WO2023131637A1 - Improved silage grasses - Google Patents

Abstract

The present invention relates to silage plants having a decreased combined expression of the CCR1 and CCR3 genes which results in a silage plant having an increased digestibility. The invention also provides methods which can be used to decrease the CCR1 and CCR3 gene expression.

Description

IMPROVED SILAGE GRASSES

Field of the invention

The present invention belongs to the field of agricultural biology. In particular the present invention relates to improved silage crops which have a gene disruption in CCR1 and CCR3 genes which results in silage crops with an improved digestibility.

Introduction

Silage is pasture grass that has been 'pickled'. It is a method used to preserve the pasture for cows and sheep to eat later when natural pasture isn't good, like in the dry season. The fermentation and storage process is called ensilage, ensiling or silaging. The grasses are cut and then fermented to keep as much of the nutrients (such as sugars and proteins) as possible. The fermentation process must be carried out under acidic conditions (around pH 4-5) in order to keep nutrients and provide a form of food that cows and sheep will like to eat. Fermentation at higher pH results in silage that has a bad taste, and lower amounts of sugars and proteins. The crops most often used for ensilage are the ordinary grasses, clovers, alfalfa, vetches, oats, rye and maize. Fiber digestibility has been a key characteristic of quality silage grasses. It is generally thought that silage higher in fiber digestibility will produce more milk. Maize is one of the most important fodder crops. Genetically engineering low-lignin plants would improve the digestibility of maize and increase its sugar yield. One of the main factors that limits digestibility is the presence of lignin in the biomass (Barriere et al., 2016; Barriere, 2017). Lignin is an aromatic heteropolymer made of a variety of phenolic monomers (Mottiar et al., 2016; Vanholme et al., 2019). After their biosynthesis, these are translocated over the plasma membrane to the cell wall, where they are oxidized to radicals. These radicals couple in a combinatorial fashion, resulting in the deposition of a polymer that strengthens the wall, and at the same time protects the carbohydrates from digestion by insects and microbes. For this reason, also the digestibility of fodder by the digesting microbes in the cattle digestive system is hindered by the presence of lignin. Consequently, lowering lignin or changing its composition can have an important beneficial effect on cell-wall digestibility. Thanks to our current insight into the core biosynthetic pathway of the lignin building blocks, metabolic engineering to steer the biosynthesis of the monolignols or the structure of the polymer itself have become possible (Mottiar et al., 2016; Ralph et al., 2019). Several low-lignin mutants with improved digestibility have been described in maize, among which the well-known brown midrib mutants (bm mutants; Ali et al, 2010; Halpin et al., 1998; Tang et al., 2014; Vignols et al., 1995). Unfortunately, the mutants suffer from lodging and have a yield drag. The availability of genomic information and efficient gene-editing tools for maize must make it possible to optimize lignin amount to improve digestibility, while avoiding the lodging and yield penalty. We hypothesize that the negative consequences of the low lignin maize varieties can be overcome by choosing the right genes or by fine-tuning the lignin reduction to specific cells or developmental stages.

CINNAMOYL CoA-REDUCTASE (CCR) catalyzes an essential metabolic conversion in the biosynthesis of lignin monomers (Goffner et al., 1994; Vanholme et al., 2019). It has been shown before that a Mutator insertion in the first intron of the CCR1 gene in maize resulted in a 31% reduction in CCR1 expression, a 12% reduction in Klason lignin content and an 18% improved digestibility, without biomass yield penalty when grown in the greenhouse (Tamashloukht et al., 2011). This mutant still had significant residual expression and is not a complete KO, likely because the Mutator transposon is present in the intron and can be spliced out. The mutant also did not display a brown midrib phenotype. CCR1 has also been downregulated in maize using an RNAi strategy (Park et al., 2012). This resulted in plants with 7-8% reduction in Klason lignin content and a 7-8% increased conversion to fermentable sugars upon ammonia fiber explosion. Six out of the twenty generated RNAi lines had a brown midrib phenotype and these had a normal biomass yield (Park et al., 2012). A third study examined the lignin and saccharification of a maize line with a Mutator insertion in the 4th exon of CCR1 (Smith et al., 2017). This maize line had about 20% lower lignin amount and up to 53% increased digestibility in a limited-extend digestibility test. These maize plants did not have a brown midrib phenotype. When grown in the field, this mutant had a 16% reduction in seed weight but an 8% increase in total biomass yield. Taken together, these data indicate that the downregulation and mutation of CCR1 in maize can result in large improvements in digestibility without yield penalty.

In the present invention we investigated whether the digestibility of maize can be further improved via stacking of CCR1 with a mutation in CCR3. The protein sequences of both CCR1 and CCR3 have a high similarity to CCR enzymes described to be involved in lignification in other plant species (AtCCRl, PotriCCR2, LpCCRl, Figure). CCR1 is expressed in all organs examined and has the highest expression levels in leaves. CCR3 is expressed in many organs, and has the highest expression in leaves and roots. The function of CCR3 has not yet been described, prohibiting its valorization in improving digestibility. To investigate the potential role of the CCR3 gene in lignin biosynthesis, we generated ccrl and ccr3 single mutants and ccrl ccr3 double mutants via CRISPR/Cas9 technology. Surprisingly this double mutant combination leads to a 7% reduction of lignin in the leaves in contrast to the single mutants which do not display a reduction of lignin in the leaves. Figure legends

Figure 1: Phylogenetic tree of the CCR proteins in different species. CCR1 in maize is known to be involved in lignification and CCR3 is its closest paralog. Both CCR enzymes have the conserved NWYCY amino acid sequence and an NADPH binding motif (Lacombe et al., 1997; Park et al., 2017; van Parijs et al., 2012). The maize CCR paralogs are clustered according to their amino-acid sequence similarity and are indicated in red. As comparison, known CCR orthologs are added: Arabidopsis thaliana CCR1, AtCCRl; Populus trichocarpa CCR2, PotriCCR2; Lolium perenne CCR1, LpCCRl.

Figure 2: Representation of the CCR1 and CCR3 gene. The grey rectangles represent the exons, the black triangles indicate the target of the gRNAs and the arrows indicate the location of the primers.

Figure 3: Phenotype of the ccr mutants. (A) Phenotype of the ccrl single and ccr3 single mutants and ccrl ccr3 double mutant next to WT. (B) Reddish-brown coloration of the stem in the ccrl mutants (left) compared to WT (right).

Figure 4: Graphical representation of the Klason and acid-soluble lignin (ASL) results of 10-week old ccrl single, ccr3 single and ccrl ccr3 double mutants. (A) Klason lignin in stem tissue. (B) Acid-soluble lignin fraction in stem tissue. (C) Klason lignin in leaf tissue (D) Acid-soluble lignin in leaf tissue. Biological replicates = 5; One-way ANOVA and Tukey post hoc test; ****p<0.0001, ***0.0001<p<0.001, **0.001<p< 0.01, *0.01<p< 0.05. Error bars indicate the standard deviation.

Figure 5: Graphical representation of the total lignin content of 15-week old ccrl single, ccr3 single and ccrl ccr3 double mutants. The total lignin content is determined the acid-insoluble (Klason) and acidsoluble lignin (ASL). (A) Total lignin content in stem tissue. (B) Total lignin content in leaf tissue. Biological replicates = 5; One-way ANOVA and Tukey post hoc test; ***0.0001<p<0.001, **0.001<p< 0.01, *0.01<p< 0.05. Error bars indicate the standard deviation.

Figure 6: Alignment of amino acid sequences of CCR1 and CCR3 with their conserved domains. The signature CCR sequence (NWYCYGK, SEQ ID NO: 23) and active site residues are indicated in grey; the NADP binding domain residues are indicated in yellow; the substrate binding pocket is indicated in green (Li etal., 2016). Figure 7: Graphical representation of the Klason and acid-soluble lignin (ASL) results of 15-week old ccrl single, ccr3 single and ccrl ccr3 double mutants. (A) Klason lignin in stem tissue. (B) Acid-soluble lignin fraction in stem tissue. (C) Klason lignin in leaf tissue (D) Acid-soluble lignin in leaf tissue. Biological replicates (WT: n = 13; ccrl: n = 13; ccr3 n = 15 and ccrl ccr3: n = 7); One-way ANOVA and Tukey post hoc test; ***0.0001<p<0.001, **0.001<p< 0.01, *0.01<p< 0.05. Error bars indicate the standard deviation.

Figure 8: Graphical representation of the total lignin content of 15-week old ccrl single, ccr3 single and ccrl ccr3 double mutants. The total lignin content is determined the acid-insoluble (Klason) and acidsoluble lignin (ASL). (A) Total lignin content in stem tissue. (B) Total lignin content in leaf tissue. Biological replicates (WT: n = 13; ccrl: n = 13; ccr3 n = 15 and ccrl ccr3: n = 7); One-way ANOVA and Tukey post hoc test; **0.001<p< 0.01, *0.01<p< 0.05. Error bars indicate the standard deviation.

Figure 9: Graphical representation of the enzymatic cellulose-to-glucose conversion of 10-week old greenhouse-grown ccrl single, ccr3 single and ccrl ccr3 double mutants. Released glucose expressed as the percentage of the total cellulose in the stem and leaf tissue after no pretreatment (A, D), acid pretreatment (B, E) and alkaline pretreatment (C, F). Significance levels indicated for the ccrl single (*) and the ccrl ccr3 double mutants (•) as compared to WT plants. Significance levels indicated for the ccrl ccr3 double mutants (★) as compared to the ccrl single mutants. Saccharification was ended after 48h. Biological replicates = 5; One-way ANOVA and Tukey post hoc test; **** and •••• p<0.0001, *** and • •• 0.0001<p<0.001, ** and •• 0.001<p< 0.01, *, • and ★ 0.01<p< 0.05. Error bars indicate the standard deviation.

Figure 10: Graphical representation of the enzymatic cellulose-to-glucose conversion of 15-week old greenhouse-grown ccrl single, ccr3 single and ccrl ccr3 double mutants. Released glucose expressed as the percentage of the total cellulose in the stem and leaf tissue after no pretreatment (A, D), acid pretreatment (B, E) and alkaline pretreatment (C, F). Significance levels indicated for the ccrl single (*) and the ccrl ccr3 double mutants (•) as compare to WT plants. Significance levels indicated for the ccrl ccr3 double mutants (★) as compared to the ccrl single mutants. Saccharification was ended after 48h. Biological replicates (WT: n = 13; ccrl: n = 13; ccr3 n = 15 and ccrl ccr3: n = 7); One-way ANOVA and Tukey post hoc test; **** and •••• p<0.0001, ***, ••• and ★ ★ ★ 0.0001<p<0.001, **, •• and ★ ★ 0.001<p< 0.01, *, • and ★ 0.01<p< 0.05. Error bars indicate the standard deviation. 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 silage plants which have a combined disruption in the genome of the CCR1 and CCR3 genes. The corn CCR1 polynucleotide sequence is depicted in SEQ ID NO: 3 and the corresponding encoded polypeptide sequence is depicted in SEQ ID NO: 1. The corn CCR3 polynucleotide sequence is depicted in SEQ ID NO: 4 and the corresponding encoded polypeptide sequence is depicted in SEQ ID NO: 2.

Thus in a first embodiment the invention provides a silage 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 and having a gene disruption in a polynucleotide encoding for SEQ ID NO: 2 or having a gene disruption in a polynucleotide encoding a plant orthologous polypeptide of SEQ ID NO: 2.

In a particular embodiment a silage plant is a grass such as Lolium, clover, alfalfa, corn, oats, rye and vetches.

In a particular embodiment a plant orthologous polypeptide sequence of SEQ ID NO: 1 comprises SEQ ID NO: 23 and SEQ ID NO: 24.

SEQ ID NO: 23 is a conserved amino acid signature (NWYCYGK, see Figure 6) which is present in plant CCR polypeptide sequences.

SEQ ID NO: 24 is a conserved signature which is typical for CCR1 sequences. SEQ ID NO: 24 was identified based on a polypeptide alignment of SEQ ID NO: 5 to 13.

In another embodiment a plant orthologous polypeptide sequence of SEQ ID NO: 2 comprises SEQ ID NO: 23 and SEQ ID NO: 25.

SEQ ID NO: 23 is a conserved amino acid signature (NWYCYGK, see Figure 6) which is present in plant CCR polypeptide sequences. SEQ ID NO: 25 is a conserved signature which is typical for CCR3 sequences. SEQ ID NO: 25 was identified based on a polypeptide alignment of SEQ ID NO: 14 to 22.

In yet another particular embodiment plant orthologous polypeptide sequences of SEQ ID NO: 1 are depicted in SEQ ID NO: 5, , 8, 9, 10, 11, 12 and 13.

In yet another particular embodiment plant orthologous polypeptide sequences of SEQ ID NO: 2 are depicted in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21 and 22.

In another particular embodiment the invention provides a seed or a plant cell derived from a silage plant having a combined gene disruption in a polynucleotide encoding a CCR1 polypeptide and a polynucleotide encoding a CCR3 polypeptide.

A method for increasing digestibility of a silage plant, the method comprising disrupting the expression of a polynucleotide in the plant encoding a CCR1 polypeptide and disrupting the expression of a polynucleotide in the plant encoding a CCR3 polypeptide.

The term "silage plant digestibility" as used herein generally refers to a measurable characteristic from a silage plant, particularly a grass. Increased silage plant digestibility correlates with reduced lignin levels in the silage plant, and correlates with increased saccharification. Silage plant digestibility can be measured directly by methods such as measurements of biomass digestibility and chemical composition using near-infrared reflectance (NIR) spectroscopy. Other methods of measuring digestibility are enzymatic methods. Also in vivo methods to directly measure digestibility or the use of rumen juice can be used to measure digestibility.

Thus, the activity of a CCR1 and a CCR3 protein may be reduced or eliminated by disrupting the genes encoding the CCR1 and CCR3 genes. The disruption inhibits expression or activity of CCR1 and CCR3 proteins compared to a corresponding control plant cell lacking the disruption. In one embodiment, the endogenous CCR1 (or CCR3) gene comprises two or more endogenous CCR1 (or CCR3) genes. Similarly, in another embodiment, in particular plants the endogenous CCR1 (or CCR3) gene comprises three or more endogenous CCR1 (or CCR3) genes. The wording "two or more endogenous CCR1 (or CCR3) genes" or "three or more endogenous CCR1 (or CCR3) genes" refers to two or more or three or more homologs of CCR1 (or CCR3) but it is not excluded that two or more or three or more combinations of homologs of CCR1 (or CCR3) 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 CCR1 and CCR3 genes. In yet another embodiment, the disruption comprises one or more point mutations in the endogenous CCR1 and CCR3 genes. The disruption can be a homozygous disruption in the CCR1 and CCR3 genes. Alternatively, the disruption is a heterozygous disruption in the CCR1 and CCR3 genes. In certain embodiments, when more than one CCR1 and CCR3 genes are 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 CCR1 and CCR3 or measuring the amount of lignin reduction in a plant or in a population of plants.

Thus, many methods may be used to reduce or eliminate the activity of CCR1 and CCR3 genes. More than one method may be used to reduce the activity of a single plant CCR1 and CCR3 gene. In addition, combinations of methods may be employed to reduce or eliminate the activity of two or more different CCR1 and CCR3 genes. Non-limiting examples of methods of reducing or eliminating the expression of a plant CCR1 and CCR3 gene 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 CCR1 and CCR3 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 CCR1 and CCR3 polypeptide is an expression cassette capable of producing an RNA molecule that inhibits the transcription and/or translation of a CCR1 and CCR3 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. Typically, 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 CCR1 and a zinc finger that binds to a gene encoding a CCR3 polypeptide, resulting in reduced expression of the genes. In particular embodiments, the zinc finger protein binds to a regulatory region of a CCR1 and a CCR3 gene. In other embodiments, the zinc finger protein binds to a messenger RNA encoding a CCR1 and a CCR3 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 CCR1 and a CCR3 polypeptide, resulting in reduced expression of the genes. In particular embodiments, the TALE protein binds to a regulatory region of a CCR1 and a CCR3 gene. In other embodiments, the TALE protein binds to a messenger RNA encoding a CCR1 and a CCR3 polypeptide and prevents their 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 CCR1 and CCR3 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 CCR1 and the CCR3 polypeptides. 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.

Silage is generally made from grass crops including sorghum, maize, barley, oats, millet or other cereals. Typically entire green plant material is used for making silage.

Silage plants that are particularly useful in the methods of the invention include plants selected from the list comprising Agrostis stolonifera, Asparagus officinalis, Avena spp. (e.g. Avena sativa, Avena fatua, Avena byzantina, Avena fatua var. sativa, Avena hybrida), Cannabis sativa, Carex elata, Hibiscus spp., Hordeum spp. (e.g. Hordeum vulgare), Juglans spp., Lathyrus spp., Lens culinaris, Linum usitatissimum, Lupinus spp., Miscanthus sinensis, Oryza spp. (e.g. Oryza sativa, Oryza latifolia), Panicum miliaceum, Panicum virgatum, Pennisetum sp., Persea spp., Petroselinum crispum, Phalaris arundinacea, Phaseolus spp., Phleum pratense, Phoenix spp., Phragmites australis, Physalis spp., Poa spp., Secale cereale, Solanum spp. (e.g. Solanum tuberosum, Solanum integrifolium or Solanum lycopersicum), Sorghum bicolor, Trifolium spp., Triticosecale rimpaui, Triticum spp. (e.g. Triticum aestivum, Triticum durum, Triticum turgidum, Triticum hybernum, Triticum macha, Triticum sativum, Triticum monococcum or Triticum vulgare), Vicia, Zea mays, 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

1. Generation of ccrl and ccr3 single mutants and ccrl ccr3 double mutants using CRISPR/Cas9

To study the role of CCR1 and its closest homolog CCR3 in maize inbred line B104, we aimed to knockout these genes using the CRISPR/Cas9-mediated genome-editing toolbox. CCR1 and CCR3 share 86% amino acid identity and the typical conserved domains and amino acid residues are present in both genes (Figure 6). Two gRNAs were designed to target both genes in their third exon, allowing to screen for deletion mutants. The distance between the target sites of the two gRNAs is 135 bp in both CCR1 and CCR3. Immature maize embryos of inbred B104 were transformed with the CRISPR/Cas9-construct using Agrobacterium and fifteen shoots derived from ten independent calli that survived the hygromycin selection phase were obtained. The shoots were analyzed using PCR followed by Sanger sequencing. Two out of the fifteen shoots were simultaneously edited in CCR1 and CCR3, resulting in frameshift mutations in both genes (Table 1). Subsequently, the shoots were cross-pollinated with a B104 WT and the T-DNA free ccrl ccr3 dihybrid plants were selected using PCR followed by Sanger sequencing. Finally, the ccrl ccr3 dihybrids were self-pollinated to obtain the ccrl single mutant, ccr3 single mutant, ccrl ccr3 double mutants and WT control plants by Mendelian segregation. We selected and grew two independent lines in the greenhouse. The ccrl-1 ccr3-l double mutant line was further analyzed and is referred to as the ccrl ccr3 double mutant hereafter.

Table 1: Mutations introduced in the CCR1 (SEQ. ID NO: 3) and CCR3 (SEQ ID NO: 4) gene, that resulted in two independent double mutants: ccrl-1 ccr3-l and ccrl-2 and ccr3-2. The gRNA target sites are underlined. The mutations are depicted in bold.

2. ccrl and ccr3 single mutants and ccrl ccr3 double mutants have no yield penalty in the greenhouse

To evaluate the effect of the mutation in either CCR1 or CCR3 or both genes simultaneously, we selected the homozygous single and double mutants and control plants from a segregating seed stock. Normal seed germination, growth and development was observed in all mutant lines compared to WT. We harvested WT plants, ccrl single, ccr3 single and ccrl ccr3 double mutants and measured biomass yield parameters after 10 weeks and 15 weeks of growth (Table 2 and Table 3). The 10-week old mutant plants showed no differences compared to WT in terms of tassel height, height of the highest leaf, ear height, ear leaf length, the length and diameter of the ear internode length, the dry weight of the stem and leaf and the total above-ground dry weight. After 15 weeks, the tassel height, height of the highest leaf, ear leaf length and ear internode length were reduced in the ccrl single and ccrl ccr3 double mutants when compared to the WT. However, the dry weight was not affected. In conclusion, the growth and development appeared not to be affected in the ccrl, ccr3 and ccrl ccr3 mutants after 10 weeks but were slightly reduced in height, ear leaf length and ear internode length after 15 weeks. Notably, the ccrl single mutant and ccrl ccr3 double mutants displayed a reddish-brown coloration in the epidermis of the stem (see Figure 3B). This coloration was neither observed inside the stem tissue nor at the midrib of the leaves in these mutants.

Table 2: Biomass analysis of ccrl single, ccr3 single and ccrl ccr3 double mutants grown for 10 weeks in the greenhouse. The tassel height, height highest leaf, ear height, ear leaf length, ear internode (IN) length and ear internode diameter were determined at harvest. The dry weight (DW) of the stems and leaves was weighted after drying the plant material. No significant differences were found between WT and mutants (one-way ANOVA and Tukey post hoc test). The data represent the averages of five biological replicates for WT, ccrl single mutants, ccr3 single mutants and ccrl ccr3 double mutants ± standard deviation.

Table 3: Biomass analysis of ccrl single, ccr3 single and ccrl ccr3 double mutants grown for 15 weeks in the greenhouse. The tassel height, height highest leaf, ear height, ear leaf length, ear internode (IN) length and ear internode diameter were determined at harvest. The dry weight (DW) of the stems and leaves was weighted after drying the plant material. The data represent the averages of n biological replicates for WT, ccrl single mutants, ccr3 single mutants and ccrl ccr3 double mutants ± standard deviation. One-way ANOVA and Tukey post hoc test; data compared to WT; ***0.0001<p<0.001, **0.001<p< 0.01, *0.01<p< 0.05.

3. The lignin content in leaves of ccrl single mutants is not altered, while the lignin content in leaves of ccrl ccr3 double mutants is reduced

The lignin content was determined on dried stem and leaf material of the ccrl and ccr3 single mutants, the ccrl ccr3 double mutants and their corresponding wild-type plants (Table 4, Table 5, Figure 4, 5, 7 and 8). First, the soluble compounds were removed from grinded plant material by a sequence of washing steps resulting in the cell wall residue (CWR). No differences in CWR were observed in the mutant lines compared to WT. Next, the CWR was used to determine the lignin content via the Klason method. All measurements were performed at two developmental stages: on 10 week-old plants and 15-week old plants. After 10 weeks, the ccrl and ccrl ccr3 mutants had a reduced Klason lignin content with a relative reduction of 20% and 19% in stem tissue, respectively, as compared to the Klason lignin content of the WT plants. In contrast, the acid-soluble lignin fraction was increased in both the ccrl single and ccrl ccr3 double mutants. Furthermore, the acid-soluble lignin fraction was 9% higher in the ccrl ccr3 double mutants as compared to the ccrl single mutants (Figure 7). The total amount of lignin in stem tissue, calculated as the sum of Klason and acid-soluble lignin was decreased in ccrl and ccrl ccr3 mutants by 16% and 14%, respectively, relative to the levels in the WT plants (Figure 8). In leaf tissue, the Klason lignin content was only reduced in the ccrl ccr3 double mutants (a relative decrease of 11% as compared to the WT plants), whereas no differences were observed for ccrl and the ccr3 single mutants. The acid-soluble lignin fraction was not significantly different between the lines. The total lignin content of the leaves was decreased in the ccrl ccr3 double mutant by 7%, as compared to that in WT. No decrease in total lignin was observed in the ccrl and the ccr3 single mutants compared to that in leaves of the WT.

After 15 weeks, stem tissue of the ccrl mutants had less Klason lignin with a relative reduction of 13% as compared to the Klason lignin content of the WT plants. Again, the acid-soluble lignin content was increased in both the ccrl single and ccrl ccr3 double mutants and with an 11% higher acid-soluble lignin fraction in the ccrl ccr3 double mutants as compared to the ccrl single mutants. The total lignin content in stem tissue was decreased in ccrl single mutants by 10% relative to the WT plants. In leaves, the Klason lignin content was only reduced in the ccrl ccr3 double mutants (a relative decrease of 15% as compared to the WT plants), whereas no differences were observed for ccrl nor the ccr3 single mutants. The acid-soluble lignin fraction showed no significant differences between the lines. The total lignin content of the leaves was decreased in the ccrl ccr3 double mutant by 12%, as compared to that in WT. No decrease in total lignin was observed in the ccrl nor the ccr3 single mutants. In conclusion, we observed a reduction in lignin content in the stem of the ccrl single mutants, while the ccrl ccr3 double mutants have reduced lignin amounts in leaves and stems.

Table 4: Lignin content in stems and leaves of WT and ccrl, ccr3 and ccrl ccr3 mutants after 10 weeks. The CWR was determined as the fraction of material retained after sequential washing steps relative to the original dry weight. The Klason lignin and acid-soluble lignin (ASL) content was determined by the Klason method and expressed in percentage DW. The data represent the averages of five biological replicates for WT, ccrl single mutants, ccr3 single mutants and ccrl ccr3 double mutants ± standard deviation. One-way ANOVA and Tukey post hoc test; data compared to WT;****p<0.0001, ***0.0001<p<0.001, **0.001<p< 0.01, *0.01<p< 0.05.

Table 5: Lignin content in stems and leaves of WT and ccrl, ccr3 and ccrl ccr3 mutants after 15 weeks. The CWR was determined as the fraction of material retained after sequential washing steps relative to the original dry weight. The Klason lignin and acid-soluble lignin (ASL) content was determined by the Klason method and expressed in percentage DW. The data represent the averages of n biological replicates for WT, ccrl single mutants, ccr3 single mutants and ccrl ccr3 double mutants ± standard deviation. One-way ANOVA and Tukey post hoc test; data compared to WT; ***0.0001<p<0.001, **0.001<p< 0.01, *0.01<p< 0.05.

4. Improved saccharification efficiency in stem tissue in the ccrl single and ccrl ccr3 double mutants with superior saccharification efficiency in leaves of the ccrl ccr3 double mutants

We investigated the saccharification efficiency of the WT, ccrl single, ccr3 single and ccrl ccr3 double mutants of the two developmental stages (after 10 weeks and after 15 weeks) under three different conditions (Figure 9 and 10). The cellulose-to-glucose conversion was calculated based on the amount of glucose released upon saccharification of stem and leaf material after acidic pretreatment (1 M HCI, 80 °C, 2 h), alkaline pretreatment (6.25 mM NaOH, 90 °C, 3 h) and no pretreatment. Prior to the saccharification assay, the cellulose content was measured of each sample and we observed no differences in cellulose and hemicellulose content in the mutant lines as compared to the WT plants (Table 6). After 48h, the cellulose-to-glucose conversion of the 10-week old stem material of the ccrl single mutants using the no pretreatment, acidic and alkaline pretreatment showed an increase of 39%, 50% and 38% as compared to WT plants, respectively. The ccrl ccr3 double mutants showed an increase in cellulose-to-glucose conversion of 43%, 57% and 46% as compared to WT plants using the no pretreatment, acidic and alkaline pretreatment, respectively. In leaf material, the cellulose-to-glucose conversion is increased by 6% and 12% using the no pretreatment and acidic pretreatment in the ccrl single mutants. In the ccrl ccr3 double mutants, the glucose release is increased by 12% and 15% as compared to WT plants using the no pretreatment and acidic pretreatment, respectively. Only the ccrl ccr3 double mutants show an increase under alkaline pretreatment with a relative increase of 10%. Furthermore, the ccrl ccr3 double mutants performed significantly better as compared to the ccrl single mutants in the no pretreatment, acidic and alkaline pretreatments with a relative increase of 6%, 4% and 7%, respectively. The ccr3 showed no increase nor decrease in glucose release in the stem or leaf material.

Stem material of 15-week old plants also showed an increase in cellulose-to-glucose conversion after 48h in the ccrl single and ccrl ccr3 double mutant in the no pretreatment, acidic and alkaline pretreatments, with 24 - 42%, 36 - 43% and 16 - 41%, respectively. Furthermore, the ccrl ccr3 double mutants performed 15% and 21% better as compared to the ccrl single mutants in the no pretreatment and alkaline pretreatment, respectively. In the leaf material, only the ccrl ccr3 double mutants showed a 14% increase glucose release in the acidic pretreatment as compared to the WT plants and 11% increase as compared to the ccrl single mutants. The ccr3 showed no increase nor decrease in glucose release in the stem or leaf material. In conclusion, the saccharification efficiency is improved in the stem material of the ccrl single and ccrl ccr3 double mutants. The ccrl ccr3 double mutants showed an increased saccharification potential in the leaves in acidic pretreatment conditions as compared to the ccrl single mutants.

Table 6: Cellulose and hemicellulose content of stems and leaves of WT and ccrl, ccr3 and ccrl ccr3 mutants after 10 and 15 weeks of growth in the greenhouse. The CWR was determined as the fraction of material retained after sequential washing steps relative to the original dry weight. The cellulose and hemicellulose content was determined by the Updegraff method and expressed in percentage DW. No significant differences were found between WT and mutants (one-way ANOVA and Tukey post hoc test). The data represent the averages of n biological replicates for WT, ccrl single mutants, ccr3 single mutants and ccrl ccr3 double mutants ± standard deviation.

In conclusion, we observed no biomass yield penalty in the ccrl single or the ccrl ccr3 double mutants of the two independent lines. In addition, we showed that ccrl single mutants have reduced lignin amounts in stems only, while ccrl ccr3 double mutants have reduced lignin amounts in leaves and stems. Cell wall processability properties were evaluated by saccharification and showed that we have improved saccharification potential in the ccrl single and ccrl ccr3 double mutants in the greenhouse. The ccrl ccr3 double mutants show superior cell-wall processability qualities in the leaves and 15-week old stem material as compared to ccrl single mutants.

Materials & methods

Design and construction of the CRISPR/Cas9 constructs

The CRISPR/Cas9 system was used to introduce knock-out mutations in the CCR1 (GRMZM2G131205; Zm00001d032152; Zm00001eb041120) and CCR3 (Zm00001d050417; Zm00001ebl79780) genes in maize. Two guide RNAs were selected in exon 3 of the CCR3 gene using the CRISP-OR webtool (http://crispor.tefor.net/) (Condordet and Haeussler, 2018; Table 5)

To construct the CRISPR vector, first the destination vector was assembled followed by introducing two guide RNAs to the vector. First, the pHb-U9Ul binary vector was first linearized by l-Scel RE for two hours. The digest was run on a mini gel and the vector backbone was isolated and extracted from gel using a the GeneJET Gel Extraction Kit (ThermoFisher). Subsequently, in a PCR tube, 1 pl of the following constructs (lOOng/ pl) were added: linearized pHb-UlU9, pGGIB-Ul-ZmUBIL-ZmCas9-NOST-U2, pGGIB- U2-AG-U3, pGGIB-U3-EFla- tDTomato-NosT-U4 and pGGIB-U4-L4-U9. Then 1 pl of l-Scel, one pl of CutSmart buffer and water up to 10 pl was added. The tube was placed in a Thermocycler for two hours at 37°C, 25 min at 65°C and 1 hour at 50°C. When the reaction reached 50°C, the machine was paused and five pl of the reaction mix was mixed with five pl NEBuilder® HiFi DNA Assembly Cloning Kit (NEB) and tube was placed in the Thermocycler for the last step. Finally, three pl of the reaction was mixed with 40 pl of ccdB survival competent cell and heat shock transformation was performed. The transformed cells were spread on the solidified LB with 100mg/l Spectomycin and incubated overnight at 37°C. The correct clone that was selected according to the restriction analysis was validated by sequencing. In the second part, the two guide RNAs (gRNAs) were cloned into two entry vectors under control of a rice U3 monocot-specific promoter (Xing et al., 2014) Subsequently, the entry vectors with a linker were cloned into the single binary destination vector using the Golden Gate strategy. Finally, the destination vector was transformed into Agrobacterium strain EH105 (Hood et al., 1993). The correct clone was selected using restriction analysis and validation by sequencing.

Table 5: Summary of oligos used in the invention

Table 6: Summary of guide RNAs used in the invention

Plant transformation and selection of the ccr knockout lines

The publicly available inbred line B104 was used throughout this study (Hallauer et al., 1997). Agrobocter/um-mediated transformation was used to transform immature embryos as previously described (Coussens etal., 2012). Briefly, plants were grown and pollinated under controlled greenhouse conditions (300 pE/m²/s light intensity, 16 hours light, 26°C and 8 hours dark, 22°C). The primary transformants (TO) were genotyped by PCR and the edited shoots were retained (Table 4). Subsequently, the primary transformants were cross-pollinated with a B104 wild-type to obtain Cos9-free isogenic control and mutants lines. The ears were harvested four weeks after pollination. In the T1 generation, two independent mutant lines were selected and the Cos9-free progeny was selected using a PCR amplifying a region in Cas9 and a leaf assay to test for hygromycin resistance. This allows to select Cas9- free plants and heterozygous for the mutation. Next, the selected plants were grown to maturity in the greenhouse and self-pollinated. In the T2 generation, control and mutants plants were selected from the segregating seed stock for two independent lines per gene. Plant material

All analysis were carried out on two independent knock-out lines for ccrl, ccr3 and ccrl ccr3, with their corresponding control line. The ccr mutants and control plants were grown together in random design under greenhouse conditions. After 10 weeks, the five plants were measured and harvested to perform metabolic profiling, anatomy, cell wall analysis and growth phenotyping. The cell wall analysis was performed on the ear leaf and ear internode. The mutant and control plants were measured from the soil using a Sola TEL500 telescope meter and harvested for cell wall analysis and growth phenotyping. Cell wall analysis was performed on the ear leaf and ear internode. The whole plant was harvested, except the bottom 10 cm and the tassel. The harvested plant material was dried for 7 days in at 50°C. The ear leaf and ear internode were grinded separately using a Fristch cutting mill with an internal sieve of 500 pm. Finally, the samples were sieved again and a homogeneous fraction of 250 - 500 pm was retained and used for cell wall analysis.

Determination of cell wall residue

Priorto determination the lignin content, cell wall residue (CWR) was prepared by subjecting the samples to a series of extraction steps. Two aliquots (65 mg) per sample were prepared and sequentially washed for 30 min at 98°C in milliQ water, 30 min at 76°C in ethanol, 30 min at 59°C in chloroform and 30 min at 54°C in acetone. Finally, the samples were dried under vacuum.

Lignin content

The lignin content was measured using the Klason method (Van den Bosch et al., 2015). Briefly, 1 mL of 72% sulphuric acid was added to 50 mg of CWR in 15 mL glass vials and stirred with a magnetic rod for 2h at room temperature. Subsequently, the samples were transferred to 100 mL flasks and diluted to 5% sulphuric acid with 22 mL of milliQ water. The flasks were autoclaved for lh at 121°C and afterwards incubated for 16 hours at 4°C. Next, 1 mL of the solution of each sample was collected to measure the acid-soluble lignin. The insoluble lignin was filtered and washed using pre-weighted glass microfiber filter papers (Sartorius AG) in Buchner filter system (Merck Millipore). Next, the filter papers were transferred to glass petri dishes and dried for 16h at 105°C. The filter papers contain lignin plus ash content and were weighted using an analytical balance (XPE105; Mettler-Toledo). Subsequently, the filter papers were transferred to a muffle furnace (12 min at 105°C, at 10°C/min to 250°C, 30 min at 250°C, at 20°C/min to 575°C, 180 min at 575°C, cool down to room temperature) and weighted again to calculate the ash- corrected lignin content. The acid-soluble lignin was determined using a spectrophotometer (Genesys 10 S UV-Vis, Thermo Scientific) by measuring the absorbance at 205 nm. The acid-soluble lignin content was calculated with the Beer-Lambert law (Dence, 1992). Cellulose and hemicellulose analysis

The hemicellulose fraction was first removed by incubating the CWR with 2M trifluoroacetic acid (TFA) for 2h at 99°C while shaking (750 rpm), according to Foster et al. (2010). After incubation, the samples were centrifuged (15 min at 14.000 rpm) and 800 pL of the TFA-extract was taken without disturbing the pellet. Subsequently, a myo-inositol solution (5 mg mL-1) was added to the TFA-extract after which the mixture was dried under vacuum. The resulting pellet of the TFA-extract was stored at -20°C until analysis. The remaining pellet after TFA incubation was washed using ImL of water and acetone and dried under vacuum. Subsequently, the pellet was weighted further used to measure the crystalline cellulose content using Updegraff method (Updegraff, 1969). Next, Updegraff reagent (acetic acid: nitric acid: water; 8:1:2 v/v) was added to the pellet and incubated for 30 min at 100°C. The samples were cooled on ice to room temperature and centrifuged (15 min at 10.000 rpm). The remaining pellet was washed with 1 mL of water and acetone and dried under vacuum. Subsequently, 175 pL mL 72% v/v H2SO4 was added to the pellet, vortexed and incubated for 30 min at room temperature, whereupon the samples were again vortexed and incubated for another 15 min. The samples were centrifuged (5 min at 10.000 rpm) and the supernatant was used for colorimetric determination of the cellulose content. As a standard curve, we used 1 mg D-glucose mL-1 stock solution and prepared a dilution series. To each sample, we added 200 pL of freshly prepared anthrone solution (2 mg anthrone mL ¹ pure H2SO4) and incubated for 30 min at 80°C. The 96-well plate was cooled down to room temperature before measuring the absorbance at 625 nm using a spectrophotometer.

Pretreatments and saccharification assay

Saccharification assays were performed as described by Van Acker et al. (2016). The acid pretreatment was performed with IM HCI at 80°C for 2 hours and the alkaline pretreatment with 6.25 mM sodium hydroxide at 90°C for 3 hours. To each sample Cellic® Ctec2 enzyme blend (Novozymes) was added with an activity of 0.1 FPU/mL. The glucose release as measured after 2, 6, 24 and 48h and normalized for the CWR.

Sequences

SEQ ID NO: 1

Zea mays CCRl(Zm00001d032152)

MTVVDAVVSSTDAGAPAAAATAVPAGNGQTVCVTGAAGYIASWLVKLLLEKGYTVKGTVRNPDDPKNAHLKALDG AAERLILCKADLLDYDAICRAVQGCQGVFHTASPVTDDPEQMVEPAVRGTEYVINAAAEAGTVRRVVFTSSIGAVTM DPKRGPDVVVDESCWSDLEFCEKTRNWYCYGKAVAEQAAWETARRRGVDLVVVNPVLVVGPLLQATVNASIAHILK YLDGSARTFANAVQAYVDVRDVADAHLRVFESPRASGRHLCAERVLHREDVVRILAKLFPEYPVPARCSDEVNPRKQP YKFSNQKLRDLGLQFRPVSQSLYDTVKNLQEKGHLPVLGERTTTEAADKDAPTAEMQQGGIAIRA SEQ ID NO: 2

Zea mays CCR3(Zm00001d050417)

MTVVVDAVVPTDAAGAPAAAAAAPAVRPGNGQTVCVTGAAGYIASWLVKLLLEKGYTVKGTVRNPDDPKSAHLKA

LDGAAERLVLCKADLLDYDAIRRAVHGCQGVFHTASPVTDDPEQMVEPAVRGTQYVVNAAAEAGTVRRVVFTSSIG

AVTMDPGRGPDVVVDESCWSDLEFCKRTRNWYCYGKAVAEQAARDLCRQRGLELAVVNPVLVVGPLLQPAVNASI

GHVLKYLDGSARTFANAVQAYVDVRDVADAHLRVFESPRASGGRYLCAESVLHREDVVRILAKLFPEYPVPTRCSDEV

NPRKRPYRFSNQKLRDLGLVFRPVSQSLYDTVKNLQEKGHLAVLGEQTTTTTTEAGREECPAADLQQQGGIAIRA

SEQ ID NO: 3

Zea mays, CCR1 polynucleotide sequence

ATGACCGTCGTCGACGCCGTCGTCTCCTCCACCGATGCCGGCGCCCCTGCTGCCGCCGCCACCGCGGTACCGGCG

GGGAACGGGCAGACCGTGTGCGTGACCGGCGCGGCCGGGTACATCGCCTCGTGGTTGGTGAAGCTGCTGCTCG

AGAAGGGATACACTGTGAAGGGCACCGTCAGGAACCCAGGCATGCATGCCTCCTGCTTGTTGTTATATATAAGC

AGTCTACTATGTCGACCTGCGCGAGCGTGGCAGCAGATGGAGCGATTAACGTGTTTGTACATGTTCATGGCAGC

AGATGACCCGAAGAACGCGCACCTCAAGGCGCTGGACGGCGCCGCCGAGCGGCTGATCCTCTGCAAGGCCGAT

CTGCTGGACTACGACGCCATCTGCCGCGCCGTGCAGGGCTGCCAGGGCGTCTTCCACACCGCCTCCCCCGTCACC

GACGACCCGGTGAGTTCCGTCGGCCGGGTTAGTTCCAGCACTCGTCGACTGACTGATCAGTAAAGTAGTAACAA

GGGGACCGCATGCACCATGCATGTGTGCGCGTGCAGGAGCAAATGGTGGAGCCGGCGGTGCGCGGCACCGAG

TACGTGATCAACGCGGCGGCGGAGGCCGGCACGGTGCGGCGGGTGGTGTTCACGTCGTCCATCGGCGCCGTGA

CCATGGACCCCAAGCGCGGGCCCGACGTCGTGGTCGACGAGTCGTGCTGGAGCGACCTCGAGTTCTGCGAGAA

AACCAGGGTGGGTGTGCTTGCTTGCTCACTTTTATTTGATCGATCGTCTCCATCCATCCATCATCTGATCTACTACT

AAGTACAGTAGCTTGTAGCTAGCTCCTGCTATACCGTCCGCTGCACCACGTACGCCAGCACCATATATTAAATTAG

TGTTTCCGATCCTTTAATTTGATGCATACGTTTTCATTTCTTGCAAGTAAGGACGATCAAAGGAAAGGGTGAAAG

AAACACTAATAAAGGTAGCTGTGACGAGATAGGCGAATCATTACCTGCTAGTATATTGGCATGCATGCACTAGCT

AGCTACCAACCACCGAGAGCCCTAGAAGAGACTAGACTAGAGTACGTTCGAGTACTTTTGAGGCCGCCAAATAG

AACCAACTAAGTGCTCATCGTCATCGATGGTGCCTGTCCAAAGCACACAGAGAGCAGCACTAGCTAGCTAGGAT

TTGAACCACAGCTTTTTCTAGCGTGACCAACAGCACTAGCTAGGCAAGCAGCCGAAATAACGCATATATATGAAA

AGGAATTTGGTTCCGCAAAAAAAAAAACGGAGAACGGAAAAGGAGCAAATCATGCATGTGGACGGAGAACGC

ACGCACCACGCGAATTAATCCTGCCTCTGCATGGGGCCACGCACGCCGTCGCCGATGGACACATGTATGGCATG

CAGCGCTTGAGCTACGACCTGCTTAATTATCAGTAGCGAAGAATCTCATCCCACATGCGTGTTTCTTCAACACGTA

CGCATGGACACTCTTTAGTGTCAAAGCTAAAGCTGAGAATTCAAATTAACCTTGCTATTTTGATCGCGGTGGGCT

CTTAAAATGATTGGACAGATGCAGCACCGTACCCACGCCTTACAACTCTCCTAGCTAGCTAGCCGCCCCGCAACC ACACTAGAATTGTTCTAGCCTAGTAGCCTGTGTCTGTGTCTGTGTGTGCGTGTAGCGTGTCCTATGGAAGACGGA AATTTCAGCTGCCCAGAAAAACACACACATGCACGACGACGACGCCACCAGTTTGCCGGTCGACACATGCTAGC AGTGATGGGCAGGCCTTTGTCGATCGCCATTTATTCTGTGCAGCAAACTCTGCTGGCTTTAATTTGCGGAGGAGC GAGCAAATTCTACTCTCCCCGGCTTTAATTTGCGGATTTATTACAAGTCGTCATCCCAACTTCATTGGACCAACTTT TATAGAATATTTTTATTTAAAAAAAACTGTAGTAATTTACGATATCAAACGAGTAATACTATATTTTTTATTAATTG TGTTTTTATAGTGTATTTATTTGATGGCATGTTTTTTTTTGCTCCTATAGTTTTAGTTAAATTTGATGTGCTACCTTT GACCCACTAATAATATTTTTAAAAAAATGATTTATAATTCTGAACCGAGGGAAGGATTTTATATATATATATATAT AT AT AT AT AT AT AT AT AT AT AT AT AT AT AT AT AT AT AT AT AT AT AT AT AT AT AT AT AT ATT AT AT AT AT AT AT AT ATG GCAACGTATCCTGTGTAAAAGGTTCTCCAGTGTAAATATGCTAAAATGCCTTAACAACCTTTGGATCCAGAATCA AAGGCTAATTTTAATCCTACCTAACCGCCACCGCACCAGATTACCAGGAGGGGGTTCTTTAGAAAAAACACAAGA TTATCAGGTGGGATTAAGATTAGCCTTTGAATCTGGATCCAAAGGTTGTTAAGACGTTTTAGCATGTTTACACAG GAGAACCTTTTACACAGGAGACGTTGCCCCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTATATATATATATATA TATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATATA TATATATATATATATATATATATATATATATGGTACGACTTTGGTGGGTCGCTCGTCGCCTCTCCCGACGGCCCAA CCTCCAACCAACGCAAAATTTACTTGCATTGCATGGCCGCCATCCTCATTATTTTTTTGTCTGTTTCTGACGCAACA ATCACGTCAGCATGACACACAGCATATATCATTACTGTGTTCTAAGTGCACGCCCATAACGCATATGCACGCTGC AGAACTGGTACTGCTACGGCAAGGCGGTGGCGGAGCAGGCGGCGTGGGAGACGGCCCGGCGGCGGGGCGTG GACCTGGTGGTGGTGAACCCCGTGCTGGTGGTGGGCCCCCTGCTGCAGGCGACGGTGAACGCCAGCATCGCGC ACATCCTCAAGTACCTGGACGGCTCGGCCCGCACCTTCGCCAACGCCGTGCAGGCGTACGTGGACGTGCGCGAC GTGGCCGACGCGCACCTCCGCGTCTTCGAGAGCCCCCGCGCGTCCGGCCGCCACCTCTGCGCCGAGCGCGTCCT CCACCGCGAGGACGTCGTCCGCATCCTCGCCAAGCTCTTCCCCGAGTACCCCGTCCCAGCCAGGTCTGATTTCATA GTACTCATTGCTTGCTTGCTTGCTTAGGAACCGGTGGATAGGAATCCACTGGTGGTATTAATTAGGTGACGCGAG CAATTAAGCAAAGCGTGGTAGTACTCGTACTACACTGCAAATTAAACTCCGGTGGATCCCAGCGTAGTAGGTGA ACGATGGACAGGGCCCGGCCGGTCACTTACGCGTACACTGCTCGATCCACCTCATCGGCCGGCTCTTGTGCTGCA CACGAAAGCAAAGCGGCCGTCAGCTGAAGAAGGGCGAACGTATGGGGGGCGGTTCAGGTGAACCACTCTTTTG TGTGTATCATCACTGACGCTGACACTGGCTAGAGCGCGTCCTGTGCAAGCTAGCTAGTTGGTAGACGCGGCTAG TACACACCACACGCCACCAATATTTGCATGCTAGTGCAAGTCAGCTAGCTAGCTAGCTAGCTCACGGCGGTGGCC AATACTCTAGACCTCAATTACTGCCACACGAAGCGGTAGCTAGATAGAGTACGTGCGACCACTTCGAAACGTATC

TGTCCGGAGCGGTAGAAATCTGTAGCGTACGTCAGCGTCGTCTCGGAGACGGAGAAGTAGCGTGTAGCTACTAT TATACTACGCGTACTAGATGCCATCCGTGTTCGCTTTGGCCAATCGGGGCAAGGCCGTTTTTCTGCCATACATCAC GTCGTCATCGCCGTCACCACCATTCGTGACTTGTGTAGTAGGTAGTAGTATACCCGGAGGCCGGAGCAGATGCA TTTGCTCGCGTCTGCGTTTTGTGCTGCAACCTGCAACCATGTCACAGCTGCTAGCTGTTCGGTTCCTCCGTTGCAG GCCTGCGTGTGCCATGTGGAGCAGAGAGTTGGGTTCTTTCTACCTAGCACTATAAGAGAAACCGCTCGGTCACG GAGGTACAGGCAACGCACGTATGTCTAGAACTTAACTTCTCGAATATTTGTCGACCACTAGTTTATTTTTTTTAACT AAAACGCGATAAATAAAAAAAACAAAGAAATACTAGTTTCTATCAAGTAAGCAGCTTAATCAAATCTACGAAACA

GAGGAAGGAACGCCGCAGAAACTTCCTAGGAATTTTCTTCATCAACTCGCAGCAAATTTATTTTTCTTCAGGCTTG

GTGGGGGAGGGGGTGACCTCTGCGGTAGGTCATCTCCAAGGTCCTACCTAGATCGATCATCATTTACCACACAA

GAAGTAACCATGCATGCATCTCTCTTACCACATCATTACATGTAGAATAAGAATATTGGCGAGTCATGCAAGCAT

CTATTACGAAACATGTAGAATATTTACACTACTACTCCACCATCCGGTATTTTCAAACAAAGAACATGCATGGGCA

AGAAAAGGTCAGTTTCGCTCGACTGCTGACTGCAAGATGATCTTTACCAACCGACCTCTAATCACGCGATGGAAT

ATTCAACTTCCCGATTCGACGGCGGCGGGCAATCCTGGTTGTAACAGCCGTGGACTGCAACTGCAATGGGTCCTC

CGTCCGTTCGTTGTTAGTTTACGGCTTTTCCCATGACAAATTCCGTATGATTCTTTCAAAGATACTACCCTCCTCGT

CCTCGACCACTTGCTTTGCTGTCGTAATCTCACCATACCAATGAAAACACCTAGCCTTAGCGTATGCTCTGGGCCG

CCCATGCAAGCGGGAAGAATCTCACCTGCCCACAGGACAACCATGTGTTTACACTAGAGTTGATCGATACTGTAA

CTGAGAACCAAAAATGAATATGAACGAAACGGTCTTGACCTTCTCGCTCCTTTTTTTTATTAGGACGACCTTGACT

GACTGAGATCCTTGACTGACTGTGTGCGTATGCTATTATTAGCGTTTGCTGCTTTGCTTCACTGACTCTGTGTGTG

TGTGTGAGCGCTTGGTGCAGGTGCTCCGACGAGGTGAATCCGCGGAAGCAGCCGTACAAGTTCTCCAACCAGAA

GCTCCGGGACCTGGGGCTGCAGTTCCGGCCGGTCAGCCAGTCGCTTTACGACACGGTGAAGAACCTCCAGGAGA

AGGGACACCTGCCGGTGCTCGGAGAGCGGACGACGACGGAGGCCGCCGACAAGGATGCCCCCACGGCCGAGA

TGCAGCAGGGAGGGATCGCCATCCGTGCCTGA

SEQ ID NO: 4

Zea mays, CCR3 polynucleotide sequence

ATGACCGTCGTCGTCGACGCCGTCGTCCCCACCGATGCGGCCGGCGCCCCTGCTGCTGCGGCCGCGGCACCGGC

GGTGCGGCCGGGGAACGGGCAGACCGTGTGCGTCACCGGCGCGGCCGGCTACATCGCCTCGTGGCTGGTCAAG

CTGCTGCTCGAGAAGGGGTACACTGTCAAGGGCACCGTCAGGAACCCAGGCACGTAGGCCTTTTAATCTGGTGT

ATATATATGCATTTATACATACATATACGACGACGACGACGCGAGCTGAGCGTGGCAGGGATTAACAACGTTTGT

GTCATTGGTCGCCATATGCATGCACATGGTGGCACTGGCAGATGACCCGAAGAGCGCGCACCTCAAGGCGCTGG

ACGGCGCCGCCGAGCGGCTGGTCCTCTGCAAGGCCGACCTGCTGGACTACGACGCCATCCGCCGAGCCGTGCAC

GGCTGCCAGGGCGTCTTCCACACCGCCTCCCCCGTCACCGACGACCCGGTGAGCTAGTGCATGGCTGCTCCGATG

ACCCCCGGCCCCTTTTTGTCGCTAGCTGTGCCACCGTGTGACCTACGCACGCACGCACGCACACATCATCGGATC

GACTAACCACTAACCGACGATGCGTCATGCATGCATGCATGCATGCAGGAGCAAATGGTGGAGCCGGCGGTGC

GCGGCACACAGTACGTGGTCAACGCGGCGGCGGAGGCCGGCACGGTGCGGCGGGTGGTGTTCACGTCCTCCAT

CGGCGCGGTCACCATGGACCCCGGCCGCGGGCCCGACGTCGTTGTCGACGAGTCGTGCTGGAGCGACCTCGAG

TTCTGCAAGAGAACGAGGGTGAGTGGGCCGGCCCTAGTAGCTTTCGTCTTCTCACGTACTACTACGCAACAACTG

CGTTGCGTCACCGTGGCGACGACGACACACCATTAGTATTAGTATTATTGTTGTTATTCTTCTTCACTGGACACAG

TTTCACCTGCCCACATCTCTCATCCGGCCACGCACGCACGACGAGAGAGGATATGATGCTAATATATACGCATGC AGAACTGGTACTGCTACGGCAAGGCGGTGGCGGAGCAGGCGGCGCGGGACTTGTGCCGGCAGCGCGGGCTGG

AGCTGGCGGTGGTGAACCCGGTGCTGGTGGTGGGCCCGCTGCTGCAGCCGGCGGTGAACGCCAGCATCGGGCA

CGTGCTCAAGTACCTGGACGGCTCCGCCCGCACCTTCGCCAACGCTGTGCAGGCGTACGTGGACGTGCGCGACG

TGGCCGACGCGCACCTCCGCGTCTTCGAGAGCCCCCGCGCGTCCGGCGGCCGCTACCTCTGCGCCGAGTCCGTCC

TCCACCGCGAGGACGTCGTCCGCATCCTCGCCAAGCTCTTCCCCGAGTACCCCGTACCCACAAGGTATAGCTAGC

TACAGGGCATCTCGAACGGTACCTCAAAATAAAATAGGATTCAACTTATACAAAAAACATACATACGCGTCTCCG

ATATTATCTTAAACGTGTGTTCTATTGTAGACTCTTGACCCCTAAATTAAAACAGCTCCAGCAATAATCTATTTTAC

ACATATATTCTTATCAAACAATATAGGCATTATAATAGAAAGTGACCCAAATATACATACATACCGGATTAATGTC

ATATTCTTATTTTCTACATGATCATCAACCTTTTTTAAATAATATAGTTTATTTCCATAGACATGCTTATTTATAGTTT

AACACAGTTGGAATTGAATTTATATTTAGTAGTATCCTAAACACTTTAAAATATAGCGAGTAGTATTTTGATTTGC

GGGACACTATTTTATGGCACATGCATGGTTGAAATAATGCTCTTATATATATGTGTGTCTTCATGACCTAGCTAGC

TAATACTAATGTCCATCCGACCATCCGTCCGTTCCTCGATCGGCCTCTCTGTGCTTGTACCGGCCGACCACATTTTT

GTTAATGGCACACGCGTCTAATTCCGCACGTATATACTAATGAATGCGCATAAATTATACCGCCACATTTTTTTTG

TTGATGGCATTTAGCGGTTGGCATGTCCATCCCAAGCCTATCTCAGGTCAGCTAGCTAACAATAACACTAGGTCT

AATACACACATACACACTGACACACAACTACACAAGTAATCAAGAGCAGATATCTTGTAGGTATATTATATATATC

TTCATGACCTAGCTAATAATATCCGCCCAGCCTTGCTTTGATCCGTGTTTATACCGACCACATTTTCTTGTTTTAAC

GGCATTTAGCGGTTGGCATGTCCATCCCAATTAGCCTGTCTCAGGTCAGCTACAACAACACTTGGTCTATACACAA

GCTAGCAATCAAGAGCAGATATGGGAAACAGTTTGATATTGGTCACAAAAATAGCTTGGAGGTATATTATATATA

TACCTTCATGACCTAGCTAGCTAATAATAATATCTTTTTTTAACGGCACTTAGCGGTTGGCATGTCCATCAGAATA

AGCCTGTCTCAGCCGGCCAGCTGCAACAACATCTATATAGATAGATAGATATCATCGCCTGGTCTGCTTGAGTGT

TCCTACCTTTTGTAGGATGACGTACGTACGGATGGCCTTGACATGTACTAGCCGGCGATGATCACGACTGGAACC

TCACTACAAGAAAACCCACATTTTTATCTATATTAAGAGTGTCGGTACAGTCAAACCCACACTTTTATCTATATTAA

AAGTGTTGGGGCTGATACCTTCTTCGACGTTTTCTTGTAATGTCTGCTCTGCCTAGCTGACTGTGCGAGCGTTTGG

TGCGTGTGGTGCAGGTGCTCCGACGAGGTGAACCCGCGGAAGCGCCCGTACAGGTTCTCGAACCAGAAGCTCC

GGGACCTGGGCTTGGTGTTCCGCCCGGTGAGCCAGTCGCTGTACGACACGGTGAAGAACCTCCAGGAGAAGGG

CCACCTAGCGGTGCTGGGAGAGCAAACGACGACGACGACGACGGAGGCCGGCCGCGAGGAGTGCCCCGCCGC

CGACCTGCAGCAGCAGGGAGGAATCGCCATCCGCGCGTGA

SEQ ID NO: 5

Hordeum vulgare CCRl(Horvu_MOREX_5H01G375600)

MTVVDAAAAVAQELPGHGQTVCVTGAAGYIASWLVKLLLERGYTVKGTVRNPDDPKNAHLKALDGAAERLVLCKAD

LLDYDAICAAVEGCHGVFHTASPVTDDPEQMVEPAVRGTEYVIDAAADAGTVRRVVFTSSIGAVTMDPNRGPDVVV

DESCWSDLEFCKKTKNWYCYGKAVAEQAAWEKARARGVDLVVVNPVLVVGPLLQPTVNASAAHILKYLDGSARKYA NAVQAYVDVRDVAGAHLRVFEAPQASGRYLCAERVLHRQDVVHILAKLFPEYPVPTRCSDEVNPRKQPYKMSNQKL

QDLGLKFTPVNDSLYETVKSLQEKGHLPVPRKDILAPQLDGATA

SEQ ID NO: 14

Hordeum vulgare CCR3(Horvu_MOREX_7H01G166900)

MTVGGEATGHGQTVCVTGAGGYIGSWIVKLLLEKGYAVRGTVRNPDDAKNAHLRALAGAAERLVLCKADLLDADAL

RAAIAGCHGVFHTASPVTDDPEEMVEPAVRGTRYVIDAAAESGTVRRVVLTSSIGAVAMDPSRAPDAVVDESCWSD

LEFCKKTKNWYCYGKTVAEREAWEAAAARGVDLVVVNPVLVQGPALQPAVNASLTHVLKYLDGSAKTYANAVQAYV

HVRDTAAAHVLVFEAPAAAGRYLCVADGAVLHREDVVTILRKFFPEYPIPSRCSDSVNPRKRPYKMSNRRLRELGLEFT

PVAQCLYDTVVSFQEKGILPVPPAPAQPAMKEIN

SEQ ID NO: 6

Lolium perenne CCRl(V3.Lp_chr5_0G13010)

MTVVNTVTQQLPGHGQTVCVTGAAGYIASWLVKLLLERGYTVKGTVRNPDDPKNAHLKALDGAVERLILCKADLLDY

DAICAAAEGCHGVFHTASPVTDDPEQMVEPAVRGTEYVINAAADAGTVRRVVFTSSIGAVTMDPNRGPDVVVDESC

WSDLEFCKKTKNWYCYGKAVAEQAAWEAARKRGIDLVVVNPVLVVGPLLQPTVNASAAHILKYLDGSAKKYANAVQ

SYVDVRDVADAHIRVFEAPEASGRYLCAERVLHRGDVVQILGKLFPEYPVPTRCSDEVNPRKQPYKMSNQKLQDLGL

QFTPVNDSLYETVKSLQEKGHLLVPSKNIPEGLNGVTA

SEQ ID NO: 15

Lolium perenne CCR3(V3.Lp_chr7_0.1G5370)

RRLAHPLSLLFFSSLSPRRRPSSRLYKFRTTRTNIAQAFAFYDESYQTELPDPSLLIELPTLLRLSSFPPHRRPVLRSKLRPSV

HIYSIDMTIAEVVAAGDTAAAVVQPAGNGQTVCVTGAAGYIASWLVKLLLEKGYTVKGTVRNPDDPKNAHLRALDG

AADRLVLCKADLLDYDAIRRAIDGCHGVFHTASPVTDDPEQMVEPAVRGTQYVIDAAAEAGTVRRMVLTSSIGAVTM

DPNRGPDVVVDESCWSDLDFCKKTRNWYCYGKAVAEQAASELARQRGVDLVVVNPVLVIGPLLQPTVNASIGHILKY

LDGSASKFANAVQAYVDVRDVADAHLRVFECAAASGRHLCAERVLHREDVVRILAKLFPEYPVPTRCSDETNPRKQPY

KMSNQKLQDLGLEFRPVSQSLYETVKSLQEKGHLPVLSEQAEADKETLAAELQAGVTIRA

SEQ ID NO: 7

Miscanthus sinensis CCRl(Misin07G299100)

MTVVDAVSTDAGAPAAAAALVQQPAGNGQTVCVTGAAGYIASWLVKLLLQKGYTVKGTVRNPDDPKNAHLKALD

GAAERLILCKADLLDYDAICRAVQGCQGVFHTASPVTDDPEQMVEPAVRGTEYVINAASEAGTVRRVVFTSSIGAVT

MDPSRGPDVVVDESCWSDLEFCKKTRNWYCYGKAVAEQAAWDAGRQRGVDLVVVNPVLVVGPLLQPTVNASIAH VVKYLDGSARTFANAVQAYVDVRDVADAHLRVFESPRASGRYLCAERVLHREDVVRILAKLFPEYPVPTRCSDEVNPR

KQPYKLSNQKLRDLGLEFRPVSQSLYDTVKNLQEKGHLPVLGEQTTEADDKEAAPAAAELQQGGIAIRA

SEQ ID NO: 16

Miscanthus sinensis CCR3(Misinl8G071300)

MTVVGGDDAAAAPGRGQTVCVTGAGGYVGSWIVKLLLERGYAVRGTVRNPDDAKNAHLRALPGAAERLALCKADL

LDYDALRAAVAGCHGVFHTASPVTDDPEEMVEPAVTGTRYVIDAAAEAGTVRRVVLTSSIGAVAMDPNRAPDAVVD

ESCWSDLEFCKKTKNWYCYGKAVAEQAAWEAAAARGVDLVVVNPVLVQGPALQPTVNASLMHVLKYLNGSAKTYA

NAVQAYVHVRDAADAHVRVFEAPHAAGRYICADAVLHREDVVRTLRKFFPDYPVPERCSDEVNPRKQPYKISNQKLR

DLGLEFTPAAQALYDTVICFQEKGIIPIPAPTPSPEPDQA

SEQ ID NO: 8

Oryza sativa spp japonica CCRl(Os08g0441500)

MTVIDGAVAADAGGAAAAVVQPGNGQTVCVTGAAGYIASWLVKLLLEKGYTVKGTVRNPDDPKNAHLKALDGAGE

RLVLCKADLLDYDAICRAVAGCHGVFHTASPVTDDPEQMVEPAVRGTEYVINAAAEAGTVRRVVFTSSIGAVTMDPN

RGPDVVVDESCWSDLDYCKETRNWYCYGKAVAEQAAWEAARRRGVELVVVNPVLVIGPLLQPTVNASVAHILKYLD

GSASKFANAVQAYVDVRDVAAAHLLVFESPSAAGRFLCAESVLHREGVVRILAKLFPEYPVPTRCSDEKNPRKQPYKM

SNQKLRDLGLEFRPASQSLYETVKCLQEKGHLPVLAAEKTEEEAGEVQGGIAIRA

SEQ ID NO: 17

Oryza sativa spp japonica CCR3(Os09g0419200)

MTVVVVADDAAAAAAAAQQQEELPPGHGQTVCVTGAAGYIASWLVKLLLERGYTVKGTVRNPDDPKNAHLKALDG

ADERLVLCKADLLDYDSIRAAVDGCHGVFHTASPVTDDPEQMVEPAVRGTEYVIKAAAEAGTVRRVVFTSSIGAVTM

DPNRGPDVVVDESCWSDLEFCKKTKNWYCYGKAVAEQEACKAAEERGVDLVVVSPVLVVGPLLQPTVNASAVHILK

YLDGSAKKYANAVQAYVDVRDVAAAHVRVFEAPEASGRHLCAERVLHREDVVHILGKLFPEYPVPTRCSDEVNPRKQ

PYKMSNKKLQDLGLHFIPVSDSLYETVKSLQEKGHLPVLSKEIPEELNGVPA

SEQ ID NO: 9

Panicum hallii CCRl(Pahal.6G217000)

MTVIDAVSESAAAAAAVQQPAGNGQTVCVTGAAGYIASWLVKLLLEKGYTVKGTVRNPDDPKNAHLKAMDGAAER

LILCKADLLDYDAICRAVQGCQGVFHTASPVTDDPEQMVEPAVRGTEYVISAAAEAGTVRRVVFTSSIGAVTMDPNR

GPDVVVDESCWSDLDFCKKTRNWYCYGKAVAEQAAWEAARQRGVDLVVVNPVLVVGPLLQPTVNASIAHILKYLD

GSARTFTNAVQAYVDVRDVAAAHVRVFESPAASGRHLCAERVLHREDVVRILAKLFPEYPVPTRCSDEVNPRKQPYKF

SNQKLRDLGLEFRPVSQSLYDTVKSLQEKGHLPVLAEQTPEAEEEAAPAAEVQQGGIAIRA SEQ ID NO: 18

Panicum hallii CCR3(PahaL4G301100)

MTVVDGGGAAAEAAPGRGQTVCVTGAGGYIGSWIVKLLLERGYAVRGTVRNPDDAKNAHLRALPGAAERLALCRA

DLLDYEALRAAVAGCHGVFHTASPVTDDPEQMVEPAVRGTRHVIDAAAEAGTVRRVVLTSSIGAVAMDPGRAPDAV

VDESCWSDLDFCKSTRNWYCYGKAAAERAAWEAAAARGVDLVAVVPVLVQGPALQPAVNASLAHVLKYLDGSVAT

FANAVQAYVHVRDVADAHVRVFEAPGAAGRYLCADAVLHREDVVRTLRKFFPEYPVPERCSDEVNPRKQPYKISNHR

LRDLGLEFTPAAQALYETVVCFQEKGILPVPATATPSSPPSLP

SEQ ID NO: 10

Secale cereale CCR1(GWHGASIYO26344)

MTVVDAAATAAVAQELPGHGQTVCVTGAAGYIASWLVKLLLERGYTVKGTVRNPDDPKNAHLKVLDGAAERLVLCK

ADLLDYGAICAAVEGCHGVFHTASPVTDDPEQMVEPAVRGTEYVINAAADAGTVRRVVFTSSIGAVTM DPNRGPDV

VVDESCWSDLEFCKKTKNWYCYGKAVAEQAAWEKARARGVDLVVVNPVLVVGPLLQPTVNASAAHILKYLDGSAKK

YANAVQAYVDVRDVAAAHVRVFEAPGASGRHLCAERVLHREDVVHILAKLFPEYPVPTRCSDEVNPRKQPYKMSNQ

KLQDLGLQFTPVNDSLYETVRSLQEKGHLPAPRKDILPAELDGATA

SEQ ID NO: 19

Secale cereale CCR3(GWHGASIY021375)

MTVGGEAMATATATGHGQTVCVTGAGGYIGSWIVKLLLEKGYAVRGTVRNPDDAKNAHLRGLAGAAERLVLCKAD

LLDGDALRAAIAGCHGVFHTASPVTDDPEEMVEPAVRGTRYVIDAAAESGTVRRVVLTSSIGAVAMDPSRAPDAVVD

ESCWSDLEFCKKTKNWYCYGKTVAEREAWEAAAARGVDLVVVNPVLVQGPALQPAVNASLTHVLKYLDGSAKTYAN

AVQAYVHVRDTAAAHVLVFESPAAAGRYLCAADGAVLHREDVVTILRKFFPEYPIPSRCSDEVNPRKQPYKMSNQRLR

ELGLEFTPVAQCLYDTVVSFQEKGILPVPPAPAQPAMREIN

SEQ ID NO: 11

Setaria viridis CCRl(Sevir.6G169100)

MTVVDAVSAAAAAVVPPAGNGQTVCVTGAAGYIASWLVKLLLEKGYTVKGTVRNPDDPKNAHLKALDGAAERLVLC

KADLLDYDAICRAVQGCQGVFHTASPVTDDPEQMVEPAVRGTEYVLSAAAEAGTVRRVVFTSSIGAVTMDPNRGPD

VVVDESCWSDLDFCKKTRNWYCYGKAVAEQAAWDAARQRGVDLVVVNPVLVVGPLLQPTVNASIAHILKYLDGSA

RTFANAVQAYVDVRDVAAAHLAVFESPAASGRHLCAERVLHREDVVRILAKLFPEYPVPTRCSDETNPRKQPYKFSNQ

KLRDLGLEFRPVSQSLYDTVKSLQEKGHLPVLGDGEQTPEAEKEEQAPAATEVQQGGIAIRA

SEQ ID NO: 20

Setaria viridis CCR3(Sevir.4G046000) MTPVDGGDAAAAEEEVVPAAPPGCGQTVCVTGAGGYIGSWIVKLLLERGYAVRGTVRNPDDAKNAHLRALPGAAE

RLELCRADLLDYDAIRAAVAGCHGVFHTASPVTDDPEQMVEPAVRGTRHVIDAAAEAGGTVRRVVLTSSIGAVAMD

PNRAADAVVDESCWSDLDFCKATRNWYCYGKAAAEKAAWEAAAARGVDLVVVNPVLVQGPALQPAVNASLMHV

LKYLDGSVSTYANAVQAYVHVRDAADAHVRVFEAPGAAGRYLCADAVLHREDVVRTLRKFFPEYPVPERCSDEVNPR

KKPYKISNQRLRDLGLEFTPTAQALYETVICFQEKGILPVPAAAPALSSSPQP

SEQ ID NO: 12

Sorghum bicolor CCRl(Sobic.007G141200)

MTVVDAVSTDAAGAAPAAAAAPVVVAQPGNGQTVCVTGAAGYIASWLVKMLLEKGYTVKGTVRNPDDPKNAHLK

ALDGAAERLILCKADLLDYDAICRAVQGCQGVFHTASPVTDDPEQMVEPAVRGTEYVINAAAEAGTVRRVVFTSSIGA

VTM DPSRGPDVVVDESCWSDLEFCKKTRNWYCYGKAVAEQAAWDAARQRGVDLVVVNPVLVVGPLLQPTVNASI

AHVLKYLDGSARTFANAVQAYVDVRDVADAHLRVFESPAASGRYLCAERVLHREDVVRILAKLFPEYPVPTRCSDEVN

PRKQPYKFSNQKLRDLGLEFRPVSQSLYDTVKNLQEKGHLPVLGEQTTEADKEEANAAAEVQQGGIAIRA

SEQ ID NO: 21

Sorghum bicolor CCR3(Sobic.010G066000)

MTVVGGDDAAAAAAGRGQTTTVCVTGAGGYVGSWIVKLLLERGYAVRGTVRNPDDAKNAHLRALPGATERLALCK

ADLLDYDTLRAAIAGCHGVFHTASPVTDDPEEMVEPAVTGTRYIIDAAAEAGTVRRVVLTSSIGAVAMDPNRSPDAV

VDESCWSDLDFCKKTKNWYCYGKAVAEQAAWEEAAARGVDLVVVNPVLVQGPALQPSVNASLMHVLKYLNGSAK

TYANAVQAYVHVRDAADAHVRVFEAPHAAGRYICADGAVLHREDVVRTLRKFFPDYPVPERCSDEVNPRKQPYKISN

QKLRDLGLEFTPAAQALYDTVICFQEKGIIPIPAPTPSPSPEA

SEQ ID NO: 13

Triticum aestivum CCRl(TraesCS5D03G0540300)

MTVVAAAAAAAAQELPGHGQTVCVTGAAGYIASWLVKLLLERGYTVKGTVRNPGTHDPKNAHLKALDGAAERLVLC

KADLLDYDAICAAVEGCHGVFHTASPVTDDPEQMVEPAVRGTEYVINAAADAGTVRRVVFTSSIGAVTMDPNRGPD

VVVDESCWSDLEFCKKTKNWYCYGKAVAEQAAWEKAAARGVDLVVVNPVLVVGPLLQPTVNASAAHILKYLDGSAK

KYANAVQAYVDVRDVAAAHVRVFEAPGASGRHLCAERVLHREDVVHILGKLFPEYPVPTRCSDEVNPRKQPYKMSN

QKLQDLGLQFTPVNDSLYETVKSLQEKGHLPAPRKDILPAELDGATA

SEQ ID NO: 22

Triticum aestivum CCR3(TraesCS7D03G0337500)

MTVGGEATGHGQTVCVTGAGGYIGSWIVKLLLEKGYAVRGTVRNPDDAKNAHLRGLAGAAERLVLCKADLLDGDAL

RAAIAGCHGVFHTASPVTDDPEEMVEPAVRGTRYVIDAAAESSTVRRVVLTSSIGAVAMDPSRAPDAVVDESCWSDL EFCKKTKNWYCYGKTVAEREAWEAAAARGVDLVVVNPVLVQGPALQPAVNASLTHVLKYLDGSAKTYANAVQAYV

HVRDTAAAHVLVFESPAAAGRYLCVADGAVLHREDVVTILRKFFPEYPIPSRCSDEVNPRKQPYKMSNQRLRELGLEFT

PVAQCLYDTVVSFQEKGVLPAPPAPAQPATKEIN SEQ ID NO: 23

Conserved signature sequence typical for CCR sequences

NWYCYG

SEQ ID NO: 24

Conserved signature for CCR1 sequences DAICX1AX2, wherein Xi is R or A, X2 is V or A

SEQ ID NO: 25

Conserved signature for CCR3 sequences

DAX1RX2AX3, wherein Xi is L or I, X2 is A or R and X3 is I or V

References

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Claims

Claims

1. A silage plant having a combined gene disruption in a polynucleotide encoding a cinnamoyl-CoA- reductase 1 (CCR1) polypeptide and in a polynucleotide encoding a cinnaomoyl-CoA-reductase 3 (CCR3) polypeptide.

2. A silage plant according to claim 1 wherein said CCR1 polypeptide is depicted in SEQ ID NO: 1 or is a plant orthologous CCR1 polypeptide of SEQ ID NO: 1 comprising SEQ ID NO: 23 and SEQ ID NO: 24 and wherein said CCR3 polypeptide is depicted in SEQ ID NO: 2 or a plant orthologous CRR3 polypeptide of SEQ ID NO: 2 comprising SEQ ID NO: 23 and SEQ ID NO: 25.

3. A plant according to claim 2 wherein the plant orthologous polypeptide sequence of SEQ ID NO: 1 is depicted in SEQ ID NO: 5, 6, 7 , 8, 9, 10, 11, 12 or 13 and wherein the plant orthologous polypeptide sequence of SEQ ID NO: 2 is depicted in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21 or 22.

4. A silage plant according to any one of claims 1 to 3 wherein the plant is a grass such as Lolium, or clover, alfalfa, corn, oats, rye or vetches.

5. A seed or plant cell derived from a plant according to any one of claims 1 to 4.

6. A method for increasing digestibility of a silage plant, the method comprising disrupting a polynucleotide encoding a cinnamoyl-CoA-reductase 1 (CCR1) polypeptide and a polynucleotide encoding a cinnaomoyl-CoA-reductase 3 (CCR3) polypeptide.

7. A method according to claim 6 wherein the CCR1 disruption is a polynucleotide encoding SEQ ID NO: 1 or a polynucleotide encoding a plant orthologous sequence of SEQ ID NO: 1 and wherein the CCR3 disruption is a polynucleotide encoding SEQ ID NO: 2 or a polynucleotide encoding a plant orthologous sequence of SEQ ID NO: 2.

8. The method according to claims 6 or 7 wherein the polynucleotide encodes a CCR1 polypeptide sequence comprising SEQ ID NO: 23 and 24 and wherein the polynucleotide encodes a CCR3 polypeptide sequence comprising SEQ ID NO: 23 and 25.

9. The method according to any one of claims 6 to 8 wherein the polynucleotide encodes a CCR1 polypeptide sequence depicted in SEQ ID NO: 5, 6, 7, 8, 9, 10, 11, 12 or 13 and wherein the polypeptide encodes a CCR3 polypeptide depicted in SEQ ID NO: 14, 15, 16, 17, 18, 19, 20, 21 or 22.

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