus spp., Malpighia emarginata, Mammea americana, Mangifera indica, Manihot spp., Manilkara zapota, Medicago sativa, Melilotus spp., Mentha spp., Miscanthus sinensis, Momordica spp., Morus nigra, Musa spp., Nicotiana spp., Olea spp., Opuntia spp., Ornithopus
spp., Oryza spp. (e.g. Oryza sativa, Oryza latifolia), Panicum miliaceum, Panicum virgatum, Passiflora edulis, Pastinaca sativa, Pennisetum sp., Persea spp., Petroselinum crispum, Phalaris arundinacea, Phaseolus spp., Phleum pratense, Phoenix spp., Phragmites australis, Physalis spp., Pinus spp., Pistacia vera, Pisum spp., Poa spp., Populus spp., Prosopis spp., Prunus spp., Psidium spp., Punica granatum, Pyrus communis, Quercus spp., Raphanus sativus, Rheum rhabarbarum, Ribes spp., Ricinus communis, Rubus spp., Saccharum spp., Salix sp., Sambucus spp., Secale cereale, Sesamum spp., Sinapis sp., Solanum spp. (e.g. Solanum tuberosum, Solanum integrifolium or Solanum lycopersicum), Sorghum bicolor, Spinacia spp., Syzygium spp., Tagetes spp., Tamarindus indica, Theobroma cacao, Trifolium spp., Tripsacum dactyloides, Triticosecale rimpaui, Triticum spp. (e.g. Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum, Triticum monococcum or Triticum vulgare), Tropaeolum minus, Tropaeolum majus, Vaccinium spp., Vicia spp., Vigna spp., Viola odorata, Vitis spp., Zea mays, Zizania palustris, Ziziphus spp., amongst others.
In some embodiments, the plant cell according to the invention is non-propagating or cannot be regenerated into a plant.
All publications and patent applications in this specification are indicative of the level of ordinary skill in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated by reference.
Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.
Examples l.Generation of HISTONE1-LIKE CRISPR-Cas9 mutants
To investigate the role of HISTONE1-LIKE (H1L) under drought conditions, knock-out mutants were generated in the B104 inbred line using the CRISPR-Cas9 system. The polynucleotide sequence of the corn H1L gene is depicted in SEQ ID NO: 21 and this gene was knocked out. SEQ ID NO: 21 encodes for the polypeptide depicted in SEQ ID NO: 1. Two independent constructs were made, each containing two
guide RNAs (gRNAs) that specifically target the linker histone domain of the H1L gene and have no off targets in other (linker histone) genes (see Figure 3). Several lines per construct were obtained in the TO generation. TO plants were backcrossed with WT B104 plants and selfed in the T1 generation to obtain a segregating T2 generation that is free of T-DNA. guide RNA sequences binding on the H1L polynucleotide (SEQ ID NO: 21) encoding SEQ ID NO: 1 are depicted in SEQ ID NO: 22-25
HlL_gRNA_l = AGCAGGAAGCCCAAGTCCGC (SEQ ID NO: 22)
HlL_gRNA_2 = GCCCAATTACCGCAAGGTGC (SEQ ID NO: 23)
HlL_gRNA_3 = CGATCCTGTCGCAGGACGGC (SEQ ID NO: 24)
HlL_gRNA_4 = GGCCAGCACCTTGCGGTAAT (SEQ ID NO: 25)
2.Histonel-like CRISPR mutants perform better under drought stress
To study the role of H1L in growth repression under drought, a mild drought stress experiment was set up using the automatic watering WIWAM-line. Plant growth was studied by daily measurements of leaf 4 of a segregating hll CRISPR T2 population under well-watered (WW) and mild drought (water deficit, WD) conditions. Two independent lines were chosen for this experiment, line Da (construct 1) and line C (construct 2). Both lines have a fragment deletion between the gRNAs, a -266 bp deletion for line Da and a -79 bp deletion for line C. Under mild drought, hll plants have a higher LER and a lower LED compared to WT plants (see Figure 4). Although no significant differences were found for final leaf 4 length, hll plants tend to have larger leaves compared to their segregating WT siblings under drought stress. Even more, hll plants under drought have one to two leaves more compared to WT resulting in a lower yield penalty and a more vigorous growth under water deficit conditions. No significant differences were found between hll and WT seedlings under well-watered conditions.
3.The HISTONE1- LIKE subclade can be recognized as a distinct group of linker histones in monocots
In the leaf 4 division zone, H1L is upregulated under drought and downregulated upon re-watering. Under drought, hll CRISPR mutants show a more vigorous growth compared to their WT siblings. In Arabidopsis (AtH1.3, AT2G18050 - polypeptide sequence depicted in SEQ ID NO: 3) and tomato (SIH1-S, Solyc02g084240.3), Hl variants that belong to the same subclade of H1L, have been shown to be induced upon drought stress (Rutowicz et al., 2015; Scippa et al., 2004). In maize, H1L has one close homolog, namely HISTONE1 (Hl) (Figure 5). Although Hl and H1L are closely related, Hl is downregulated under drought and upregulated upon rewatering, suggesting an opposite role for Hl compared to H1L in response to drought (see Figure 6).
To further investigate the opposite responses of Hl and H1L under drought and the divergence of Hl variants in monocots, protein sequences of rice, wheat and maize in the Hl-subclade and the H1L- subclade were aligned, together with AtH1.3 (SEQ. ID NO: 3) (Arabidopsis) and SIH1-S (tomato) and other dicot orthologs in tobacco (Nitab), soya bean (Glyma) and cotton (Gohir) (see Figure 7). Protein alignment of the dicot orthologs and the proteins belonging to the Hl and H1L subclade, reveal a high degree of conservation of the linker domain and a high variability in the N- and C-terminal domains. Linker histones typically have a tripartite structure which is composed of a conserved central globular domain flanked by a highly variable short N-terminal domain and a longer highly basic C-terminal domain. The N- and C-terminal domains are prone to post-translational modifications which can cause alterations in chromatin condensation, resulting in differential gene expression (Gibbs and Kriwacki, 2018). Most linker histones are known to have a N-terminal domain ranging from 20 to 35 amino acids (AA) (Vyas and Brown, 2012). Interestingly, the N-terminal domain of the proteins belonging to the H1L subclade, have an extremely long N-terminal domain, ranging from 67 to 113 AA (counted until the start of the linker histone domain). In contrast, the longest N-terminal domain in the Hl subclade consists of 36 AA (TraesCS2A03G0957600), suggesting that the heterogeneity in drought response among subclades/variants may arise because of the variability in their terminal domains. In dicots, Hl variants can be divided into two groups according to the length of the N-terminal domain, the first group has a short N- terminal domain consisting of 8 to 9 AA, the second group has a longer N-domain ranging from 25 to 47 AA. Visualizing the amino acids of the N-terminal domain according to polarity and charge, could predict which homologs might be similarly regulated post-translational since polarity and charge are of critical importance to the structure and function of proteins (see Figure 8) (Zhou and Pang, 2018). Monocot proteins belonging to the Hl subclade have a basic N-terminal domain containing no acidic amino acid residues. In contrast, dicot proteins and proteins belonging to the H1L subclade have a mix of acidic and basic AA residues, most of them being acidic.
Blasting the protein sequence of AtH1.3 at NCBI, yields Hl as a first hit in Zea mays (see Figure 9). If we blast the sequence of H1L and Hl in the species described above in the protein alignment (see figure 9), the ranking of the monocot proteins is identical to their divergence into H1L and Hl subclades. For example, blasting H1L gives Sobic.010G021300, TraesCS7A03G0150000 and 0s06g0130800 as best hits in Sorghum, wheat and rice, respectively (see Figure 10). For Hl, the best hits in Sorghum, wheat and rice are Sobic.006G186700, TraesCS2A03G0956900 and 0s04g0253000 (see Figure 11). In dicots, the divergence in Hl and H1L initially was more difficult. Based on the blast results for H1L, Solyc02g084240.2, Gohir.AllG176800, Nitab4.5_0002880g0020 and Nitab4.5_0005721g0020 give higher hits than the first Hl ortholog we encounter in the table, namely 0s04g0253000. Although these genes are also present in the blast results for Hl.
Based on best blast hits, proteins from the H1L subclade were aligned to search for conserved amino acid residues, using ClustalO (Figure 12). For monocot species belonging to the H1L subclade, conserved residues were found in the N- and C-terminal domain of the proteins. In the N-terminal domain, there is conservation of a KPR-motif, which presumably functions as a nuclear localization signal. Next to this motif there is a conserved RKP(K/R)SAG (SEQ ID NO: 18) motif which, according to a protein domain search using the SMART database, refers to an AT-hook motif that allows the protein to bind to AT rich sequences in the genome. In the C-terminal domain there is conservation of a (S/A)EE(K/R)K (SEQ ID NO: 19) and a (A/V)RxKRA(R/K)(R/K) (SEQ ID NO: 20) motif for which no function was found searching multiple databases. Orthologous protein sequences of corn H1L of different monocotyledonous plants are depicted in SEQ I D NO: 2, 4, 5, 6, 7, 8 and 9. Alignment of monocot species belonging to the H1L subclade with the dicot proteins from tomato (Solyc02g084240.2), tobacco (Nitab4.5_0002880g0020, Nitab4.5_0005721g0020) and cotton (Gohir.AllG176800) that were found in the H1L blast, shows conserved substitutions in the RKP(K/R)SAG motif, in the N-terminal domain (see Figure 13). In tomato and tobacco, the RKP(K/R)SAG motif changes into a (K/R)KP(K/R)SA motif by conserved substitution of residues. This conserved motif is not found in cotton, Arabidopsis thaliana and Glycine max, nor in proteins belonging to the monocot Hl subclade. The (A/V)RxKRA(R/K)(R/K) motif shows a low degree of conservation whereas the (S/A)EE(K/R)K motif is conserved as a AGKKE motif by amino acid substitutions in tomato (Solyc02g084240.2), tobacco (Nitab4.5_0002880g0020, Nitab4.5_0005721g0020) and cotton (Gohir.AllG176800, Gohir.DllG184700). The AGKKE and the (S/A)EE(K/R)K motif were not found in monocot proteins belonging to the Hl subclade, nor in Arabidopsis thaliana, Glycine max and cotton (with the exception of Gohir.AllG176800, Gohir.DllG184700). Dicotyledonous orthologous protein sequences of corn H1L (SEQ ID NO: 1) are depicted in SEQ ID NO: 10-17.
Sequences
SEQ ID NO: 1: Zea mays polypeptide sequence of ZmHlL
MATVTEEVAPVVTVSEEPAPEAAKEVVEKPEEGKKPDEEGDERKKADPAAEKEKKADPAAEKEKKARKPRSRKPKSAG LHHPPYFEMIKEAILSQDGGKVGASPYAIAKHMGEKHRDVLPPNYRKVLAVQLRGFAAKGRLVKVKASFKLAAAEERK PSAAAKAKKKASAGMAKRKRAAAPAKMKPAAAASAPSREARKVRAKRARKVAPAPAQPKPKPARAAASGAGKKVN KASA
SEQ ID NO: 2 : Zea mays polypeptide sequence of ZmHIL (orthologue of SEQ ID NO: 1)
MATVTEVAPVVTVSEEPAPEAAKEVVEKPEEGKKPDEEGDERKKADPAAEKEKKADPAAEKEKKARKPRSRKPKSAGL HHPPYFEVSKDAVVVVVVLSVTHPCLDSHSDDRSLCLLGTWGDRGQMIKEAILSQDGGKVGASPYAIAKHMGEKHR
DVLPPNYRKVLAVQLRGFAAKGRLVKVKASFKLAAAEERKPSAAAKAKKKASAGMAKRKRAAAPAKMKPAAAASAP SREARKVRAKRARKVAPAPAQPKPKPARAAASGAGKKVNKASA
SEQ ID NO: 3 (Arabidopsis thaliana Hl.3 polypeptide, encoded from gene AT2G18050)
MAEDKILKKTPAAKKPRKPKTTTHPPYFQMIKEALMVLKEKNGSSPYAIAKKIEEKHKSLLPESFRKTLSLQLKNSVAKGK
LVKIRASYKLSDTTKM ITRQQDKKNKKNMKQEDKEITKRTRSSSTRPKKTVSVNKQEKKRKVKKARQPKSIKSSVGKKK AMKASAA
SEQ ID NO: 4 (Sorghum bicolor, H1L orthologue of SEQ ID NO: 1, NCBI accession: OQU75744)
MATVTEEVAPAVAQEPAPEEAKGVMETPEKVEEGKKPEEGVEGKKAAAEKEKKARKPRSRKPKSAGPHHPPYFEM IK
EAILSQDVGKVGASPYAIAKHIGEKHRDVLPPNYRKVLAVQLRGFAAKGRLVKVKASFKLAASEEKKAAAASAAAKTKK
TPPAKRKRAAAPAKKKPAVAAAAPPREARKARAKRARKVAPAPVQPKPKAARAAAGGGGKKANKASA
SEQ ID NO: 5 (Oryza sativa, orthologue of SEQ ID NO: 1, NCBI accession: XP_015644353)
MATATEEVAAAAAAAGEAPPPPPPAVVEEAKEALEAPKPEEAPKAEEGEEKKAEGEKEKEKAKKERKPRARKPRSAGP
HHPPYFEM IKEAIMALDGNGKAGSSPYAIAKYMGEQHMGVLPANYRKVLAVQLRNFAAKGRLVKVKASFKLSAAEE
KKATAAKAARSKAAKGVVGGAKRKRTPRPSAAAAKKPASSAEAKKAVPPARPARAKRARKAAPAKPMQPPKSIRSAI SKKANKASA
SEQ ID NO: 6 (Triticum aestivum, orthologue of SEQ ID NO: 1, NCBI accession: KAF7093214)
MATVMEEAGAVVMGGTGEEEVVAAPEKVEEVKEAGAGGVDVEVAGGEAKKAEEEQGEQGKGTEKKPRRRKPRSA
GPHHPPYFEMIKEAIMAAGDGKAGASAYAIAIAKRVGERHGEALPGNYRKVLATQLRGFAAKGRLVQVKASFRLAPA EEKKALQAATPKSKKRTTTAKKTASKNVAPAPARAKRAKKANKASA
SEQ ID NO: 7 (Triticum aestivum, orthologue of SEQ ID NO: 1, NCBI accession: KAF7093216)
MATLTEEVAAAAVVGAGEKAEAVATPEKVDEVKEAGAGGEEMEVAGGEAKKAEEEQGEQGKETEKKPRSRKPRSA
GPHHPPYFEMIKEAIMAAGDGKAGASAYAIAKRVGERHGEALPGNYRKVLGAQLRGFAAKGRLVRVKASFRLAPAEE KKAAPKSKKRTATTKKAASKKAAPAPARQKRAKKAGPPTAKPKPKQPKSIRGRKANKASA
SEQ ID NO: 8 (Triticum aestivum, orthologue of SEQ ID NO: 1, NCBI accession: KAF7045985)
MATVMEEAAAKAVVGAGEKAEAVATPEKVDEVKQAGAGGEEMEVAGGEAKKAEEGGEVKKAEGEEKKPRSRKPR
SAGPHHPPYFEM IKEAIMAAGDGQAGASAYAIAKRVGERHGEALPGNYRKVLAAQLRSFAAKGRLVRVKASFRLAPA EEKKALPAKKRTTKKAASKKARPKRAKKAGPPPAKPKPKQPKSIRARKANKASA
SEQ ID NO: 9 (Triticum aestivum, orthologue of SEQ ID NO: 1, NCBI accession: XP_044442749)
MATVMAAAAPAMVGAGEEVKEAVAAPEKVEEVKEAVAAPEKVEEVKEAGAGEEVMEVAAGEAKEAGAGEEAME
VAGGEAKKAEEGGEVKKTEGGQGKGEEKKPRSRKPRSAGPHHPPYFEMIKEAIMAAGDGKAGASAYAIAKRVGERH
GEALPGNYRKVLAAQLRSFAAKGRLVRVKASFRLAPAEEKKALPAKKTTTNKAASKKARPKRAKRAGPPPAKPKPKQP KSIRARKANKASA
SEQ ID NO: 10 (Solanum lycopersicum, orthologue of SEQ ID NO: 1, NCBI accession: NP_001234389)
MTAIGEVENPAVVQRPTEASKVKEQASATEKAVKEKKPRAPKEKKPKSAKAVTHPPYFQM IKEALLSLNEKGGSSPYA
VAKYMEDKHKDELPANFRKILGLQLKNSAAKGKLIKIKASYKLSEAGKKETTTKTSTKKLPKADSKKKPRSTRATSTAAKK
TEVPKKAKATPKPKKVGAKRTRKSTPAKAKQPKSIKSPAAKRAKKIAV
SEQ ID NO: 11 (Gossypium hirsutum, orthologue of SEQ ID NO: 1, NCBI accession: XP_040937395)
MATAEPEVPVTEQQPAAAEEPKPAEKPVKEKRPRAPKEKKPKQPKSAAHPPYFQMIKEALLALKEKSGSSPYAIAKYM
EEKHKAVLPANFRKILGLQLKNSAARGKLIKIKASYKLSEAGKKERAPVTKAKM EKKAKPASKPKKAEATKKPTKRVGAK KKSTPAKPKQPKSIKSPAAKKAKKAAA
SEQ ID NO: 12 (Nicotiana tabacum, orthologue of SEQ ID NO: 1, NCBI accession: AAN37904)
MSATGKVESSAVEQPPAKAPKAEDQPPATKKSVKEKKPRAPREKKPKSAKTVTHPPYFQM IKEALLALNEKGGSSPYAI
AKYMEDKHKDELPANFRKILGLQLKNSAAKGKLMKIKASYKLSVAGKKERTTASTKKVPKADTKKKPRSTRSTTATAKK TEVAKKAKPTQKPKKVGAKKIRKSTPAKAKQPKSIKSPAAKRAKKVAA
SEQ ID NO: 13 (Glycine max, orthologue of SEQ ID NO: 1, NCBI accession: XP_003537627)
MSAAEEAKVPAVEKPVEEVKAPKLAKEKKPKAPKEKKPKQAKTASHPPYLQMIKDALIALNEKGGSSPYAIAKYM EEK
HKAVLPANFKKILGLQLKNQAARGKLVKIKASYKLAEAAKKVKESTAKATKESRPKRNKIATAVAPKKTEAVKKPAKKV GPKKTKKVSTPAKPKQPRSIRSPTKRARKAAVAAA
SEQ ID NO: 14 (Glycine max, orthologue of SEQ ID NO: 1, NCBI accession: KAG5089416)
MSAAAEEANAPAVEKPVEEVKAPNPAKEKKPKAPKEKKPKQAKTASHPPYFQM IKEALIALNEKGGSSPYAIAKYMEE
KHKAVLPANFKKILGLQLKNQAARGKLVKIKASYKLTEAAKKENTAKVTKANAEKKESRPKRSKTATAAAPKKTEAVKR
AAKKVVPKKTKKVSTPAKPKQPKSIRSPTKRARKAAVAAA
SEQ ID NO: 15 (Glycine max, orthologue of SEQ ID NO: 1, NCBI accession: NP_001237870)
MSAAAEEANAPAVEKPVEEVKAPNPAKEKKPKAPKEKKPKQAKTASHPPYFQM IKEALIALNEKGGSSPYAIAKYMEE
KHKAVLPANFKKILGLQLKNQAARGKLVKIKASYKLTEAAKKENTAKVTKANAEKKESRPKRSKTATAAAPKKTEAVKR
AAKKVGPKKTKKVSTPAKPKQPKSIRSPTKRARKAAVAAA
SEQ ID NO: 16 (Glycine max, orthologue of SEQ ID NO: 1, NCBI accession: KAG5069708)
MSAAAEEANAPAVEKPVEEVKAPKPAKEKKPKAPKEKKPKQAKTASHPPYFQM IKEALIALNEKGGSSPYAIAKYMEE
KHKAVLPANFKKILGLQLKNQAARGKLVKIKASYKLTEAAKKENTAKVTKANAEKKESRPKRSKAATAAAPKKTEAVKR
AAKKVGPKKTKKVSTPAKPKQPKSIRSPTKRARKAAVAAA
SEQ ID NO: 17 (Glycine max, orthologue of SEQ ID NO: 1, NCBI accession: ACU15107)
MSAAAEEANAPAVEKPVEEVKAPNPAKEKKPKAPKEKKPKQAKTASHPPYFQM IKEALIALNEKGGSSPYAIAKYMEE
KHKAVLPANFKKILGLQLKNQAARGKLVKIKASYKLTEAAKKENTAKVTKATAEKKESRPKRSKTATAAAPKKTEAVKR
AAKKVGPKKTKKVSTPAKPKQPKSISLQPRGPGKQPLLRLDTCLV
SEQ ID NO: 21: polynucleotide sequence encoding SEQ ID NO: 1
ZmHl-like (Zea mays)
Variety B104 - gene name Zm00007a00000971
ATGGCAACCGTAACTGAGGAAGTTGCTCCGGTCGTCACCGTCTCCGAGGAGCCGGCGCCGGAAGCGGCGAAGG
AGGTGGTGGAGAAGCCGGAGGAGGGGAAGAAGCCCGACGAGGAGGGTGACGAGAGGAAGAAGGCCGACCC
CGCCGCCGAGAAGGAGAAGAAGGCCGACCCCGCCGCCGAGAAGGAGAAGAAGGCGAGGAAGCCGCGGAGCA
GGAAGCCCAAGTCCGCCGGGCTGCACCACCCGCCCTATTTCGAGGTGAGCAAAGATGCCGTCGTCGTCGTCGTC
GTCTTGTCGGTTACTCATCCGTGCTTGGATTCTCATTCTGATGATCGGTCGCTGTGTCTGCTGGGGACCTGGGGT
GATCGAGGTCAGATGATCAAGGAGGCGATCCTGTCGCAGGACGGCGGGAAGGTCGGGGCGAGCCCGTACGCG
ATCGCCAAGCACATGGGGGAGAAGCACCGGGACGTGCTCCCGCCCAATTACCGCAAGGTGCTGGCCGTGCAGC
TGCGCGGCTTCGCCGCCAAGGGCCGCCTCGTCAAGGTCAAGGCCTCCTTCAAGCTCGCCGCCGCCGAGGAGAGG
AAGCCCTCCGCGGCCGCGAAGGCGAAGAAGAAGGCGTCGGCGGGAATGGCCAAGCGCAAGAGGGCCGCCGCG
CCGGCCAAGATGAAGCCGGCGGCCGCTGCCTCGGCGCCGTCGAGGGAGGCACGTAAGGTGCGCGCGAAGCGA
GCGAGGAAGGTGGCCCCGGCGCCCGCGCAGCCCAAGCCCAAGCCAGCCCGCGCCGCCGCTTCCGGTGCCGGCA
AGAAGGTCAACAAGGCCAGCGCCTGA
References
Baute, J., Herman, D., Coppens, F., De Block, J., Slabbinck, B., Dell'Acqua, M., ... & Inze, D. (2015). Correlation analysis of the transcriptome of growing leaves with mature leaf parameters in a maize RIL population. Genome biology, 16(1), 1-26.
Gibbs, E. B., & Kriwacki, R. W. (2018). Linker histones as liquid-like glue for chromatin. Proceedings of the National Academy of Sciences, 115(A7), 11868-11870.
Nelissen, H., Sun, X. H., Rymen, B., Jikumaru, Y., Kojima, M., Takebayashi, Y., ... & Inze, D. (2018). The reduction in maize leaf growth under mild drought affects the transition between cell division and cell expansion and cannot be restored by elevated gibberellic acid levels. Plant biotechnology journal, 16(2), 615-627.
Przewloka, M. R., Wierzbicki, A. T., Slusarczyk, J., Kuras, M., Grasser, K. D., Stemmer, C., & Jerzmanowski, A. (2002). The" drought-inducible" histone His of tobacco play no role in male sterility linked to alterations in Hl variants. Planta, 215(3), 371-379.
Rutowicz, K., Puzio, M., Halibart-Puzio, J., Lirski, M., Kotliriski, M., Kroteri, M. A., ... & Jerzmanowski, A. (2015). A specialized histone Hl variant is required for adaptive responses to complex abiotic stress and related DNA methylation in Arabidopsis. Plant Physiology, 169(3), 2080-2101.
Scippa, G. S., Di Michele, M., Onelli, E., Patrignani, G., Chiatante, D., & Bray, E. A. (2004). The histone-like protein Hl-S and the response of tomato leaves to water deficit. Journal of Experimental Botany, 55(394), 99-109.
Trivedi, I., Ranjan, A., Sharma, Y. K., & Sawant, S. (2012). The histone Hl variant accumulates in response to water stress in the drought tolerant genotype of Gossypium herbaceum L. The protein journal, 31(6), 477-486.
Vyas, P., & Brown, D. T. (2012). N-and C-terminal domains determine differential nucleosomal binding geometry and affinity of linker histone isotypes H10 and Hlc. Journal of Biological Chemistry, 287(15), 11778-11787.
Zhou, H. X., & Pang, X. (2018). Electrostatic interactions in protein structure, folding, binding, and condensation. Chemical reviews, 118(4), 1691-1741.
Claims
1. A plant having a gene disruption in a polynucleotide encoding for SEQ ID NO: 1 or having a gene disruption in a polynucleotide encoding a plant orthologous polypeptide sequence of SEQ ID NO: 1.
2. A plant according to claim 1 which is a cultivated crop plant.
3. A plant according to claims 1 or 2 wherein the plant orthologous polypeptide sequence comprises SEQ ID NO: 18, 19 and 20.
4. A plant according to any one of claims 1 to 3 wherein the plant orthologous polypeptide sequence is depicted in SEQ ID NO: 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17.
5. A seed or plant cell derived from a plant according to any one of claims 1 to 4.
6. A method for increasing tolerance to abiotic stress in a plant, the method comprising: disrupting the expression of a polynucleotide in the plant encoding SEQ ID NO: 1 or disrupting the expression of a polynucleotide encoding a plant orthologous sequence of SEQ ID NO: 1.
7. The method according to claim 6, wherein the polynucleotide encodes a polypeptide sequence comprising SEQ ID NO: 18, 19 and 20.
8. The method according to claims 6 or 7 wherein the polynucleotide encodes a polypeptide sequence depicted in SEQ ID NO: 1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or 17.
22
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