Peptidase compositions, methods, and uses for viral disease resistance in plants

Abstract

Provided are plants, cells, tissues, and germplasm thereof comprising one or more targeted alterations of sequences encoding a peptidase associated with increased resistance to viral plant disease. Also provided are methods and compositions to make peptidase gene variants, as well as breeding methods and methods of identifying and selecting plant materials having peptidase gene variants. Further provided are methods of improving resistance to maize lethal necrosis (MLN) and / or providing increased resistance to maize chlorotic mottle virus (MCMV), tombusvirus, or other viruses in maize and other plant species.

Description

PEPTIDASE COMPOSITIONS, METHODS, AND USES FOR VIRAL DISEASE RESISTANCE IN PLANTS REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0001] The official copy of the sequence listing is submitted electronically as an xml-formatted sequence listing file named 315085-US-PRV-l-ST26.xml created on January 24, 2025 and having a size of 97,589 bytes which is filed concurrently with the specification. The sequence listing comprised in this xml-formatted document is part of the specification and is herein incorporated by reference in its entirety.FIELD OF THE DISCLOSURE

[0002] The disclosure relates to compositions, polynucleotides, constructs, plants, plant breeding and methods for creating plants with resistance to viral diseases, including maize lethal necrosis (MLN). The disclosed polynucleotides, constructs and molecular tools are useful in the production of disease resistant plants through breeding, transgenic modification, and / or genome editing.BACKGROUND

[0003] Diseases and environmental stresses suppress crop yields below their potential levels. Narrowing this gap has been, and will continue to be, the target of plant scientists to ensure food security, particularly in food-insecure regions (Duvick, 2005, Advances in Agronomy 86, 83-1452005; Wulff and Dhugga, 2018, Science 361, 451-452).

[0004] Maize lethal necrosis (MLN), a viral disease, emerged in Kenya in 2011 and quickly spread to the surrounding countries. It significantly reduces grain yield, on average by 25%; though the loss can be complete at farms with high disease pressure (De Groote et al., 2016, Crop Protect.82, 30-35). Nearly all commercial germplasm was susceptible when the MLN disease initially broke out (Boddupalli et al., 2020, Virus Res. 282, 197943). Some hybrids with varying levels of resistance have since been developed through conventional breeding but the disease continues to pose a serious challenge to maize production in Eastern Africa. Sub-Saharan Africa (SSA), where maize is a staple crop, already has the lowest grain yield in the world (Erenstein et al., 2022). MLN has thus further exacerbated food insecurity in this region.

[0005] Maize chlorotic mottle virus (MCMV) along with any of the potyviruses (commonly sugarcane mosaic virus (SCMV)) causes MLN (Boddupalli et al., 2020). Although several QTLs are known for SCMV resistance, they are not effective against MLN, as none of these QTLseliminates SCMV from the plant. If SCMV is present, infection by MCMV leads to development of MLN. Resistance against MCMV, as well as against poty viruses such as SCMV, is thus required for MLN resistance (Carino et al., 2020).

[0006] Few natural sources for MLN resistance are known. An exotic maize line, KS23-6, developed at Kasetsart University in Thailand, exhibits strong resistance to MLN. This resistance is attributed to a single QTL located on chromosome 6 (C6QTL) (Murithi et al., 2021, Fronters in Genetics 12, 767883). The C6QTL was introgressed into a diverse range of inbred lines, which were subsequently made available to breeders (Awata et al., 2021, Frontiers in Plant Science 12, 699). However, incorporating resistance from exotic sources is a resource-intensive process that requires many years to complete and often results in the retention of undesirable genes from the donor parent in the final product (Dhugga, 2022, Frontiers in Plant Science 13, 889995). Developing a method to overcome these challenges would significantly accelerate the creation of MLN-resistant maize lines.

[0007] There is a need to identify genes and gene editing technologies that can help quickly introduce MLN resistance directly in elite lines and avoid more time consuming and resource intensive breeding strategies.SUMMARY OF THE DISCLOSURE

[0008] The disclosed compositions and methods are based, at least in part, on the discovery that variants of a maize gene annotated as a peptidase (hereafter the peptidase, disclosed peptidase, or peptidase disclosed herein) provide effective disease resistance against maize lethal necrosis (MLN) and can provide resistance against maize chlorotic mottle virus (MCMV) infection, which is a Tombusvirus. Additional disclosed compositions and methods are based on further discovery that there are orthologs of the peptidase disclosed in other plant species, including those that were screened and disclosed herein, indicating that variants of such peptidase orthologs can be used to provide disease resistance against viruses (e.g., Tombusviruses) in multiple plant species.

[0009] The term “altered” or “variant” used in connection with a peptidase gene (or coding sequence) herein refers to a peptidase gene (or coding sequence)that differs from the wild-type version of the gene (or the coding sequence) that encodes the peptidase in susceptible plants. A wild type version of the gene is hereinafter referred to as the “base peptidase gene”, and it encodes a wild type peptidase or a “base peptidase” in susceptible plants. Thus, the term “altered” or “variant” used in connection with a peptidase refers to a peptidase that differs from the basepeptidase in susceptible plants (the base peptidase encoded by the base peptidase gene). In some examples, the base peptidase gene encodes a base peptidase that comprises a canonical or non-canonical peroxisomal targeting signal (PTS) and / or the base peptidase localizes to the peroxisome of the plant cell. In particular examples, the base peptidase comprises one or more of the of the following targeting signal sequences: SKI, SKL, SRL, AKL, SRM, SRI, SSL, SKM, ARL, PRL, SNL, PKL, SYM, ASL, SML, SFM, SNM, CKL, SGL, SRV, TKL, AKI, STL, SAL, SHL, ALL, SSM, SLM, PSL, ARM, SLL, SNI, SCI, AKM, ANL, SFL, SKF, SYI, SQL, PRM, or CRL. The term peptidase is used herein without regards to any particular protease activity or level of proteolytic activity.

[0010] The altered peptidase gene (peptidase gene variant) or variant peptidase disclosed herein is an engineered variant, deliberately created by genome editing (e.g., with Cas protein such as Cas9, Cas 12 or another Cas) or transgenic modification.

[0011] An example of such altered peptidase variants provided herein are G250 variants. “G250 variant” is a variant of a base peptidase that comprises a glycine at the amino acid position corresponding to position 250 of SEQ ID NO:7, when the base peptidase sequence is aligned with SEQ ID NO:7. A G250 variant differs from the base peptidase in that the variant comprises an amino acid other than glycine (e.g., a substitution or deletion) at the position corresponding to position 250 of SEQ ID NOY. Also provided are genomic or cDNA sequences that encode a G250 variant.

[0012] Another example of such altered peptidase variants provided herein is a “truncation variant”, which are variants comprising a C-terminal truncation of the base peptidase. In particular examples, a truncation variant is encoded by a variant peptidase gene that comprises a premature stop codon or a missense or frameshift mutation at the codon corresponding to the codon encoding any one of positions 250-450 of SEQ ID NOY. In some examples, a truncation variant lacks at least 10%, 20%, 30%, 40%, 50%, 60% or 65% of the C-terminal amino acid sequence of the base peptidase. For example, a truncation variant can comprise 90%, 80%, 70%, 60%, 50%, 40%, 35% or less of it’s the base peptidase N-terminal sequence and lack 10%, 20%, 30%, 40%, 50%, 60%, 65%, or more, respectively, of its C-terminal amino acid sequence relative to the base peptidase. Also provided are genomic or cDNA sequences that encode a truncation variant.

[0013] In other examples, such an altered peptidase gene variant can be a gene deletion variant or an inactivated expression variant. A “gene deletion variant” is a deletion of genomic sequencecomprising sequence encoding the base peptidase. An “inactivated expression variant” refers to genomic sequence which has been altered (e.g., by deleting or substituting genomic sequences) to remove sequences required for normal expression of the base peptidase. Accordingly, plants comprising a gene deletion variant or an inactivated expression variant do not express the base peptidase or express reduced levels of the base peptidase as compared to plants comprising a base peptidase gene.

[0014] In a first aspect, provided is a method of altering a virus susceptible plant, cell, seed, tissue or germplasm thereof (e.g., the plant is susceptible to MLN and / or to MCMV, a tombusvirus, or another virus). The method comprises altering a base peptidase gene (e.g., its endogenous base peptidase gene) encoding a base peptidase in the susceptible plant, cell, seed, tissue or germplasm genome to thereby produce an altered plant, cell, seed, tissue or germplasm comprising an altered gene associated with improved resistance to viral disease relative to the susceptible plant, cell, seed, tissue or germplasm thereof. The improved resistance can be resistance to diseases (at least partly) caused by a tombusvirus such as MCMV. In some examples of the first aspect, the method comprises identifying a base peptidase gene in the susceptible plant that encodes a base peptidase comprising a canonical or non-canonical peroxisomal targeting signal (PTS) (e.g., SKI or another PTS disclosed herein); then altering the base peptidase gene to (i) encode a G250 variant, (ii) encode a truncation variant, (iii) create a gene deletion variant or (iv) create an inactivated expression variant as disclosed in paragraph

[0013] herein. In particular examples of the first aspect, the glycine at the base peptidase position corresponding to position 250 of SEQ ID NO:7 is replaced with a different amino acid e g., with aspartate. In other examples, the base peptidase glycine at position corresponding to position 250 of SEQ ID NO:7 is replaced with glutamate (another negatively charged amino acid) or with a positively charged amino acid such as arginine, histidine, or lysine. In particular examples of the first aspect involving a truncation variant, the base peptidase gene is altered to encode a variant peptidase that differs from the C-terminal portion of the base peptidase beginning at amino acid positions corresponding to one or more of positions 289, 290, or 291 of SEQ ID NO:7. In each of the foregoing examples of the first aspect, the base peptidase gene encodes a base peptidase that comprises at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs:25-38.

[0015] In one embodiment of the first aspect, the disclosed method comprises altering the genome of the plant, cell, seed, tissue or germplasm thereof by altering one allele of itsendogenous base peptidase gene. In another embodiment of the second aspect, the alterations are homozygous so that both copies of the base peptidase gene are altered in the plant, cell, seed, tissue or germplasm thereof.

[0016] In a second aspect, provided herein is a method of altering viral disease susceptible corn plant, cell, seed, tissue or germplasm thereof. The method comprises altering a base peptidase gene (e.g., its endogenous base peptidase gene) encoding a base peptidase in the susceptible com plant, cell, seed, tissue or germplasm genome to thereby produce an altered com plant, cell, seed, tissue or germplasm comprising an altered peptidase gene associated with improved resistance to viral disease relative to the susceptible corn plant, cell, seed, tissue or germplasm thereof. The improved resistance can be resistance to diseases (at least partly) caused by a tombusvirus such as MCMV, e.g., improved resistance to MLN. In some examples of the second aspect, the method comprises altering a base peptidase gene comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO:7, such that the altered base peptidase gene (i) encodes a G250 variant, (ii) encodes a truncation variant, (iii) is a gene deletion variant or (iv) is an inactivated expression variant as disclosed in paragraph

[0013] herein. In particular examples of this method of altering a base peptidase gene in corn, the glycine at the base peptidase position corresponding to position 250 of SEQ ID NO:7 is replaced with a different amino acid e.g., with aspartate. In other examples of this method of altering a base peptidase gene in com, the base peptidase glycine at position corresponding to position 250 of SEQ ID NO:7 is replaced with glutamate (another negatively charged amino acid) or with a positively charged amino acid such as arginine, histidine, or lysine. In particular examples of this method of altering a base peptidase gene in corn, the base peptidase gene is altered to encode a variant peptidase that differs from the C-terminal portion of the base peptidase beginning at amino acid positions corresponding to one or more of positions 289, 290, or 291 of SEQ ID NO:7. In each of the foregoing examples of a method of altering a base peptidase gene in corn, the base peptidase gene is a coding sequence that comprises at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 3, 6, 9, 12, 15, or 18. Alternatively, in each of the foregoing examples of a method of altering a base peptidase gene in corn, the base peptidase gene is a genomic sequence that comprises at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 2, 5, 8, 11, 14, or 17.

[0017] In one embodiment of the second aspect, the disclosed method comprises altering the genome of the corn plant, cell, seed, tissue or germplasm thereof by altering one allele of its endogenous base peptidase gene. In another embodiment of the second aspect, the alterations are homozygous so that both copies of the base peptidase gene are altered in the com plant, cell, seed, tissue or germplasm thereof.

[0018] In particular examples of each of the foregoing first and second aspects, the method involves altering the genome of a plant, cell, seed, tissue or germplasm thereof that is susceptible to MLN. The method can thereby generate an altered plant, cell, seed, tissue or germplasm thereof that is (i) more resistant to viral disease (e.g., tombusvirus and / or MLN) than the susceptible plant material was prior to the alteration and / or (ii) useful for introducing altered base peptidase gene alleles in a breeding program to improve resistance to viral disease (e.g., tombusvirus and / or MLN) in the breeding population.

[0019] Plants that are susceptible to tombusviruses, MLN and other viral diseases include, but are not necessarily limited to maize, Cucurbitaceae (e.g., squash, pumpkin, zucchini, calabash, watermelon, cucumber, and melons), stone fruit trees (e.g., plum, peach, cherry, apricots, nectarines, mango, olive, lychee, and coconut trees) pome trees (e.g., apple, pear, and quince trees), soybean, oilseed rape (Brassica napus), sunflower, wheat, or cotton.

[0020] In each of the foregoing first aspect method and second aspect method, the step of altering a plant, cell, seed, tissue or germplasm genome can be accomplished by mutagenesis, use of double-strand-break inducing agents (DSB Agents), base editing and the like. For example, base peptidase gene alterations can be induced by genome editing, e.g., with a Cas endonuclease. Therefore techniques such as gene editing or base editing make it possible to alter a peptidase base gene.

[0021] In a third aspect, provided herein is plant (other than maize), cell, seed, tissue or germplasm thereof comprising peptidase gene variant that (i) encodes a G250 variant, (ii) encodes a truncation variant, (iii) is a gene deletion variant or (iv) is an inactivated expression variant as disclosed in paragraph

[0013] herein. In particular examples of the third aspect, the glycine at the base peptidase position corresponding to position 250 of SEQ ID NO:7 is replaced with a different amino acid e.g., with aspartate. In other examples, the base peptidase glycine at position corresponding to position 250 of SEQ ID NO:7 is replaced with glutamate (another negatively charged amino acid) or with a positively charged amino acid such as arginine, histidine, or lysine.In particular examples of the first aspect involving a truncation variant, the base peptidase gene is altered to encode a variant peptidase that differs from the C-terminal portion of the base peptidase beginning at amino acid positions corresponding to one or more of positions 289, 290, or 291 of SEQ ID NO:7. In each of the foregoing examples of the first aspect, the base peptidase gene encodes a base peptidase that comprises at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs:25-38.

[0022] In a fourth aspect, provided herein is an altered maize plant, cell, seed, tissue or germplasm comprising an altered base peptidase gene comprising at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 7, such that the altered base peptidase gene (i) encodes a G250 variant, (ii) encodes a truncation variant, (iii) is a gene deletion variant or (iv) is an inactivated expression variant as disclosed in paragraph

[0013] herein. In particular examples of this fourth aspect, the glycine at the base peptidase position corresponding to position 250 of SEQ ID NO:7 has been replaced with a different amino acid e.g., with aspartate. In other examples of this fourth aspect, the base peptidase glycine at position corresponding to position 250 of SEQ ID NO:7 is replaced with glutamate (another negatively charged amino acid) or with a positively charged amino acid such as arginine, histidine, or lysine. In particular examples of this fourth aspect, the base peptidase gene is altered to encode a variant peptidase that differs from the C-terminal portion of the base peptidase beginning at amino acid positions corresponding to one or more of positions 289, 290, or 291 of SEQ ID NO:7. In each of the foregoing examples of the fourth aspect, the base peptidase gene is a coding sequence that comprises at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 3, 6, 9, 12, 15, or 18. Alternatively, in each of the foregoing examples of the fourth aspect, the base peptidase gene is a genomic sequence that comprises at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 2, 5, 8, 11, 14, or 17..

[0023] In each of the foregoing third and fourth aspect, the plant, cell, seed, tissue or germplasm comprises one altered allele of its endogenous base peptidase gene. In another embodiment of the second aspect, the alterations are homozygous so that both copies of the base peptidase gene are altered in the plant, cell, seed, tissue or germplasm thereof.

[0024] In a fifth aspect, provided herein is a method of identifying a plant, seed, tissue or germplasm thereof comprising a peptidase gene variant associated with increased resistance to MLN and / or increased resistance to maize chlorotic mottle virus (MCMV), a tombusvirus, or other viruses. The method comprises providing one or more plant, seed, tissue or germplasm thereof comprising an base peptidase gene variant; obtaining a sample comprising nucleic acid from each of the plant, seed, tissue or germplasm thereof; screening the sample for the peptidase gene variant; and detecting the one or more peptidase gene variant in the sample and thereby identifying the plant or plant material as comprising a peptidase gene variant associated with increased resistance to MLN and / or increased resistance to maize chlorotic mottle virus (MCMV), a tombusvirus, or another virus. In one embodiment of this fifth aspect, screening can include screening the sample for one or more of the following peptidase gene variants:i. variant genomic sequence or cDNA sequence encoding a peptidase variant that comprises an amino acid other than glycine at the position corresponding to position 250 of SEQ ID NO:7, when the peptidase variant sequence is aligned with SEQ ID NO:7,ii. variant genomic sequence or cDNA sequence encoding a peptidase variant that comprises an insertion, deletion, substitution, or stop codon relative to the base peptidase and thereby encodes an variant peptidase comprising an altered C-terminal portion relative to the base peptidase beginning at amino acid positions corresponding to one or more of positions 289, 290, or 291 of SEQ IDNO:7,iii. variant genomic sequence or cDNA sequence encoding a peptidase variant that is truncated and comprises a premature stop codon at the codon corresponding to the codon encoding any one of positions 250-450 of SEQ ID NO: 7,iv. variant genomic sequence or cDNA sequence encoding a peptidase variant that is truncated and comprises a deletion of at least 10%, 20%, 30%, 40%, 50%, 60% or 65% of the C-terminal amino acid sequence of the base peptidase,v. variant genomic sequence comprising a deletion of genomic sequence (x’) comprising the base gene coding sequence for the base peptidase, (y’)required to express the base peptidase, or (z) express the base peptidase at wild type level,vi. any of the foregoing i.-v., wherein the base peptidase comprises at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 7,vii. any of the foregoing i.-v., wherein the base gene coding sequence comprises at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 3, 6, 9, 12, 15, or 18,viii. any of the foregoing i.-v., wherein the base gene genomic sequence comprises at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 2, 5, 8, 11, 14, or 17,ix. any of the foregoing i.-v., wherein base peptidase comprises at least 40%, at least 50% at least 60% or at least 70% sequence identity to SEQ ID NO: 7, orx. any of the foregoing i.-v., wherein the base peptidase comprises at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs:25-37, andUpon detecting one or more of the foregoing peptidase gene variant sequences in the sample, the corresponding plant or plant material can be identified as comprising a peptidase gene variant associated with increased resistance to MLN and / or increased resistance to MCMV, a tombusvirus, or another virus.

[0025] In a sixth aspect, provided herein is a method of improving resistance to MLN and / or to MCMV, a tombusvirus, or another virus. The method comprises using the method of the fifth aspect disclosed herein to identify a first parent plant as comprising a peptidase gene variant associated with increased resistance to MLN and / or to MCMV, a tombusvirus, or another virus; and crossing the identified first parent plant with a second parent plant (that does not include the peptidase gene variant) to produce one or more progeny plants comprising the peptidase gene variant. In the method of this sixth aspect, the second parent plant is susceptible to MLN (and / or to MCMV, a tombusvirus, or another virus) and preferably the progeny plants are more resistantto MLN (and / or to MCMV, a tom busvirus, or another virus) than the second parent plant. It should be noted, however, that even if progeny plants are not themselves, significantly more resistant to virus than the second parent plant, such progeny plants can still be useful for introducing peptidase gene variant alleles into a breeding program for the purpose of improving resistance to viral disease (e g., tombusvirus, MCMV and / or MLN) in the breeding population.

[0026] In one embodiment of the sixth aspect, the first parent plant comprises a variant peptidase gene that (i) encodes a G250 variant, (ii) encodes a truncation variant, (iii) is a gene deletion variant or (iv) is an inactivated expression variant as disclosed in paragraph

[0013] herein. In particular examples, the variant peptidase gene encodes a variant peptidase in which the glycine at the base peptidase position corresponding to position 250 of SEQ ID NO:7 is replaced with a different amino acid e.g., with aspartate. In other examples, the variant peptidase gene encodes a variant peptidase in which the glycine at position corresponding to position 250 of SEQ ID NO:7 is replaced with glutamate (another negatively charged amino acid) or with a positively charged amino acid such as arginine, histidine, or lysine. In particular examples of this embodiment of the sixth aspect, the base peptidase gene encodes a variant peptidase that differs from the C-terminal portion of the base peptidase beginning at amino acid positions corresponding to one or more of positions 289, 290, or 291 of SEQ ID NO:7. In each of the foregoing examples the variant peptidase gene can be a variant of a base peptidase gene coding sequence that comprises at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 3, 6, 9, 12, 15, or 18. Alternatively, in each of the foregoing examples of this embodiment of the sixth aspect, the variant peptidase gene can be a variant of a base peptidase gene genomic sequence that comprises at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 2, 5, 8, 11, 14, or 17. In this embodiment, the resulting progeny plant with peptidase gene variant can be more resistant to MLN (and / or to MCMV, a tombusvirus, or another virus) than the second parent plant.

[0027] In certain embodiments of the sixth aspect, the foregoing method can further comprise crossing one or more of the progeny plants to the second parent plant to thereby produce backcross progeny plants comprising the peptidase gene variant from the first parent plant. In particular examples, one or more backcross progeny plants comprising the peptidase gene variant can be crossed with the second parent plant to produce next generation backcross progeny plants comprising the peptidase gene variant. This backcrossing scheme can be repeated to generateadditional plants that introgress the peptidase gene variant into the genetic background of the second parent plant and thereby produce new plants that include the peptidase gene variant but are otherwise substantially isogenic to the second parent plant and include desirable agronomic properties of the second parent plant.

[0028] In each embodiment and example of the foregoing first aspect, second aspect, third aspect, fourth aspect, fifth aspect, sixth aspect, the plant, plant, cell, seed, tissue or germplasm thereof is preferably a reference to a plant or plant material. Alternatively it can also refer to plant or plant material of Cucurbitaceae (e.g., squash, pumpkin, zucchini, calabash, watermelon, cucumber, and melons), stone fruit tree (e.g., plum, peach, cherry, apricots, nectarines, mango, olive, lychee, and coconut trees) or pome tree (e.g., apple, pear, and quince trees). In other alternatives, it can refer to soybean, oilseed rape (Brassica napus sunflower, wheat, or cotton.

[0029] Provided herein is an isolated recombinant nucleic acid encoding one or more of SEQ ID NOs: 1, 4, 7, 10, 13, or 16. Also provided herein is an isolated recombinant nucleic acid comprising one or more of SEQ ID NOs: 5, 6, 8, 9, 11, 12, 14, 15, 17, or 19. In some examples, the isolated recombinant nucleic acid is a gene editing construct comprising one or more guide RNA sequences (e.g., one or more of SEQ ID NOs:20, 21, 23, or 24) for introducing a peptidase gene variant disclosed herein.

[0030] Methods for introducing Cas endonucleases and guide RNAs are described in more detail herein.BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTING

[0031] Figure 1 is a series of schematic diagrams showing the multi-year process of fine mapping the locus for MLN resistance in maize line KS23 Chromosome 6. Figure 1 A shows how the original 137-170 Mb region of Chr 6 known to confer MLN resistance was progressively narrowed by analysis of segregating markers in mapping populations obtained from crosses with susceptible lines: first it was narrowed to a 2.3 Mb region in 2017 using markers M147 and M373 in populations from crosses with CML548 and CML537, then narrowed to a 218 kb region based on markers M165 and M170 in in populations from crosses with CML548 and CKDHL0186, and then further refined to a 105 kb region based on markers M167 and M343 in populations from crosses with CKDHL0186 and CKDHL0221; Figure IB shows the four genes (and other gene elements) in the 105 kb region as well as the relative positions of gene editing target sites for guide RNAs (gRNAs) CR8, CR2, CR3, CR4, CR13, and CR 14 which were used to selectively inactivateone or more of the four genes; Figure 1 C shows the further fine mapping to a 33 kb region and this region contained only the peptidase gene; Figure ID shows the 5’- and 3 ’ -untranslated regions (UTRs), introns, and exons of the peptidase gene and also shows portions of peptidase coding sequences of CML536 and KS23-6 (positions 724-774 of SEQ ID NO:6 and SEQ ID NO:3, respectively) as amino acid sequences encoded thereby (positions 242-258 of SEQ ID NO:4 and SEQ ID NO:1, respectively).

[0032] Figure 2 is an alignment of genomic sequences from genome edited Variantsl-3 with sequences of viral disease susceptible line CML536 (corresponding to positions 3709-3760 of SEQ ID NO:5, dashes indicate deleted nucleotides in each of Variants 1-3); Figure 2 also shows target sequence of gRNA CR8 (corresponding to positions 3722-3739 of SEQ ID NO: 5), PAM sequence (underlined) used for genome editing, as well as nucleotides of Intron 9 and Exon 10 in CML536.

[0033] Figure 3 A shows the target sequences of gRNAs CR13 and CR14 and respective PAM sequences (underlined) used to generate the targeted deletion of 39,885 bp within the 105 kb region of Chromosome 6 (shown in Fig. IB) containing ribosomal LI and helicase genes; Figure 3B shows the target sequences of gRNAs CR3 and CR4 and respective PAM sequences (underlined) used to generate targeted deletion of 2,907 bp within the 105 kb region of Chromosome 6 (shown in Fig. IB) containing eIF4A gene; Figure 3C shows the target sequences of gRNA CR2 and PAM sequence (underlined) used to generate targeted deletion in 5’UTR of the eIF4A gene.

[0034] Figure 4 is a bar graph showing viral disease performance in greenhouse assays of disease resistant control line (KS23-6); disease susceptible control line CML536; genome edited Variant 1, Variant 2, and Variant 3 (each line deleted for the peptidase); CR2 variant and CR3+CR4 variant (each line a deleted variant of elFlA); and CR13+CR14 variant (~40 kb deletion of region including Ribosomal LI and Helicase genes). For each indicated plant line, parenthetical numbers indicate number of screened plants, and each bar indicates MLN disease score assessed at each of the following time points after virus inoculation (starting closest to y-axis): 8 days, 12 days, 16 days, 22 days, and 26 days after inoculation.

[0035] Figure 5 shows consensus sequence (SEQ ID NO:7) for the maize peptidase disclosed herein; the bold G at amino acid position 250 is altered to a D in disease resistant lines KS23 and lines derived therefrom; the three amino acids at positions 289-291 which are deleted in Variant 3 are shown in bold and underlined; and C-terminal tripeptide signal (SKI) is underlined.

[0036] Figure 6 comprises two panels: the upper panel shows phylogenetic analysis of the peptidase disclosed herein in a wide range of plants, including monocots, dicots, magnoliids, and others; the lower panel shows an alignment of peptidase sequences disclosed herein and indicates (arrow) the glycine corresponding to position 250 of SEQ ID NO:7, which is found in all plant versions of the peptides disclosed herein and which provides disease resistance when altered in maize. In addition to the maize sequences disclosed herein, also disclosed is the corresponding peptidase from a wide range of other plants including sorghum, rice, barley, wheat, soybean banana, pineapple, asparagus, camphor, nelumbo, macleaya, tetracentron, peanut, chickpea, common bean, oak, citrus, and poplar.

[0037] Figure 7 is a bar graph showing grain yield of field trials of the indicated elite maize inbred lines, genome edited Variant 1 and Variant 2 (edited peptidase), as well as two Null lines that underwent genome-editing process but did not contain edited peptidase. Grain was harvested at crop maturity after controlled, artificial viral inoculation at MLN screening facility in Naivasha, Kenya.

[0038] Figure 8 is a bar graph showing grain yield of field trials in Kiboko, Kenya of the indicated elite maize inbred lines, genome edited Variant 1 and Variant 2 (edited peptidase), as well as two Null lines that underwent genome-editing process but did not contain edited peptidase. Trials at Kiboko provided optimal growing conditions to assess agronomic performance in the absence of disease pressure.

[0039] Figure 9 is a bar graph showing the effect of genome edited peptidase on virus replication; for each indicated maize line, the first bar (closer to y-axis) indicates the relative amount of SCMV and second bar indicates relative amount of MCMV - both assessed two weeks after inoculation with MLN virus. The bar graph shows that MCMV replication in KS23 and genome edited variants was negligible.

[0040] Figure 10 is a schematic diagram showing the range of structural variability (darker carats indicate insertions and lighter carats indicate deletions) revealed by sequencing the peptidase genomic region of indicated lines.

[0041] Nucleic acid sequences listed in the accompanying sequence listing and referenced herein are shown using standard letter abbreviations for nucleotide bases. While only one strand of each nucleic acid sequence is shown, the complementary strand is understood to be included in any reference to the displayed strand. Sequence listings are described in the following Table 1.Table 1DETAILED DESCRIPTION

[0042] As used herein the singular forms “a”, “and”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a cell” includes a plurality of such cells and reference to “the protein” includes reference to one or more proteins andequivalents thereof, and so forth. All technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this disclosure belongs unless clearly indicated otherwise.

[0043] As used herein, the term “maize” means Zea mays or corn and includes all plant varieties that can be bred with corn, including wild maize species.

[0044] A gene or allele is “associated with” a trait when it is part of or linked to a DNA sequence or allele that affects the expression of the trait. The presence of the allele is an indicator of how the trait will be expressed.

[0045] “Backcrossing” refers to the process whereby hybrid progeny are repeatedly crossed back to one of the parents. In a backcrossing scheme, the “donor” parent refers to the parental plant with the desired gene or locus to be introgressed. The “recipient” parent (used one or more times) or “recurrent” parent (used two or more times) refers to the parental plant into which the gene or locus is being introgressed.

[0046] “CRISPR” (Clustered Regularly Interspaced Short Palindromic Repeats) loci refers to certain genetic loci encoding components of DNA cleavage systems, for example, used by bacterial and archaeal cells to destroy foreign DNA (Horvath and Barrangou, 2010, Science 327:167-170; International Application Publication W02007 / 025097, published 01 March 2007). A CRISPR locus can consist of a CRISPR array, comprising short direct repeats (CRISPR repeats) separated by short variable DNA sequences (called spacers), which can be flanked by diverse Cas (CRISPR-associated) genes.

[0047] The term “Cas protein” refers to a polypeptide encoded by a Cas (CRISPR-associated) gene. A Cas protein includes but is not limited to: a Cas9 protein, a Cpfl (Casl2) protein, a C2cl protein, a C2c2 protein, a C2c3 protein, Cas3, Cas3-HD, Cas 5, Cas7, Cas8, CaslO, or combinations or complexes of these. A Cas protein may be a “Cas endonuclease” or “Cas effector protein”, that when in complex with a suitable polynucleotide component, is capable of recognizing, binding to, and optionally nicking or cleaving all or part of a specific polynucleotide target sequence. A Cas endonuclease described herein comprises one or more nuclease domains. The endonucleases of the disclosure may include those having one or more RuvC nuclease domains. A Cas protein is further defined as a functional fragment or functional variant of a native Cas protein, or a protein that shares at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%,at least 98%, at least 99%, or 100% sequence identity with a native Cas protein, and retains at least partial activity.

[0048] A “Cas endonuclease” may comprise domains that enable it to function as a doublestrand-break-inducing agent. A “Cas endonuclease” may also comprise one or more modifications or mutations that abolish or reduce its ability to cleave a double-strand polynucleotide (dCas). In some aspects, the Cas endonuclease molecule may retain the ability to nick a single-strand polynucleotide (for example, a D10A mutation in a Cas9 endonuclease molecule) (nCas9).

[0049] The term “crossed” or “cross” refers to a sexual cross and involved the fusion of two haploid gametes via pollination to produce diploid progeny (e.g., cells, seeds or plants). The term encompasses both the pollination of one plant by another and selfing (or self-pollination, e.g., when the pollen and ovule are from the same plant).

[0050] An “elite line” is any line that has resulted from breeding and selection for superior agronomic performance.

[0051] “Gene” includes a nucleic acid fragment that expresses a functional molecule such as, but not limited to, a specific protein, including regulatory sequences preceding (5’ non-coding sequences) and following (3’ non-coding sequences) the coding sequence, as well as intervening intron sequences. “Native gene” refers to a gene as found in its natural endogenous location with its own regulatory sequences.

[0052] “Germplasm” refers to genetic material of or from an individual (e.g., a plant), a group of individuals (e.g., a plant line, variety or family), or a clone derived from a line, variety, species, or culture, or more generally, all individuals within a species or for several species (e.g., maize germplasm collection or Andean germplasm collection). The germplasm can be part of an organism, cell, or can be separate from the organism or cell. In general, germplasm provides genetic material with a specific molecular makeup that provides a physical foundation for some or all of the hereditary qualities of an organism or cell culture. As used herein, germplasm includes cells, seed or tissues from which new plants may be grown, or plant parts, such as leaves, stems, pollen, or cells, that can be cultured into a whole plant.

[0053] The term “genome” as it applies to a prokaryotic and eukaryotic cell or organism cells encompasses not only chromosomal DNA found within the nucleus, but organelle DNA found within subcellular components (e.g., mitochondria, or plastid) of the cell.

[0054] As used herein, a “genomic sequence” or “genomic region” is a segment of a chromosome in the genome of a cell that is present on either side of the target site or, alternatively, also comprises the target site or a portion thereof. An “endogenous genomic sequence” refers to native genomic sequence within a plant cell, (e.g. native sequence of a peptidase gene present within the genome of a maize plant cell).

[0055] A “genomic locus” as used herein refers to the genetic or physical location on a chromosome of a gene. As used herein, “gene” includes a nucleic acid fragment that expresses a functional molecule such as, but not limited to, a specific protein coding sequence and regulatory elements, such as those preceding (5’ non-coding sequences) and following (3’ non-coding sequences) the coding sequence.

[0056] As used herein, “genotype” is the actual nucleic acid sequence at one or more loci in an individual plant. As used herein, “phenotype” means the detectable characteristics (e g. increased free phosphate) of a cell or organism which can be influenced by genotype.

[0057] As used herein, the term “guide polynucleotide”, relates to a polynucleotide sequence that can form a complex with a Cas endonuclease and that enables the Cas endonuclease to recognize, optionally bind to, and optionally cleave a DNA target site. The guide polynucleotide sequence can be a RNA sequence, a DNA sequence, or a combination thereof (a RNA-DNA combination sequence).

[0058] The terms “single guide RNA” and “sgRNA” are used interchangeably herein and relate to a synthetic fusion of two RNA molecules, a crRNA (CRISPR RNA) comprising a variable targeting domain (linked to a tracr mate sequence that hybridizes to a tracrRNA), fused to a tracrRNA (trans-activating CRISPR RNA). The single guide RNA can comprise a crRNA or crRNA fragment and a tracrRNA or tracrRNA fragment of the type II CRISPR / Cas system that can form a complex with a type II Cas endonuclease, wherein said guide RNA / Cas endonuclease complex can direct the Cas endonuclease to a DNA target site, enabling the Cas endonuclease to recognize, optionally bind to, and optionally nick or cleave (introduce a single or double-strand break) the DNA target site.

[0059] As used herein, the terms “guide polynucleotide / Cas endonuclease complex”, “guide polynucleotide / Cas endonuclease system”, “ guide polynucleotide / Cas complex”, “guide polynucleotide / Cas system” and “guided Cas system” “Polynucleotide-guided endonuclease” , “PGEN” are used interchangeably herein and refer to at least one guide polynucleotide and at leastone Cas endonuclease, that are capable of forming a complex, wherein said guide polynucleotide / Cas endonuclease complex can direct the Cas endonuclease to a DNA target site, enabling the Cas endonuclease to recognize, bind to, and optionally nick or cleave (introduce a single or double-strand break) the DNA target site. A guide polynucleotide / Cas endonuclease complex herein can comprise Cas protein(s) and suitable polynucleotide component(s) of any of the known CRISPR systems (Horvath and Barrangou, 2010, Science 327:167-170; Makarova et al. 2015, Nature Reviews Microbiology Vol. 13:1-15; Zetsche et al., 2015, Cell 163, 1-13; Shmakov et al., 2015, Molecular Cell 60, 1-13).

[0060] The term “hybrid” refers to the progeny obtained between the crossing of at least two genetically dissimilar parents.

[0061] The term “inbred” refers to a line that has been bred for genetic homogeneity.

[0062] The term “introgression” refers to the transmission of a desired allele of a genetic locus from one genetic background to another. For example, introgression of a desired peptidase gene variant or peptidase gene variant allele at a specified locus can be transmitted to at least one progeny via a sexual cross between two parents of the same species, where at least one of the parents has the desired allele in its genome. Alternatively, for example, transmission of an allele can occur by recombination between two donor genomes, e.g., in a fused protoplast, where at least one of the donor protoplasts has the desired allele in its genome. The desired allele can be, e.g., detected by a marker that is associated with a phenotype, at a QTL, a transgene, or the like. Offspring comprising the desired allele may be repeatedly backcrossed to a line having a desired genetic background and selected for the desired allele, to result in the allele becoming fixed in a selected genetic background.

[0063] The process of “introgressing” is often referred to as “backcrossing” when the process is repeated two or more times.

[0064] A “line” or “strain” is a group of individuals of identical parentage that are generally inbred to some degree and that are generally homozygous and homogeneous at most loci (isogenic or near isogenic). A “subline” refers to an inbred subset of descendants that are genetically distinct from other similarly inbred subsets descended from the same progenitor.

[0065] The term “plant material” includes whole plants, plant cells, plant protoplast, plant cell or tissue culture from which plants can be regenerated, plant calli, plant clumps and plant cells that are intact in plants, or parts of plants, such as seeds, flowers, cotyledons, leaves, stems, buds, roots,root tips and the like. As used herein, a “modified plant” means any plant that has a genetic change due to human intervention. A modified plant may have genetic changes introduced through plant transformation, genome editing, mutagenesis, or conventional plant breeding.

[0066] A “marker” is a means of finding a position on a genetic or physical map, or else linkages among markers and trait loci (loci affecting traits). The position that the marker detects may be known via detection of polymorphic alleles and their genetic mapping, or else by hybridization, sequence match or amplification of a sequence that has been physically mapped. A marker can be a DNA marker (detects DNA polymorphisms), a protein (detects variation at an encoded polypeptide), or a simply inherited phenotype (such as a MLN resistance). A DNA marker can be developed from genomic nucleotide sequence or from expressed nucleotide sequences (e.g., from a spliced RNA or a cDNA). Depending on the DNA marker technology, the marker may consist of primers complementary to sequence flanking the locus and / or probes that hybridize to polymorphic alleles at the locus. A DNA marker, or a genetic marker, may also be used to describe the gene, DNA sequence or nucleotide on the chromosome itself (rather than the components used to detect the gene or DNA sequence) and is often used when that DNA marker is associated with a particular trait in human genetics (e.g. a marker for breast cancer). The term marker locus is the locus (gene, sequence or nucleotide) that the marker detects.

[0067] As used herein, a ‘nucleic acid molecule” is a polymeric form of nucleotides, which can include both sense and anti-sense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. A nucleotide refers to a ribonucleotide, deoxynucleotide, or a modified form of either type of nucleotide. A “nucleic acid molecule” as used herein is synonymous with “nucleic acid”, “nucleotide sequence”, “nucleic acid sequence”, and “polynucleotide.” The term includes single- and double-stranded forms of DNA. A nucleic acid molecule can include either or both naturally occurring and modified nucleotides linked together by naturally occurring and / or engineered nucleotide linkages.

[0068] Nucleic acid molecules may be modified chemically or biochemically, or may contain non-natural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, intemucleotide modifications, such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc ), charged linkages (e.g., phosphorothi oates, phosphorodithioates, etc ), pendent moieties (e.g.,peptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.). The term “nucleic acid molecule” also includes any topological conformation, including single-stranded, double-stranded, partially duplexed, triplexed, hairpinned, circular, and padlocked conformations. An “endogenous nucleic acid sequence” refers to a nucleic acid sequence within a plant cell, (e.g. an endogenous allele of an peptidase gene present within the genome of a maize plant cell).

[0069] A “protospacer adjacent motif’ (PAM) herein refers to a short nucleotide sequence adjacent to a target sequence (protospacer) that is recognized (targeted) by a guide polynucleotide / Cas endonuclease system described herein. The Cas endonuclease may not successfully recognize a target DNA sequence if the target DNA sequence is not followed by a PAM sequence. The sequence and length of a PAM herein can differ depending on the Cas protein or Cas protein complex used. The PAM sequence can be of any length but is typically 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleotides long.

[0070] As used herein, the term “plant material” refers to any processed or unprocessed material derived, in whole or in part, from a plant. For example, and without limitation, a plant material may be a plant part, a seed, a fruit, a leaf, a root, a plant tissue, a plant tissue culture, a plant explant, or a plant cell.

[0071] The terms “target site”, “target sequence”, “target site sequence, “target DNA”, “target locus”, “genomic target site”, “genomic target sequence”, “genomic target locus”, and “target polynucleotide”, can be used interchangeably herein and refer to a polynucleotide sequence such as, but not limited to, a nucleotide sequence on a chromosome, episome, a locus, or any other DNA molecule in the genome (including chromosomal, chloroplastic, mitochondrial DNA, plasmid DNA) of a cell, at which a guide polynucleotide / Cas endonuclease complex can recognize, bind to, and optionally nick or cleave . The target site can be an endogenous site in the genome of a cell, or alternatively, the target site can be heterologous to the cell and thereby not be naturally occurring in the genome of the cell, or the target site can be found in a heterologous genomic location compared to where it occurs in nature. As used herein, terms “endogenous target sequence” and “native target sequence” are used interchangeable herein to refer to a target sequence that is endogenous or native to the genome of a cell and is at the endogenous or native position of that target sequence in the genome of the cell.

[0072] A “targeted alteration” or “variant” is a gene (e.g., peptidase gene) sequence that has been altered through human intervention of genome editing or transgenic modification. Such an “altered” or “modified” gene has a sequence that differs from the sequence of the corresponding native or non-altered gene by at least one nucleotide (i) insertion (i.e., addition of a nucleotide in a sequence), (ii) deletion, (iii) substitution (i.e., replacement of at least one nucleotide), or (iv) a combination of the foregoing alterations. An “altered” or “modified” plant is a plant comprising an altered gene sequence, e.g., a deletion. As used herein, a “targeted alteration” in a gene (referred to as the target gene) can be made by altering a target sequence within the target gene using any method known to one skilled in the art, including a method involving a guided Cas9 endonuclease system as described herein or a method involving the human controlled use of mutagenesis and subsequent recovery of an altered target sequence (e.g., peptidase gene variant). The disclosed targeted alterations or variants of an peptidase gene can be human-made, intentionally produced and selected nucleic acid changes that can be generated by any known methods, including the use of targeted mutagenesis.

[0073] A virus or vector “transforms” or “transduces” a cell when it transfers nucleic acid molecules into the cell. A cell is “transformed” by a nucleic acid molecule transduced into the cell when the nucleic acid molecule becomes stably replicated by the cell, either by incorporation of the nucleic acid molecule into the cellular genome, or by episomal replication. As used herein, the term “transformation” encompasses all techniques by which a nucleic acid molecule can be introduced into such a cell. Examples include, but are not limited to, transfection with viral vectors, transformation with plasmid vectors, electroporation (Fromm et al., 1986, Nature 319:791-3), lipofection (Feigner et al., 1987, Proc. Natl. Acad. Set. USA 84:7413-7), microinjection (Mueller et al., 1978, Cell 15:579-85), Agrobacteriu -mediated transfer (Fraley et al., 1983, Proc. Natl. Acad. Sci. USA 80:4803-7), direct DNA uptake, and microprojectile bombardment (Klein et al., 1987, Nature 327:70).

[0074] The term “variants” refer to substantially similar sequences which have been altered by genome editing or transgenic modification. For polynucleotides, a variant comprises a deletion and / or addition of one or more nucleotides at one or more internal sites within the native polynucleotide and / or a substitution of one or more nucleotides at one or more sites in the polynucleotide (e.g. an peptidase gene variant disclosed herein). As used herein, a “native”polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively.

[0075] Double-Strand-Break (DSB) Inducing Agents (DSB Agents). Double-strand breaks can be induced by agents such as endonucleases that cleave the phosphodiester bond within a polynucleotide chain, can result in the induction of DNA repair mechanisms, including the non-homologous end-joining pathway, and homologous recombination. Endonucleases include a range of different enzymes, including restriction endonucleases (see e.g. Roberts et al., 2003 Nucleic Acids Res 1:418-20, Roberts et al., 2003, Nucleic Acids Res 31:1805-12, and Belfort et al., 2002 mMobile DNA II, pp. 761-783, Eds. Craigie etal., (ASMPress, Washington, DC)), meganucleases (see e.g., International Application Publication WO 2009 / 114321; Gao et al., 2010, Plant Journal 1:176-187), and TAL effector nucleases or TALENs (see e.g., US Application Publication US 20110145940 and Christian et al., 2010, Genetics 186(2): 757-61). Methods of targeting DNA double-strand breaks have been described for TALENs (Christian et al., 2010, Genetics 186(2): 757-61 and Boch et al., 2009, Science 326(5959): 1509-12), zinc finger nucleases (see e.g. Kim, et al., 1996, Proc. Nat Acad. Sci USA 93(3)1156-1160) and CRISPR-Cas endonucleases (see e.g. International Application Publication W02007 / 025097).

[0076] Any DSB or -nick or -modification inducing agent may be used for the methods described herein, including for example but not limited to: Cas endonucleases, recombinases, TALENs, zinc finger nucleases, restriction endonucleases, meganucleases, and deaminases.

[0077] Methods and compositions are provided for polynucleotide modification with a CRISPR Associated (Cas) endonuclease. Class I Cas endonucleases comprise multi-subunit effector complexes (Types I, III, and IV), while Class 2 systems comprise single protein effectors (Types II, V, and VI) (Makarova et al., 2015, Nature Reviews Microbiology 13:1-15; Zetsche et al., 2015, Cell 163:1-13; Shmakov et al., 2015, Molecular Cell 60, 1-13; Haft et al., 2005, Computational Biology, PLoS Comput Biol 1(6)'. e60; and Koonin et al., 2017, Curr Opinion Microbiology 37:67-78). In Class 2 Type II systems, the Cas endonuclease acts in complex with a guide RNA (gRNA) that directs the Cas endonuclease to cleave the DNA target to enable target recognition, binding, and cleavage by the Cas endonuclease. The gRNA comprises a Cas endonuclease recognition (CER) domain that interacts with the Cas endonuclease, and a Variable Targeting (VT) domain that hybridizes to a nucleotide sequence in a target DNA. In some aspects, the gRNA comprises a CRISPR RNA (crRNA) and a trans-activating CRISPR RNA (tracrRNA)to guide the Cas endonuclease to its DNA target. The crRNA comprises a spacer region complementary to one strand of the double strand DNA target and a region that base pairs with the tracrRNA, forming an RNA duplex. In many systems, the Cas endonuclease-guide polynucleotide complex recognizes a short nucleotide sequence adjacent to the target sequence (protospacer), called a “protospacer adjacent motif’ (PAM).

[0078] Examples of a Cas endonuclease include but are not limited to Cas9, Casl2f, Casl2a or Cpfl, and variants thereof (See e.g., US Patent No. 10,934,536 and International Application Publication WO 2022 / 082179). Cas9 (formerly referred to as Cas5, Csnl, or Csxl2) is a Class 2 Type II Cas endonuclease (Makarova et al., 2015, Nature Reviews Microbiology 13:1-15). For Cas9 and Casl2f, a Cas-gRNA complex recognizes a 3’ PAM sequence at the target site, permitting the spacer of the guide RNA to invade the double-stranded DNA target, and, if sufficient homology between the spacer and protospacer exists, generate a DSB cleavage. Cas9 endonucleases comprise RuvC and HNH domains that together produce DSBs, and separately can produce single strand breaks. For the S. pyogenes Cas9 endonuclease, the DSB leaves a blunt end. Cpfl is a Class 2 Type V Cas endonuclease, and comprises a nuclease RuvC domain but lacks an HNH domain (Yamane et al., 2016, Cell 165:949-962). Casl2f can generate 5’ staggered overhangs at DSB sites (Karvelis et al., Nucl Acids Res 48( 12): 5016-5023 ). Cpfl endonucleases create “sticky” overhang ends.

[0079] Some uses for Cas-gRNA systems at a genomic target site include but are not limited to insertions, deletions, substitutions, or modifications of one or more nucleotides at the target site; modifying or replacing nucleotide sequences of interest (such as a regulatory elements); insertion of polynucleotides of interest; gene dropout; gene knock-out; gene knock in; modification of splicing sites and / or introducing alternate splicing sites; modifications of nucleotide sequences encoding a protein of interest; amino acid and / or protein fusions; and gene silencing by expressing an inverted repeat into a gene of interest. Genome editing using DSB-inducing agents, such as Cas9-gRNA complexes, has been described, for example in U.S. Patent Application No.2015 / 0082478, US Patent No. 10,934,536, International Application Publication WO2015 / 026886 Al, International Application Publication W02016007347, International Application Publication WO201625131, and International Application Publication WO 2022 / 082179 all of which are incorporated by reference herein.

[0080] In some aspects of the disclosure, a targeted genomic modification is introduced in a maize plant cell, wherein the targeted modification includes a targeted alteration of the genomic sequence of a peptidase gene in plant cell. In a further aspect, the targeted genomic modification is induced by a DSB Agent, such as a CRISPR-associated (Cas) nuclease. A Cas nuclease is introduced into the maize cell with a first and second guide RNAs as Cas-gRNA complexes that recognizes peptidase gene target sequences in the genome of the cell and is able to induce DSBs in the genomic sequence, e.g., thereby altering the endogenous target peptidase gene.

[0081] Recombinant Constructs and Transformation of Cells. The disclosed guide polynucleotides can be introduced into a cell with the disclosed DSB agents e.g., CRISPR-Cas endonucleases. Cells include, but are not limited to, human, non-human, animal, bacterial, fungal, insect, yeast, non-conventional yeast, and plant cells as well as plants and seeds produced by the methods described herein. In a preferred aspect of the disclosure, the cells are maize cells.

[0082] Standard recombinant DNA and molecular cloning techniques used herein are known in the art and are described more fully in Sambrook et al., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory: Cold Spring Harbor, NY (1989). Transformation methods are well known to those skilled in the art and are described infra.

[0083] Vectors and constructs include circular plasmids, and linear polynucleotides, comprising a polynucleotide of interest and optionally other components including linkers, adapters, regulatory or analysis. In some examples a recognition site and / or target site can be comprised within an intron, coding sequence, 5' UTRs, 3' UTRs, and / or regulatory regions.

[0084] In one aspect, the constructs of the disclosure comprise a promoter operably linked to a nucleotide sequence encoding a DSB Agent, such as a CAS nuclease (e.g., gene encoding a Streptococcus pyrogenes Cas9 gene or Casl2f gene) and a promoter operably linked to a guide RNA of the present disclosure. The promoter is capable of driving expression of an operably linked nucleotide sequence in a prokaryotic or eukaryotic cell / organism. In some aspects, target specific guide RNAs are built as a fusion of CRISPR RNA (crRNA) fused to trans-activating CRISPR RNA (tracrRNA) of Streptococcus pyrogenes.

[0085] In accordance with the methods disclosed herein, a guide RNA comprising any of SEQ ID NOs:20-24) can be used to introduce an peptidase gene variant disclosed herein.

[0086] The isolated polynucleotides, constructs and vectors disclosed herein (e.g., for expression of endonucleases or guide RNAs) can comprise a selectable marker to identify or selectfor or against a molecule or a cell that comprises the construct or vector. Examples of selectable or scorable markers that can be used in a construct or vector disclosed herein include DsRed and phosphinothricin acetyltransferase gene (pat) for herbicide resistance.

[0087] Isolated Nucleic Acid Molecules and Variants and Fragments Thereof. Isolated or recombinant nucleic acid molecules comprising peptidase gene variants disclosed herein as well as nucleic acid molecules sufficient for use as hybridization probes to identify peptidase gene variants by sequence homology are provided. As used herein, the term “nucleic acid molecule” refers to DNA molecules (e.g., recombinant DNA, cDNA, genomic DNA, plastid DNA, mitochondrial DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. In some examples, the nucleic acid molecule can be singlestranded. In some examples, the nucleic acid molecule can be double-stranded.

[0088] An “isolated” nucleic acid molecule (e.g., RNA or DNA) is used herein to refer to a nucleic acid sequence (e.g., RNA or DNA) that is no longer in its natural environment, for example in vitro. A “recombinant” nucleic acid molecule (e.g., RNA or DNA) is used herein to refer to a nucleic acid sequence (e.g., RNA or DNA) that is in a recombinant bacterial or plant host cell; has been edited from its native sequence; or is located in a different location than the native sequence. In some embodiments, an “isolated” or “recombinant” nucleic acid is free of sequences (preferably protein encoding sequences) that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For purposes of the disclosure, “isolated” or “recombinant” when used to refer to nucleic acid molecules excludes isolated chromosomes. For example, in various embodiments, the recombinant nucleic acid molecules can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleic acid sequences that naturally flank a peptidase gene variant in the genome of the cell.

[0089] In some embodiments, an isolated nucleic acid molecule comprising peptidase gene variant has one or more change in the nucleic acid sequence encoding the C-terminal portion of its protein gene product as compared to the native or genomic nucleic acid sequence. In some embodiments, the change in the native or genomic nucleic acid sequence includes but is not limited to: changes in the nucleic acid sequence due to the degeneracy of the genetic code; changes in the nucleic acid sequence due to the amino acid substitution, insertion, deletion and / or addition compared to the native or genomic sequence; removal of one or more intron / exon.

[0090] A variety of polynucleotides comprising peptidase gene variants disclosed herein are contemplated. Such polynucleotides are useful for production of encoded polypeptides in host cells when operably linked to a suitable promoter, transcription termination and / or polyadenylation sequences. Such polynucleotides are also useful as probes for isolating homologous or substantially homologous polynucleotides that are peptidase gene variants disclosed herein.

[0091] Provided herein are nucleic acid molecules (i) encoding one or more of SEQ ID NOs: 1, 4, 7, 10, 13, or 16, (ii) comprising one or more of SEQ ID NOs: 5, 6, 8, 9, 11, 12, 14, 15, 17, or 19, or (iii) comprising one or more of SEQ ID NOs:20, 21, 23, or 24. “Complement” is used herein to refer to a nucleic acid sequence that is sufficiently complementary to a given nucleic acid sequence such that it can hybridize to the given nucleic acid sequence to thereby form a stable duplex. A reverse complement is a complement formed by exchanging each A with T, T with A, C with G, and G with C in a sequence and then reversing the 5’ to 3’ order of the exchanged sequence, such that the reverse complement of 5’-ACCTGAG-3’ is 5’-CTCAGGT-3’. “Polynucleotide sequence variants” is used herein to refer to a nucleic acid sequence that except for the degeneracy of the genetic code encodes the same polypeptide.

[0092] “Percent (%) sequence identity” with respect to a reference sequence (subject) is determined as the percentage of amino acid residues or nucleotides in a candidate sequence (query) that are identical with the respective amino acid residues or nucleotides in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any amino acid conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent sequence identity can be achieved in various ways, for instance, using publicly available computer software such as BLAST, BLAST-2. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (e.g., percent identity of query sequence = number of identical positions between query and subject sequences / total number of positions of query sequence *100).

[0093] Nucleotide Constructs, Expression Cassettes and Vectors. The use of the term “construct” in connection with isolated and / or heterologous polynucleotides herein is not intended to limit the disclosure to constructs comprising DNA. Polynucleotide constructs, particularlypolynucleotides and oligonucleotides composed of ribonucleotides and combinations of ribonucleotides and deoxyribonucleotides, may also be employed in the methods disclosed herein. The isolated polynucleotide constructs, nucleic acids, and nucleotide sequences disclosed herein additionally encompass all complementary forms (e.g., the reverse complement) of each sequence disclosed for such a construct. Further, polynucleotide constructs and nucleotide sequences disclosed herein can encompass any such constructs, molecules, and sequences suitable for use in a method for transforming plant material disclosed herein. Such constructs can include naturally occurring molecules and / or synthetic analogues. The disclosed nucleotide constructs, nucleic acids, and nucleotide sequences also encompass all forms of nucleotide constructs including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-and-loop structures and the like.

[0094] Transformed organisms disclosed herein include plant cells, bacteria, yeast, baculovirus, protozoa, nematodes and algae. The transformed organism comprises a disclosed sequence (e.g., as part of a construct, expression cassette, or vector comprising the nucleotide sequence disclosed herein which are associated with increased disease resistance.

[0095] The disclosed sequences can be used in constructs for transformation and, in some cases, expression in the organism of interest. Constructs can include 5’ and 3’; regulatory sequences operably linked to an R gene sequence, variant or fragment disclosed herein. The term “operably linked” as used herein refers to a functional linkage between a promoter and / or a regulatory sequence and a second sequence, wherein the promoter and / or regulatory sequence initiates, mediates, and / or affects transcription of the DNA sequence corresponding to the second sequence. Generally, operably linked means that the nucleic acid sequences being linked are contiguous and, where necessary, to join two protein coding regions in the same reading frame. The construct may additionally contain at least one additional gene to be cotransformed into the organism. Alternatively, the additional gene(s) can be provided on multiple DNA constructs.

[0096] Such a DNA construct is provided with a plurality of restriction sites for insertion of the polypeptide gene sequence of the disclosure to be under the transcriptional regulation of the regulatory regions. The DNA construct may additionally contain selectable marker genes.

[0097] The DNA construct will generally include in the 5' to 3' direction of transcription: a transcriptional and translational initiation region (e.g., a promoter), a DNA sequence of the embodiments, and a transcriptional and translational termination region (e.g., termination region)functional in the organism serving as a host. The transcriptional initiation region (e.g., the promoter) may be native, analogous, foreign or heterologous to the host organism and / or to the sequence of the embodiments. Additionally, the promoter or regulatory sequence may be the natural sequence or alternatively a synthetic sequence. The term “foreign” as used herein indicates that the promoter is not found in the native organism into which the promoter is introduced. As used herein, the term “heterologous” in reference to a sequence means a sequence that originates from a foreign species or, if from the same species, is substantially modified from its native form in composition and / or genomic locus by deliberate human intervention. As used herein, a chimeric gene comprises a coding sequence operably linked to a transcription initiation region that is heterologous to the coding sequence. Where the promoter is a native or natural sequence, the expression of the operably linked sequence is altered from the wild-type expression, which results in an alteration in phenotype.

[0098] In some embodiments the DNA construct comprises a polynucleotide comprising a one or more of a sequence (i) encoding one or more of SEQ ID NOs: 1, 4, 7, 10, 13, or 16, (ii) comprising one or more of SEQ ID NOs: 5, 6, 8, 9, 11, 12, 14, 15, 17, or 19, or (iii) comprising one or more of SEQ ID NOs:20, 21, 23, or 24; or a fragment or variant of (i) to (iii).

[0099] A DNA construct may also include a transcriptional enhancer sequence. An “enhancer” refers to a DNA sequence which can stimulate promoter activity, and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. Various enhancers include, for example, introns with gene expression enhancing properties in plants (US Patent Application Publication Number 2009 / 0144863, the ubiquitin intron (i.e., the maize ubiquitin intron 1 (see, for example, NCBI sequence S94464)), the omega enhancer or the omega prime enhancer (Gallie et al. 1989 Molecular Biology ofRNA ed. Cech (Liss, New York) 237-256 and Gallie et al. 1987 Gene 60: 217-25), the CaMV 35S enhancer (see, e.g., Benfey et al. 1990 EMBO J. 9:1685-96) and the enhancers of US Patent Number 7,803,992 may also be used. The above list of transcriptional enhancers is not meant to be limiting. Any appropriate transcriptional enhancer can be used in the embodiments.

[0100] The termination region may be native with the transcriptional initiation region, may be native with the operably linked DNA sequence of interest, may be native with the plant host or may be derived from another source (i.e., foreign or heterologous to the promoter, the sequence of interest, the plant host or any combination thereof).

[0101] Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also, Guerineau et al. 1991 Mol. Gen. Genet. 262:141-144; Proudfoot 1991 Cell 64:671-674; Sanfacon et al. 1991 Genes Dev. 5:141-149; Mogen et al. 1990 Plant Cell 2:1261-1272; Munroe et al. 1990 Gene 91:151-158; Ballas et al. 1989 Nucleic Acids Res. 17:7891-7903 and Joshi et al. 1987 Nucleic Acid Res. 15:9627-9639.

[0102] Where appropriate, a nucleic acid may be optimized for increased expression in the host organism. Thus, where the host organism is a plant, the synthetic nucleic acids can be synthesized using plant-preferred codons for improved expression. See, for example, Campbell and Gowri 1990 Plant Physiol. 92:1-11 for a discussion of host-preferred usage. For example, although nucleic acid sequences of the embodiments may be expressed in both monocotyledonous and dicotyledonous plant species, sequences can be modified to account for the specific preferences and GC content preferences of monocotyledons or dicotyledons as these preferences have been shown to differ (Murray et al. 1989 Nucleic Acids Res. 17:477-498). Thus, the plantpreferred for a particular amino acid may be derived from known gene sequences from plants.

[0103] Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exonintron splice site signals, transposon-like repeats, and other well-characterized sequences that may be deleterious to gene expression. The GC content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. The term “host cell” as used herein refers to a cell which contains a vector and supports the replication and / or expression of the expression vector is intended. Host cells may be prokaryotic cells such as E. coli or eukaryotic cells such as yeast, insect, amphibian or mammalian cells or monocotyledonous or dicotyledonous plant cells. An example of a monocotyledonous host cell is a maize host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures.

[0104] In preparing the expression cassette, the various DNA fragments may be manipulated so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the DNA fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites or the like. For this purpose, in vitro mutagenesis,primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved.

[0105] A number of promoters can be used in the practice of the embodiments. The promoters can be selected based on the desired outcome. The nucleic acids can be combined with constitutive, tissue-preferred, inducible, or temporally-regulated promoters. “Promoter” refers to a nucleotide sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3' to a promoter sequence. The promoter sequence comprises proximal and more distal upstream elements, the latter elements are often referred to as enhancers. Accordingly, an “enhancer” is a nucleotide sequence that can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. Promoters may be derived in their entirety from a native gene or be composed of different elements derived from different promoters found in nature, or even comprise synthetic nucleotide segments. It is understood by those skilled in the art that different regulatory elements may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, nucleic acid fragments of different lengths may have identical promoter activity.

[0106] Plant Transformation. Plant Transformation. Any suitable techniques known in the art for introduction of transgenes into plants may be used to produce a transformed plant or plant cell disclosed herein. Suitable methods for transformation of plants may include virtually any method by which DNA can be introduced into a cell, such as: by electroporation as illustrated in U.S. Patent No. 5,384,253; by microprojectile bombardment, as illustrated in U.S. Patent Nos.5,015,580, 5,550,318, 5,538,880, 6,160,208, 6,399,861, and 6,403,865; by Agrobacterium-mediated transformation as illustrated in U.S. Patent Nos. 5,635,055, 5,824,877, 5,591, 616; 5,981,840, and 6,384,301; and by protoplast transformation, as set forth in U.S. Patent No.5,508,184, etc. These techniques can be used to transform plant cells and these cells may be developed into transgenic plants by techniques known to those of skill in the art.

[0107] After effecting delivery of exogenous DNA to recipient cells, transformed cells are identified for further culturing and plant regeneration. In order to improve the ability to identify transformants, one may desire to employ a selectable marker gene with the transformation vectorused to generate the transformant. In this case, the potentially transformed cell population can be assayed by exposing the cells to a selective agent or agents, or the cells can be screened for the desired marker.

[0108] Cells that survive the exposure to the selective agent, or cells that have been scored positive in a screening assay, may be cultured in media that supports regeneration of plants. In some embodiments, any suitable plant tissue culture media may be modified by including further substances, such as growth regulators. Tissue may be maintained on a basic media with growth regulators until sufficient tissue is available to begin plant regeneration efforts, or following repeated rounds of manual selection, until the morphology of the tissue is suitable for regeneration (e.g., at least 2 weeks), then transferred to media conducive to shoot formation. Cultures are transferred periodically until sufficient shoot formation has occurred. Once shoots are formed, they are transferred to media conducive to root formation. Once sufficient roots are formed, plants can be transferred to soil for further growth and maturity.

[0109] The alteration (e.g., introduction of a stop codon, mutation or deletion) of an endogenous gene (e.g., peptidase gene) in regenerating plants can be confirmed by one or more assays, for example, a molecular biological assay, such as Southern blotting, Northern blotting, or PCR; a biochemical assay, such as detecting the absence of a protein product by immunoassay (ELISA or Western blot) or by screening for reduced enzymatic function; plant part assays, such as leaf or root assays; and analysis of the phenotype of the whole regenerated plant.

[0110] Using the methods disclosed herein, peptidase gene variant-containing plants are generated. For example, a plant comprising an altered endogenous genomic sequence containing one or more of the following variants: a G250 variant, (ii) encodes a truncation variant, (iii) is a gene deletion variant or (iv) is an inactivated expression variant, each of the foregoing as disclosed in paragraph

[0013] herein.

[0111] “Stable transformation” as used herein means that the nucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by the progeny thereof. “Transient transformation” as used herein means that a polynucleotide is introduced into the plant and does not integrate into the genome of the plant or a polypeptide is introduced into a plant. “Plant” as used herein refers to whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, propagules, embryos and progeny of the same. Plant cells can be differentiatedor undifferentiated (e.g. callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells and pollen).

[0112] Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation. Suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. 1986 Proc. Natl. Acad. Sci. USA 83:5602-5606), Agrobacterium-mediated transformation (US Patent Numbers 5,563,055 and 5,981,840), direct gene transfer (Paszkowski et al. 1984 IM BO J. 3:2717-2722) and ballistic particle acceleration (see, for example, US Patent Numbers 4,945,050; 5,879,918; 5,886,244 and 5,932,782; Tomes et al. 1995 in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin) and McCabe et al. 1988 Biotechnology 6:923-926) and Led transformation (WO 00 / 28058). For potato transformation see, Tu et al. 1998 Plant Molecular Biology: 37:829-838 and Chong et al. 2000 Transgenic Research 9:71-78. Additional transformation procedures can be found in Weissinger et al. 1988 Ann. Rev. Genet. 22:421-477; Sanford et al. 1987 Particulate Science and Technology 5 :27 -37 (onion); Christou et al. 1988 Plant Physiol. 87:671-674 (soybean); McCabe et al. 1988 Bio technology 6:923-926 (soybean); Finer and McMullen 1991 In Vitro Cell Dev. Biol. 27P:175-182 (soybean); Singh et al. 1998 Theor. Appl. Genet. 96:319-324 (soybean); Datta et al. 1990 Biotechnology 8:736-740 (rice); Klein et al.1988 Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. 1988 Biotechnology 6:559-563 (maize); US Patent Numbers 5,240,855; 5,322,783 and 5,324,646; Klein et al. (1988) Plant Physiol. 91 :440-444 (maize); Fromm et al. 1990 Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren et al. 1984 Nature (London) 311:763-764; US Patent Number 5,736,369 (cereals); Bytebier et al. 1987 Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al. 1985 in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, New York), pp.197-209 (pollen); Kaeppler et al. 1990 Plant Cell Reports 9:415-418 and Kaeppler et al. 1992 Theor. Appl. Genet. 84:560-566 (whisker-mediated transformation); D'Halluin et al. 1992 Plant Cel! 4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports 12:250-255 and Christou and Ford 1995 Annals of Botany 75:407-413 (rice); Osjoda et al. 1996 Nature Biotechnology’ 14:745-750 (maize via Agrobacterium tumefaciens)'.

[0113] Marker assisted selection. Molecular markers can be used in a variety of plant breeding applications (e g. see Staub et al. 1996 Hortscience 31:729-741; Tanksley 1983 Plant Molecular Biology Reporter. 1:3-8). One of the main areas of interest is to increase the efficiency of backcrossing and introgressing genes using marker-assisted selection (MAS). Thus, MAS can be used for backcrossing and introgressing the peptidase gene variants disclosed herein.

[0114] A molecular marker that demonstrates linkage with a locus affecting a desired phenotypic trait provides a useful tool for the selection of the trait in a plant population. This is particularly true where the phenotype is hard to assay. Since DNA marker assays are less laborious and take up less physical space than field phenotyping, much larger populations can be assayed, increasing the chances of finding a recombinant with the target segment from the donor line moved to the recipient line. The closer the linkage, the more useful the marker, as recombination is less likely to occur between the marker and the gene causing the trait, which can result in false positives. Having flanking markers decreases the chances that false positive selection will occur as a double recombination event would be needed. In the most preferred case, a marker is located within the gene itself, so that recombination cannot occur between the marker and the gene. In some embodiments, the methods targeted alteration disclosed herein produce one or more markers that can be used to identify the peptidase gene variant(s).

[0115] Gene introgression by MAS can be used to diminish linkage drag and yield drag. Gepts 2002 Crop Sc , 42:1780-1790; Young et al. 1998 Genetics 120:579-585; Tanksley et al. 1989 Biotechnology 7: 257-264. Markers disclosed herein, as well as other marker types such as SSRs and FLPs, can be used in marker assisted selection protocols. SSRs can be defined as relatively short runs of tandemly repeated DNA with lengths of 6 bp or less, which are often highly suited to mapping and MAS. Tautz 1989 Nucleic Acid Research 17: 6463-6471; Wang et al. 1994 Theoretical and Applied Genetics 88: 1-6; Levinson and Gutman 1987 Mol Biol Evol 4: 203-221; Weber and May 1989 Am J Hum Genet. 44:388-396; Rafalski et al. 1996 Generating and using DNA markers in plants. In: Non-mammalian genomic analysis: a practical guide. Academic press, pp 75-135). FLP markers refer to fragment length polymorphisms that are in many ways similar to SSR markers, except that the region amplified by the primers is not typically a highly repetitive region. Bhattramakki et al. 2002 Plant Mol Biol 48, 539-547; Rafalski 2002b, supra.

[0116] SNP markers detect single base pair nucleotide substitutions, which can be assayed at an even higher level of throughput than SSRs, in so-called “ultra-high-throughput” fashion, asSNPs do not require large amounts of DNA and automation of the assay may be straight-forward. SNPs also have the promise of being relatively low-cost systems. These three factors together make SNPs highly attractive for use in MAS. Several methods are available for SNP genotyping, including but not limited to, hybridization, primer extension, oligonucleotide ligation, nuclease cleavage, mini sequencing, and coded spheres. Such methods have been reviewed in: Gut 2001 HumMutat 17: 475-492; Shi 2001 Clin Chem 47: 164-172; Kwok 2000 Pharmacogenomics 1 : 95- 100; and Bhattramakki and Rafalski 2001 Discovery and application of single nucleotide polymorphism markers in plants. In: R. J. Henry, Ed, Plant Genotyping: The DNA Fingerprinting of Plants, CABI Publishing, Wallingford. A wide range of commercially available technologies utilize these and other methods to interrogate SNPs including Masscode. TM. (Qiagen), INVADER®. (Third Wave Technologies) and Invader PLUS®, SNAPSHOT®. (Applied Biosystems), TAQMAN®. (Applied Biosystems) and BEAD ARRAYS®. (Illumina).

[0117] In addition to SSR's, FLPs and SNPs, as described above, other types of molecular markers are also widely used, including but not limited to expressed sequence tags (ESTs), SSR markers derived from EST sequences, randomly amplified polymorphic DNA (RAPD), and other nucleic acid-based markers. Isozyme profiles and linked morphological characteristics can, in some cases, also be indirectly used as markers. Even though they do not directly detect DNA differences, they are often influenced by specific genetic differences. However, markers that detect DNA variation are far more numerous and polymorphic than isozyme or morphological markers (Tanksley 1983 Plant Molecular Biology Reporter 1: 3-8).

[0118] The following are specific examples of some aspects of the invention. The examples are offered for illustrative purposes only and are not intended to limit the scope of the invention in any way.EXAMPLESExample 1 : Fine mapping of C6QTL in maize populations.

[0119] Donor line KS23 (Accession Ames 30911, Zea mays L. subsp. mays, plant name KS23-6) was procured from the GRIN-Global, the global Germplasm Resource Information Network GRIN. Large-scale field screening of the segregating populations derived from crosses between MLN-susceptible lines to the resistance donor line KS23 was required for fine genetic mapping of the C6QTL for MLN resistance. To accomplish this, International Maize and Wheat Improvement Center (CIMMYT), in collaboration with the Kenyan Agricultural and LivestockResearch Organization (KALRO), designed a protocol for artificial MLN inoculation and symptom screening in a dedicated, isolated field nursery in Naivasha, Kenya. Maize Chlorotic Mottle Virus (MCMV) and Sugarcane Mosaic Virus (SCMV) were mass-produced in separate greenhouses using established inoculum preparation and inoculation protocols available through CIMMYT web site. The first inoculation was done at the V5 stage (four weeks after planting), followed by a second inoculation one week later. MLN disease severity was assessed every 10 days, beginning 2 weeks after the second inoculation, with 4 total scorings. Ratings were on a 1-9, resistance to susceptibility, scale. Harvested ears from each plot were weighed, and grain yield was calculated using field weight and grain moisture content adjusted to 12.5%. Data analysis for individual sites was performed using META-R software. Similar practices and conditions were used for all the fine mapping experiments. Testing of maize lines in this nursery produced reproducible and consistent results, enabling the genetic mapping of MLN resistance (Boddupalli et al., 2020 and Murithi et al., 2021).

[0120] Before fine mapping the C6QTL, its locus was confirmed by marker trait association using marker and phenotypic data available at CIMMYT for screening of segregating populations derived from crosses between elite MLN-susceptible lines and resistant KS23 donor lines for response to MLN artificial inoculation (Table 2). Resistant donor lines KS23-5 and KS23-6 were developed at Kasetsart University in Thailand (Jampatong et al., 2010, Agriculture and Natural Resources 44, 523-528).Table 2

[0121] For fine mapping, seven distinct F2 populations (shown in Table 3) were developed by crossing MLN-susceptible inbred lines adapted to SSA with the MLN resistance donors. These parents and several others, along with 40-50 F2 individual plants from each population, were screened using approximately 70 markers spanning the C6QTL region (137 Mb-170 Mb) to identify the most suitable polymorphic markers for fine mapping. Fifteen to twenty markers foreach F2 population were then employed to detect recombination events across the 33 Mb genetic interval (Fig. 1A).Table 3

[0122] All selected F2 plants were self-pollinated for progeny testing. Phenotyping F3 progenies enabled narrowing of the target interval to approximately 3 Mb (Fig. 1A).

[0123] Additional populations (Table 4) were also used to find additional recombination events. Recurrent parents are MLN susceptible lines.Table 4

[0124] Custom markers were designed from SNPs within this interval, to further reduce the region to 105 kb.Example 2: Germplasm for gene validation and genome sequencing

[0125] For subsequent experiments, four commercial lines were selected: CML536, CML543, CKL05022, and CKL05004 (Table 2). These lines serve as parents for two, three-way cross hybrids that were widely cultivated in Bomet County, Kenya, prior to the emergence of MLN: (CKL05004 x CML543) x CML536 and (CKL05022 x CML543) x CML536. The goal was to reintroduce the MLN-resistant versions of these hybrids into the epicenter of the disease outbreak.

[0126] These four commercial lines, resistance donor KS23-6, and another elite line CKDHL0186 used to develop the mapping populations, were subjected to whole genome sequencing. The genome sequences later proved valuable in assessing potential off-target effects of guide RNA molecules. The fine-mapped interval was structurally variable among the sequenced lines but was relatively similar between KS23-6 and CML536. Both these lines, the interval region was approximately 22 kb smaller than B73. CML543 and CKL05004 were nearly identical whereas CKL05022 was an outlier that contained different genomic insertions and deletions as compared to all other lines (Fig. 10).Example 3: Genome editing for identification of gene for MLN resistance

[0127] To expedite confirmation of the resistance gene, various parts of the entire 105 kb interval were selected as targets for simultaneous genome editing. Transformation and regeneration of TO events require approximately nine months, followed by an additional five to six months to propagate T1 to T2 seed.

[0128] To avoid revisiting the identified target in case of failure, candidate sub-regions were identified within the 105 kb interval for simultaneous targeted editing. Targeted regions included open reading frames (ORF) and other regions which had been shown to regulate other genes. These edits included introducing frameshift mutations or completely removing regions covering each target using two flanking guide RNA molecules, ensuring comprehensive coverage of the region. Fig. IB, Fig. 2, and Figs. 3A-C show the regions targeted by gRNAs indicated as CR2, CR3-4, CR13-14 or CR8.

[0129] At the outset of this project, transformation of tropical germplasm had not yet been established. Briefly, each guide sequence was inserted into a gene editing construct containing aZmAxiglpro:ZmWus2 cassette, a ZmUbilpro:SpCas9 cassette and a ZmU6pro:gRNA cassette and transformed into 2 mm immature maize embryos extracted from ears approximately two weeks after pollination. LBA4404(THY-) strain of A. tumefaciens containing the pVIR9 plasmid was used (Lowe et al., 2018). This was followed by selection and then recovery of at least about 100 TO plants per line. Thus it was confirmed that all four tropical lines could be transformed with high efficiency using the proprietary genotype-independent transformation approach that includes the use of morphogenetic factors (Lowe et al, 2016, Plant Cell 28, 1998-2015; Laliberte and Sanfaqon, 2010, Annual Review of Phytopathology, Vol 48, VanAlfen et al, Eds., pp. 69-91).

[0130] In the process of genome editing to identify the causal sub-region within the fine-mapped 105 kb interval, phenotyping of additional populations identified two additional recombinants, thereby narrowing the interval to 33 kb (Fig. 1C). The only annotated gene in this interval was annotated as a peptidase.Example 4: Peptidase facilitates MLN susceptibility

[0131] Various guide RNAs targeting the 105 kb region in the MLN-susceptible CML536 line were designed (Fig. IB). Genotyping with NextGen sequencing confirmed the production of genome-edited variants (Variant 1, Variant 2, and Variant 3) using the CR8 gRNA targeting the peptidase shown in Fig.2 (genomic sequences of CML536 and variants) and Table 5 (cDNA and encoded peptides sequences of CML536 and gene-edited variants). Fig. 3A shows an approximately 40 kb deletion created using CR.I3 and CR14 gRNAs; Fig. 3B shows a 2.9 kb deletion created using CR3 and CR4 gRNAs, and Fig. 3C shows a frameshift variant created using CR2 gRNA. These edited TO plants were outcrossed with wild-type plants of the parental line. Table 5 shows cDNA sequence corresponding to SEQ ID NO:6 (positions 859-921). The sequence of Exon 10 is underlined and deleted nucleotides in each of the variants are indicated by dashes (-); Table 5 also shows amino acid sequences corresponding to SEQ ID NO:4 (positions 287-307), including changes in encoded amino acids relative to SEQ ID NO:4 for each of the variants.Table 5

[0132] The transformation vectors for gene editing incorporated an amylase gene under the control of a pollen-specific promoter. This prevented pollen from germinating. Whereas this blocked transmission of transgenes to the next generation via the male gametophyte, helping avoid cross-contamination in the greenhouse, an additional generation to obtain homozygous edited alleles.

[0133] Three edited variants for the peptidase gene in CML536 were identified (Fig. 2 and Table 5). Two of the variants resulted from a single nucleotide deletion in the 10th exon, causing a frameshift mutation. The third variant was a 9 nt deletion with 3 nt in the 9th intron and 6 nt in the 10th exon, which eliminated the intron / exon junction motif ...AG... (Fig. 2). Sequencing of the RT-PCR product of the mRNA from this variant revealed that the 9th intron was processed at the next available ... AG... motif, which was 3 nt distal to the deleted nucleotides in the genomic sequence. This resulted in a predicted in-frame deletion of three amino acids (Table 5). Aside from the peptidase variants, two edited alleles, one with a frameshift mutation and the second with the entire coding region deleted for elFlA, and a single allele with an ~40 kb deletion that included several open reading frames and a MiTE were carried to homozygosity (Fig. IB, Fig. 3).

[0134] MLN growth chamber assays (Gentzel et al., 2024, Phytopathology, 114(2), 484-495) were used for phenotyping of edits (initial T1 characterization and for qPCR for viral assays on the edits) with modifications. The Ohio SCMV isolate was used with a Hawaii isolate of MCMV (unpublished) in a 4 parts SCMV to 1 part MCMV ratio in potassium phosphate, pH 7, buffer containing 0.1% Tween-20. Carborundum was mixed into the buffer to a concentration of 1 mg / ml and plants were directly rub inoculated with 20 strokes per leaf 21-23 days after sowing. At USDA in Wooster, Ohio, phenotypic evaluations were done as previously described (Gentzel et al., 2024; Jones et al., 2018, Phytopathology, 108(6), 748-758) every 3 days for a total of 4-5 times 35-50 days after sowing.

[0135] Testing the T2 plants homozygous for the edited variants of the elFlA gene and the nearly 40 kb deletion (CR13+CR14) revealed no significant impact on MLN resistance (Fig. 4). However, the T2 plants homozygous for each of the three variants of the peptidase gene (Variant1, Variant 3 and Variant 3) exhibited comparable MLN tolerance to KS23-6 in greenhouse screening (Fig. 4). The wild type peptidase gene thus emerged as a potential contributor to MLN susceptibility.

[0136] The peptidase gene encodes a predicted protein of 722 amino acids, with a molecular mass of 78.64 kD and an isoelectric point of 7.73. A single nucleotide change, from guanosine in the MLN-susceptible maize line CML536 to adenine in the resistant line KS23-6, resulted in a corresponding nonsynonymous amino acid change. This change replaced glycine at position 250 with aspartate (Fig. 5). Glycine, a neutral amino acid, has only a hydrogen as its side chain, while aspartic acid has a relatively bulky side chain that is negatively charged (pKa 3.9) at cellular pH.

[0137] Upon structural homology modeling against known structures in the databases, the peptidase protein appears to contain two domains that are similar to those found in serine proteases. The amino acid glycine that determines susceptibility to MLN is conserved in all plant species we have examined, except for KS23-6 (Fig. 6). It is also conserved in all the proteases with resolved structures. Although this disclosure is not limited to any particular mechanism, it is possible that substituting a neutral amino acid with a negatively charged one may disrupt the function of the peptidase.

[0138] The in-frame deletion of three amino acids in variant 3 also apparently disrupted the peptidase function as these plants exhibited equivalent resistance to the variants with frameshift mutations (Fig. 4). In a recent report where the edits in eukaryotic translation initiation factors (eIF4E) were targeted to the C-terminal domain, the MLN viruses were unable to multiply regardless of whether the edits caused frameshift or in-frame mutations (Wen et al., 2024, Plant Biotechnol. J. 22, 3523-3535).Example 5: Field evaluation of the gene-edited variants of peptidase

[0139] Two peptidase knockout variants were selected for field testing. Their genotypes, seed purity and the absence of vector components were confirmed. These lines were tested in Naivasha, Kenya for response to MLN and in Kiboko, Kenya, to evaluate agronomic performance and increase seed.

[0140] Plants derived from genome edited seeds exhibited remarkable resistance to MLN compared to those from wildtype (null) seeds (Table 6). The wildtype (null) plants derived from the segregating seeds of edited plants succumbed 4-6 weeks after inoculation, whereas thehomozygous edited plants appeared normal. CKDHL05004, CKDHL05022, 9YCML442, CML536, and CML543 are MLN susceptible elite lines.Table 6

[0141] None of the elite, MLN-susceptible lines survived to flowering, resulting in no seeds being produced and thus no grain yield (Fig. 7). In Kiboko, an MLN-free nursery, the agronomic performance of the edited lines was found to be comparable to the control CML536 line, indicating that peptidase knockouts were not agronomically detrimental under normal growing conditions (Fig. 8).

[0142] Peptidase edits have been generated in the remaining three lines — CML543, CKL05022, and CKL05023 — using the CR8 guide RNA. Testing of these edits and their null seeds in open fields in Kenya can confirm MLN resistance in Naivasha, having already demonstrated that peptidase knockout confers MLN resistance in CML536.

[0143] Single-cross and three-way-cross hybrids can be developed and tested at multiple locations in Kenya. Three-way cross hybrids having suitable agronomics can be released to farmers in regions severely impacted by MLN disease.Example 6: Intracellular localization of peptidase

[0144] The peptidase protein disclosed herein contains a C-terminal tripeptide signal, serine-lysine-isoleucine (SKI) (Fig. 5, indicated by underline at C-terminus). The well-establisheddiagnostic signal for peroxisome targeting is serine-lysine-leucine (SKL) (Trelease et al., 1996, Protoplasma 195: 156-167), and SKI has been reported to target proteins to this organelle with a lower probability (Deng et al., 2022, The Plant Journal 111: 567-582.). Vectors were designed containing green and red fluorescent proteins (GFP and RFP), each fused to an SKI or SKL tripeptide at the C-terminus. A second set included both, RFP with either SKI or SKL tripeptide fused to the C-terminus and GFP fused to the N-terminus of the native peptidase protein, as well as one with an SKL signal fused at the C-terminus. In all cases, the fluorescent signal was detected in particulate intracellular compartments. Overlay of the signal confirmed that both SKI and SKL targeted the respective proteins to the same particulate compartment, which previous studies indicate correspond to peroxisomes, thereby confirming intracellular localization of the peptidase disclosed herein.Example 7: Possible mode of action of peptidase

[0145] Viruses are known to form invaginations in the plasma membrane and the membranes of subcellular compartments to shield themselves from the host's defense, creating an environment where viral factories can operate unhindered. For instance, SCMV forms spherules in the endoplasmic reticulum and MCMV in the peroxisomal membranes. Since the peptidase is confined to the peroxisomes, it is likely that it confers MLN susceptibility by specifically facilitating MCMV replication. As all the maize lines would have been selected in the presence of the globally endemic SCMV, they would be variably tolerant to its presence. MLN develops only when MCMV infects these plants. The diversity of the SCMV strains in Kenya was attributed to its numerous hosts and the long duration since its first report there in the nineteen seventies.

[0146] To assess whether the knocked-out peptidase protected the plant against either or both viruses, we screened the susceptible line, CML536, the MLN-tolerant line, KS23-6, and three variants of the edited peptidase for their response to MLN inoculation in the greenhouse.

[0147] Viral quantitation was done by extracting RNA from maize leaf tissue using the Qiagen RNeasy Plant Mini Kit (Qiagen, Germantown, MD USA) and cDNA was synthesized using the Applied Biosystems (Waltham, MA USA) High-Capacity cDNA Reverse Transcription Kit. Quantitative PCR was performed using MCMV primers targeting the TGI Coat Protein (unpublished) and SCMV primers designed against the genetic region corresponding to the Coat Protein. Technical replicates along with negative controls and positive controls that were validated against a standard curve were run using standard two step cycling conditions at 95°C for 3 minutes,(95°C for 15 s, 60°C for 30 s) x 45. Cq values were averaged across technical reps and converted to estimated copy numbers.

[0148] As shown in Fig. 9, SCMV was equally abundant in the leaves of the edited variants and the parental line and KS23-6. In contrast, MCMV was almost entirely eliminated in the edited lines, and was similarly negligible in KS23-6, indicating that the peptidase only assisted with MCMV replication. SCMV could still multiply regardless of the presence or absence of MCMV (Fig. 9). This observation explains the persistence of mild mosaic-like symptoms in the leaves of the edited variants and KS23-6.

[0149] MCMV sequestered in the spherules would occur on the cytosolic side of the peroxisomal membrane. Although this disclosure is not limited by any particular mechanism or theory of action, it is possible that the peptidase enzyme, located in the lumen, mediates the formation of the MCMV-containing spherules. The observation that an edited variant with a 3-aa in-frame deletion also eliminated MCMV from the infected cells suggested that a complete peptidase knockout was not required to block replication of this virus. Whereas an apparent knockout of the peptidase caused strong MLN resistance, it did not affect plant performance, suggesting that the enzyme is not essential for plant viability.Example 8: Genome editing strategies for viral resistance

[0150] A wheat (Triticum aestivum or Triticum durum) plant is identified that comprises a gene sequence (base peptidase gene) encoding a peptidase (base peptidase) that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:25. The base peptidase gene sequence is altered by genome editing to create an altered gene, e.g., (A) an altered gene encoding a peptidase variant that (i) comprises an amino acid other than glycine at the position corresponding to position 250 of SEQ ID NO:7 or position 249 of SEQ ID NO:25, when the peptidase variant sequence is aligned with SEQ ID NO:7 or SEQ ID NO:25, respectively, (ii) comprises an insertion, deletion, substitution, or stop codon relative to the base peptidase and thereby encodes an variant peptidase comprising a an altered C-terminal portion relative to the base peptidase beginning at amino acid positions corresponding to one or more of positions 289, 290, or 291 of SEQ ID NO:7, (iii) is truncated and comprises a premature stop codon at the codon corresponding to the codon encoding any one of positions 250-450 of SEQ ID NO:7, or (iv) is truncated and comprises a deletion of at least 10%, 20%, 30%, 40%, 50%, 60% or 65% of the C-terminal amino acid sequence of the base peptidase; or (B) comprising a deletion of the codingsequence for the base peptidase or a deletion of sequence required to properly express the base peptidase.

[0151] A soybean (Glycine max') plant is identified that comprises a gene sequence (base peptidase gene) encoding a peptidase (base peptidase) that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:26. The base peptidase gene sequence is altered by genome editing to create an altered gene, e.g., (A) an altered gene encoding a peptidase variant that (i) comprises an amino acid other than glycine at the position corresponding to position 250 of SEQ ID NO:7 or position 251 of SEQ ID NO:26, when the peptidase variant sequence is aligned with SEQ ID NO:7 or SEQ ID NO:26, respectively, (ii) comprises an insertion, deletion, substitution, or stop codon relative to the base peptidase and thereby encodes an variant peptidase comprising a an altered C-terminal portion relative to the base peptidase beginning at amino acid positions corresponding to one or more of positions 289, 290, or 291 of SEQ ID NO:7, (iii) is truncated and comprises a premature stop codon at the codon corresponding to the codon encoding any one of positions 250-450 of SEQ ID NO:7, or (iv) is truncated and comprises a deletion of at least 10%, 20%, 30%, 40%, 50%, 60% or 65% of the C-terminal amino acid sequence of the base peptidase; or (B) comprising a deletion of the coding sequence for the base peptidase or a deletion of sequence required to properly express the base peptidase.

[0152] A sorghum (e.g., sorghum bicolor) plant is identified that comprises a gene sequence (base peptidase gene) encoding a peptidase (base peptidase) that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:27. The base peptidase gene sequence is altered by genome editing to create an altered gene, e.g., (A) an altered gene encoding a peptidase variant that (i) comprises an amino acid other than glycine at the position corresponding to position 250 of SEQ ID NO:7 or position 245 of SEQ ID NO:27, when the peptidase variant sequence is aligned with SEQ ID NO:7 or SEQ ID NO:27, respectively, (ii) comprises an insertion, deletion, substitution, or stop codon relative to the base peptidase and thereby encodes an variant peptidase comprising a an altered C-terminal portion relative to the base peptidase beginning at amino acid positions corresponding to one or more of positions 289, 290, or 291 of SEQ ID NO:7, (iii) is truncated and comprises a premature stop codon at the codon corresponding to the codon encoding any one of positions 250-450 of SEQ ID NO:7, or (iv) is truncated and comprises a deletion of at least 10%, 20%, 30%, 40%, 50%, 60% or 65% of the C-terminal amino acid sequence of the base peptidase; or (B) comprising a deletion of the coding sequence for the base peptidase or a deletion of sequence required to properly express the base peptidase.

[0153] A rice (Oryza meyeriana or Oryza sativa) plant is identified that comprises a gene sequence (base peptidase gene) encoding a peptidase (base peptidase) that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:30. The base peptidase gene sequence is altered by genome editing to create an altered gene, e.g., (A) an altered gene encoding a peptidase variant that (i) comprises an amino acid other than glycine at the position corresponding to position 250 of SEQ ID NO:7 or position 248 of SEQ ID NO:30, when the peptidase variant sequence is aligned with SEQ ID NO:7 or SEQ ID NO:30, respectively, (ii) comprises an insertion, deletion, substitution, or stop codon relative to the base peptidase and thereby encodes an variant peptidase comprising a an altered C-terminal portion relative to the base peptidase beginning at amino acid positions corresponding to one or more of positions 289, 290, or 291 of SEQ ID NO:7, (iii) is truncated and comprises a premature stop codon at the codon corresponding to the codon encoding any one of positions 250-450 of SEQ ID NO:7, or (iv) is truncated and comprises a deletion of at least 10%, 20%, 30%, 40%, 50%, 60% or 65% of the C-terminal amino acid sequence of the base peptidase; or (B) comprising a deletion of the coding sequence for the base peptidase or a deletion of sequence required to properly express the base peptidase.

[0154] A bean (Phaseolus vulgaris) plant is identified that comprises a gene sequence (base peptidase gene) encoding a peptidase (base peptidase) that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 28. The base peptidase gene sequence is altered by genome editing to create an altered gene, e.g., (A) an altered gene encoding a peptidase variant that (i) comprises an amino acid other than glycine at the position corresponding to position 250 of SEQ ID NO:7 or position 237 of SEQ ID NO:28, when the peptidase variant sequence is aligned with SEQ ID NO: 7 or SEQ ID NO: 28, respectively, (ii) comprises an insertion, deletion, substitution, or stop codon relative to the base peptidase and thereby encodes an variant peptidase comprising a an altered C-terminal portion relative to the base peptidase beginning at amino acid positions corresponding to one or more of positions 289, 290, or 291 of SEQ ID NO:7, (iii) is truncated and comprises a premature stop codon at the codon corresponding to the codon encoding any one of positions 250-450 of SEQ ID NO:7, or (iv) istruncated and comprises a deletion of at least 10%, 20%, 30%, 40%, 50%, 60% or 65% of the C-terminal amino acid sequence of the base peptidase; or (B) comprising a deletion of the coding sequence for the base peptidase or a deletion of sequence required to properly express the base peptidase.

[0155] A peanut (Arachis hypogaea) plant is identified that comprises a gene sequence (base peptidase gene) encoding a peptidase (base peptidase) that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:29. The base peptidase gene sequence is altered by genome editing to create an altered gene, e.g., (A) an altered gene encoding a peptidase variant that (i) comprises an amino acid other than glycine at the position corresponding to position 250 of SEQ ID NO:7 or position 238 of SEQ ID NO:29, when the peptidase variant sequence is aligned with SEQ ID NO: 7 or SEQ ID NO: 29, respectively, (ii) comprises an insertion, deletion, substitution, or stop codon relative to the base peptidase and thereby encodes an variant peptidase comprising a an altered C-terminal portion relative to the base peptidase beginning at amino acid positions corresponding to one or more of positions 289, 290, or 291 of SEQ ID NO:7, (iii) is truncated and comprises a premature stop codon at the codon corresponding to the codon encoding any one of positions 250-450 of SEQ ID NO:7, or (iv) is truncated and comprises a deletion of at least 10%, 20%, 30%, 40%, 50%, 60% or 65% of the C-terminal amino acid sequence of the base peptidase; or (B) comprising a deletion of the coding sequence for the base peptidase or a deletion of sequence required to properly express the base peptidase.

[0156] A chickpea (Cicer arietinum) plant is identified that comprises a gene sequence (base peptidase gene) encoding a peptidase (base peptidase) that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:31. The base peptidase gene sequence is altered by genome editing to create an altered gene, e.g., (A) an altered gene encoding a peptidase variant that (i) comprises an amino acid other than glycine at the position corresponding to position 250 of SEQ ID NO:7 or position 237 of SEQ ID NO:31, when the peptidase variant sequence is aligned with SEQ ID NO:7 or SEQ ID NO:31, respectively, (ii) comprises an insertion, deletion, substitution, or stop codon relative to the base peptidase and thereby encodes an variant peptidase comprising a an altered C-terminal portion relative to the base peptidase beginning at amino acid positions corresponding to one or more of positions 289, 290, or 291 of SEQ ID NO:7, (iii) is truncated and comprises a premature stop codon at the codoncorresponding to the codon encoding any one of positions 250-450 of SEQ ID NO:7, or (iv) is truncated and comprises a deletion of at least 10%, 20%, 30%, 40%, 50%, 60% or 65% of the C-terminal amino acid sequence of the base peptidase; or (B) comprising a deletion of the coding sequence for the base peptidase or a deletion of sequence required to properly express the base peptidase.

[0157] A banana (Musasp.) plant is identified that comprises a gene sequence (base peptidase gene) encoding a peptidase (base peptidase) that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:38. The base peptidase gene sequence is altered by genome editing to create an altered gene, e.g., (A) an altered gene encoding a peptidase variant that (i) comprises an amino acid other than glycine at the position corresponding to position 250 of SEQ ID NO:7 or position 243 of SEQ ID NO:38, when the peptidase variant sequence is aligned with SEQ ID NO:7 or SEQ ID NO:38, respectively, (ii) comprises an insertion, deletion, substitution, or stop codon relative to the base peptidase and thereby encodes an variant peptidase comprising a an altered C-terminal portion relative to the base peptidase beginning at amino acid positions corresponding to one or more of positions 289, 290, or 291 of SEQ ID NO:7, (iii) is truncated and comprises a premature stop codon at the codon corresponding to the codon encoding any one of positions 250-450 of SEQ ID NO:7, or (iv) is truncated and comprises a deletion of at least 10%, 20%, 30%, 40%, 50%, 60% or 65% of the C-terminal amino acid sequence of the base peptidase; or (B) comprising a deletion of the coding sequence for the base peptidase or a deletion of sequence required to properly express the base peptidase.

[0158] An orange (Citrus sinensis) plant is identified that comprises a gene sequence (base peptidase gene) encoding a peptidase (base peptidase) that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:34. The base peptidase gene sequence is altered by genome editing to create an altered gene, e.g., (A) an altered gene encoding a peptidase variant that (i) comprises an amino acid other than glycine at the position corresponding to position 250 of SEQ ID NO:7 or position 252 of SEQ ID NO:34, when the peptidase variant sequence is aligned with SEQ ID NO:7 or SEQ ID NO:34, respectively, (ii) comprises an insertion, deletion, substitution, or stop codon relative to the base peptidase and thereby encodes an variant peptidase comprising a an altered C-terminal portion relative to the base peptidase beginning at amino acid positions corresponding to one or more of positions 289, 290, or 291 of SEQ ID NO:7, (iii) is truncated and comprises a premature stop codon at the codoncorresponding to the codon encoding any one of positions 250-450 of SEQ ID NO:7, or (iv) is truncated and comprises a deletion of at least 10%, 20%, 30%, 40%, 50%, 60% or 65% of the C-terminal amino acid sequence of the base peptidase; or (B) comprising a deletion of the coding sequence for the base peptidase or a deletion of sequence required to properly express the base peptidase.

[0159] A pineapple (Ananas comosus) plant is identified that comprises a gene sequence (base peptidase gene) encoding a peptidase (base peptidase) that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:35. The base peptidase gene sequence is altered by genome editing to create an altered gene, e.g., (A) an altered gene encoding a peptidase variant that (i) comprises an amino acid other than glycine at the position corresponding to position 250 of SEQ ID NO:7 or position 251 of SEQ ID NO:35, when the peptidase variant sequence is aligned with SEQ ID NO:7 or SEQ ID NO:35, respectively, (ii) comprises an insertion, deletion, substitution, or stop codon relative to the base peptidase and thereby encodes an variant peptidase comprising a an altered C-terminal portion relative to the base peptidase beginning at amino acid positions corresponding to one or more of positions 289, 290, or 291 of SEQ ID NO: 7, (iii) is truncated and comprises a premature stop codon at the codon corresponding to the codon encoding any one of positions 250-450 of SEQ ID NO:7, or (iv) is truncated and comprises a deletion of at least 10%, 20%, 30%, 40%, 50%, 60% or 65% of the C-terminal amino acid sequence of the base peptidase; or (B) comprising a deletion of the coding sequence for the base peptidase or a deletion of sequence required to properly express the base peptidase.

[0160] An Arabidopsis thaliana plant is identified that comprises a gene sequence (base peptidase gene) encoding a peptidase (base peptidase) that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:36. The base peptidase gene sequence is altered by genome editing to create an altered gene, e.g., (A) an altered gene encoding a peptidase variant that (i) comprises an amino acid other than glycine at the position corresponding to position 250 of SEQ ID NO:7 or position 240 of SEQ ID NO:36, when the peptidase variant sequence is aligned with SEQ ID NO:7 or SEQ ID NO:36, respectively, (ii) comprises an insertion, deletion, substitution, or stop codon relative to the base peptidase and thereby encodes an variant peptidase comprising a an altered C-terminal portion relative to the base peptidase beginning at amino acid positions corresponding to one or more of positions 289,290, or 291 of SEQ ID NO:7, (iii) is truncated and comprises a premature stop codon at the codon corresponding to the codon encoding any one of positions 250-450 of SEQ ID NO:7, or (iv) is truncated and comprises a deletion of at least 10%, 20%, 30%, 40%, 50%, 60% or 65% of the C-terminal amino acid sequence of the base peptidase; or (B) comprising a deletion of the coding sequence for the base peptidase or a deletion of sequence required to properly express the base peptidase.

[0161] A poplar, oak, or camphor tree is identified that comprises a gene sequence (base peptidase gene) encoding a peptidase (base peptidase) that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:32, 33, or 37, respectively. The base peptidase gene sequence is altered by genome editing to create an altered gene, e.g., (A) an altered gene encoding a peptidase variant that (i) comprises an amino acid other than glycine at the position corresponding to position 250 of SEQ ID NO:7 or position 255 ,239, or 263 of SEQ ID NO:32, 33, or 37, respectively, when the peptidase variant sequence is aligned with SEQ ID NO:7 or SEQ ID NO:32, 33, or 37, respectively, (ii) comprises an insertion, deletion, substitution, or stop codon relative to the base peptidase and thereby encodes an variant peptidase comprising a an altered C-terminal portion relative to the base peptidase beginning at amino acid positions corresponding to one or more of positions 289, 290, or 291 of SEQ ID NO:7, (iii) is truncated and comprises a premature stop codon at the codon corresponding to the codon encoding any one of positions 250-450 of SEQ ID NO:7, or (iv) is truncated and comprises a deletion of at least 10%, 20%, 30%, 40%, 50%, 60% or 65% of the C-terminal amino acid sequence of the base peptidase; or (B) comprising a deletion of the coding sequence for the base peptidase or a deletion of sequence required to properly express the base peptidase.

[0162] In particular examples of each of the foregoing, the plant comprising the disclosed altered gene as a result of genome editing has greater resistance to a viral disease (e.g., a Tombusvirus) as compared to the plant comprising base peptidase gene.

Claims

Claims1. A method of altering a virus susceptible plant, cell, seed, tissue or germplasm thereof, the method comprising altering by genome editing a base peptidase gene encoding a base peptidase in the susceptible plant, cell, seed, tissue or germplasm genome to thereby produce an altered plant, cell, seed, tissue or germplasm comprising an altered gene and improved resistance to viral disease relative to the susceptible plant, cell, seed, tissue or germplasm thereof.

2. The method of claim 1, wherein altering the base peptidase gene comprises causing a targeted alteration of the base peptidase gene in the susceptible plant, cell, seed, tissue or germplasm thereof.

3. The method of claim 1 or 2, wherein the base peptidase comprises a canonical or non-canonical peroxisomal targeting signal (PTS).

4. The method of any one of claims 1-3, wherein the base peptidase localizes to the peroxisome.

5. The method of any one of claims 1-4, wherein the base peptidase comprises a serine-lysine-isoleucine (SKI) targeting signal sequence.

6. The method of any one of claims 1-5, wherein the base peptidase comprises peptidase activity in the susceptible plant, cell, seed, tissue or germplasm.

7. The method of any one of claims 1-6, wherein the base peptidase comprises a glycine at amino acid position corresponding to position 250 of SEQ ID NO:7, when the base peptidase sequence is aligned with SEQ ID NO: 7.

8. The method of any one of claims 1-7, wherein the altered gene encodes a variant peptidase that comprises an amino acid other than glycine or a deletion at amino acid position corresponding to position 250 of SEQ ID NO:7, when the variant peptidase is aligned with SEQ ID NO:7.

9. The method of any one of claims 1-7, wherein the altered gene (i) comprises a premature stop codon at the codon corresponding to the codon encoding any one of positions 250-450 of SEQ ID NO: 7 or (ii) encodes a variant peptidase which is truncated and lacks at least 10%, 20%, 30%, 40%, 50%, 60% or 65% of the C-terminal amino acid sequence of the base peptidase.

10. The method of any one of claims 1-9, wherein the altered gene encodes a variant peptidase that differs from the C-terminal portion of the base peptidase beginning at amino acid positions corresponding to one or more of positions 289, 290, or 291 of SEQ ID NO:7.

11. The method of any one of claims 1 -6, wherein altering the base peptidase gene comprises a deletion of genomic sequence comprising the base peptidase gene coding sequence for the base peptidase, deletion of sequence required to express the base peptidase, or deletion sequence required to express the base peptidase at wild type levels.

12. The method of any one of claims 1-11, wherein the base peptidase comprises at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:7.

13. The method of any one of claims 1-11, wherein the base peptidase gene coding sequence comprises at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 3, 6, 9, 12, 15, or 18.

14. The method of any one of claims 1-11, wherein the base peptidase gene genomic sequence comprises at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 2, 5, 8, 11, 14, or 17.

15. The method of any one of claims 1-11, wherein the base peptidase comprises at least 40%, at least 50% at least 60% or at least 70% sequence identity to SEQ ID NO:7.

16. The method of any one of claims 1-11, wherein the base peptidase comprises at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs:25-38.

17. The method of any one of claims 1-16, wherein the susceptible plant, cell, seed, tissue or germplasm is susceptible to maize chlorotic mottle virus (MCMV), a tombusvirus, or another virus, and the altered plant, cell, seed, tissue or germplasm thereof has increased resistance to MCMV, tombusvirus, or another virus, relative to the susceptible plant, cell, seed, tissue or germplasm.

18. The method of any one of claims 1-17, wherein the susceptible plant, cell, seed, tissue or germplasm is susceptible to maize lethal necrosis (MLN) and the altered plant, cell, seed, tissue or germplasm thereof has increased resistance to MLN relative to the susceptible plant, cell, seed, tissue or germplasm.

19. The method of any one of claims 1-18, wherein the targeted alteration is introduced by genome editing20. The method of claim 19, wherein the genome editing includes the use of a CAS endonuclease.

21. A plant, cell, seed, tissue or germplasm thereof, comprising a peptidase gene variant generated by genome editing, derived through the method of any one of claims 1-20, comprising an altered version (altered nucleotide sequence) of a base gene or base coding sequence encoding a base peptidase, whereina. the altered nucleotide sequence encodes an altered peptidase that (i) comprises an amino acid other than glycine or a deletion of the glycine at amino acid position corresponding to position 250 of SEQ ID NO:7, when the variant peptidase is aligned with SEQ ID NO:7 (ii) differs from the C-terminal portion of the base peptidase beginning at amino acid positions corresponding to one or more of positions 289, 290, or 291 of SEQ ID NO:7, (iii) comprises a premature stop codon at the codon corresponding to the codon encoding any one of positions 250-450 of SEQ ID NO: 7, or (iv) is truncated and comprises a deletion of at least 10%, 20%, 30%, 40%, 50%, 60% or 65% of the C-terminal amino acid sequence of the base peptidase; or b. the altered nucleotide sequence is an altered genomic sequence that comprises a deletion of genomic sequence comprising the base peptidase gene coding sequence, deletion of sequence required to express the base peptidase, or deletion sequence required to express the base peptidase at wild type levels.

22. The plant, cell, seed, tissue or germplasm thereof of claim 21, wherein the base peptidase comprises at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:7.

23. The plant, cell, seed, tissue or germplasm thereof of claim 21, wherein the altered nucleotide sequence is an altered version of a base coding sequence that comprises at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 3, 6, 9, 12, 15, or 18.

24. The plant, cell, seed, tissue or germplasm thereof of claim 21, wherein the altered nucleotide sequence is an altered version of a base gene sequence that comprises at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 2, 5, 8, 11, 14, or 17.

25. The plant, cell, seed, tissue or germplasm thereof of claim 21, wherein the base peptidase comprises at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs:25-38.

26. A method of identifying a plant, seed, tissue or germplasm thereof, developed through the method of any one of claims 1-20, and comprising a peptidase gene variant generated by genome editing, associated with increased resistance to MLN and / or increased resistance to maize chlorotic mottle virus (MCMV), tombusvirus, or another virus, the method comprisinga. providing one or more plant, seed, tissue or germplasm thereof comprising an engineered altered gene relative to a base peptidase gene encoding a base peptidase; b. obtaining a sample comprising nucleic acid from each plant, seed, tissue or germplasm thereof;c. screening the sample for any of the following altered variant sequences:i. a variant genomic sequence or cDNA sequence encoding a peptidase variant that comprises an amino acid other than glycine at the position corresponding to position 250 of SEQ ID NO:7, when the peptidase variant sequence is aligned with SEQ ID NO:7,ii. a variant genomic sequence or cDNA sequence encoding a peptidase variant that comprises an insertion, deletion, substitution, or stop codon relative to the base peptidase and thereby encodes an variant peptidase comprising an altered C-terminal portion relative to the base peptidase beginning at amino acid positions corresponding to one or more of positions 289, 290, or 291 of SEQ ID NO: 7,iii. a variant genomic sequence or cDNA sequence encoding a peptidase variant that is truncated and comprises a premature stop codon at the codon corresponding to the codon encoding any one of positions 250-450 of SEQ ID NO: 7,iv. a variant genomic sequence or cDNA sequence encoding a peptidase variant that is truncated and comprises a deletion of at least 10%, 20%, 30%, 40%, 50%, 60% or 65% of the C-terminal amino acid sequence of the base peptidase,v. a variant genomic sequence comprising a deletion of genomic sequence (x’) comprising the base peptidase gene coding sequence or (y’) required to express the base peptidase,vi. any of the foregoing i.-v., wherein the base peptidase comprises at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 7,vii. any of the foregoing i.-v., wherein the base peptidase gene coding sequence comprises at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 3, 6, 9, 12, 15, or 18,viii. any of the foregoing i.-v., wherein the base peptidase gene genomic sequence comprises at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 2, 5, 8, 11, 14, or 17,ix. any of the foregoing i.-v., wherein base peptidase comprises at least 40%, at least 50% at least 60% or at least 70% sequence identity to SEQ ID NO: 7, orx. any of the foregoing i.-v., wherein the base peptidase comprises at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs:25-38, andd. detecting one or more of altered variant sequences (i)-(x) in the sample and thereby identifying the plant or plant material as comprising a peptidase gene variant associated with increased resistance to MLN and / or increased resistance to maize chlorotic mottle virus (MCMV), tombusvirus, or another virus.

27. A method of improving plant’s resistance to MLN and / or to maize chlorotic mottle virus (MCMV), tombusvirus, or another virus, developed through the method of any one of claims 1-20, the method comprising:a. identifying a first parent plant as comprising a peptidase gene variant associated with increased resistance to MLN and / or to maize chlorotic mottle virus (MCMV), tombusvirus, or another virus in accordance with claim 26; andb. crossing the first parent plant with a second parent plant to produce one or more progeny plants comprising the peptidase gene variant, wherein the second parent plant is susceptible to MLN, MCMV, tombusvirus, or other virus and wherein the one or more progenyplants are more resistant to the MLN, MCMV, tombusvirus, or other virus than the second parent plant.

28. The method of claim 27, wherein the second parent plant comprises the base peptidase gene andxi. the base peptidase gene comprises at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO:7,xii. the base peptidase gene comprises at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 3, 6, 9, 12, 15, or 18,xiii. the base peptidase gene comprises at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 2, 5, 8, 11, 14, or 17,xiv. the base peptidase comprises at least 40%, at least 50% at least 60% or at least 70% sequence identity to SEQ ID NO:7, orxv. the base peptidase comprises at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs:25-38.

29. A method of improving a plant’s resistance to MLN and / or to maize chlorotic mottle virus (MCMV), tombusvirus, or another virus, developed through the method of any one of claims 1-20, the method comprising:a. crossing a first parent plant with a second parent plant to produce progeny plant(s), wherein at least the first parent comprises a peptidase gene variant associated with increased resistance to MLN, MCMV, tombusvirus, or other virus; and b. identifying at least one progeny plant comprising the peptidase gene variant associated with increased resistance to MLN, MCMV, tombusvirus, or other virus that is a plant in accordance with claim 26; andc. selecting one or more progeny plants identified in step (b).

30. The method of claim 29 further comprising:c. crossing the selected one or more progeny plants with the second parent plant to produce backcross progeny plants;d. identifying at least one backcross progeny plant comprising the peptidase gene variant in accordance with claim 26; ande. selecting one or more backcross progeny plants identified in step (d).

31. The method of any one of claims 1-30, wherein the plant is a maize plant.

32. The method of any one of claims 1-30 wherein the plant is sorghum, rice, barley, wheat, soybean banana, pineapple, asparagus, camphor, nelumbo, macleaya, tetracentron, peanut, chickpea, common bean, oak, citrus, or poplar.

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