Eif4e compositions and methods for viral resistance in maize

Abstract

Provided are plants, cells, tissues, and germplasm thereof comprising one or more targeted alterations of the genomic sequences of eIF4E1, eIF4E2 or both eIF4E1 and eIF4E2 genes. Also provided are methods and compositions to make eIF4E1 and eIF4E2 gene variants; breeding methods and methods of identifying and selecting plant materials having the disclosed eIF4E1 and eIF4E2 variants. Further provided are methods of improving resistance to MLN and / or increased resistance to maize chlorotic mottle virus (MCMV), potyvirus, or sugarcane mosaic virus (SCMV) in maize.

Description

EIF4E COMPOSITIONS AND METHODS FOR VIRAL RESISTANCE IN MAIZE 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 212657-US-PRV-1 ST26 created on September 24, 2024, and having a size of 100,888 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 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; 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. On average, MLN reduces grain yield by a quarter although complete loss in some fields is common (De Groote et al., 2016). 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 QTL are known for SCMV resistance, they are not effective against MLN, as none of these QTLs eliminates SCMV from the plant. If SCMV is present, infection by MCMV leads to development of MLN. Resistance against MCMV, as well as against potyvirus such as SCMV, is thus required for MLN resistance (Carino et al., 2020).

[0006] Few natural sources for MLN resistance are known. Additionally, introgression of resistance QTL by backcrossing takes many cycles and, at the end, many undesirable genes from the donor parent continue to persist in the converted line, unpredictably affecting its agronomic performance (Dhugga, 2022).

[0007] A more efficient and precise tool to alter a single gene is gene editing directly in elite lines. A hurdle in taking this route has been the difficulty in transforming elite germplasm. Genotype-independent transformation, which involves including cell cycle morphogenesis genes in the transformation vector, has enabled direct transformation of elite germplasm, expediting trait improvement (Lowe et al., 2018a; Lowe et al., 2018b; Lowe et al., 2016). Methods using eukaryotic translation initiation factors (elFs) to provide resistance to certain viruses in certain plants have been described. See, e.g., PCT Application Publications WO 2024 / 023207 and WO 2024 / 023208, which describe the targeting of elF genes on chromosome 3 to confer viral resistance. However, the commercial viability of the approaches described in these publications is unclear, particularly in light of results described herein.

[0008] There is a need to identify genes and gene editing technologies that can help quickly introduce MLN resistance directly in elite lines through gene editing.SUMMARY OF THE DISCLOSURE

[0009] The disclosed compositions and methods are based, in part, on the discovery that C-terminal truncated versions of eIF4E proteins provide effective resistance against viruses that cause maize lethal necrosis (MLN) (e.g., maize chlorotic mottle virus (MCMV), which is a Tombusvirus, and a potyvirus, generally sugarcane mosaic virus (SCMV). This was surprising, at least partly, because of the results described herein showing that viable double homozygous knockouts of eIF4El and eIF4E2 genes were impossible to generate. By contrast, disclosed herein is the discovery that combining a knockout of eIF4El with a previously unknown natural variant of eIF4E2 (where the protein encoded by the latter had a 22 amino acids long in-frame deletion 16 amino acids proximal to the C-terminus) conferred complete resistance against both MCMV and SCMV. Thus disclosed herein are combination of eIF4E gene variants that allow for normal growth and block replication of viruses responsible for MLN.

[0010] Accordingly, the term “variant” used in connection with an eIF4E2 gene herein refers to an eIF4E2 gene that differs from wild type eIF4E2, such that the variant eIF4E2 encodes variant eIF4E2 protein with a C-terminal domain that differs from wild type in the region corresponding to C-terminal 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24,23, 22, 21, or 20 amino acids of wild type eIF4E2 and such that the variant eIF4E2 retains function in the protein region preceding (N-terminal to) the altered C-terminal domain. Similarly, the term “variant” used in connection with an eIF4El gene herein refers to an eIF4El gene that differs from wild type eIF4El, such that the variant eIF4El encodes variant eIF4El protein with a C-terminal domain that differs from wild type in the region corresponding to C- terminal 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, or 20 amino acids of wild type eIF4El and such that the variant eIF4El retains function in the protein region preceding (N-terminal to) the altered C-terminal domain. The variant eIF4El gene and variant eIF4E2 gene disclosed herein can be a naturally occurring variant or it can be an engineered variant, deliberately created using scientific techniques such as genome editing, allele replacement, mutagenesis, or the like.

[0011] In a first aspect, provided is a method of altering a plant, cell, seed, tissue or germplasm thereof, that comprises introducing a targeted alteration to the sequence of one or more eIF4E genes to produce an altered genome in the plant, cell, seed, tissue or germplasm thereof, wherein the altered genome comprises an eIF4El variant (producing an eIF4El protein variant), an eIF4E2 variant (producing an eIF4E2 protein variant), or a combination of an eIF4El variant and an eIF4E2 variant, wherein each variant produces an eIF4E protein comprising an inactivated C-terminal region.

[0012] In a first embodiment of the first aspect, the method can include altering the genome of a plant, cell, seed, tissue or germplasm thereof by altering its endogenous eIF4E2 gene that encodes endogenous eIF4E2 protein (preferably by altering both copies of the eIF4E2 gene so that the alteration is homozygous). The alteration generates an eIF4E2 gene variant that (i) is altered in the region comprising the fourth exon of endogenous eIF4El gene or (ii) encodes an eIF4E2 protein variant which differs from the endogenous protein in the region corresponding to the C-terminal 38 amino acids of wild type eIF4E2 protein. For example, the altered eIF4E2 gene variant can encode an eIF4E2 protein variant that comprises a deletion of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 , 35, 36, or 37 amino acids in the region corresponding to the C-terminal 38 amino acids of wild type eIF4E2 protein. In some examples, the altered eIF4E2 gene variant encodes an eIF4E2 protein variant is truncated and comprises a deletion of the (a) first 22 amino acids of the C-terminal 38 amino acids of wildtype eIF4E2 protein or (b) C-terminal 38 amino acids of the wild-type eIF4E2 protein. In certain examples, the altered eIF4E2 gene variant encodes an eIF4E2 protein variant of any one of SEQ ID NOs:61-66.

[0013] In a second embodiment of the first aspect, the method can include altering the genome of a plant, cell, seed, tissue or germplasm thereof by altering its endogenous eIF4El gene that encodes endogenous eIF4El protein (preferably by altering both copies of the eIF4El gene so that the alteration is homozygous). The alteration generates an eIF4El gene variant that (i) is altered in the region comprising the fourth exon of endogenous eIF4El gene or (ii) encodes an eIF4El protein variant which differs from the endogenous protein in the region corresponding to the C-terminal 38 amino acids of wild type eIF4El protein. For example, the altered eIF4El gene variant can encode an eIF4El protein variant that comprises a deletion of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 , 35, 36, or 37 amino acids in the region corresponding to the C-terminal 38 amino acids of wild type eIF4El protein. In some examples, the altered eIF4El gene variant encodes an eIF4El protein variant is truncated and comprises a deletion of the (a) first 22 amino acids of the C-terminal 38 amino acids of wildtype eIF4El protein or (b) C-terminal 38 amino acids of the wild-type eIF4El protein.

[0014] In a third embodiment of the first aspect, the method involves altering the genome of a plant, cell, seed, tissue or germplasm thereof by altering its endogenous eIF4El (e.g., as described in the first embodiment) and altering its endogenous eIF4E2 gene (e.g., as described in the second embodiment. Preferably the alterations are homozygous so that both copies of the eIF4El gene and eIF4E2 gene are altered.

[0015] In particular examples of each of the foregoing first, second, and third embodiments of the first aspect, 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 MLN than the susceptible plant material was prior to the alteration and / or (ii) useful for introducing altered eIF4E gene alleles in a breeding program to improve resistance to MLN in the breeding population.

[0016] Also, in particular examples of each of the foregoing first, second, and third embodiments of the first aspect, the method involves altering the genome of a plant, cell, seed, tissue or germplasm thereof that is susceptible to maize chlorotic mottle virus (MCMV) and a potyvirus or other viruses. The method can thereby generate an altered plant, cell, seed, tissue or germplasm thereof that is (i) more resistant to MCMV and a potyvirus (or other viruses) than the susceptible plant material was prior to the alteration and / or (ii) useful for introducing altered eIF4E gene alleles in a breeding program to improve resistance to MCMV and a potyvirus (or other viruses) in the breeding population.

[0017] Plants that are susceptible to 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.

[0018] In a second aspect, provided is a method of altering a plant, cell, seed, tissue or germplasm thereof genome that comprises an endogenous variant eIF4E2 gene (e.g., the Mini Maize eIF4E2 gene, which encodes a truncated eIF4E2 deletion of the first 22 amino acids of the C-terminal 38 amino acids of wild-type eIF4E2 protein). The method includes altering the genome’s endogenous eIF4El gene to generate an eIF4El knockout that does not encode functional eIF4El protein (preferably by altering both copies of the eIF4El gene so that the knockout is homozygous). In some examples, the method includes introducing a nonsense mutation, missense mutation, and / or deletion so that no functional eIF4El protein is produced. In certain examples the eIF4El knockout comprises any one of SEQ ID NOs:26-40.

[0019] In a third aspect, provided is a method of altering a plant, cell, seed, tissue or germplasm thereof genome that comprises an endogenous variant eIF4El gene. The method includes altering the genome’s endogenous eIF4E2 gene to generate an eIF4E2 knockout that does not encode functional eIF4E2 protein (preferably by altering both copies of the eIF4E2 gene so that the knockout is homozygous). In some examples, the method includes introducing a nonsense mutation, missense mutation, and / or deletion so that no functional eIF4E2 protein is produced. In certain examples, the eIF4E2 knockout comprises any one of SEQ ID NOs:41- 55.

[0020] In each of the foregoing first aspect method, second aspect method, and third 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, gene alterations can be induced by genome editing, e.g., with a Cas endonuclease. In this regard, the closely spaced eIF4El and eIF4E2 genes are located near the centromere on chromosome 3, in a low recombination region. Therefore techniques such as gene editing or base editing make it possible to alter both these genes, which would be otherwise challenging if the mutations in each gene had to be stacked genetically. The process of introgressing this edited gene pair into other lines is also simpler as they would predominantly segregate as a single locus.

[0021] In a fourth aspect, provided herein is plant, cell, seed, tissue or germplasm thereof comprising (i) a first eIF4E gene variant that encodes a first eIF4E protein comprising afunctional N-terminal region and an inactivated C-terminal region and a second eIF4E gene variant that that encodes a second eIF4E protein comprising a functional N-terminal region and an inactivated C-terminal region or (ii) a first eIF4E gene knockout and a second eIF4E gene variant that encodes a second eIF4E protein comprising a functional N-terminal region and an inactivated C-terminal region. The plant, cell, seed, tissue or germplasm thereof can comprise (i) two first eIF4E variant alleles at the same locus and two second eIF4E variant alleles at the same locus or (ii) two first eIF4E gene knockout alleles at the same locus and two second eIF4E gene variant alleles at the same locus.

[0022] In a first embodiment of the fourth aspect, the plant, cell, seed, tissue or germplasm comprises an eIF4El gene variant encoding an eIF4El protein whose inactivated C-terminal region is altered in the region corresponding to the C-terminal 38 amino acids of wild type eIF4El protein and an eIF4E2 gene variant encoding an eIF4E2 protein whose inactivated C-terminal region is altered in the region corresponding to the C-terminal 38 amino acids of wild type eIF4E2 protein. In some examples, the eIF4El gene variant is altered in the region corresponding to the fourth exon of wild type eIF4El gene and the eIF4E2 gene variant is altered in the region corresponding to the fourth exon of wild type eIF4E2 gene. In some examples (A) the eIF4El gene variant encodes an eIF4El protein variant which differs from the endogenous protein in the region corresponding to the C- terminal 38 amino acids of wild type eIF4El protein (e.g., the eIF4El protein variant that comprises a deletion of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 , 35, 36, or 37 amino acids in the region corresponding to the C-terminal 38 amino acids of wild type eIF4El protein) and (B) the eIF4E2 gene variant encodes an eIF4El protein variant which differs from the endogenous protein in the region corresponding to the C-terminal 38 amino acids of wild type eIF4E2 protein (e.g., the eIF4E2 protein variant that comprises a deletion of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 , 35, 36, or 37 amino acids in the region corresponding to the C-terminal 38 amino acids of wild type eIF4E2 protein).

[0023] In a particular example, (A) the altered eIF4El gene variant encodes an eIF4El protein variant that is truncated and comprises a deletion of the sequence corresponding to the first 22 amino acids of the C-terminal 38 amino acids of wild-type eIF4El protein and (B) the eIF4E2 gene variant (i) encodes an eIF4E2 protein variant which differs from the endogenous protein in the region corresponding to the C-terminal 38 amino acids of wild type eIF4E2 protein (e.g., the eIF4E2 protein variant that comprises a deletion of 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 , 35, 36, or 37 amino acids in the region corresponding to the C-terminal 38 amino acids of wild type eIF4E2 protein) or (ii) encodes an eIF4E2 protein variant of any one of SEQ ID NOs:68-73 or (ii) comprises one of SEQ ID NOs:62-67. In each of the foregoing examples, the plant, cell, seed, tissue or germplasm preferably comprises two altered eIF4El gene variant alleles at the same locus (e.g., is homozygous for the altered eIF4El gene variant) and comprises two altered eIF4E2 gene variant alleles at the same locus (e.g., is homozygous for the altered eIF4E2 gene variant).

[0024] In another particular example, (A) the altered eIF4El gene variant encodes an eIF4El protein variant is truncated and comprises a deletion of the sequence corresponding to the C-terminal 38 amino acids of the wild-type eIF4El protein and (B) the eIF4E2 gene variant (i) encodes an eIF4E2 protein variant which differs from the endogenous protein in the region corresponding to the C-terminal 38 amino acids of wild type eIF4E2 protein (e.g., the eIF4E2 protein variant that comprises a deletion of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 , 35, 36, or 37 amino acids in the region corresponding to the C-terminal 38 amino acids of wild type eIF4E2 protein) or (ii) encodes an eIF4E2 protein variant of any one of SEQ ID NOs:68-73 or (iii) comprises one of SEQ ID NOs:62-67 or (iv) comprises one of SEQ ID NOs:41-55. In each of the foregoing examples, the plant, cell, seed, tissue or germplasm preferably comprises two altered eIF4El gene variant alleles at the same locus (e.g., is homozygous for the altered eIF4El gene variant) and comprises two altered eIF4E2 gene variant alleles at the same locus (e.g., is homozygous for the altered eIF4E2 gene variant).

[0025] In an additional example, (A) the altered eIF4E2 gene variant encodes an eIF4E2 protein variant is truncated and comprises a deletion of the sequence corresponding to the first 22 amino acids of the C-terminal 38 amino acids of wild-type eIF4E2 protein and (B) the eIF4El gene variant (i) encodes an eIF4El protein variant which differs from the endogenous protein in the region corresponding to the C-terminal 38 amino acids of wild type eIF4El protein (e.g., the eIF4El protein variant that comprises a deletion of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 , 35, 36, or 37 amino acids in the region corresponding to the C-terminal 38 amino acids of wild type eIF4El protein) or (ii) comprises an eIF4El gene knockout (e.g., comprises any one of SEQ ID NOs:26-40). In another additional example, (A) the altered eIF4E2 gene variant encodes an eIF4E2 protein variant is truncated and comprises a deletion of the sequence corresponding to the C-terminal 38 amino acids of wild-type eIF4E2 protein and (B) theeIF4El gene variant encodes an eIF4El protein variant which differs from the endogenous protein in the region corresponding to the C-terminal 38 amino acids of wild type eIF4El protein (e.g., the eIF4El protein variant that comprises a deletion of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 , 35, 36, or 37 amino acids in the region corresponding to the C-terminal 38 amino acids of wild type eIF4El protein) or (ii) comprises an eIF4El gene knockout (e.g., comprises any one of SEQ ID NOs:26-40). In each of the foregoing examples, the plant, cell, seed, tissue or germplasm preferably comprises two altered eIF4El gene variant alleles at the same locus (e.g., is homozygous for the altered eIF4El gene variant) and comprises two altered eIF4E2 gene variant alleles at the same locus (e.g., is homozygous for the altered eIF4E2 gene variant).

[0026] In certain examples of another embodiment of the fourth aspect, the plant, cell, seed, tissue or germplasm comprises an eIF4El gene knockout comprising any one of SEQ ID NOs:26-40 (and is preferably homozygous for the knockout) and comprises any two altered eIF4E2 gene variant alleles disclosed herein at the same locus (e.g., is homozygous for the altered eIF4E2 gene variant). In other examples of the fourth aspect, the plant, cell, seed, tissue or germplasm comprises an eIF4E2 gene knockout comprising any one of SEQ ID NOs:41-55 (and is preferably homozygous for the knockout) and comprises any two altered eIF4El gene variant alleles disclosed herein at the same locus (e.g., is homozygous for the altered eIF4El gene variant).

[0027] In a fifth aspect, provided herein is a method of identifying a plant, seed, tissue or germplasm thereof comprising an eIF4E variant associated with increased resistance to MLN and / or increased resistance to maize chlorotic mottle virus (MCMV) and a potyvirus such as sugarcane mosaic virus (SCMV), or other viruses. The method comprises providing one or more plant, seed, tissue or germplasm thereof comprising an eIF4E variant; obtaining a sample comprising nucleic acid from each of the plant, seed, tissue or germplasm thereof; screening the sample for the eIF4E variant; and detecting the one or more an eIF4E variant in the sample and thereby identifying the plant or plant material as comprising a eIF4E variant associated with increased resistance to MLN and / or increased resistance to MCMV and a potyvirus such as SCMV, or other viruses. In one embodiment of this fifth aspect, screening can include screening the sample for one or more of the following eIF4E variants:(i) variant genomic sequence or cDNA sequence encoding an eIF4El protein variant sequence that is altered (comprises an insertion, deletion, or substitution) in the region corresponding to the C-terminal 38 amino acids of wild type eIF4El protein,(ii) variant genomic sequence or cDNA sequence encoding an eIF4El protein variant that comprises a deletion of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 , 35, 36, or 37 amino acids in the region corresponding to the C-terminal 38 amino acids of wild type eIF4El protein,(iii) variant genomic sequence or cDNA sequence encoding an eIF4El protein variant that is truncated and comprises a deletion of the sequence corresponding to the C- terminal 38 amino acids of wild-type eIF4El,(iv) variant genomic sequence or cDNA sequence encoding an eIF4El protein variant that is truncated and comprises a deletion of the sequence corresponding to the first 22 amino acids of the C-terminal 38 amino acids of wild-type eIF4El protein,(v) variant genomic sequence or cDNA sequence encoding eIF4E2 protein variant sequence that is altered (comprises an insertion, deletion, or substitution) in the region corresponding to the C-terminal 38 amino acids of wild type eIF4El protein,(vi) variant genomic sequence or cDNA sequence encoding an eIF4E2 protein variant that comprises a deletion of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 , 35, 36, or 37 amino acids in the region corresponding to the C-terminal 38 amino acids of wild-type eIF4E2 protein,(vii) variant genomic sequence or cDNA sequence encoding eIF4E2 protein variant that is truncated and comprises a deletion of the sequence corresponding to the C-terminal 38 amino acids of wild-type eIF4E2 protein,(viii) variant genomic sequence or cDNA sequence encoding eIF4E2 protein variant that is truncated and comprises a deletion of the sequence corresponding the first 22 amino acids of the C-terminal 38 amino acids of wild-type eIF4E2 protein,(ix) variant eIF4El protein sequence that is altered (comprises an insertion, deletion, or substitution) in the region corresponding to the C-terminal 38 amino acids of wild type eIF4El protein,(x) variant eIF4El protein that comprises a deletion of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 , 35, 36, or 37 amino acids in the region corresponding to the C-terminal 38 amino acids of wild type eIF4El protein,(xi) variant eIF4El protein that is truncated and comprises a deletion of the sequence corresponding to the C-terminal 38 amino acids of wild-type eIF4El,(xii) variant eIF4El protein that is truncated and comprises a deletion of the sequence corresponding to the first 22 amino acids of the C-terminal 38 amino acids of wildtype eIF4El protein,(xiii) variant eIF4E2 protein sequence that is altered (comprises an insertion, deletion, or substitution) in the region corresponding to the C-terminal 38 amino acids of wild type eIF4E2 protein,(xiv) variant eIF4E2 protein that comprises a deletion of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 , 35, 36, or 37 amino acids in the region corresponding to the C-terminal 38 amino acids of wild type eIF4E2 protein,(xv) variant eIF4E2 protein that is truncated and comprises a deletion of the sequence corresponding to the C-terminal 38 amino acids of wild-type eIF4E2,(xvi) variant eIF4E2 protein that is truncated and comprises a deletion of the sequence corresponding to the first 22 amino acids of the C-terminal 38 amino acids of the wild-type eIF4E2 protein, or(xvii) variant nucleotide sequence comprising any of SEQ ID NOs: 16, 17, or 62- 67.Upon detecting one or more of the foregoing eIF4E variant sequences in the sample, the corresponding plant or plant material can be identified as comprising a eIF4E variant associated with increased resistance to MLN and / or increased resistance to MCMV and potyvirus (such as SCMV), or other viruses.

[0028] In a sixth aspect, provided herein is a method of improving resistance to MCMV and a potyvirus (such as SCMV), or other viruses in a plant. The method comprises using the method of the fifth aspect disclosed herein to identify a first parent plant as comprising a eIF4E variant associated with increased resistance to MLN and / or increased resistance to MCMV and a potyvirus (such as SCMV); and crossing the identified first parent plant with a second parent plant (that does not include the eIF4E variant) to produce one or more progeny plants comprising the eIF4E variant. In the method of this sixth aspect, the second parent plant is susceptible to the MLN (and / or to MCMV and potyvirus (such as SCMV), or other viruses) and preferably the progeny plants are more resistant to MLN (and / or to the MCMV and potyvirus (such as SCMV), or other viruses) than the second parent plant.

[0029] In one embodiment of the sixth aspect, the first parent plant comprises a (i) variant genomic sequence or cDNA sequence encoding an eIF4El protein variant sequence that is altered (comprises an insertion, deletion, or substitution) in the region corresponding to theC-terminal 38 amino acids of wild type eIF4El protein, (ii) variant genomic sequence or cDNA sequence encoding an eIF4El protein variant that comprises a deletion of 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 , 35, 36, or 37 amino acids in the region corresponding to the C-terminal 38 amino acids of wild type eIF4El protein, (iii) variant genomic sequence or cDNA sequence encoding an eIF4El protein variant that is truncated and comprises a deletion of the sequence corresponding to the C-terminal 38 amino acids of wild-type eIF4El, (iv) variant genomic sequence or cDNA sequence encoding an eIF4El protein variant that is truncated and comprises a deletion of the sequence corresponding to the first 22 amino acids of the C- terminal 38 amino acids of wild-type eIF4El protein, (v) variant genomic sequence or cDNA sequence encoding eIF4E2 protein variant sequence that is altered (comprises an insertion, deletion, or substitution) in the region corresponding to the C-terminal 38 amino acids of wild type eIF4El protein, (vi) variant genomic sequence or cDNA sequence encoding an eIF4E2 protein variant that comprises a deletion of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 , 35, 36, or 37 amino acids in the region corresponding to the C-terminal 38 amino acids of wild-type eIF4E2 protein,(vii) variant genomic sequence or cDNA sequence encoding eIF4E2 protein variant that is truncated and comprises a deletion of the sequence corresponding to the C-terminal 38 amino acids of wild-type eIF4E2 protein, (viii) variant genomic sequence or cDNA sequence encoding eIF4E2 protein variant that is truncated and comprises a deletion of the sequence corresponding to the first 22 amino acids of the C-terminal 38 amino acids of the wild-type eIF4E2 protein, (ix) variant eIF4El protein sequence that is altered (comprises an insertion, deletion, or substitution) in the region corresponding to the C-terminal 38 amino acids of wild type eIF4El protein, (x) variant eIF4El protein that comprises a deletion of 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 , 35, 36, or 37 amino acids in the region corresponding to the C-terminal 38 amino acids of wild type eIF4El protein, (xi) variant eIF4El protein that is truncated and comprises a deletion of the sequence corresponding to the C-terminal 38 amino acids of wild-type eIF4El, (xii) variant eIF4El protein that is truncated and comprises a deletion of the sequence corresponding to the first 22 amino acids of the C-terminal 38 amino acids of the wild-type eIF4El protein, (xiii) variant eIF4E2 protein sequence that is altered (comprises an insertion, deletion, or substitution) in the region corresponding to the C-terminal 38 amino acids of wild type eIF4E2 protein, (xiv) variant eIF4E2 protein that comprises a deletion of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34 , 35, 36, or 37 amino acids in the region corresponding to the C-terminal 38 amino acids of wild type eIF4E2 protein, (xv) variant eIF4E2 protein that is truncated and comprises a deletion of the sequence corresponding to the C-terminal 38 amino acids of wildtype eIF4E2, (xvi) variant eIF4E2 protein that is truncated and comprises a deletion of the sequence corresponding to the first 22 amino acids of the C-terminal 38 amino acids of wildtype eIF4E2 protein, or (xvii) variant nucleotide sequence comprising any of SEQ ID NOs: 16, 17, or 62-67. In this embodiment, the resulting progeny plant with the eIF4E variant preferably more resistant to MLN (and / or to MCMV and a potyvirus such as SCMV, or other viruses) than the second parent plant.

[0030] 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 eIF4E variant from the first parent plant. In particular examples, one or more backcross progeny plants comprising the eIF4E variant can be crossed with the second parent plant to produce next generation backcross progeny plants comprising the eIF4E variant. This backcrossing scheme can be repeated to generate additional plants that introgress the eIF4E variant into the genetic background of the second parent plant and thereby produce new plants that include the eIF4E variant but are otherwise substantially isogenic to the second parent plant and include desirable agronomic properties of the second parent plant.

[0031] In a seventh aspect, disclosed herein is a method of generating viable double mutants of eIF4El and eIF4E2 in plants. The method comprises crossing a first parent plant with a second parent plant to produce progeny plants, wherein the first parent and the second parent genomes collectively comprise (i) a first eIF4E gene variant that encodes a first eIF4E protein comprising a functional N-terminal region and an inactivated C-terminal region and a second eIF4E gene variant that that encodes a second eIF4E protein comprising a functional N-terminal region and an inactivated C-terminal region or (ii) a first eIF4E gene knockout and a second eIF4E gene variant that encodes a second eIF4E protein comprising a functional N- terminal region and an inactivated C-terminal region; and selecting at least one progeny plant that is a plant in accordance with the fourth aspect disclosed herein. Thus, the selected one or more progeny plants can be any of the embodiments and particular examples of the fourth aspect described herein. In one embodiment of the seventh aspect, the progeny plant comprises two altered eIF4El gene variant alleles at the same locus (e.g., is homozygous for the altered eIF4El gene variant) and comprises two altered eIF4E2 gene variant alleles at the same locus (e.g., is homozygous for the altered eIF4E2 gene variant). In another embodiment of theseventh aspect, the progeny plant comprises two altered eIF4El gene knockout alleles at the same locus (e.g., is homozygous for the altered eIF4El knockout) and comprises two altered eIF4E2 gene variant alleles at the same locus (e.g., is homozygous for the altered eIF4E2 gene variant). In yet another embodiment, the progeny plant comprises two altered eIF4E2 gene knockout alleles at the same locus (e.g., is homozygous for the altered eIF4E2 knockout) and comprises two altered eIF4El gene variant alleles at the same locus (e.g., is homozygous for the altered eIF4E2 gene variant).

[0032] In some embodiments of the seventh aspect, the first parent plant comprises two eIF4El gene variant alleles disclosed herein at the same locus (e.g., is homozygous for the eIF4El gene variant) and comprises two eIF4E2 gene variant alleles disclosed herein at the same locus (e.g., is homozygous for the eIF4E2 gene variant) and the second parent plant comprises at least one eIF4El or eIF4E2 gene allele that is wild type and is susceptible to MLN (and / or to MCMV and a potyvirus such as SCMV, or other viruses). In these embodiments, the selected each of the one or more selected progeny plants preferably comprises two eIF4El gene variant alleles disclosed herein at the same locus (e.g., is homozygous for the eIF4El gene variant) and comprises two eIF4E2 gene variant alleles disclosed herein at the same locus (e.g., is homozygous for the eIF4E2 gene variant). Thus, in some examples of these embodiments, the selected progeny plant(s) have improved resistance to MLN (and / or to MCMV and a potyvirus such as SCMV, or other viruses) than the second parent plant.

[0033] In certain embodiments of the seventh aspect, the 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 eIF4E variant(s) from the first parent plant. In particular examples, one or more backcross progeny plants comprising the eIF4E variant(s) can be crossed with the second parent plant to produce next generation backcross progeny plants comprising the eIF4E variant(s). This backcrossing scheme can be repeated to generate additional plants that introgress the eIF4E variant(s) into the genetic background of the second parent plant and thereby produce new plants that include the eIF4E variant(s) but are otherwise substantially isogenic to the second parent plant and include desirable agronomic properties of the second parent plant.

[0034] In each embodiment and example of the foregoing first aspect, second aspect, third aspect, fourth aspect, fifth aspect, sixth aspect, and seventh 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.

[0035] Provided herein is an isolated recombinant nucleic acid comprising one or more of SEQ ID NOs: 16, 17, 19-25, and 37-67. In particular example, 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: 19 and 20, or SEQ ID NOs:21 and 22, or SEQ ID NOs:23 and 24 or SEQ ID NO:25) for introducing an eIF4E variant disclosed herein.

[0036] Methods for introducing Cas endonucleases and guide RNAs are described in more detail herein.BRIEF DESCRIPTION OF THE DRAWINGS AND SEQUENCE LISTING

[0037] Figure 1 is a schematic diagram of a genomic copy of the eIF4El gene showing exons and genome edits in elite maize lines CKL05022 and CML536, and Mini Maize. Guide RNAs were targeted to target sequence indicated by bold text in the first exon of each gene. Nucleotide deletions are indicated by dashes and the position of insertions are indicated by a caret symbol and corresponding inserted base(s) under each sequence. PAM sequences are underlined and start ATG codons shown underneath an arrow. Each tested event was (i) screened for response to MLN after artificial inoculation and (ii) was an apparent knockout from frameshift mutations.

[0038] Figure 2 is a schematic diagram of a genomic copy of the eIF4E2 gene showing exons and genome edits in elite maize lines CKL05022 and CML536, and Mini Maize. Guide RNAs, nucleotide deletions, nucleotide insertions, PAM sequences and start ATG codons are indicated in same way as in Figure 1. Each tested event was (i) screened for response to MLN after artificial inoculation and (ii) was an apparent knockout from frameshift mutations.

[0039] Figure 3 is a schematic diagram of a genomic copy of eIF(iso)4El gene showing exons and genome edits in elite maize lines CKL05022 and CML536 e. Guide RNAs, nucleotide deletions, nucleotide insertions, PAM sequences and start ATG codons are indicated in same way as in Figure 1. Each tested event was (i) screened for response to MLN after artificial inoculation and (ii) was an apparent knockout from frameshift mutations.

[0040] Figure 4 is a schematic diagram of a genomic copy of eIF(iso)4E2 gene showing exons and genome edits in elite maize lines CKL05022 and CML536. Guide RNAs, nucleotide deletions, nucleotide insertions, PAM sequences and start ATG codons are indicated in same way as in Figure 1. Each tested event was (i) screened for response to MLN after artificial inoculation and (ii) was an apparent knockout from frameshift mutations.

[0041] Figure 5 is a set of bar graphs showing the severity of MLN symptoms in the indicated plant lines at 14-, 16-, 19-, 21-, and 24-days following MLN inoculation. Panel (a) shows wild-type and gene-edited versions of elite corn lines CKL05022 and CML536; and panel (b) shows wild-type and gene-edited versions of Mini Maize line. The y-axis of each graph indicates symptom severity score on a scale of 1 (no symptoms, resistant or R) to 9 (plant death, susceptible or S). Error bars represent standard error estimates and the numbers in parentheses the biological replicates (plants). Molecular details of gene editing for each event are shown in Figures 1-4. Method for scoring MLN is described and available from the Maize Lethal Necrosis (MLN) Information Portal hosted by the CIMMYT organization web site. The lowest score for MLN screening is set to 1, which corresponds to no disease symptoms. The SE scores for eIF4El-KO in Mini Maize result from minor damage to the leaves, which can be physical in nature, but is recorded as a score higher than 1.

[0042] Figure 6 panel (a) is an image showing the response to MLN of CKL05022 (WT) and CKL05022 knocked out for eIF4el (eif4e l-KO) at 45 days after either inoculation (dai) with MLN virus (+) or uninfected control (-). Figure 6 panel (b) is an image showing the response of Mini Maize WT, eif4el-KO or knocked out for eif4el eif4e2-KO') to MLN (40 dai). Figure 6 panel (c) is a bar graph showing SCMV and MCMV quantification by ELISA in MLN-inoculated Mini Maize plants.

[0043] Figure 7 is a schematic exon diagram comparing genomic sequence of eIF4E2 among CKL05022, CML536, and Mini Maize. The deletion across the intron / exon junction in Mini Maize is indicated by dashes. Regulatory motifs present in CKL05022 CML536 lines for RNA splicing between the 3rd intron and 4th exon is indicated.

[0044] Figure 8 is a bar showing SCMV and MCMV quantification by ELISA in MLN- the following inoculated plants: single eIF4el knockouts (ez / e7-KO) and doubled edited plants carrying eif4el-KO and disrupted fourth exon of eIF4E2 (eIF4E2-exon-4ED).

[0045] Figure 9 is a set of schematic diagrams comparing eIF4E2 wild type protein with natural and gene-edited variants disclosed herein. Figure 9 panel (a) shows wildtype eIF4E2 in maize is 220 amino acids long, naturally occurring variant in Mini Maize has an in-frame deletion of 22 amino acids corresponding to the 4th exon (i.e., deletion of the first 22 amino acids of the C-terminal 38 amino acids of wild-type eIF4E2 protein), and some of the edited variants disclosed herein in elite line CKL05022 are shorter by nearly the entire stretch of C- terminal 38 amino acids of eIF4E2. Panel (b) illustrates the effect of different combinations of eIF4El-KO and eIF4E2ED variants on plant growth and viral replication / MLN resistance. In panel (b), KO indicates knockout; X indicates no growth; dash (-) indicates no host; checkmark ( ) indicates normal growth. The numbers in the respective horizonal bars are for the amino acids. In addition to the naturally occurring variant of eIF4E2 in Mini Maize, any frameshift mutation in the 4th exon resulted in complete MLN resistance (when combined with eif4el-KO). Besides frameshift mutants, we obtained at least two in-frame mutants that lacked either the first four amino acids (181-184) in CKL05022 (Figure 6, event 2) or the first six amino acids (183-188) in CML536 (Figure S6, event 3) corresponding to the 4th exon that were completely resistant to MLN. The C-terminal 38 amino acids behaved like a domain required for recognition by the viruses to translate their proteins but are not necessary for the translation of the maize proteins.

[0046] Figure 10 is a diagram comparing the gene structure of eIF4E genes across a variety of monocot and dicot plant species. Notably the third, fourth, and fifth exon, which encode the C-terminal region of each eIF4E gene product, are nearly perfectly conserved across these widely divergent species; in particular the length of the fourth exon is perfectly conserved in all of these species. Therefore, selective disruption of the region comprising of the fourth exon of eIF4E gene (as exemplified herein in maize) is expected to provide resistance to viral infection in a wide range of monocot and dicot plant species.

[0047] Figure 11 is a sequence alignment of eIF4E proteins across the same monocot and dicot plant species shown in Figure 10. The C-terminal region of each eIF4E protein encoded by the fourth exon (Exon 4) of each corresponding is indicated. The Exon-4 encoded regions of eIF4E proteins are highly conserved; and amino acid substitutions in this region are conservative. This further supports the use of selective disruption of Exon-4 can provide resistance to viral infection in a wide range of monocot and dicot plant species.

[0048] 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

[0049] 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 and equivalents 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.

[0050] As used herein, the term “maize” means Zea mays or com and includes all plant varieties that can be bred with corn, including wild maize species.

[0051] 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.

[0052] “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.

[0053] “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.

[0054] 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.

[0055] A “Cas endonuclease” may comprise domains that enable it to function as a double- strand-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).

[0056] 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).

[0057] An “elite line” is any line that has resulted from breeding and selection for superior agronomic performance.

[0058] “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’ noncoding 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.

[0059] “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.

[0060] 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.

[0061] 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 genomic sequence within a plant cell, (e.g. an endogenous genomic sequence of a eIF4El or eIF4E2 gene present within the genome of a maize plant cell).

[0062] 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’ noncoding sequences) the coding sequence.

[0063] 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.

[0064] 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).

[0065] 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.

[0066] 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 least one 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).

[0067] The term “hybrid” refers to the progeny obtained between the crossing of at least two genetically dissimilar parents.

[0068] The term “inbred” refers to a line that has been bred for genetic homogeneity.

[0069] 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 eIF4El variant or eIF4E2 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 canoccur 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.

[0070] The process of “introgressing” is often referred to as “backcrossing” when the process is repeated two or more times.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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, internucleotide modifications, such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, 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 eIF4El or eIF4E2 gene present within the genome of a maize plant cell).

[0076] 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.

[0077] 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.

[0078] 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.

[0079] A “targeted alteration” or “variant” is a gene (e.g., eIF4El or eIF4E2 gene) sequence that has been altered through human intervention. 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 Cas 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., eIF4El or eIF4E2 gene variant). The disclosed targeted alterations or variants of an eIF4El or eIF4E2 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, Targeting Induced Local Lesions IN Genomes or TILLING (see e.g., McCallum et al., 2000, Nat Biotechnol 18:455-457), or the use of double-strand-break inducing agents (DSB Agents).

[0080] 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), Agr()bacterium-mQd\a.iQ transfer (Fraley et al., 1983, Proc. Natl. Acad. Set. USA 80:4803-7), direct DNA uptake, and microprojectile bombardment (Klein et al., 1987, Nature 327:70).

[0081] The term “variants” refer to substantially similar sequences. 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 native polynucleotide (e.g. an eIF4El or eIF4E2 gene variant disclosed herein). As used herein, a “native” polynucleotide or polypeptide comprises a naturally occurring nucleotide sequence or amino acid sequence, respectively.

[0082] 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 in Mobile DNA II, pp. 761-783, Eds. Craigie et al., (ASM Press, 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’l Acad. Sci USA 93(3)1156-1160) and CRISPR-Cas endonucleases (see e.g. International Application Publication W02007 / 025097).

[0083] 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.

[0084] 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 7(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 transactivating 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).

[0085] 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 doublestranded 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.

[0086] 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 WO2016007347, International Application Publication WO201625131, and International Application Publication WO 2022 / 082179 all of which are incorporated by reference herein.

[0087] In some aspects of the disclosure, a targeted genomic modification is introduced in a B. napus plant cell, wherein the targeted modification includes a targeted alteration of the genomic sequence of DFR and / or F3H gene in the 7>. napus 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 B. napus cell with a first and second guide RNAs as Cas-gRNA complexes that recognizes target sequences in the genome of the B. napus cell and is able to induce DSBs in the genomic sequence, e.g., thereby altering the endogenous target DFR and / or F3H gene.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] In accordance with the methods disclosed herein, a guide RNA comprising any of SEQ ID NOs: 19-25) can be used to introduce an eIF4E variant disclosed herein.

[0093] 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 select for 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 Glyphosate N-Acetyltransferase (GAT) gene variant 4621 for herbicide resistance.

[0094] Isolated Nucleic Acid Molecules and Variants and Fragments Thereof. Isolated or recombinant nucleic acid molecules comprising eIF4El or eIF4E2 gene variants disclosed herein as well as nucleic acid molecules sufficient for use as hybridization probes to identify eIF4El or eIF4E2 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 single-stranded. In some examples, the nucleic acid molecule can be doublestranded.

[0095] 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 eIF4El or eIF4E2 gene variant in the genome of the cell.

[0096] In some embodiments, an isolated nucleic acid molecule comprising an eIF4El or eIF4E2 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 ofthe 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. In some embodiments, the nucleic acid molecule comprising one of SEQ ID NOs:26-40, SEQ ID NOs:41-55, or SEQ ID NOs:62-67 is non-genomic.

[0097] A variety of polynucleotides comprising eIF4El or eIF4E2 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 eIF4El or eIF4E2 gene variants or related to DFR or F3H variants disclosed herein.

[0098] Provided herein are nucleic acid molecules comprising one or more of SEQ ID NOs:26-40, SEQ ID NOs:41-55, or SEQ ID NOs:62-67. “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.

[0099] “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).

[0100] Nucleotide Constructs, Expression Cassettes and Vectors. The use of the term “construct” in connection with isolated and / or heterologous polynucleotides herein is notintended to limit the disclosure to constructs comprising DNA. Polynucleotide constructs, particularly polynucleotides 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, doublestranded forms, hairpins, stem-and-loop structures and the like.

[0101] 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.

[0102] The disclosed sequences can be used in constructs for 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.

[0103] 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.

[0104] 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 / orto 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.

[0105] In some embodiments the DNA construct comprises a polynucleotide comprising one or more of SEQ ID NOs:26-40, SEQ ID NOs:41-55, or SEQ ID NOs:62-67 or a fragment or variant thereof.

[0106] 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 tissuespecificity 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 of RNA 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.

[0107] 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).

[0108] 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.

[0109] 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 plant-preferred for a particular amino acid may be derived from known gene sequences from plants.

[0110] Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron 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.

[0111] 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.

[0112] 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

[0113] 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 virtuallyany 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. Techniques for transforming Brassica plants in particular are disclosed, for example, in U.S. Patent No. 5,750,871.

[0114] 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 vector used 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.

[0115] 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.

[0116] The alteration (e.g., introduction of a stop codon, mutation or deletion) of an endogenous gene (e.g., eIF4El or eIF4E2 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.

[0117] Using the methods disclosed herein, eIF4E variant-containing plants are generated. For example, a plant comprising an altered endogenous genomic sequence containing one ormore of the following variants: SEQ ID NOs:26-40, SEQ ID NOs:41-55, or SEQ ID NOs:62- 67. In some examples, the modified maize plant comprises double homozygous variants of eIF4El or eIF4E2, or both eIF4El and eIF4E2.

[0118] “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 differentiated or undifferentiated (e.g. callus, suspension culture cells, protoplasts, leaf cells, root cells, phloem cells and pollen).

[0119] 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), dgrotocterzwm-mediated transformation (US Patent Numbers 5,563,055 and 5,981,840), direct gene transfer (Paszkowski et al. 1984 EMBO 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 roc. 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 ExperimentalManipulation 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 Cell 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).

[0120] 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 eIF4El or eIF4E2 gene variants disclosed herein.

[0121] 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 eIF4El or eIF4E2 gene variant(s).

[0122] Gene introgression by MAS can be used to diminish linkage drag and yield drag. Gepts 2002 Crop Sei,' 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 thatare 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.

[0123] 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, as SNPs do not require large amounts of DNA and automation of the assay may be straightforward. 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 Chem164-172; 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 BEADARRAYS®. (Illumina).

[0124] 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).

[0125] 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 : Identification of Maize elF Paralogs.

[0126] To identify maize paralogs of elF proteins that have been associated with virus resistance, phylogenetic analysis of the maize elF proteins was performed in context of those from other plant species. This approach proved useful in assigning specific function to geneswithin a family. Four of the 17 maize proteins, two each from the eIF4E and eIF(iso)4E subfamilies, were found to group in a clade with the highest frequency of elF proteins from non-maize plant species where their mutants were known to confer virus resistance. These are referred to herein as eIF4El, eEF4E2, eIF(iso)4El, and eIF(iso)4E2.Example 2: Editing genes for eIF4E and eIF(iso)4E factors

[0127] Guide RNAs (sgRNAs) were targeted to the two genes in the eIF4E subfamily in two elite MLN-susceptible CIMMYT maize inbred lines, CKL05022 and CML536. Additionally, the eIF(iso)4El and eIF(iso)4E2 genes were edited in CML536. Guide RNA sequences are shown in Table 2.Table 2

[0128] Selected gene edited events of each target gene included SEQ ID NOs: 26-40, SEQ ID NOs: 56-61 and are shown in Figures 1, 2, 3, and 4.

[0129] Edited lines homozygous for respective alleles were screened in the greenhouse for response to controlled MLN inoculation (Gowda et al., 2015, Theor. AppL Genet. 128, 1957-1968). Single knockouts (KOs) of each of the genes conferred partial resistance in both the lines. See Figure 5(a). Although the edited plants produced several additional leaves as compared to wildtype after MLN inoculation, none of the plants survived to flowering. No double knockouts for the eif4el / eif4e2 combination were recovered despite multiple attempts with either co-transformation using the guides for both the genes or using the eIF4E2 guide to transform the eif4el-KO events and vice versa. This suggested that at least one or the other paralog from the eIF4E subfamily was required for plant survival.

[0130] The virus may prefer one or the other paralog (or member) of the eIF4E or eIF(iso)4E subfamilies, so knocking out various members may provide different levels of resistance (Wang et al., 2013, PLoS One 8, e50627). However, as long as one of the family members, which is required for plant survival, is functional, the virus can still replicate. Thus, single knockouts are expected to result in only partial resistance, if any.Example 3 : Complete MLN Resistance by Editing eIF4E genes in Fast Flowering Mini Maize

[0131] Fast Flowering Mini Maize (“Mini Maize”) is a dwarf experimental line with a short lifecycle, making for relatively quick testing of the effects of gene alteration on phenotype (McCaw et al., 2016, Genetics 204, 35-42). The same guideRNAs used for CKL0522 (see Table 2 - SEQ ID NOs: 19 and 21) were used to create knockouts of eIF4El and eIF4E2. The eIF4El knockouts in Mini Maize plants were highly resistant to MLN (See Figures 5b and 6b). For additional evidence confirming the resistant phenotype, we did not detect either MCMV or SCMV in the leaf tissue of MLN-inoculated eIF4El-KO Mini Maize plants (Figure 6c). Mini Maize plants with the edited eif4el gene went on to produce normal-looking ears like the wild-type plants. In contrast, the eif4e2-KO plants exhibited only marginal improvement in resistance (Figure 6b and 6c). This suggested that eIF4E2 protein was altered in Mini Maize, which allowed for normal plant growth but no longer facilitated the translation of the viral genome in the absence of eIF4El.

[0132] The foregoing results demonstrated that knocking out different eif4e genes had different effects on resistance against maize lethal necrosis (MLN) in Mini Maize plants and also indicated that knocking out eIF4El in certain genetic backgrounds (presumably backgrounds comprising a compromised, but not full knock-out of, eIF4E2') provides much better resistance to MLN.Example 4: Analysis of eIF4E2 in Mini Maize

[0133] A comparison of the genomic sequences of eIF4E2 revealed a novel deletion encompassing the 3’ region of the third intron and 5’ region of the fourth exon in Mini Maize as compared to CKL05022 and CML536 (Figure 7). The deletion included the pyrimidine-rich motif that preceded the splicing motif, AG, at the intron-3 / exon-4 junction along with five nucleotides from the fourth exon (Lim and Burge, 2001). Additionally, the cDNA for the eIF4E2 gene from Mini Maize was distinctly smaller than that from CKL05022 and Sanger sequencing revealed that the entire exon 4 was spliced out from the Mini Maize eIF4E2 cDNA. The destruction of the intron / exon junction motif apparently caused alternative RNA processing where the remainder of exon-4 was spliced out as part of the larger intron that encompassed the third and fourth introns. The excision of the fourth exon shortened the predicted eIF4E2 protein by 22 amino acids but without affecting its isoelectric point, which is a feature involved in certain protein-protein interactions (size of the truncated protein ~22 kDa, pl 5.8). The native protein is 220 amino acids long (24.6 kDa, pl 5.8 for CML536).

[0134] Further screening was done of more than 40 genetically diverse CIMMYT maize lines to identify any containing the same eIF4E2 in-frame deletion as Mini Maize. The deletion was not found in any of the other screened lines.Example 5: Validating the Contribution of Mutant eIF4E2 to MLN Resistance

[0135] To determine whether the modified eIF4E2 gene in Mini Maize was responsible for MLN resistance described above, full-length eIF4E2 cDNA was overexpressed in the inbred line CML536 containing the eif4el-KO as described above. Expression of the transgenic cDNA was confirmed by RT-PCR in multiple events and also by Sanger sequencing. The resulting CML536 eif4el-KO plants overexpressing eIF4E2 cDNA were highly susceptible to MLN, relative to CML536 eif4el-KO plants expressing only the native eIF4E2 comprising the deletion discussed above. The foregoing indicates that the in-frame deletion of eIF4E2 was indeed responsible for MLN resistance in Mini Maize plants that were also edited to knock out eIF4El.Example 6: Structural analysis of eIF4E2 Protein that Contributes to MLN Resistance

[0136] It appears that truncated eIF4E2 protein was apparently sufficient for normal plant growth in the absence of eIF4El, but that the truncated protein did not facilitate the interaction of the MCMV or SCMV genome with the elF complex. We threaded the structures of the full- length and truncated versions of the maize eIF4E2 proteins based on the structure from the human eIF4E (Coutinho de Oliveira et al., 2019; Peter et al., 2015; Volpon et al., 2006). The binding of the viral genome-linked protein (VPg) from potato virus Y involves the C-terminal domain of the eIF4E protein, which does not overlap with the binding site of eIF4G, another protein that is involved in forming the translation initiation complex. The truncation or elimination of the C-terminal folds in the maize eIF4E2 protein makes it unrecognizable to both SCMV, a poty virus, and MCMV, a Tombusvirus.

[0137] Unlike SCMV, MCMV does not bind the eIF4E protein through a VPg but does so directly via the 3’-CITEs, which form various types of secondary structures (Carino et al., 2020; Simon and Miller, 2013). Viral VPg-bound RNAs are known to compete with the 5’- m7G-capped eukaryotic mRNAs for binding to the eIF4E protein (Simon and Miller, 2013). The maize mRNAs, however, must still be able to bind the truncated eIF4E. The VPg from SCMV has a molecular mass of approximately 21 kDa (accession: AZM65781.1) as compared to 0.3 kDa of m7G, so would require a larger surface to form a stable complex with the eIF4E protein. Based on the crystal structure of the murine eIF4E bound to 7-methyl-GDP, a number of amino acid residues were identified that were conserved across diverse species, including wheat (Marcotrigiano et al., 1997). None of these conserved residues is found in the C-terminaldomain that is partially or completely deleted in Mini Maize or edited variants in the elite lines. In contrast, two of the conserved residues, a histidine and a lysine, required for VPg binding to the eIF4E protein happen to be in this domain. Without wishing to be bound by any theory or mechanism of action, this may explain why truncated eIF4E is still able to recognize the maize mRNA but not the viral RNA. It is possible that the C-terminal domain interacts with the genomes of other viruses as well. That would make it a suitable target to exploit for introducing broad-spectrum resistance. This discovery should bring other plant species, even the ones that contain only a single copy of the eIF4E gene, within the scope of gene editing to confer virus resistance.Example 7: Generating Complete MLN-Resistance in Elite Maize Lines

[0138] To further confirm results discussed in the foregoing examples and to confer MLN- resistance in additional elite maize lines, sgRNA was designed to target the 5’ end of the fourth exon of the eIF4E2 gene in CKL05022 and CML536 lines comprising eIF4el knockouts discussed above. Table 3 shows the sgRNA sequence,Table 3

[0139] Different edited eIF4E2 variants were created and the expression of edited eIF4E2 in these events was confirmed by RT-PCR. Most variants contained frameshift mutations close to the 5’-end of the fourth exon. Table 4 shows Edited Events Relative to CKL5022 eIF4E2 genomic sequence (SEQ ID NO: 10), added nucleotides are bolded and underlined, deletions are indicated by dashes, and the number of inserted nucleotides relative to WT type sequence is indicated by (+_), number of deleted nucleotides by (- > ).Table 4

[0140] Table 5 shows Edited Events Relative to CML536 eIF4E2 genomic sequence (SEQ ID NO: 13), added nucleotides are bolded and underlined, deletions are indicated by dashes, and the number of inserted nucleotides relative to WT type sequence is indicated by (+_), number of deleted nucleotides by (- > ).Table 5

[0141] Thus, in one of the CKL5022 edited events selected for further study caused a single nucleotide deletion; in a different CKL5022 edited event, a 12-nucleotide deletion resulting in an in-frame removal of 4 amino acids from the translated protein, and in yet another CKL5022 edited event, a 27-nucleotide insertion that shifted the reading frame as well as introduced a premature stop codon. For CML536, one of the selected edited events had a single nucleotide addition, the second a single-nucleotide deletion, and the third one had an eight-nucleotide deletion that included the G of the AG intron / exon junction. Disruption of the intron / exon splice junction suggested the next available AG motif in the fourth exon might serve as the new splice junction.

[0142] Instead of relying on theoretical predictions, we performed RT-PCR followed by Sanger sequencing to determine whether the NextGen sequences of the genomic amplicons of the edited events matched the cDNA sequences. For CKL05022, the deletion of 12 nucleotides beginning with the first nucleotide after the AG intron / exon splice junction was preserved in the cDNA. However, for CML536, where the splice junction was disrupted, instead of the next AG downstream of the disrupted intron-3 / exon-4 splice junction, it was the second AG where splicing occurred. The mature transcript was shorter by 18 nucleotides, causing an in-frame deletion of six amino acids. Excision of the whole exon-4 in Mini Maize where only five nucleotides from exon-4 were included in the naturally occurring deletion may be attributed to the deletion of the polypyrimidine-rich motif that preceded the AG splice site (Lim and Burge,2001). This motif was intact in the CML536 edit. It is not clear, however, why splicing occurred at the second AG motif and not the first available one.

[0143] The double-edited plants (comprising eIF4El-KO and disrupted fourth exon of the eIF4E2 gene) grew normally. Given the inability described herein to obtain double knockouts of the eIF4E genes, all the variants of eIF4E2 edited in the fourth exon must be functional proteins that allow normal plant growth. Thus, the double edits described in this example that grew normally are referred as eif4el-KO!eIF4E2-exon-4ED.

[0144] Several double-edited eif4el-KO!eIF4E2-exon-4ED variants were screened for resistance against MLN in the greenhouse. Whereas the unedited parental lines died at 7-8 leaf stage, frameshift mutations, as previously discussed, in either eIF4El or eIF4E2 produced several additional leaves before the plants died. For reference, the ear is born at the 9thor 10thnode aboveground in tropical maize lines which generally have 17-18 total above ground leaves. The plants with the eif4el-KO!eIF4E2-exon-4ED edits looked normal with no sign of the disease upon MLN inoculation and went on to produce normal-looking ears that were similar to the uninoculated parental lines. Neither MCMV nor SCMV could be detected in the MLN-inoculated, double-edited plants with ELISA assays whereas leaves of the unedited plants or the ones with only eif4el-KO contained high levels of these viruses (Figure 8).

[0145] The eif4el-KOIeIF4E2-exon-4ED double edits in CML536 were just as resistant to MLN as the double edits of CKL05022. While not desiring to be bound by theory or any mechanism of action, these results independently confirm that the truncated eIF4E2 protein may not interact with MLN viruses and / or prevents translation of their respective genomes.

[0146] In the absence of a functional eIF4El, the eIF4E2 protein lacking the C-terminal 38 amino acids blocked the replication of both the viruses (Figure 9). Regardless of whether it was a frameshift mutation or an in-frame deletion of a few amino acids, any alteration in eIF4E2 starting with the 4thexon in the background of eif4el-KO sufficed to block virus replication without affecting plant growth. From the translated product of eIF4E2, which is 220-aa long, the C-terminal 38-aa are apparently dispensable for normal plant growth (Figure 9).

[0147] This stretch of amino acids corresponds to a domain known to interact with the VPg in human eIF4E (Coutinho de Oliveira et al., 2019). A tyrosyl group in VPg covalently binds the 5’ end of the poty virus RNA and competes with the m7G-capped host mRNA. This may account for SCMV resistance, however, our results show that this domain may also be required for the recognition of 3’-CITEs from the MCMV genome (Coutinho de Oliveira et al., 2019; Simon and Miller, 2013; Sorokin et al., 2021).Example 8: Inactivating the Fourth Exon in Both eIF4El and eIF4E2

[0148] The C-terminal 38-amino acid domain is nearly identical between eIF4El and eIF4E2, indicating that an alteration in this domain in eIF4El should also make it unrecognizable for viral genome replication. Therefore, instead of knocking out one of the eIF4E genes and modifying the other in the fourth exon, both eIF4E genes could be simultaneously targeted and edited in the fourth exon to generate double edits for MLN resistance directly in commercial and elite MLN-susceptible lines.

[0149] Elite maize lines that are susceptible to MLN are edited using sgRNAs that target the fourth exon regions and introduce mutations and / or deletions in the C-terminal 38-aa of both eIF4El and eIF4E2. The resulting double edited lines display normal plant growth and provide robust resistance against MLN.

[0150] Inbred parents of drought tolerant maize hybrids are edited introduce mutations and / or deletions in the C-terminal 38-aa of both eIF4El and eIF4E2. The resulting double edited lines display normal plant growth, are drought tolerant, and provide strong resistance against MLN.Example 9: Improving MLN resistance by inactivating the Fourth Exon in Both eIF(iso)4El and eIF(iso)E2

[0151] Elite maize lines that are susceptible to MLN are edited using sgRNAs that target the fourth exon regions and introduce mutations and / or deletions in the C-terminal 38-aa of both eif(iso)4El and eif(iso)E2. The resulting homozygous double edited lines display normal plant growth and provide robust resistance against MLN.

[0152] Elite maize lines that are susceptible to MLN are edited using sgRNAs to generate altered plants comprising (i) homozygous eif(iso)4El knockout and homozygous mutations and / or deletions in the C-terminal 38-aa of eif(iso)E2, (ii) homozygous mutations and / or deletions in the C-terminal 38-aa of both eif(iso)4El and homozygous eif(iso)4El knockout or (iii) homozygous mutations and / or deletions in the C-terminal 38-aa of both eif(iso)4El and eif(iso)E2 genes. Each of the foregoing altered maize plants provide robust resistance against MLN.

Claims

Claims1. A method of altering a virus susceptible plant, cell, seed, tissue or germplasm thereof, the method comprising introducing a targeted alteration to the sequence of one or more eIF4E genes in the susceptible plant, cell, seed, tissue or germplasm to thereby produce an altered plant, cell, seed, tissue or germplasm comprising an altered genome, wherein the altered genome encodes one or more altered eIF4E proteins and each altered eIF4E protein comprises a functional N-terminal region and an inactivated C-terminal region.

2. The method of claim 1 , wherein the altered genome comprises an altered eIF4El gene, an altered eIF4E2 gene, or a combination of altered eIF4El and eIF4E2 genes, which respectively encode an eIF4El protein variant, an eIF4E2 protein variant, or a combination of altered eIF4El and eIF4E2 protein variants.

3. The method of claim 1 , wherein the altered genome comprises an altered eIF4El gene and the eIF4El protein variant is truncated and comprises a deletion of the sequence corresponding to the (i) C-terminal 38 amino acids of the wild-type eIF4El protein or (ii) the first 22 amino acids of the C-terminal 38 amino acids encoded by exon 4 of the wild-type eIF4El gene.

4. The method of claim 3, wherein the inactivated C-terminal region comprises a deletion of (i) at least four amino acids, (ii) at least six amino acids, or (iii) at least eight amino acids in the region corresponding to the C-terminal 38 amino acids of wild type eIF4El protein.

5. The method of claim 3, wherein the eIF4El protein variant is truncated and comprises a deletion of the sequence corresponding to (i) C-terminal 38 amino acids of the wild-type eIF4El protein or (ii) the first 22 amino acids of the C-terminal 38 amino acids of the wildtype eIF4El protein.

6. The method of any one of claims 3-4, wherein the altered genome comprises an eIF4El gene knockout.

7. The method of any one of claims 3-5, wherein the altered genome comprises an eIF4E2 gene knockout.

8. The method of any one of claims 2-6, wherein the altered genome comprises an altered eIF4E2 gene that encodes an eIF4E2 protein sequence that is altered in the region corresponding to (i) C-terminal 38 amino acids of the wild-type eIF4E2 protein or (ii) the first 22 amino acids of the C-terminal 38 amino acids of the wild-type eIF4E2 protein.

9. The method of claim 1, wherein the altered genome comprises an altered eIF4E2 gene and the eIF4E2 protein variant’s inactivated C-terminal region is altered in the region corresponding to the C-terminal 38 amino acids of wild type eIF4E2 protein.

10. The method of claim 9, wherein the inactivated C-terminal region comprises a deletion of (i) at least four amino acids, (ii) at least six amino acids, or (iii) at least eight amino acids in the region corresponding to the C-terminal 38 amino acids of wild type eIF4E2 protein.

11. The method of claim 9, wherein the eIF4E2 protein variant is truncated and comprises a deletion of the sequence corresponding to the (i) C-terminal 38 amino acids of the wild-type eIF4E2 protein or (ii) the first 22 amino acids of the C-terminal 38 amino acids of the wildtype eIF4E2 protein.

12. The method of any one of claims 9-11, wherein the altered genome comprises a eIF4El gene knockout.

13. The method of any one of claims 9-11, wherein the altered genome comprises an altered eIF4El gene that encodes an eIF4El protein sequence altered relative the region corresponding to the C-terminal 38 amino acids of wild type eIF4El protein.

14. The method of any one of claims 9-13, wherein the eIF4E2 protein variant comprises one of SEQ ID NOs:41-55 or SEQ ID NOs:62-67.

15. The method of any one of claims 1-14, wherein the susceptible plant, cell, seed, tissue or germplasm is susceptible to maize chlorotic mottle virus (MCMV) or a potyvirus and the altered plant, cell, seed, tissue or germplasm thereof has increased resistance to MCMV or the potyvirus, relative to the susceptible plant, cell, seed, tissue or germplasm.

16. The method of any one of claims 1-14, 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.

17. The method of any one of claims 1-16, wherein the targeted alteration to the sequence of one or more eIF4E genes is introduced by genome editing18. The method of claim 17, wherein the genome editing includes the use of a CAS endonuclease.

19. The method of claim 18, wherein the genome editing further includes the use of a guide RNA comprising one of SEQ ID NOs: 19-25.

20. Plant, cell, seed, tissue or germplasm comprising (i) a first eIF4E gene variant that encodes a first eIF4E protein comprising a functional N-terminal region and an inactivated C- terminal region and a second eIF4E gene variant that that encodes a second eIF4E proteincomprising a functional N-terminal region and an inactivated C-terminal region or (ii) a first eIF4E gene knockout and a second eIF4E gene variant that encodes a second eIF4E protein comprising a functional N-terminal region and an inactivated C-terminal region.

21. The plant, cell, seed, tissue or germplasm of claim 20, wherein the plant, cell, seed, tissue or germplasm comprises an eIF4El gene variant encoding an eIF4El protein whose inactivated C-terminal region is altered in the region corresponding to the C-terminal 38 amino acids of wild type eIF4El protein and an eIF4E2 gene variant encoding an eIF4E2 protein whose inactivated C-terminal region is altered in the region corresponding to the C-terminal 38 amino acids of wild type eIF4E2 protein.

22. The plant, cell, seed, tissue or germplasm of claim 20, wherein the plant, cell, seed, tissue or germplasm comprises an eIF4El gene knockout and an eIF4E2 gene variant encoding an eIF4E2 protein is altered in the region corresponding to the C-terminal 38 amino acids of wild type eIF4E2 protein.

23. The plant, cell, seed, tissue or germplasm of any one of claims 20-22, wherein the eIF4E2 protein comprises a deletion of (i) at least four amino acids, (ii) at least six amino acids, or (iii) at least eight amino acids in the region corresponding to the C-terminal 38 amino acids of wild type eIF4E2 protein.

24. The plant, cell, seed, tissue or germplasm of any one of claims 20-23, wherein the eIF4E2 protein is truncated and comprises a deletion of the sequence corresponding to the (i) C-terminal 38 amino acids of the wild-type eIF4E2 protein or (ii) the first 22 amino acids of the C-terminal 38 amino acids of the wild-type eIF4E2 protein.

25. The plant, cell, seed, tissue or germplasm of any one of claims 20-24, wherein the eIF4E2 protein comprises one of SEQ ID NOs:41-55 or SEQ ID NOs:62-67.

26. The plant, cell, seed, tissue or germplasm of claim 20, wherein the plant, cell, seed, tissue or germplasm comprises an eIF4El gene variant encoding an eIF4El protein whose inactivated C-terminal region is altered in the region corresponding to the C-terminal 38 amino acids of wild type eIF4El protein and an eIF4E2 gene knockout.

27. The plant, cell, seed, tissue or germplasm of claim 20 or 26, wherein the eIF4El protein’s inactivated C-terminal region comprises a deletion of (i) at least four amino acids, (ii) at least six amino acids, or (iii) at least eight amino acids in the region corresponding to the C- terminal 38 amino acids of wild type eIF4El protein.

28. The plant, cell, seed, tissue or germplasm of any one of claims 20, 26, or 27, wherein the eIF4El protein is truncated and comprises a deletion of the comprises a deletion of thesequence corresponding to the (i) C-terminal 38 amino acids of the wild-type eIF4E2 protein or (ii) C-terminal 22 amino acids of the wild-type eIF4El protein.

29. The plant, cell, seed, tissue or germplasm of claims 20, or 26-28, wherein first eIF4E gene knockout sequence comprises one of SEQ ID NOs:26-40.

30. A method of identifying a plant, seed, tissue or germplasm thereof comprising an eIF4E variant associated with increased resistance to MLN and / or increased resistance to maize chlorotic mottle virus (MCMV), potyvirus, or sugarcane mosaic virus (SCMV), the method comprising a. providing one or more plant, seed, tissue or germplasm thereof comprising an eIF4E variant encoding an eIF4E comprising an inactivated C-terminal region; b. obtaining a sample comprising nucleic acid from each plant, seed, tissue or germplasm thereof; c. screening the sample for any of the following variant sequences: i. variant genomic sequence or cDNA sequence encoding eIF4El protein variant sequence that is altered (comprises an insertion, deletion, or substitution) in the region corresponding to the C-terminal 38 amino acids of wild type eIF4El protein, ii. variant genomic sequence or cDNA sequence encoding eIF4El protein variant that comprises a deletion of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 , 35, 36, or 37 amino acids in the region corresponding to the C- terminal 38 amino acids of wild type eIF4El protein, iii. variant genomic sequence or cDNA sequence encoding an eIF4El protein variant that is truncated and comprises a deletion of the sequence corresponding to the C-terminal 38 amino acids of wild-type eIF4El, iv. variant genomic sequence or cDNA sequence encoding an eIF4El protein variant that is truncated and comprises a deletion of the sequence corresponding to the first 22 amino acids of the C-terminal 38 amino acids of wild-type eIF4El, v. variant genomic sequence or cDNA sequence encoding eIF4E2 protein variant sequence that is altered (comprises an insertion, deletion, or substitution) in the region corresponding to the C-terminal 38 amino acids of wild type eIF4El protein,vi. variant genomic sequence or cDNA sequence encoding an eIF4E2 protein variant that comprises a deletion of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 , 35, 36, or 37 amino acids in the region corresponding to the C-terminal 38 amino acids of wild-type eIF4E2 protein, vii. variant genomic sequence or cDNA sequence encoding eIF4E2 protein variant that is truncated and comprises a deletion of the sequence corresponding to the C-terminal 38 amino acids of wild-type eIF4E2 protein, viii. variant genomic sequence or cDNA sequence encoding eIF4E2 protein variant that is truncated and comprises a deletion of the sequence corresponding to the first 22 amino acids of the C-terminal 38 amino acids of wild-type eIF4E2 protein, ix. variant eIF4El protein sequence that is altered (comprises an insertion, deletion, or substitution) in the region corresponding to the C-terminal 38 amino acids of wild type eIF4El protein, x. variant eIF4El protein that comprises a deletion of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 , 35, 36, or 37 amino acids in the region corresponding to the C-terminal 38 amino acids of wild type eIF4El protein, xi. variant eIF4El protein that is truncated and comprises a deletion of the sequence corresponding to the C-terminal 38 amino acids of wild-type eIF4El, xii. variant eIF4El protein that is truncated and comprises a deletion of the sequence corresponding to the first 22 amino acids of the C-terminal 38 amino acids of the wild-type eIF4El protein, xiii. variant eIF4E2 protein sequence that is altered (comprises an insertion, deletion, or substitution) in the region corresponding to the C-terminal 38 amino acids of wild type eIF4E2 protein, xiv. variant eIF4E2 protein that comprises a deletion of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 , 35, 36, or 37 amino acids in the regioncorresponding to the C-terminal 38 amino acids of wild type eIF4E2 protein, xv. variant eIF4E2 protein that is truncated and comprises a deletion of the sequence corresponding to the C-terminal 38 amino acids of wild-type eIF4E2, or xvi. variant eIF4E2 protein that is truncated and comprises a deletion of the sequence corresponding to the first 22 amino acids of the C-terminal 38 amino acids of the wild-type eIF4E2 protein, d. detecting the one or more altered variant sequences in the sample and thereby identifying the plant or plant material as comprising a eIF4E variant associated with increased resistance to MLN and / or increased resistance to maize chlorotic mottle virus (MCMV), potyvirus, or sugarcane mosaic virus (SCMV).

31. A method of improving plant’s resistance to MLN and / or to maize chlorotic mottle virus (MCMV), potyvirus, or sugarcane mosaic virus (SCMV), the method comprising: a. identifying a first parent plant as comprising a eIF4E variant associated with increased resistance to MLN and / or increased resistance to maize chlorotic mottle virus (MCMV), potyvirus, or sugarcane mosaic virus (SCMV) in accordance with claim 27; and b. crossing the first parent plant with a second parent plant to produce one or more progeny plants comprising the eIF4E variant, wherein the second parent plant is susceptible to MLN and / or to MCMV, potyvirus, or SCMV and wherein the progeny plants are more resistant to MLN and / or to MCMV, potyvirus, or SCMV than the second parent plant.

32. The method of claim 28, wherein the first parent plant comprises: i. variant genomic sequence or cDNA sequence encoding eIF4El protein variant sequence that is altered relative the region comprising the C- terminal 38 amino acids of wild type eIF4El protein; ii. variant genomic sequence or cDNA sequence encoding eIF4El protein variant comprises a deletion of at least four amino acids, at least six amino acids, or at least eight amino acids in its C-terminal 38 amino acid region, relative to wild type eIF4El protein; iii. variant genomic sequence or cDNA sequence encoding an eIF4El protein variant is truncated and comprises a deletion of the C-terminal38 amino acids of wild-type eIF4El protein or the first 22 amino acids of the C-terminal 38 amino acids of wild-type eIF4El protein; iv. variant genomic sequence or cDNA sequence encoding eIF4E2 protein variant sequence that is altered relative the region comprising the C- terminal 38 amino acids of wild type eIF4El protein; v. variant genomic sequence or cDNA sequence encoding eIF4E2 protein variant comprises a deletion of at least four amino acids, at least six amino acids, or at least eight amino acids in its C-terminal 38 amino acid region, relative to wild type eIF4E2 protein or the first 22 amino acids of the C-terminal 38 amino acids of wild-type eIF4E2 protein; or vi. variant genomic sequence or cDNA sequence encoding an eIF4E2 protein variant is truncated and comprises a deletion of the C-terminal 38 amino acids of wild-type eIF4E2.

33. The method of claim 28, wherein the first parent plant comprises a variant eIF4El gene and the second parent plant comprises: i. variant genomic sequence or cDNA sequence encoding eIF4E2 protein variant sequence that is altered relative the region comprising the C- terminal 38 amino acids of wild type eIF4El protein; ii. variant genomic sequence or cDNA sequence encoding eIF4E2 protein variant comprises a deletion of at least four amino acids, at least six amino acids, or at least eight amino acids in its C-terminal 38 amino acid region, relative to wild type eIF4E2 protein; or iii. variant genomic sequence or cDNA sequence encoding an eIF4E2 protein variant is truncated and comprises a deletion of the C-terminal 38 amino acids of wild-type eIF4E2 or the first 22 amino acids of the C-terminal 38 amino acids of the wild-type eIF4E2 protein.

34. The method of claim 28, wherein the first parent plant comprises a variant eIF4E2 gene and the second parent plant comprises: i. variant genomic sequence or cDNA sequence encoding eIF4El protein variant sequence that is altered relative the region comprising the C- terminal 38 amino acids of wild type eIF4El protein; ii. variant genomic sequence or cDNA sequence encoding eIF4El protein variant comprises a deletion of at least four amino acids, at least sixamino acids, or at least eight amino acids in its C-terminal 38 amino acid region, relative to wild type eIF4El protein; or iii. variant genomic sequence or cDNA sequence encoding an eIF4El protein variant is truncated and comprises a deletion of the C-terminal 38 amino acids of wild-type eIF4El or the first 22 amino acids of the C-terminal 38 amino acids of the wild-type eIF4E2 protein.

35. The method of claim 28, wherein the second parent plant does not comprise a eIF4E gene variant and the first parent plant comprises one of the variant eIF4El genes and one of the variant eIF4E2 genes in the following list: i. variant genomic sequence or cDNA sequence encoding eIF4El protein variant sequence that is altered relative the region comprising the C- terminal 38 amino acids of wild type eIF4El protein; ii. variant genomic sequence or cDNA sequence encoding eIF4El protein variant comprises a deletion of at least four amino acids, at least six amino acids, or at least eight amino acids in its C-terminal 38 amino acid region, relative to wild type eIF4El protein; iii. variant genomic sequence or cDNA sequence encoding an eIF4El protein variant is truncated and comprises a deletion of the C-terminal 38 amino acids of wild-type eIF4El or the first 22 amino acids of the C-terminal 38 amino acids of wild-type eIF4El protein; iv. variant genomic sequence or cDNA sequence encoding eIF4E2 protein variant sequence that is altered relative the region comprising the C- terminal 38 amino acids of wild type eIF4El protein; v. variant genomic sequence or cDNA sequence encoding eIF4E2 protein variant comprises a deletion of at least four amino acids, at least six amino acids, or at least eight amino acids in its C-terminal 38 amino acid region, relative to wild type eIF4E2 protein; or vi. variant genomic sequence or cDNA sequence encoding an eIF4E2 protein variant is truncated and comprises a deletion of the C-terminal 38 amino acids of wild-type eIF4E2 or the first 22 amino acids of the C-terminal 38 amino acids of wild-type eIF4E2 protein.

36. The method of any one of claims 28-32, further comprising crossing one or more of the progeny plants to the second parent plant to thereby produce backcross progeny plants comprising the eIF4E variant from the first parent plant.

37. A method of improving plant’s resistance to MLN and / or to maize chlorotic mottle virus (MCMV), potyvirus, or sugarcane mosaic virus (SCMV), the method comprising: a. crossing a first parent plant with a second parent plant to produce progeny plants, wherein the first parent and the second parent genomes collectively comprise (i) a first eIF4E gene variant that encodes a first eIF4E protein comprising a functional N-terminal region and an inactivated C-terminal region and a second eIF4E gene variant that that encodes a second eIF4E protein comprising a functional N-terminal region and an inactivated C-terminal region or (ii) a first eIF4E gene knockout and a second eIF4E gene variant that encodes a second eIF4E protein comprising a functional N-terminal region and an inactivated C-terminal region; and b. selecting at least one progeny plant that is a plant in accordance with any one of claims 17-26.

38. The method of claim 33 further comprising: c. crossing the selected one or more progeny plants with the second parent plant to produce backcross progeny plants; and d. selecting at least one backcross progeny plant, wherein the selected backcross progeny that is a plant in accordance with any one of claims 17-26.

39. The method of any one of claims 1-19 and 30-38 wherein the plant is a maize plant.

40. The method of any one of claims 1-19 and 30-38 wherein the plant is a Cucurbitaceae, stone fruit tree, pome tree, soybean, oilseed rape, sunflower, wheat, or cotton.

41. The plant, cell, seed, tissue or germplasm thereof of any one of claims 20-29, wherein the plant is a maize plant.

42. The plant, cell, seed, tissue or germplasm thereof of any one of claims 20-29, wherein the plant is a Cucurbitaceae, stone fruit tree, pome tree, soybean, oilseed rape, sunflower, wheat, or cotton.

43. The method of any one of claims 1-19 and 30-38 wherein the plant is a maize plant that is viable and / or capable of reproducing.

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