EIF (ISO) 4E protein variants resistant to corn virus disease

Abstract

The present invention relates to an eIF (iso) 4E protein variant capable of conferring resistance to at least one corn virus, in particular to corn fatal necrosis (MLND). The protein variants comprise at least one mutation compared to the corresponding native corn eIF (iso) 4E protein. The invention also relates to a nucleic acid encoding at least one eIF (iso) 4E protein variant as defined above, a vector comprising said nucleic acid and a genetically modified or edited corn plant or seed resistant to at least one corn virus, said corn plant or seed comprising at least one eIF (iso) 4E protein variant as defined above. The invention also relates to a method for obtaining a genetically modified or edited maize plant that is resistant to at least one maize virus.

Description

Field of the Invention

[0001] The present invention relates to resistance of corn to viral diseases. Background Art

[0002] Corn can be affected by many viral diseases, especially those caused by several potato viruses Y, such as Sugarcane Mosaic Virus (SCMV), Maize Dwarf Mosaic Virus (MDMV), Wheat Stripe Mosaic Virus (WSMV), and Maize Lethal Necrosis Disease (MLND) caused by a combination of the following two viruses: MCMV (Maize Chlorotic Mottle Virus) and a potato virus Y of the SCMV or MDMV type. MCMV belongs to the genus Machlomovirus of the family Tombusviridae.

[0003] Insecticide treatments are effective in protecting maize crops from MNLD, and until recently, little research has been done to find alternatives to these treatments. As insecticide controls become more stringent in some parts of the world, this can lead to increased insect pressure and, indirectly, an increase in plant viruses, as insects are important vectors of viruses.

[0004] Plants do have genetic resistance to certain viruses in nature. The introgression of this genetic resistance in commercial varieties is an alternative to chemical treatments and has no impact on the environment. Resistance to MLND viruses can be obtained by exploiting this natural variation by introgressing QTLs known to confer resistance to such diseases (Murithi et al., Frontiers in Genetics, 2021, 12, 767883: 1-17) or, for example, using favorable alleles of genes known to be involved in SCMV resistance (Leng et al., Molecular Plant, 2017, 10: 1357-1359).

[0005] Certain crops do not always have natural resistance. However, similar resistance can be produced through mutagenesis, genetic modification or new breeding techniques.

[0006] eIF4E protein has been used as a target to improve genetic resistance in many vegetable crops. eIF4E protein is indeed known to play a role in viral replication in plants. It was first demonstrated that vegetable plants with eIF4E knockout (KO) do confer resistance to several viruses of the Potyviridae family. They were also evaluated in tomatoes (Gauffier et al., 2016, The Plant Journal, 85: 717-729). In addition, point mutations that can destroy the interaction between certain plant viruses and eIF4E proteins have been found in vegetable plants (such as peppers) (Charron et al., 2008, The Plant Journal, 54: 56-58).

[0007] Therefore, there is a need to provide corn plants that are resistant to viral diseases, such as those caused by potato virus Y and / or maize chlorotic mottle virus, as well as other viruses capable of infecting corn, as an alternative to treatment with insecticides. Summary of the invention

[0008] The present inventors have now discovered novel eIF4E and eIFiso4E protein variants which are capable of conferring resistance to viruses in maize, in particular resistance to at least one potato virus Y and / or maize chlorotic mottle virus.

[0009] These eIF4E and eIFiso4E protein variants contain at least one mutation compared to the corresponding native maize eIF4E and eIFiso4E proteins.

[0010] Advantageously, the eIF4E and eIFiso4E protein variants comprising at least two mutations have increased resistance to maize potato virus Y and / or maize chlorotic mottle virus.

[0011] Furthermore, to facilitate long-term viral resistance, it is preferred to combine at least two mutations, preferably the mutations are not located in the same region of the protein to prevent the virus from circumventing resistance.

[0012] Therefore, the eIF4E and eIFiso4E protein variants according to the invention preferably combine one, two or at least three mutations, which are selected from the newly identified mutations and mutations already identified in eIF4E proteins of other plant species.

[0013] Useful combinations of mutations include amino acid substitutions transposed from different plant species.The eIF4E and eIFiso4E protein variants of the present invention are not the result of simple transposition of known eIF4E and eIFiso4E mutations from a single given species.

[0014] A first object of the present invention is an eIF(iso)4E protein variant capable of conferring resistance to at least one maize virus, wherein said protein variant comprises at least one mutation compared to the corresponding native maize eIF(iso)4E.

[0015] The eIF(iso)4E protein variant is preferably an eIF4E1 protein variant, an eIF4E2 protein variant, an eIFiso4E1 protein variant or an eIFiso4E2 protein variant.

[0016] The native corn eIF(iso)4E protein is, for example, the protein eIF4E1 of SEQ ID NO:1, the protein eIF4E2 of SEQ ID NO:2, the protein eIFiso4E1 of SEQ ID NO:3, or the protein eIFiso4E2 of SEQ ID NO:4.

[0017] The maize virus is preferably potato virus Y or maize chlorotic mottle virus.

[0018] The at least one mutation preferably disrupts the interaction between the protein variant and at least one corn virus, preferably a virus selected from the group consisting of MCMV (maize chlorotic mottle virus), SCMV (sugarcane mosaic virus), MDMV corn (dwarf mosaic virus) and WSMV (wheat streak mosaic virus).

[0019] For example, the at least one mutation may be selected from:

[0020] - a group consisting of V96D, G97R, T7S, A19V, L33I, N55D / K, P56S / T, A63D, A64D, S67A, D99G, S159K, Q164K and G211L / D / S, with reference to the native zein sequence of SEQ ID NO: 1,

[0021] - the group consisting of D101G, A65D, A66D, N57D / K, P58S / T, S69A, G98D, G99R, S161K, Q166K and G213L / D / S, with reference to the native zein sequence of SEQ ID NO: 2,

[0022] - the group consisting of A60D, A61D, N94R, W108L, S157K and R161E / L, with reference to the native zein sequence of SEQ ID NO: 3, or

[0023] - The group consisting of A54D, A55D, T88R, W102L, S151K and R155E / L, with reference to the native zein sequence of SEQ ID NO: 4.

[0024] A protein variant as defined above preferably comprises at least two mutations compared to the corresponding native maize eIF(iso)4E protein.

[0025] For example, a variant of the eIF(iso)4E protein as defined above may comprise:

[0026] - the mutations (i) A63D and A64D, (ii) V96D and G97R, (iii) D99G and / or (iv) G211L / D / S, with reference to the native zein of SEQ ID NO: 1,

[0027] - the mutations (i) A65D and A66D, (ii) G98D and G99R, (iii) D101G and / or (iv) G213L / D / S, with reference to the native zein of SEQ ID NO: 2,

[0028] - mutations (i) A60D and A61D, (ii) N94R, (iii) W108L and R161E / L and / or (iv) S157K, with reference to the native zein of SEQ ID NO: 3, or

[0029] - the mutations (i) A54D, A55D, (ii) T88R, (iii) W102L and R155E / L and / or (iv) S151K, with reference to the native zein sequence of SEQ ID NO: 4.

[0030] For example, the eIF(iso)4E protein variant as defined above comprises or consists of the sequence SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 or SEQ ID NO:8.

[0031] Another object of the invention is a nucleic acid encoding at least one eIF(iso)4E protein variant as defined above.

[0032] Another object of the invention is a vector comprising at least one nucleic acid encoding at least one eIF(iso)4E protein variant as defined above.

[0033] Another object of the present invention is a genetically modified or edited corn plant resistant to at least one corn virus, or a genetically modified or edited corn seed resistant to at least one corn virus, wherein the plant or seed comprises at least one eIF(iso)4E protein variant as defined above. The plant is not obtained by means of an essentially biological process.

[0034] Another object of the present invention is the offspring of a genetically modified or edited corn plant as defined above that is resistant to at least one corn virus, or the offspring of a genetically modified or edited corn seed as defined above that is resistant to at least one corn virus, wherein the plant or seed comprises at least one eIF(iso)4E protein variant as defined above.

[0035] Another object of the present invention is a method for obtaining a genetically modified or edited corn plant resistant to at least one corn virus, wherein said method comprises:

[0036] - transforming a maize cell or maize tissue using at least one vector as defined above to obtain a transformed maize cell or a transformed maize tissue, and

[0037] - Regenerating a maize plant from said transformed maize cell or transformed maize tissue.

[0038] Another object of the present invention is a method for obtaining an edited modified corn plant resistant to at least one corn virus, wherein the method comprises introducing at least one mutation in an endogenous corn gene encoding the eIF(iso)4E protein to obtain a mutant gene encoding a variant of the eIF(iso)4E protein as defined above.

[0039] Another object of the present invention is the use of at least one eIF(iso)4E protein variant as defined above for preventing at least one viral disease of maize, wherein the viral disease is preferably caused by a potato virus Y and / or a maize chlorotic mottle virus.

[0040] Another object of the present invention is a method for identifying a corn plant that is resistant to at least one corn virus, wherein the method comprises detecting the expression of an eIF(iso)4E protein variant as defined above and / or the presence of a nucleic acid encoding an eIF(iso)4E protein variant as defined above in the cells of the corn plant.

[0041] Native maize eIF(iso)4E protein and eif4e gene

[0042] The eIF(iso)4E gene encodes the eukaryotic translation initiation factor 4E (eIF4E) protein and its isoforms.

[0043] The term "eIF(iso)4E" as used herein refers to eIF4E (eukaryotic translation initiation factor 4E) or its isoform eIFiso4E (eukaryotic translation initiation factor isoform 4E).

[0044] There are two maize eIF4E proteins: the eIF4E1 protein encoded by the eif4e1 and the eIF4E2 protein encoded by the eif4e2 gene.

[0045] "Native zein" herein refers to wild-type zein.

[0046] "Native maize gene" herein refers to a wild-type maize gene.

[0047] The reference sequence of the native zein eIF4E1 is the sequence SEQ ID NO: 1. Said sequence is also known as Zm00001d041682_eIF4E1 in the B73 genome version 4 (identified as ZmA188v1aHC012309_eIF4E1 in the J1A genome).

[0048] The reference sequence of the native zein eIF4E2 is the sequence SEQ ID NO: 2. Said sequence is also known as Zm00001d041973_eIF4E2 in the B73 genome version 4 (identified as ZmA188v1aHC012641_eIF4E2 in the J1A genome).

[0049] The reference sequence of the native maize eif4e1 gene is SEQ ID NO:9.

[0050] The eif4e1 gene is located on chromosome 3, more precisely at chr3:133075625-133080137 in the B73 genome version 4. The eif4e1 gene contains 5 exons. Exon 1 is located at chr3:133075814-133076061 in the B73 strain. Exon 2 is located at chr3:133077529-133077694 in the B73 strain.

[0051] The reference sequence of the native maize eif4e2 gene is SEQ ID NO:10.

[0052] The eif4e2 gene is located on chromosome 3, more precisely at chr3:146285064-146288191 (minus strand) in the B73 genome version 4. The eif4e2 gene contains 5 exons. Exon 1 is located at chr3:146288074-146287821 in the B73 strain. Exon 2 is located at chr3:146286386-146286221 in the B73 strain (minus strand).

[0053] Maize contains a single copy of the eif4e1 gene and a single copy of the eif4e2 gene.

[0054] The skilled person knows how to easily locate the eif4e1 and eif4e2 genes, and the corresponding exons 1 and 2, in a given maize line, for example by sequence alignment with the sequences of the corresponding genes and exons described for line B73 as defined above.

[0055] There are two maize eIFiso4E proteins: proteins eIFiso4E1 and eIFiso4E2.

[0056] The reference sequence of the native zein eIFiso4E1 is the sequence SEQ ID NO:3.

[0057] The reference sequence of the natural corn protein eIFiso4E2 is SEQ ID NO:4.

[0058] eIF(iso)4E protein variant

[0059] The present invention particularly relates to an eIF(iso)4E protein variant capable of conferring resistance to at least one virus capable of infecting corn.

[0060] The eIF(iso)4E protein variant may be an eIF4E1 protein variant, an eIF4E2 protein variant, an eIFiso4E1 protein variant or an eIFiso4E2 protein variant.

[0061] "Maize virus" as used herein refers to a virus capable of infecting maize.

[0062] The maize virus as defined above is preferably potato virus Y or maize chlorotic mottle virus.

[0063] For example, the potato virus Y may be selected from the group consisting of Sugarcane Mosaic Virus (SCMV), Maize Dwarf Mosaic Virus (MDMV) and Wheat Streak Mosaic Virus (WSMV).

[0064] For example, the maize chlorotic mottle virus (machlomovirus) is MCMV (Maize Chlorotic Mottle Virus).

[0065] Therefore, the eIF(iso)4E protein variant capable of conferring resistance to at least one corn virus is capable of conferring resistance to diseases caused by infection with the at least one corn virus.

[0066] For example, the disease may be maize lethal necrosis disease (MLND), which is caused by a combination of (i) MCMV and (ii) SCMV, MDMV, and WSMV.

[0067] The expression "capable of conferring resistance to at least one corn virus" herein means that expression of the eIF(iso)4E protein variant in a corn plant confers resistance to the corn plant to the corn virus.

[0068] A skilled artisan can readily assess the ability of eIF(iso)4E protein variants to confer resistance to maize viruses in vitro or in vivo.

[0069] For example, resistance to corn viruses can be assessed by monitoring the development (or absence of development) of corn diseases caused by infection with the corn virus in corn plants that have been genetically transformed or edited to express the eIF4E or eIFiso4E protein variant to be assessed. The corn virus is preferably inoculated onto the plant, for example by mechanical inoculation. The results are preferably compared with wild-type corn plants placed under the same conditions. Compared to wild-type corn plants, a reduction in the development of corn virus diseases or the absence of corn virus diseases in corn plants expressing the eIF (iso) 4E protein variant to be assessed indicates that the eIF (iso) 4E protein variant confers resistance to the corn virus.

[0070] Resistance to corn viruses can also be assessed by virus quantification, for example using ELISA or qPCR methods, which allow the presence or absence of viruses and / or the amount of viruses in corn plants expressing the eIF(iso)4E protein variant to be assessed to be determined by comparison with wild-type corn plants. ELISA can measure the extent of viral accumulation, for example using antibodies specific to the corn virus. For example, anti-SCMV, anti-MDMV and anti-MCMV antibodies can be used.

[0071] Resistance to corn virus diseases can also be evaluated in vitro, for example, by a yeast two-hybrid system, in which the potential binding of viral VPg to the eIF4E or eIFiso4E protein variant to be evaluated is determined by a yeast two-hybrid assay. Protein variants that are unable to bind to corn virus VPg in this in vitro assay are able to confer resistance to the corn virus when expressed.

[0072] The (viral protein genome-linked) VPg protein is actually a potato virus Y protein that binds to the eIF(iso)4E protein, and the binding is required for viral infection. Therefore, if the VPg protein cannot bind to the eIF(iso)4E protein, the viral cycle is inhibited.

[0073] The eIF(iso)4E protein variant as defined above comprises at least one mutation, preferably at least two or at least three mutations compared to the corresponding native maize eIF(iso)4E protein.

[0074] "Corresponding native maize eIF(iso)4E protein" refers to the native maize eIF4E1 protein of the eIF4E1 protein variant, the native maize eIF4E2 protein of the eIF4E2 protein variant, the native maize eIF(iso)4E1 protein of the eIF(iso)4E1 protein variant, and the native maize eIF(iso)4E2 protein of the eIF(iso)4E2 protein variant.

[0075] The term "mutation" herein refers to a substitution of an amino acid, a deletion of an amino acid or an addition of an amino acid.

[0076] The at least one mutation, preferably the at least two or the at least three mutations, are preferably amino acid substitutions.

[0077] The at least one mutation, preferably the at least two or the at least three mutations, disrupt the interaction between (i) the eIF(iso)4E protein variant as defined above and (ii) at least one corn virus, such as potato virus Y (preferably SCMV, MDMV or WSMV) and / or maize chlorotic mottle virus (such as MCMV). The at least one mutation, preferably the at least two or the at least three mutations, in particular disrupt the interaction between (i) the eIF(iso)4E protein variant as defined above and (ii) the VPg protein (viral protein genome linked) of at least one corn virus as defined above.

[0078] The eIF(iso)4E protein variant is functional as defined above.

[0079] "Functional" herein means that when present or expressed in a corn plant, the eIF(iso)4E protein variant has at least one biological function of the corresponding native eIF(iso)4E protein.

[0080] One example of a biological function of the corresponding native eIF(iso)4E protein is protein synthesis.

[0081] Whether the eIF(iso)4E protein variant is functional may be assessed using any suitable method, such as a complementation assay, preferably in yeast.

[0082] In the yeast complementation test, yeast is made deficient in eIF(iso)4E protein. The deficient yeast will only grow if the eIF(iso)4E protein variant is functional.

[0083] One, two or three consecutive amino acids may be mutated, preferably substituted, to disrupt the interaction.

[0084] When the protein variant comprises at least two mutations, at least two of the mutations may be located in (i) two consecutive amino acids or (ii) two non-consecutive amino acids.

[0085] When the protein variant comprises at least three mutations, the at least three substitutions may be located in (i) three consecutive amino acids, (ii) two consecutive amino acids and one non-consecutive amino acid, or (iii) three non-consecutive amino acids.

[0086] The eIF4E protein variant as defined above may be an eIF4E1 protein variant or an eIF4E2 protein variant.

[0087] The eIFiso4E protein variant as defined above may be an eIFiso4E1 protein variant or an eIFiso4E2 protein variant.

[0088] The native corn eIF4E protein is preferably the protein eIF4E1 of SEQ ID NO:1 or the protein eIF4E2 of SEQ ID NO:2.

[0089] The native corn eIFiso4E protein is preferably the protein eIFiso4E1 of SEQ ID NO:3 or the protein eIFiso4E2 of SEQ ID NO:4.

[0090] For example, the eIF(iso)4E protein variant as defined above may comprise at least one mutation, preferably at least two mutations or at least three mutations, such as at least four, at least five or at least six mutations selected from:

[0091] - a group consisting of V96D, G97R, T7S, A19V, L33I, N55D / K, P56S / T, A63D, A64D, S67A, D99G, S159K, Q164K and G211L / D / S, with reference to the native zein sequence of SEQ ID NO: 1,

[0092] - the group consisting of D101G, A65D, A66D, N57D / K, P58S / T, S69A, G98D, G99R, S161K, Q166K and G213L / D / S, with reference to the native zein sequence of SEQ ID NO: 2,

[0093] - the group consisting of A60D, A61D, N94R, W108L, S157K and R161E / L, with reference to the native zein sequence of SEQ ID NO: 3, or

[0094] - The group consisting of A54D, A55D, T88R, W102L, S151K and R155E / L, with reference to the native zein sequence of SEQ ID NO: 4.

[0095] The mutation "XNY" refers to the substitution of amino acid X at position N by amino acid Y.

[0096] The mutation "XNY / Z" means that the amino acid X at position N is replaced by the amino acid Y or Z.

[0097] For example, the eIF4E protein variant as defined above may comprise at least one mutation selected from the group consisting of:

[0098] - the group consisting of P56T, V96D, G97R, D99G and S159K, with reference to the native zein sequence of SEQ ID NO: 1, or

[0099] - The group consisting of A66D, G99R, D101G and S161K, with reference to the native zein sequence of SEQ ID NO: 2.

[0100] Each of the mutations P56T, V96D, G97R, D99G, S159K of the native zein with reference to the sequence SEQ ID NO: 1 specifically confers resistance to SCMV. The mutation D99G of the native zein with reference to the sequence SEQ ID NO: 1 also confers resistance to MDMV.

[0101] With reference to the native zein sequence of SEQ ID NO: 2, each of the mutations G99R, D101G, S161K specifically confer resistance to SCMV.

[0102] With reference to the native zein sequence of SEQ ID NO: 2, each of the mutations A66D and D101G specifically confer resistance to MDMV.

[0103] For example, the eIF4E1 protein variant as defined above may comprise at least two mutations selected from the group consisting of:

[0104] -N55K and P56S / T, mutations that specifically confer resistance to SCMV,

[0105] -N55D and P56T, mutations that specifically confer resistance to SCMV,

[0106] - A63D and A64D, mutations that specifically confer resistance to SCMV and MDMV,

[0107] - V96D and G97R, mutations that specifically confer resistance to SCMV and MDMV,

[0108] -S67A and S159K, mutations that specifically confer resistance to SCMV,

[0109] -D99G and Q164K, mutations that specifically confer resistance to SCMV and MDMV, and

[0110] -S159K and Q164K, mutations that specifically confer resistance to SCMV,

[0111] By reference to the native zein sequence SEQ ID NO:1.

[0112] For example, the eIF4E2 protein variant as defined above may comprise at least two mutations selected from the group consisting of:

[0113] - A65D and A66D, mutations that specifically confer resistance to SCMV and MDMV,

[0114] - G98D and G99R, mutations that specifically confer resistance to SCMV,

[0115] -S69A and S161K, mutations that specifically confer resistance to SCMV,

[0116] -D101G and Q166K, mutations that specifically confer resistance to SCMV and MDMV, and

[0117] -S161K and Q166K, mutations that specifically confer resistance to SCMV,

[0118] By reference to the native zein sequence SEQ ID NO:2.

[0119] For example, the eIF4E1 protein variant as defined above may comprise at least three mutations selected from the group consisting of:

[0120] -T7S, A19V and L33I, mutations that specifically confer resistance to SCMV,

[0121] -N55K, P56T and G211S, mutations that specifically confer resistance to SCMV,

[0122] -N55K, P56T and S159K, mutations that specifically confer resistance to SCMV,

[0123] - A63D, A64D and G97R, mutations that confer resistance specifically to SCMV and MDMV,

[0124] - A63D, A64D and D99G, mutations that confer resistance specifically to SCMV and MDMV,

[0125] - A63D, A64D and S159K, mutations that confer resistance specifically to SCMV and MDMV,

[0126] - S67A, V96D and G97R, mutations that specifically confer resistance to SCMV,

[0127] - V96D, G97R and D99G, mutations that confer resistance specifically to SCMV and MDMV, and

[0128] - V96D, G97R and S159K, mutations that confer resistance specifically to SCMV and MDMV,

[0129] By reference to the native zein sequence SEQ ID NO:1.

[0130] For example, the eIF4E1 protein variant as defined above may comprise at least four mutations selected from the group consisting of:

[0131] - A63D, A64D, G97R and S159K, mutations that confer resistance specifically to SCMV and MDMV,

[0132] - A63D, A64D, D99G and S159K, mutations that confer resistance specifically to SCMV and MDMV,

[0133] - A63D, A64D, S67A and S159K, mutations that confer resistance specifically to SCMV and MDMV,

[0134] - A63D, A64D, V96D and G97R, mutations that confer resistance specifically to SCMV and MDMV,

[0135] - N55K, P56T, A63D and A64D, mutations that specifically confer resistance to SCMV and MDMV, and

[0136] - N55D, P56S, A63D and A64D, mutations that confer resistance specifically to SCMV and MDMV,

[0137] Reference is made to the native zein sequence of SEQ ID NO:1.

[0138] For example, the eIF4E1 protein variant as defined above may comprise at least the following mutations:

[0139] - A63D, A64D, V96D, G97R and S159K, mutations that confer resistance specifically to SCMV and MDMV,

[0140] - N55D, P56S, A63D, A64D, G97R and S159K, mutations that confer resistance specifically to SCMV and MDMV,

[0141] - N55D, P56S, A63D, A64D, V96D, G97R and S159K, mutations that confer resistance specifically to SCMV and MDMV,

[0142] - N55D, P56S, A63D, A64D, V96D, G97R, S159K and G211S, which mutations confer resistance to SCMV and MDMV in particular, or

[0143] - N55K, P56T, A63D, A64D, V96D, G97R, S159K and G211D, mutations that confer resistance specifically to SCMV and MDMV,

[0144] By reference to the native zein sequence SEQ ID NO:1.

[0145] For example, the eIF4E2 protein variant as defined above may comprise at least three mutations selected from the group consisting of:

[0146] -N57K, P58T and S161K, mutations that specifically confer resistance to SCMV,

[0147] - A65D, A66D and G99R, mutations that confer resistance specifically to SCMV and MDMV,

[0148] - A65D, A66D and S161K, mutations that confer resistance specifically to SCMV and MDMV,

[0149] - A65D, A66D and D101G, mutations that confer resistance specifically to SCMV and MDMV,

[0150] - G98D, G99R and D101G, mutations that confer resistance specifically to SCMV and MDMV,

[0151] - G98D, G99R and S161K, mutations that specifically confer resistance to SCMV, and

[0152] - S69A, G98D and G99R, mutations that specifically confer resistance to SCMV,

[0153] By reference to the native zein sequence SEQ ID NO:2.

[0154] For example, a variant of the eIF4E2 protein as defined above may comprise at least the following mutations:

[0155] - A65D, A66D, D101G and S161K, mutations that confer resistance specifically to SCMV and MDMV,

[0156] - A65D, A66D, G99R and S161K, mutations that confer resistance specifically to SCMV and MDMV,

[0157] - A65D, A66D, S69A and S161K, mutations that confer resistance specifically to SCMV and MDMV,

[0158] - A65D, A66D, G98D and G99R, mutations that confer resistance specifically to SCMV and MDMV,

[0159] - N57K, P58T, A65D and A66D, mutations that specifically confer resistance to SCMV and MDMV,

[0160] - N57D, P58S, A65D and A66D, mutations that confer resistance specifically to SCMV and MDMV,

[0161] - D101G, S161K, Q166K and G213L, mutations that specifically confer resistance to SCMV and MDMV,

[0162] -N57D, P58S, G98D, G99R and S161K, mutations that specifically confer resistance to SCMV,

[0163] - A65D, A66D, G98D, G99R and S161K, these mutations specifically confer resistance to SCMV and MDMV, or

[0164] -N57K, P58T, G98D, G99R, S161K and G213D, mutations that specifically confer resistance to SCMV,

[0165] By reference to the native zein sequence SEQ ID NO:2.

[0166] For example, the eIF(iso)4E protein variant as defined above may comprise at least one mutation, preferably at least two mutations or at least three mutations, such as at least four, at least five or at least six mutations, selected from:

[0167] - the group consisting of A63D, A64D, V96D, G97R, D99G and G211L / D / S, with reference to the native zein sequence of SEQ ID NO: 1,

[0168] - the group consisting of A65D, A66D, G98D, G99R, D101G and G213L / D / S, with reference to the native zein sequence of SEQ ID NO: 2,

[0169] - the group consisting of A60D, A61D, N94R, W108L, S157K and R161E / L, with reference to the native zein sequence of SEQ ID NO: 3, or

[0170] - The group consisting of A54D, A55D, T88R, W102L, S151K and R155E / L, with reference to the native zein sequence of SEQ ID NO: 4.

[0171] Mutations A63D, A64D, V96D, G97R and D99G of the reference sequence SEQ ID NO: 1 disrupt the interaction between eIF4E1 and potato Y viruses (particularly both SCMV and MDMV). These mutations are homologous to mutations A65D, A66D, G98D, G99R of the reference SEQ ID NO: 2, and thus disrupt the interaction between eIF4E2 and potato Y viruses (particularly both SCMV and MDMV).

[0172] The mutation G211L / D / S of the reference sequence SEQ ID NO: 1 disrupts the interaction between eIF4E1 and MCMV. This mutation is homologous to the mutation G213L / D / S of the reference sequence SEQ ID NO: 2, thus disrupting the interaction between eIF4E2 and MCMV.

[0173] The mutations A60D, A61D, N94R, W108L, S157K and R161E / L of the reference sequence SEQ ID NO: 3 disrupt the interaction between eIFiso4E1 and potato virus Y (particularly MDMV). These mutations are homologous to the mutations A54D, A55D, T88R, W102L, S151K, R155E / L of the reference sequence SEQ ID NO: 4, thus disrupting the interaction between eIFiso4E2 and potato virus Y.

[0174] Mutations A63D and A64D of the reference sequence SEQ ID NO:1 are homologous to mutations A65D and A66D of the reference sequence SEQ ID NO:2, to mutations A60D and A61D of the reference sequence SEQ ID NO:3, and to mutations A54D and A55D of the reference sequence SEQ ID NO:4.

[0175] For example, a variant of the eIF(iso)4E protein as defined above may comprise:

[0176] - mutations (i) A63D and A64D, (ii) V96D and G97R, (iii) D99G and / or (iv) G211L / D / S, by reference to the native zein sequence SEQ ID NO: 1,

[0177] - mutations (i) A65D and A66D, (ii) G98D and G99R, (iii) D101G and / or (iv) G213L / D / S, by reference to the native zein sequence SEQ ID NO: 2,

[0178] - mutations (i) A60D and A61D, (ii) N94R, (iii) W108L and R161E / L and / or (iv) S157K, by reference to the native zein sequence of SEQ ID NO: 3, or

[0179] - the mutations (i) A54D and A55D, (ii) T88R, (iii) W102L and R155E / L and / or (iv) S151K, by reference to the native zein sequence SEQ ID NO:4.

[0180] For example, a preferred eIF(iso)4E protein variant as defined above may comprise: (i) at least one mutation that disrupts the interaction between said protein variant and at least one potato virus Y, and (ii) at least one mutation that disrupts the interaction between said protein variant and at least one maize chlorotic mottle virus.

[0181] For example, a preferred eIF4E1 protein variant as defined above may comprise the mutations G211L / D / S and at least two mutations selected from the group consisting of (i) A63D and A64D, (ii) V96D and G97R and (iii) D99G, with reference to the native zein sequence of SEQ ID NO: 1.

[0182] For example, still preferred eIF4E1 protein variants as defined above may comprise the mutations G211L / D / S, A63D and A64D, and optionally at least one further mutation selected from the group consisting of (i) V96D and G97R and (ii) D99G, with reference to the native zein sequence SEQ ID NO: 1.

[0183] For example, a preferred eIF4E2 protein variant as defined above may comprise the mutations G213L / D / S and at least two mutations selected from the group consisting of (i) A65D and A66D, (ii) G98D and G99R and (iii) D101G, with reference to the native zein sequence of SEQ ID NO:2.

[0184] For example, still preferred eIF4E2 protein variants as defined above may comprise the mutations G213L / D / S, A65D and A66D, and optionally at least one further mutation selected from the group consisting of (i) G98D and G99R and (ii) D101G, with reference to the native zein sequence of SEQ ID NO:2.

[0185] For example, a preferred eIFiso4E1 protein variant as defined above may comprise mutations A60D and A61D and at least one mutation selected from the group consisting of (i) N94R, (ii) W108L and R161E / L and (iii) S157K, by reference to the native zein sequence of SEQ ID NO: 3, or

[0186] For example, a preferred eIFiso4E2 protein variant as defined above may comprise mutations A54D and A55D and at least one mutation selected from the group consisting of (i) T88R, (ii) W102L and R155E / L and (iii) S151K, with reference to the native zein sequence of SEQ ID NO:4.

[0187] In a preferred embodiment, the eIF(iso)4E protein variant is an eIF4E1 protein variant comprising or consisting of the sequence SEQ ID NO:5, an eIF4E2 protein variant comprising or consisting of the sequence SEQ ID NO:6, an eIFiso4E1 protein variant comprising or consisting of the sequence SEQ ID NO:7, or an eIFiso4E2 protein variant comprising or consisting of the sequence SEQ ID NO:8.

[0188] Genetically modified corn cells

[0189] The present invention also relates to a genetically modified maize cell comprising at least one eIF(iso)4E protein variant capable of conferring resistance to at least one maize virus, in particular wherein said protein variant comprises at least one mutation compared to the corresponding native maize eIF(iso)4E.

[0190] The eIF(iso)4E protein variant is in particular as defined above.

[0191] As used herein, "maize cell" or "maize plant cell" refers in particular to a maize cell obtained or found from a seed, suspension culture, embryo, meristem, callus, leaf, root, shoot, gametophyte, sporophyte, pollen or microspore. Maize cells also include protoplasts, in particular protoplasts obtained from tissues as defined above, as well as cells from or found in plant cell tissue culture from which a plant can be regenerated, or plant callus.

[0192] The expression "a cell comprising at least one protein X" herein refers to a cell expressing the at least one protein X.

[0193] For example, a genetically modified maize cell as defined above may comprise at least one exogenous nucleic acid encoding said eIF(iso)4E protein variant.

[0194] "Exogenous nucleic acid" herein refers to a nucleic acid that does not naturally occur in corn cells. For example, the exogenous nucleic acid can be introduced into the cell by genetic engineering.

[0195] The genetically modified maize cell as defined above preferably comprises at least one eIF(iso)4E protein variant and does not comprise any native eIF4E1 protein nor any native eIF4E2 protein.

[0196] In one embodiment, the genetically modified maize cell as defined above preferably expresses at least one endogenous eIFiso4E protein. For example, the maize cell as defined above expresses protein eIFiso4E1 and / or protein eIFiso4E2.

[0197] For example, a genetically modified maize cell as defined above may comprise at least one eIF(iso)4E protein variant and at least one native eIFiso4E protein.

[0198] For example, a genetically modified maize cell as defined above may comprise at least one eIF(iso)4E protein variant, a native eIFiso4E1 protein and a native eIFiso4E2 protein.

[0199] The genetically modified maize cell as defined above may comprise at least one eIF(iso)4E protein variant, preferably an eIF4E2 protein variant, a native eIFiso4E1 protein, a native eIFiso4E2 protein and a native eIF4E1 protein, and does not comprise any eIF4E2 protein.

[0200] The genetically modified maize cell as defined above may comprise at least one eIF(iso)4E protein variant, preferably an eIF4E1 protein variant, a native eIFiso4E1 protein, a native eIFiso4E2 protein and a native eIF4E2 protein, and not comprise any eIF4E1 protein.

[0201] The genetically modified maize cell as defined above preferably comprises at least one eIF(iso)4E protein variant, a native eIFiso4E1 protein and a native eIFiso4E2 protein and does not comprise any native eIF4E1 protein nor any native eIF4E2 protein.

[0202] In one embodiment, the eIF(iso)4E protein variant is the result of endogenous gene editing.

[0203] Gene editing can be performed by any suitable method well known to the skilled person, such as using base editing, prime editing or targeted homologous recombination, in particular using specific nucleases, such as megabase meganucleases (such as I-SceI), transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs) or clustered regularly interspaced short palindromic repeats-associated nucleases Cas (CRISPR / Cas), such as CRISPR / Cas9 or CRISPR / Cas 12. These methods also include physical or chemical mutagenesis.

[0204] In another embodiment, the eIF(iso)4E protein variant is expressed exogenously in the cell. For example, the exogenous expression is driven by an expression vector (such as a vector comprising at least an exogenous nucleic acid as defined above).

[0205] Advantageously, the genetically modified maize cell may further comprise at least one knocked out eif4e gene and / or at least a mutated apb1 gene.

[0206] Knocking out at least one eif4e gene can be advantageous in preventing the expression of native eiF4E protein, particularly when endogenous native eif4e1 gene and / or eif4e2 gene are still present in the cell. Knocking out at least one eif4e gene can indeed further increase the viral resistance conferred by the eiF(iso)4E protein variant. In one embodiment, the genetically modified corn cell as defined above comprises at least one eiF(iso)4E protein variant as defined in the "eiF(iso)4E protein variant" section and at least one knocked out eif4e gene, particularly as described below.

[0207] In a specific embodiment, the genetically modified maize cell as defined above comprises at least one eIF(iso)4E protein variant as defined in the "eIF(iso)4E protein variants" section and a knocked-out eif4e1 gene.

[0208] In another specific embodiment, the maize cell as defined above comprises at least one eIF(iso)4E protein variant as defined in the "eIF(iso)4E protein variants" section and a knocked-out eif4e2 gene.

[0209] The knocked-out eIF4E gene is the endogenous eIF4E gene.

[0210] "Endogenous gene" herein refers to a gene that is naturally present in a maize cell.

[0211] The eif4e gene to be knocked out can be the eif4e1 gene of sequence SEQ ID NO:9 or the eif4e2 gene of sequence SEQ ID NO:10.

[0212] For example, the eif4e gene to be knocked out in the genetically modified corn cell comprises or consists of a sequence that is at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to SEQ ID NO:9 or SEQ ID NO:10.

[0213] In one embodiment, the eif4E gene knocked out in the genetically modified corn cell comprises or consists of the sequence SEQ ID NO:9 or SEQ ID NO:10.

[0214] Thus, the genetically modified maize cell as defined above may further comprise a knocked out eif4e1 gene and / or a knocked out eif4e2 gene.

[0215] "Knockout gene" refers to a non-functional gene in this article. The gene knocked out can no longer express a functional protein, which means that the full-length protein originally encoded by the gene is no longer translated, but can optionally translate a non-functional protein from the gene knocked out. Non-functional genes can also cause being unable to be transcribed into RNA and / or translated into protein." non-functional protein" refers to a protein lacking at least one function of a functional protein (particularly native protein) in this article. Therefore, gene knockout can not produce a phenotype in which protein level expression is reduced.

[0216] In the present application, knockout can be accomplished by introducing at least one mutation, at least one other gene or sequence and / or at least double-strand break in any part of the genomic sequence, including the 5'UTR, 3'UTR, introns, exons and promoter region of the eif4e gene.

[0217] The knockout is preferably accomplished by introducing at least one mutation, at least one other gene or sequence and / or at least a double strand break in any part of the eif4e gene, more preferably within an intron and / or exon.

[0218] The gene can be knocked out by any suitable method known to the skilled person, such as using:

[0219] - a nucleic acid comprising a mutation or a gene to be inserted into the eif4e gene by homologous recombination,

[0220] - site-specific nuclease for introducing double-strand breaks in the eIF4E gene,

[0221] - a zinc finger nuclease comprising a DNA binding domain that targets the eif4e gene and a restriction endonuclease, thereby causing a double-strand break in the eif4e gene,

[0222] - TALEN (transcription activator-like effector nuclease) comprising a DNA binding domain targeting the eIF4E gene and a nuclease, thereby causing a double-strand break in the eIF4E gene, and / or

[0223] -Gene editing, for example using the CRISPR / cas system, comprising a guide RNA complementary to a target sequence in the eif4e gene, which complexes with the Cas protein, thereby causing a double-strand break in the eif4e gene and optionally introducing a desired sequence.

[0224] When double-strand breaks occur in the eif4e gene, the cellular DNA repair machinery usually repairs the double-strand breaks by non-homologous end joining (NHEJ), resulting in insertion-deletions (INDELs). INDELs can change the reading frame and / or introduce nonsense mutations, resulting in a non-functional eif4e gene and, thus, a disrupted or truncated protein.

[0225] In this application, the term "gene editing" includes insertion, deletion or substitution of at least one base pair and results in knockout, for example, by changes in the reading frame, changes in specific amino acids (including the generation of stop (STOP) codons) or a combination thereof. Gene editing involves DNA strand breaks and DNA damage repair mechanisms, such as non-homologous end joining or homologous recombination.

[0226] Therefore, gene knockout is different from gene silencing.

[0227] Gene silencing is actually the regulation of gene expression in cells to prevent the expression of a given gene. Gene silencing usually reduces the expression of a gene, but does not completely (or completely) inhibit its expression into a functional protein, which is the opposite of gene knockout. The most popular gene silencing method is RNAi.

[0228] Therefore, the expression "knocked out gene" does not encompass gene mutations that result in reduced expression levels of the protein encoded by the gene.

[0229] Thus, genetically modified maize cells comprising a knocked-out eif4e1 gene do not express any eIF4E1 protein.

[0230] Thus, genetically modified maize cells comprising a knocked-out eif4e2 gene do not express any eIF4E2 protein.

[0231] The maize cell as defined above may express a non-functional protein from a knocked out eif4e gene.

[0232] "Non-functional protein encoded by the knocked-out eif4e gene" herein refers to a protein that cannot participate in protein synthesis.

[0233] Any suitable method can be used to assess whether the protein encoded by the knocked-out eif4e gene is non-functional, such as a yeast complementation assay or any known standard method.

[0234] In the yeast complementation assay, yeast is made deficient in eIF4E protein. If the protein encoded by the knocked-out eif4e gene is non-functional, the deficient yeast will not grow.

[0235] For example, the non-functional protein encoded by the knocked-out eif4e gene can be composed of an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18, or comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 or SEQ ID NO: 18.

[0236] For example, the non-functional protein encoded by the knocked-out eif4e gene can be composed of an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, or comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.

[0237] The non-functional protein as defined above encoded by the knocked-out eif4e gene preferably has a length of less than 150 amino acids, preferably less than 100 amino acids. In a preferred embodiment, the non-functional protein has a length of less than 100 amino acids and consists of an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 18, or comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 98% or at least 99% identical to SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 17 or SEQ ID NO: 18.

[0238] The non-functional protein may have a length of less than 100 amino acids, and consist of, or comprise, the amino acid sequence of SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:17 or SEQ ID NO:18.

[0239] The genetically modified corn cell may further comprise at least one apbl mutant gene.

[0240] The apb1 gene is located on chromosome 3, more precisely at position chr3:134550012-134554530 in version 4 of the B73 genome.

[0241] The reference sequence of the apb1 gene is, for example, the sequence SEQ ID NO:11.

[0242] The apb1 gene encodes the protein APB1.

[0243] The reference sequence of the native zein APB1 is the sequence SEQ ID NO: 12, also known as Zm00001d041711_APB1 in the B73 genome version 4.

[0244] The apb1 gene is located between the eif4e1 and eif4e2 loci. In some lines, regulatory sequences in the apb1 gene promoter have been identified as being responsible for resistance to Sugarcane Mosaic Virus (SCMV), a potato virus Y (see Leng et al., 2017).

[0245] The term "mutated gene" includes at least one mutation, such as insertion, deletion and / or substitution, compared to a wild-type gene (such as the apb1 gene of sequence SEQ ID NO: 11). The at least one mutation may occur in the promoter, untranslated region, coding region (exon) and / or non-coding region (intron) of the gene. The expression of the mutated apb1 gene preferably increases resistance to at least one corn virus conferred by at least one eIF (iso) 4E protein variant, or results in resistance to infection by another virus.

[0246] Preferably, the apb1 promoter comprises a BoxII motif and three SP1 motifs.

[0247] In another embodiment, the genetically modified maize cell as defined above does not comprise any apbl mutated gene.

[0248] For example, a genetically modified maize cell as defined above may comprise:

[0249] at least one eiF4E1 protein variant (in particular expressed from a gene obtained by gene editing at the eif4E1 locus), a knocked-out eif4e2 gene, a native eIFiso4E1 protein, a native eIFiso4E2 protein and optionally a mutated apb1 gene,

[0250] at least one eiF4E2 protein variant (in particular expressed from a gene obtained by gene editing at the eif4E2 locus), a knocked-out eif4e1 gene, a native eIFiso4E1 protein, a native eIFiso4E2 protein and optionally a mutated apb1 gene,

[0251] at least one eiF4E1 protein variant (in particular obtained by transformation with a DNA sequence encoding an eIF4E1 protein variant, which DNA sequence is integrated at a locus different from the eif4e1 gene locus), at least one knocked-out eif4e gene, a native eIFiso4E1 protein, a native eIFiso4E2 protein, and optionally a mutated apb1 gene,

[0252] at least one eiF4E1 protein variant (in particular obtained by transformation with a DNA sequence encoding an eIF4E1 protein variant, which DNA sequence is integrated at a locus different from the eif4e1 gene locus), a knocked-out eif4e1 gene, a knocked-out eif4e2 gene, a native eIFiso4E1 protein, a native eIFiso4E2 protein, and optionally a mutated apb1 gene,

[0253] at least one eiF4E2 protein variant (in particular obtained by transformation with a DNA sequence encoding an eIF4E2 protein variant, which DNA sequence is integrated at a locus different from the eif4e2 locus), at least one knocked-out eif4e gene, a native eIFiso4E1 protein, a native eIFiso4E2 protein, and optionally a mutated apb1 gene,

[0254] at least one eiF4E2 protein variant (in particular obtained by transformation with a DNA sequence encoding an eIF4E2 protein variant, which DNA sequence is integrated at a locus different from the eif4e2 locus), a knocked-out eif4e1 gene, a knocked-out eif4e2 gene, a native eIFiso4E1 protein, a native eIFiso4E2 protein, and optionally a mutated apb1 gene,

[0255] at least one eiF4E1 protein variant (in particular expressed from a gene obtained by gene editing at the eif4e1 locus), at least one eiF4E2 protein variant (in particular obtained by transformation with a DNA sequence encoding an eIF4E2 protein variant, which DNA sequence is integrated at a locus different from the eif4e2 locus), the native eIFiso4E1 protein, the native eIFiso4E2 protein, and optionally a mutated apb1 gene,

[0256] at least one eiF4E1 protein variant (in particular expressed from a gene obtained by gene editing at the eif4e1 locus), at least one eiF4E2 protein variant (in particular obtained by transformation with a DNA sequence encoding an eIF4E2 protein variant, which DNA sequence is integrated at a locus different from the eif4e2 locus), a knocked-out eif4e2 gene, a native eIFiso4E1 protein, a native eIFiso4E2 protein and optionally a mutated apb1 gene,

[0257] at least one eiF4E1 protein variant (in particular obtained by transformation with a DNA sequence encoding an eIF4E1 protein variant, which DNA sequence is integrated at a locus different from the eif4e1 locus), at least one eiF4E2 protein variant (in particular expressed from a gene obtained by gene editing at the eif4e2 locus), the native eIFiso4E1 protein, the native eIFiso4E2 protein, and optionally a mutated apb1 gene,

[0258] at least one eiF4E1 protein variant (in particular obtained by transformation with a DNA sequence encoding an eIF4E1 protein variant, which DNA sequence is integrated at a locus different from the eif4e1 locus), at least one eiF4E2 protein variant (in particular expressed from a gene obtained by gene editing at the eif4e2 locus), a knocked-out eif4e1 gene, a native eIFiso4E1 protein, a native eIFiso4E2 protein and optionally a mutated apb1 gene,

[0259] at least one eiF4E1 protein variant (in particular expressed from a gene obtained by gene editing at the eif4E1 locus), at least one eiF4E2 protein variant (in particular expressed from a gene obtained by gene editing at the eif4E2 locus), a native eIFiso4E1 protein, a native eIFiso4E2 protein, and optionally a mutated apb1 gene, or

[0260] - at least one eiF4E1 protein variant and at least one eiF4E2 protein variant (in particular both obtained by transformation with DNA sequences encoding eIF4E1 and eIF4E2 protein variants, which are integrated at a locus different from the eif4e1 locus and the eif4e2 locus), a knocked-out eif4e1 gene, a knocked-out eif4e2 gene, a native eIFiso4E1 protein, a native eIFiso4E2 protein, and optionally a mutant apb1 gene.

[0261] Nucleic acids and vectors

[0262] The present invention also relates to a nucleic acid encoding at least one eIF(iso)4E protein variant as defined above.

[0263] The nucleic acid as defined above may be provided in the form of a vector.

[0264] The present invention also relates to a vector.

[0265] A vector as defined above may be:

[0266] a) a vector suitable for integrating at least one nucleic acid encoding at least one eIF(iso)4E protein variant into the genome of a maize plant, or

[0267] b) A vector suitable for introducing at least one mutation into the eIF(iso)4E gene sequence, such that the obtained mutated eIF(iso)4E gene encodes an eIF(iso)4E protein variant as defined above.

[0268] For example, the vector suitable for introducing at least one mutation may be a vector suitable for targeted base editing or a vector suitable for lead editing.

[0269] In one embodiment, the vector is suitable for integrating at least one nucleic acid encoding at least one eIF(iso)4E protein variant into the genome of a maize plant, preferably at a targeted position in the genome, in particular by homologous recombination.

[0270] Vectors suitable for integration comprise at least one nucleic acid encoding an eIF(iso)4E protein variant as defined above.

[0271] The nucleic acid encoding at least one eIF(iso)4E protein variant is controlled by a promoter, preferably a plant promoter, more preferably a promoter whose expression is compatible with the expression of the wild-type eIF(iso)4E gene. In a specific embodiment, the promoter may comprise or consist of 2000 nucleotides upstream of the ATG of the eIF(iso)4E gene.

[0272] As defined above, the vector suitable for integration can further include nucleic acid encoding Cas protein (such as Cas9 or Cas12 protein), and at least one guide nucleotide, particularly designed to allow specific cutting of genome at 5' and 3' ends of eIF (iso) 4E gene to carry out homologous recombination. Nucleic acid encoding Cas protein (such as Cas9 or Cas12 protein) and guide nucleotide are preferably controlled by constitutive promoter (such as ZmUbi). As defined above, the vector suitable for integration can further include 5' and 3' homologous sequences at the ends of eIFiso4E gene to be replaced. The length of the homologous sequence is preferably at least 250 nucleotides and at most 2000 nucleotides.

[0273] As defined above, a vector suitable for targeted base editing may include a nucleic acid encoding a Cas protein (e.g., Cas9 or Cas12 protein) for a base editor module (preferably a deaminase) to convert a nucleotide into another type of nucleotide, such as A to T or A to G. As defined above, a vector suitable for targeted base editing may further include at least one guide nucleic acid homologous to the upstream or downstream sequence of the nucleotide being targeted for editing, particularly according to specific rules known to the technician. Nucleic acids encoding Cas proteins (e.g., Cas9 or Cas12 proteins) and guide nucleotides are preferably under the control of a constitutive promoter (e.g., ZmUbi).

[0274] "Cas protein" herein refers to the endonuclease of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas system.

[0275] The preferred Cas protein is Cas9 or Cas12 protein.

[0276] “Cas9 protein”, “CRISPR-associated protein 9” or “Cas9-type protein” herein refers to a dual RNA-guided DNA endonuclease.

[0277] "Cas12 protein", "CRISPR-associated protein 12" or "Cas12-type protein" refers herein to a single RNA-guided endonuclease. Non-limiting examples of Cas9 or Cas12 proteins as defined above include nickase Cas9, dead Cas9 or Cas12 (e.g., nCas9, nCas12, dCas9, dCas12), and Cas9 or Cas12 without PAM.

[0278] The base editor module allows a nucleotide to be converted into another type of nucleotide depending on the type of deaminase. For example, the base editor can be a cytidine deaminase, an adenosine deaminase, or a guanine deaminase.

[0279] A vector suitable for lead editing as defined above may comprise a nucleic acid encoding a Cas protein (e.g., Cas9 or Cas12 protein) fused to a reverse transcriptase. The vector may also comprise a lead editing guide (pegRNA) that is capable of (i) identifying a target nucleotide sequence to be edited and (ii) encoding new genetic information to replace the target nucleotide sequence.

[0280] The reverse transcriptase is preferably MMLV (Murine Leukemia Virus).

[0281] A vector suitable for prime editing as defined above may comprise a Cas9 nickase (under the control of the maize actin gene promoter) fused to a reverse transcriptase (preferably MMLV) and a prime editing guide.

[0282] The present invention also relates to a vector suitable for knocking out at least one maize eIF4E gene (eg, maize eIF4E1 gene and / or maize eIF4E2 gene).

[0283] A vector as defined above may be a plasmid.

[0284] A vector as defined above may comprise:

[0285] a) at least one CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas endonuclease expression cassette, and / or

[0286] b) at least one gRNA expression cassette.

[0287] The vector as defined above preferably comprises:

[0288] a) at least one gRNA expression cassette, wherein the gRNA expression cassette comprises a nucleic acid encoding a gRNA under the control of a promoter, and wherein the gRNA comprises a region complementary to a target region of a maize eif4E gene, and

[0289] b) Optionally, at least one CRISPR-Cas endonuclease expression cassette.

[0290] The at least one CRISPR-Cas endonuclease expression cassette and the at least one gRNA expression cassette may be provided in the same vector or in different vectors.

[0291] The CRISPR-Cas endonuclease expression cassette comprises a nucleic acid encoding a Cas endonuclease under the control of a promoter.

[0292] Cas endonucleases are enzymes that use gRNA as a guide to recognize a specific position in a DNA sequence and perform a double-strand break at that specific position. Cas endonucleases generally require the presence of a protospacer adjacent motif (PAM) sequence near a specific targeted position. The PAM sequence is preferably spaced up to 20 nucleotides from the targeted position. The PAM sequence can vary depending on the Cas endonuclease.

[0293] As the Cas endonuclease requires a PAM sequence, the eif4E gene contains the PAM sequence downstream of the targeted region.

[0294] The Cas endonuclease can be selected from the group consisting of Cas9, Cas12a, Cas12b, C2c1 and C2c2.

[0295] The Cas endonuclease is preferably a Cas9 (CRISPR-associated protein 9) endonuclease. For example, the Cas9 endonuclease may comprise or consist of the sequence SEQ ID NO: 23.

[0296] The promoter of the CRISPR-Cas endonuclease expression cassette may be a constitutive promoter selected from the group consisting of the ZmUbi promoter, the 35S promoter or the 19S promoter (Kay et al., 1987), the rice actin promoter (McElroy et al., 1990), the pCRV promoter (Depigny-This et al., 1992), the CsVMV promoter (Verdaguer et al., 1998) and the ubiquitin promoter from rice or sugarcane. The promoter of the CRISPR-Cas endonuclease expression cassette is preferably the ZmUbi promoter.

[0297] The CRISPR-Cas endonuclease expression cassette preferably comprises a terminator, such as SbHSP.

[0298] The gRNA expression cassette contains a nucleic acid encoding the gRNA under the control of a promoter.

[0299] gRNA contains:

[0300] a) a region complementary to a target region of the eif4E gene, preferably complementary to a target region in an exon of the eif4E gene, and

[0301] b) A scaffold region that allows binding to the CRISPR-Cas endonuclease encoded by the CRISPR-Cas endonuclease expression cassette.

[0302] The eif4E gene is particularly as defined above. The target region of the eif4E gene is preferably located in exon 1 or exon 2 of the eif4E gene. Exons 1 and 2 of the eIF4E1 gene and exons 1 and 2 of the eIF4E2 gene are particularly as defined above in the section "Maize eif4E Gene and Protein".

[0303] For example, the promoter of the gRNA expression cassette can be selected from the group consisting of RNA polymerase III promoters (e.g., TaU6 promoter, ZmU6 promoter, or ZmU3), or RNA polymerase II promoters, such as constitutive promoters (e.g., ZmUbi or TaUbi). The promoter of the gRNA expression cassette is preferably a ZmU6 promoter.

[0304] Therefore, the gRNA produced by the gRNA expression cassette is able to recognize the target region of the eif4E gene, and therefore, when complexed with the CRISPR-Cas endonuclease, introduces a double-strand break in the target region of the eif4E gene through the action of the CRISPR-Cas endonuclease.

[0305] The gRNA preferably comprises at least 15 nucleotides, preferably at least 17 nucleotides, more preferably at least 18 nucleotides and / or at most 25 nucleotides, preferably at most 24 nucleotides, more preferably at most 23 nucleotides.

[0306] For example, the gRNA can comprise or consist of the sequence SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, or SEQ ID NO: 22.

[0307] Genetically modified or edited corn plants or seeds that are resistant to at least one corn virus

[0308] The present invention also relates to a genetically modified or edited corn plant that is resistant to at least one corn virus or a genetically modified or edited corn seed that is resistant to at least one corn virus.

[0309] The maize virus is in particular as defined above.

[0310] Thus, the genetically modified or edited corn plant or genetically modified or edited corn seed is resistant to a disease caused by the at least one corn virus.

[0311] For example, the disease is as defined above.

[0312] For example, a genetically modified or edited corn plant or a genetically modified or edited corn seed as defined above may be resistant to MNLD.

[0313] "Genetically modified" or "GM" herein refers to the integration of the modification at any position in the genome. Thus, in a GM plant or GM seed, the wild-type gene is present.

[0314] "Gene editing" or "GE" as used herein refers to a modification at the origin site. Thus, in a GE plant or GE seed, the wild-type gene is no longer present.

[0315] The GM or GE plant resistant to at least one corn virus, or the GE or GE seed resistant to at least one corn virus, expresses at least one eIF(iso)4E protein variant as defined above. In other words, the cells of the plant or seed express at least one eIF(iso)4E protein variant as defined above.

[0316] A plant or seed as defined above comprises a genetically modified maize cell, in particular as defined in the section "Genetically modified maize cell".

[0317] The plant or seed as defined above preferably comprises a genetically modified maize cell, wherein said cell comprises at least one eIF(iso)4E protein variant and does not comprise any native eIF4E1 protein nor any native eIF4E2 protein.

[0318] The cells of a plant or seed as defined above preferably comprise at least one nucleic acid as defined above encoding at least one eIF(iso)4E protein variant as defined above.

[0319] The nucleic acid is preferably inserted into the genome of a cell of the plant or seed.

[0320] Said nucleic acid preferably replaces (in particular by allelic exchange) the corresponding endogenous eIF(iso)4E gene.

[0321] The maize plant or seed as defined above is preferably an agronomic plant or seed.

[0322] The expression "agronomic plants or seeds" refers herein to plants or seeds which are suitable for large-scale production, in particular for human and animal food or for industrial purposes.

[0323] In one embodiment, the plant or seed resistant to at least one maize virus as defined above is not a null mutant of any or all eIF(iso)4E genes, since the plant or seed expresses at least one eIF(iso)4E protein variant.

[0324] Method for obtaining a corn plant resistant to at least one corn virus

[0325] The present invention particularly relates to a method for obtaining a corn plant resistant to at least one corn virus, particularly as defined above, wherein said method comprises:

[0326] (i) introducing at least one nucleic acid encoding at least one eIF(iso)4E protein variant into the genome of a maize cell or maize tissue, or (ii) introducing at least one mutation into an endogenous eIF(iso)4E gene of a maize cell or maize tissue, so that the obtained mutant eIF(iso)4E gene encodes at least one eIF(iso)4E protein variant as defined above, and

[0327] - Regenerating a corn plant from the obtained corn cells or corn tissues.

[0328] Methods for introducing at least one nucleic acid into the genome of a maize cell or maize tissue are well known to the skilled person.

[0329] For example, introducing at least one nucleic acid encoding at least one eIF(iso)4E protein variant into the genome of a corn cell or corn tissue can comprise transforming the corn cell or corn tissue with a vector comprising at least one nucleic acid encoding at least one eIF(iso)4E protein variant.

[0330] Methods for introducing at least one mutation in an endogenous plant gene are well known to the skilled person. For example, such methods include base editing, lead editing or targeted homologous recombination, in particular using specific nucleases, such as megabase meganucleases (such as I-SceI), transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs) or regularly spaced clustered short palindromic repeats associated nucleases Cas (CRISPR / Cas), such as CRISPR / Cas9 or CRISPR / Cas12. These methods also include physical or chemical mutagenesis.

[0331] Introducing at least one mutation in an endogenous corn gene encoding an eIF(iso)4E protein as defined above may comprise transforming corn cells or corn tissues with a vector suitable for introducing at least one mutation in an endogenous eIF(iso)4E gene, such that the obtained mutated eIF(iso)4E gene encodes at least one eIF(iso)4E protein variant as defined above.

[0332] Therefore, the present invention particularly relates to a method for obtaining a corn plant that is resistant to at least one corn virus, particularly as defined above, wherein the method comprises transforming corn cells or corn tissue with (i) a vector comprising at least one nucleic acid encoding at least one eIF(iso)4E protein variant or (ii) a vector suitable for introducing at least one mutation in an endogenous eIF(iso)4E gene, such that the mutated eIF(iso)4E gene obtained encodes at least one eIF(iso)4E protein variant as defined above.

[0333] The vector may be a vector as defined above, such as a vector suitable for inserting into the maize genome at least one nucleic acid encoding at least one eIF(iso)4E protein variant as defined above, a vector suitable for targeted base editing, or a vector suitable for lead editing.

[0334] In a preferred embodiment, a nucleic acid encoding an eIF(iso)4E protein variant is introduced into a specific genomic location in a maize cell or tissue.

[0335] The method as defined above preferably allows replacing the endogenous eIF(iso)4E gene with at least one nucleic acid encoding a variant of the eIF(iso)4E protein, in particular by homologous recombination at the same genomic location.

[0336] The nucleic acid encoding the eIF(iso)4E protein variant is preferably as defined above. The nucleic acid may be provided in the form of a vector as defined above.

[0337] In another preferred embodiment, the method as defined above allows mutating at least one nucleotide of the endogenous eIF(iso)4E gene, for example by gene editing or prime editing, to obtain a mutated eIF(iso)4E gene encoding an eIF(iso)4E protein variant as defined above.

[0338] The maize virus is in particular as defined above.

[0339] Resistance to the at least one corn virus confers resistance to diseases caused by the corn virus.

[0340] The diseases are in particular as defined above.

[0341] Any method known to the skilled artisan can be used to introduce a nucleic acid of interest into the genome of a plant.

[0342] Any method known to the skilled person for replacing a nucleic acid with another nucleic acid of interest in a plant can be used, such as allelic exchange.

[0343] Any method known to the skilled artisan can be used to introduce a mutation of interest into the plant genome.

[0344] For example, a method for obtaining a corn plant resistant to at least one corn virus as defined above may comprise:

[0345] a) transforming a maize cell or maize tissue with at least one vector as defined above in the section "Vectors", in particular a vector comprising a nucleic acid encoding an eIF(iso)4E protein variant as defined above, or transforming a maize cell or maize tissue with a vector suitable for introducing at least one mutation into an endogenous eIF(iso)4E gene, so that the mutated eIF(iso)4E gene obtained encodes at least one eIF(iso)4E protein variant as defined above, thereby obtaining a transformed maize cell or a transformed maize tissue,

[0346] b) regenerating a maize plant from the transformed maize cell or transformed maize tissue, and

[0347] c) optionally, evaluating the regenerated maize plants for expression of said eIF(iso)4E protein variant.

[0348] Step a)

[0349] Step a) comprises transforming maize cells or maize tissues with the vector.

[0350] The corn cell can be a protoplast.

[0351] The corn tissue may be apical meristem, cotyledons, embryos (preferably immature embryos), pollen, and / or microspores.

[0352] The maize tissue is preferably an immature embryo at 10 days after fertilization.

[0353] The maize cells or maize tissue used as starting material comprise at least one knocked-out eif4e gene and / or a mutated apb1 gene, in particular as defined above.

[0354] Any technique suitable for transformation of plant cells or plant tissues may be used, such as biolistic particle delivery, PEG transformation, electroporation or Agrobacterium transgene delivery.

[0355] For Agrobacterium transgene delivery, the vector is first transferred into Agrobacterium to obtain transformed Agrobacterium, which is then used to transform corn cells or corn tissues. The Agrobacterium is preferably Agrobacterium tumefaciens.

[0356] For example, transformation with the transformed Agrobacterium includes co-culturing corn cells or corn tissue with the Agrobacterium, for example, for at least 5 minutes.

[0357] In addition to transformation, vectors can also be introduced using haploid inducible lines or related techniques.

[0358] When the vector comprises a nucleic acid encoding an eIF(iso)4E protein variant, the transformed maize cells or transformed maize tissues obtained in step a) thus express said eIF(iso)4E protein variant.

[0359] If desired, the corn cell or corn tissue of step a) or the transformed corn cell or transformed corn tissue of step a) can knock out at least one eIF4E gene, in particular so that the transformed corn cell or transformed corn tissue used in step b) does not express any native eIF4E1 protein, nor any native eIF4E2 protein.

[0360] Therefore, the method as defined above may optionally comprise the step of knocking out at least one eif4e gene in a corn cell or corn tissue before, during or after step a) to obtain a knocked-out corn cell or a knocked-out corn tissue.

[0361] Thus, the method as defined above may comprise:

[0362] - before step a), a step of knocking out at least one eIF4E gene in a corn cell or corn tissue to obtain a knocked-out corn cell or knocked-out corn tissue, wherein the knocked-out corn cell or knocked-out corn tissue is the corn cell or corn tissue used in step a), or

[0363] - After step a) and before step b), a step of knocking out at least one eIF4E gene in the transformed corn cell or the transformed corn tissue to obtain a knocked-out transformed corn cell or a knocked-out transformed corn tissue, wherein the knocked-out transformed corn cell or the knocked-out transformed corn tissue is the transformed corn cell or the transformed corn tissue used in step b).

[0364] Alternatively, step a) comprises: (i) transforming corn cells or corn tissues with at least one vector as defined in the "Vector" section above, in particular a vector comprising a nucleic acid encoding an eIF(iso)4E protein variant as defined above, or transforming corn cells or corn tissues with a vector suitable for introducing at least one mutation in an endogenous eIF(iso)4E gene, so that the obtained mutated eIF(iso)4E gene encodes at least one eIF(iso)4E protein variant as defined above, and (ii) knocking out at least one eif4E gene in the corn cell or corn tissue to obtain a transformed corn cell or transformed corn tissue.

[0365] The eif4E gene is an endogenous eif4E gene, in particular as defined above.

[0366] The eif4E gene can be knocked out by any suitable method known to the skilled person as defined above, such as:

[0367] - performing homologous recombination with a nucleic acid comprising a mutation or a gene to be inserted into the eIF4E gene,

[0368] - Double-strand breaks are introduced in the eif4E gene by: site-specific nucleases, zinc finger nucleases containing a DNA binding domain targeting the eif4E gene and a restriction endonuclease, TALENs (transcription activator-like effector nucleases) containing a DNA binding domain targeting the eif4E gene and a nuclease, or by gene editing.

[0369] The eIF4E gene is preferably knocked out by gene editing, more preferably using the CRISPR / Cas system.

[0370] The step of knocking out at least one eif4E gene preferably comprises introducing into said corn cell (or transformed corn cell) or corn tissue (or transformed corn tissue) at least one vector suitable for knocking out at least one corn eif4E gene.

[0371] Any method of introducing nucleic acids or vectors into cells or tissues can be used, such as transformation or the use of haploid inducible lines.

[0372] Transformation and use of haploid induction lines are as defined above.

[0373] For example, the vector suitable for knocking out at least one maize eif4E gene is as defined above.

[0374] When multiple vectors are used in step a), it is preferred to transform maize cells or maize tissues with different vectors simultaneously.

[0375] When using a vector suitable for knocking out at least one maize eif4E gene as defined above, step b) results in particular in:

[0376] - expression of a Cas endonuclease from a CRISPR-Cas endonuclease expression cassette and production of a gRNA from a gRNA expression cassette to generate a double strand break in the eif4E gene in the plant genome, and

[0377] - Double-strand break repair, in particular by non-homologous end joining (NHEJ), leading to insertions and / or deletions in the targeted eif4E gene.

[0378] When corn cells or corn tissue are transformed with: at least one vector comprising a nucleic acid encoding a variant of the eIF4E1 protein suitable for homologous recombination at the eif4e1 locus (thereby replacing the endogenous eif4e1 gene), or a vector suitable for introducing at least one mutation in the endogenous eif4e1 gene as defined above, the method as defined above preferably comprises knocking out the eif4e2 gene or using corn cells or corn tissue in which the eif4E2 gene has been knocked out (as starting material).

[0379] When corn cells or corn tissue are transformed with at least one vector comprising a nucleic acid encoding a variant of the eIF4E2 protein suitable for homologous recombination at the eif4e2 locus (thereby replacing the endogenous eif4e2 gene), or a vector suitable for introducing at least one mutation in the endogenous eif4e2 gene as defined above, the method as defined above preferably comprises knocking out the eif4e1 gene or using corn cells or corn tissue in which the eif4e1 gene has been knocked out (as starting material).

[0380] When corn cells or corn tissues are transformed with: (i) at least one vector comprising a nucleic acid encoding a variant of the eIF4E1 protein suitable for homologous recombination at the eif4e1 locus (thereby replacing the endogenous eif4e1 gene), or a vector suitable for introducing at least one mutation in the endogenous eif4e1 gene, and (ii) at least one vector comprising a nucleic acid encoding a variant of the eIF4E2 protein suitable for homologous recombination at the eif4e2 locus (thereby replacing the endogenous eif4e2 gene), or a vector suitable for introducing at least one mutation in the endogenous eif4e2 gene, the method as defined above does not comprise knocking out the eif4e1 gene, nor does it comprise knocking out the eif4e2 gene.

[0381] When a corn cell or corn tissue is transformed with: at least one vector comprising: (i) a nucleic acid encoding a variant of the eIF4E1 protein suitable for homologous recombination at the eif4e1 locus (thereby replacing the endogenous eif4e1 gene) and (ii) a nucleic acid encoding a variant of the eIF4E2 protein suitable for homologous recombination at the eif4e2 locus (thereby replacing the endogenous eif4e2 gene), or a vector suitable for introducing at least one mutation in the endogenous eif4e1 gene and at least one mutation in the endogenous eif4e2 gene, the method as defined above does not include knocking out the eif4e1 gene, nor does it include knocking out the eif4e2 gene.

[0382] Step b)

[0383] Step b) involves the regeneration of corn plants.

[0384] The regeneration of plants from plant cells or plant tissues is well known to the skilled person.

[0385] In particular, corn cells or corn tissue can be placed in a medium suitable for growing corn.

[0386] Regenerating a corn plant from a corn cell or corn tissue may include:

[0387] - growing the maize cells or maize tissue to obtain callus tissue, and

[0388] - Regeneration of shoots from callus, in particular by somatic embryogenesis.

[0389] The regeneration of corn cells into callus and shoots, particularly by somatic embryogenesis, is carried out in any suitable medium containing plant growth regulators. The induction of somatic embryogenesis can be activated by genes encoding transcription factors, such as BABY BOOM (bbm) (Horstman et al., 2017 Oct; 175 (2): 848-857. doi: 10.1104 / pp.17.00232. Epub 2017 Aug 22).

[0390] Regenerating corn plants from corn tissue may include regeneration of shoots.

[0391] Regeneration of shoots from corn tissue can be carried out in any suitable culture medium containing plant growth regulators.

[0392] Step c)

[0393] The method may further comprise the step c) of evaluating the regenerated maize plant for expression of the eIF(iso)4E protein variant.

[0394] Evaluation of the expression of the eIF(iso)4E protein variant by the regenerated maize plants can be performed as follows:

[0395] - evaluating the resistance of the regenerated corn plants to at least one corn virus (e.g., a corn virus as disclosed above),

[0396] - detecting the expression of the eIF(iso)4E protein variant by the cells of the regenerated maize plants, in particular according to any method familiar to the skilled person, such as Western blotting or immunoassay, and / or

[0397] - evaluating whether the cells of the regenerated maize plant comprise the nucleic acid encoding said eIF(iso)4E protein variant, in particular according to any method with which the skilled person is familiar, such as sequencing or PCR.

[0398] Optionally, step c) may also include assessing whether at least one eif4e gene is knocked out in the regenerated corn plant, for example by assessing whether the cells of the regenerated corn plant contain a nucleic acid encoding a non-functional eIF4E protein, in particular according to any method familiar to the skilled person, such as sequencing or PCR, or by detecting the expression of a non-functional eIF4E protein by the cells of the regenerated corn plant, in particular according to any method familiar to the skilled person, such as Western blotting or immunoassay.

[0399] The regenerated maize plant expresses the eIF(iso)4E protein variant if the following conditions are met:

[0400] - the regenerated corn plants are resistant to at least one corn virus,

[0401] - cells of the regenerated maize plant express said eIF(iso)4E protein variant, and / or

[0402] - The cells of the regenerated maize plant comprise a nucleic acid encoding said eIF(iso)4E protein variant.

[0403] At least one eIF4E gene is knocked out in the regenerated maize plant if the following conditions are met:

[0404] - cells of the regenerated maize plant express a non-functional eIF4E protein, and / or

[0405] - The cells of the regenerated maize plant comprise a nucleic acid encoding a non-functional eIF4E protein.

[0406] Use of at least one eIF(iso)4E protein variant for preventing at least one viral disease in corn

[0407] The present invention also relates to the use of at least one eIF(iso)4E protein variant for preventing at least one viral disease in corn.

[0408] The viral diseases are in particular as defined above.

[0409] The viral diseases are in particular caused by maize viruses as defined above.

[0410] The viral disease is preferably caused by potato virus Y and / or maize chlorotic mottle virus.

[0411] The invention relates in particular to the use as defined above for the prevention of MNLD, in particular MNLD caused by MCMV and at least one potato virus Y, such as SCMV or MDMV.

[0412] The eIF(iso)4E protein variant is as defined above.

[0413] The present invention particularly relates to the use as defined above, wherein the at least one eIF(iso)4E protein variant is expressed in maize, particularly in a maize plant or maize seed, in other words in the cells of said maize plant or maize seed.

[0414] The present invention also relates to the use of at least one eIF(iso)4E protein variant for conferring resistance to at least one corn virus in corn, preferably at least one potato virus Y (such as SCMV or MDMV) and / or at least one maize chlorotic mottle virus (such as MCMV), in particular wherein the at least one eIF(iso)4E protein variant is expressed in corn, in particular in a corn plant or corn seed, in other words, in the cells of the corn plant or corn seed.

[0415] The eIF(iso)4E protein variant is as defined above.

[0416] Method for identifying corn plants resistant to at least one corn virus

[0417] The present invention also relates to a method for identifying a corn plant that is resistant to at least one corn virus, wherein the method comprises detecting the expression of an eIF(iso)4E protein variant as defined above and / or the presence of a nucleic acid encoding an eIF(iso)4E protein variant as defined above, and optionally, the expression of a non-functional eIF4E protein and / or the presence of a nucleic acid encoding a non-functional eIF4E protein and / or the presence of a nucleic acid that is unable to express any eif4e gene in the cells of the corn plant.

[0418] The corn virus is as defined above.

[0419] Detecting the expression of the eIF(iso)4E protein variant or the non-functional eIF4E protein can be performed as defined above.

[0420] The nucleic acid encoding the eIF(iso)4E protein variant or the non-functional eIF4E protein may be DNA or RNA.

[0421] Detection of the presence of a nucleic acid encoding a variant of the eIF(iso)4E protein or a nucleic acid encoding a non-functional eIF4E protein, or a nucleic acid that is unable to express any eif4e gene, can be performed by any method known to the skilled person, such as using at least one pair of PCR primers.

[0422] The pair of PCR primers may be capable of amplifying a region comprising a portion of an endogenous plant genome and at least a portion of a nucleic acid encoding an eIF(iso)4E protein variant or encoding a non-functional eIF4E protein or a nucleic acid incapable of expressing any eif4e gene.

[0423] The pair of PCR primers is preferably capable of detecting the presence of the eIF(iso)4E protein variant but not the presence of the corresponding wild-type eIF(iso)4E protein.

[0424] The expression of an eIF(iso)4E protein variant as defined above and / or the presence of a nucleic acid encoding an eIF(iso)4E protein variant as defined above in a corn plant cell indicates that the corn plant is resistant to at least one corn virus.

[0425] The present invention will be further illustrated in the following figures and examples.

[0426] A brief description of the sequence

[0427] SEQ ID NO: 1 corresponds to the sequence of native maize eIF4E1 protein.

[0428] SEQ ID NO:2 corresponds to the sequence of native maize eIF4E2 protein.

[0429] SEQ ID NO: 3 corresponds to the sequence of native maize eIFiso4E1 protein.

[0430] SEQ ID NO:4 corresponds to the sequence of the native maize eIFiso4E2 protein.

[0431] SEQ ID NO:5 corresponds to the sequence of a maize eIF4E1 protein variant comprising mutations A63D, A64D, V96D, G97R, D99G and G211L.

[0432] SEQ ID NO:6 corresponds to the sequence of a maize eIF4E2 protein variant comprising mutations A65D, A66D, G98D, G99R, D101G and G213L.

[0433] SEQ ID NO:7 corresponds to the sequence of a maize eIFiso4E1 protein variant comprising mutations A60D, A61D, N94R, W108L, S157K and R161E.

[0434] SEQ ID NO:8 corresponds to the sequence of a maize eIFiso4E2 protein variant comprising mutations A54D, A55D, T88R, W102L, S151K and R155E.

[0435] SEQ ID NO:9 corresponds to the genomic sequence of the maize eif4e1 gene.

[0436] SEQ ID NO: 10 corresponds to the genomic sequence of the maize eif4e2 gene.

[0437] SEQ ID NO: 11 corresponds to the genomic sequence of the maize apb1 gene.

[0438] SEQ ID NO: 12 corresponds to the sequence of native maize APB1 protein.

[0439] SEQ ID NO: 13 corresponds to the sequence of eIF4E1 protein allele 9_16.

[0440] SEQ ID NO: 14 corresponds to the sequence of the non-functional eIF4E1 protein obtained after knockout.

[0441] SEQ ID NO: 15 corresponds to the sequence of another non-functional eIF4E1 protein obtained after knockout.

[0442] SEQ ID NO: 16 corresponds to the sequence of the non-functional eIF4E2 protein obtained after knockout.

[0443] SEQ ID NO: 17 corresponds to the sequence of another non-functional eIF4E2 protein obtained after knockout.

[0444] SEQ ID NO: 18 corresponds to the sequence of another non-functional eIF4E2 protein obtained after knockout.

[0445] SEQ ID NO: 19 corresponds to the sequence of gRNA14 (ZmeIF4E_a_SpCas9_gRNA14) targeting a region in exon 1 of eIF4E1.

[0446] SEQ ID NO:20 corresponds to the sequence of gRNA47 (ZmeIF4E_b_SpCas9_gRNA47) targeting a region in exon 1 of eIF4E2.

[0447] SEQ ID NO:21 corresponds to the sequence of gRNA ZmeIF4E_a324&b330_SpCas9_gRNA targeting a region in exon 2 of eIF4E1 and eIF4E2.

[0448] SEQ ID NO:22 corresponds to the sequence of gRNA ZmeIF4E_a367&b373_SpCas9_gRNA targeting a region in exon 2 of eIF4E1 and eIF4E2.

[0449] SEQ ID NO:23 corresponds to the sequence of V40 NLS-SpCas9_Nucleoplasmin NLS, which is a Cas9 (CRISPR-associated protein 9) endonuclease.

[0450] SEQ ID NO:24 corresponds to the U6 promoter sequence from maize.

[0451] SEQ ID NO: 25 corresponds to the ubiquitin promoter and 5'UTR sequence from maize.

[0452] SEQ ID NOs: 26 to 47 correspond to the epegRNA designs in Table 5. BRIEF DESCRIPTION OF THE DRAWINGS

[0453] Figure 1 : Growth of yeast cells co-transformed with (i) pGBKT7-SCMV-VPg and (ii) pGADT7-eIF4E1, pGADT7-eIF4E2, pGADT7-eIFiso4E1 or pGADT7-eIFiso4E2. Prey vector pGADT7 (containing the activation domain (AD) of the eIF(iso)4E protein) and bait vector pGBKT7 (containing the binding domain (BD) of the VPg protein) were used on selective media.

[0454] Figure 2 : Growth of yeast cells co-transformed with (i) pGBKT7-MDMV-VPg and (ii) pGADT7-eIF4E1, pGADT7-eIF4E2, pGADT7-eIFiso4E1 or pGADT7-eIFiso4E2. Prey vector pGADT7 (containing the activation domain (AD) of the eIF(iso)4E protein) and bait vector pGBKT7 (containing the binding domain (BD) of the VPg protein) were used on selective media.

[0455] Figure 3: Growth of yeast cells co-transformed with (i) pGBKT7-SCMV-VPg and (ii) pGADT7-eIF4E1, pGADT7-eIF4E1_varA63D and pGADT7-eIF4E1_varA63D / A64D on selective medium (SD-LW: selective medium without leucine and tryptophan; SD-LWH: selective medium without leucine, tryptophan and histidine; SD-LWHA: selective medium without leucine, tryptophan, histidine and adenine).

[0456] Figure 4 : MDMV infection rates in plant J1A, plants expressing native eiF4E1 protein (Zm00001d041682_eIF4E1_native), and plants expressing variants of eiF4E1 protein (Zm00001d041682_eIF4E1_Var1, Zm00001d041682_eIF4E1_Var4, and Zm00001d041682_eIF4E1_Var5). Example

[0457] Example 1 :Identification of the eIF(iso)4E gene in maize and selection of candidate variants resistant to viruses

[0458] Identification of the maize eIF(iso)4E gene has been accomplished by blasting using eIF(iso)4E protein sequences from other species as bait. Two eIF4E and two eIFiso4E genes were identified on chromosome 3 in the maize genome of B73, and were named eIF4E1 and eIF4E2:

[0459] Zm00001d041682 as eIF4E1,

[0460] Zm00001d041973 as eIF4E2,

[0461] Zm00001d032775 as eIFiso4E2,

[0462] Zm00001d014065 as eIFiso4E1.

[0463] Example 2 :Yeast two-hybrid results for identification of VPg-eIF(iso)4E interaction

[0464] For Y2H (yeast two-hybrid) assay, activation domain (AD) vector pGADT7 and GAL4 DNA-binding domain (BD) vector pGBKT7 (Matchmaker Gold Yeast Two-Hybrid System (Clontech, Mountain View, CA, USA) were used. The prey vector pGADT7 (containing the activation domain (AD) of eIF(iso)4E protein) and the bait vector pGBKT7 (containing the binding domain (BD) of VPg protein) carrying the gene to be tested were co-transformed into yeast (Saccharomyces cerevisiae) strains Y187 & Y2HGold.

[0465] GAL4-based Y2H was used to identify the interaction between SCMV- / MDMV-VPg and two ZmeIF4E and two ZmeIFiso4E homologs. The bait vectors pGBKT7-SCMV-VPg, pGBKT7-SrMV-VPg and pGBKT7-SCSMV-VPg were co-transformed with the prey vectors pGADT7-eIF4E1, pGADT7-eIFiso4E2, pGADT7-eIFiso4E1, and pGADT7-eIFiso4E2 into Saccharomyces cerevisiae strains Y187&Y2HGold, respectively.

[0466] As a result, yeast cells co-transformed with pGBKT7-SCMV-VPg and pGADT7-eIF4E1 or pGADT7-eIF4E2 grew well, indicating that there was an interaction between SCMV-VPg and eIF4E protein, while yeast cells with pGADT7-eIFiso4E1 and pGADT7-eIFiso4E2 did not grow, indicating that there was no interaction between SCMV-VPg and eIFiso4E protein (see Figure 1 ).

[0467] For MDMV, pGBKT7-MDMV-VPg and pGADT7-eIF4E1 or pGADT7-eIF4E2 grew well, indicating that there was an interaction between MDMV and the eif4E gene, and the same result was obtained for pGADT7-eIFiso4E1. However, when pGADT7-eIFiso4E2 was used, no yeast cells grew, indicating that there was no interaction between MDMV-VPg and the eIFiso4E2 gene (see Figure 2 ).

[0468] Example 3 :Identification of eIF(iso)4E mutations that disrupt VPg-eIF(iso)4E interaction by yeast two-hybrid

[0469] Based on previous results, amino acid modifications were introduced into the eIF(iso)4E gene from maize to determine the effect of the mutations on the VPg-eIF(iso)4E gene interaction. The goal was to identify factors that disrupt the interaction between viral and host factors. As an example, A63D and A63D / A64D altered the VPg-eIF4E1 interaction (see Figure 3 ).

[0470] Therefore, Tables 3 and 4 below summarize all mutations tested (singly or in combination), and Tables 1 and 2 below indicate the biological origin of the mutations tested when relevant.

[0471] This set of data allowed us to identify mutations of interest that disrupt the interaction with MDMV and / or SCMV.

[0472]

[0473] Table 3 : Results of Y2H experiments on the interaction between (i) MDMV or SCMV and (ii) wild-type eIF4E1 variants

[0474]

[0475]

[0476] “+”: interaction between VPg and eIF4E; “-”: no interaction was detected.

[0477] Table 4 : Results of Y2H experiments on the interaction between (i) MDMV or SCMV and (ii) wild-type and eIF4E2 variants

[0478]

[0479]

[0480] “+”: interaction between VPg and eIF4E; “-”: no interaction was detected.

[0481] Based on these results, a set of mutations from several species have been identified in the eIF(iso)4E gene that disrupt the interaction between the two viruses, SCMV and MDMV. The list of validated mutations is:

[0482]

[0483] Example 4 : Genetically modified expression of eIF(iso)4E variants in the maize J1A genotype

[0484] Validation of the maize variant of interest was accomplished by transgenic expression of the ZmeIF4E variant in a susceptible background. The scientific rationale relies on the inability of the virus to infect plants expressing the eIF4E variant, due to the abundance of the eIF4E resistance allele that the virus cannot recruit, and the inability of the virus to access the product of the native susceptible allele due to competition with the resistant version. Expression of the eIF4E1 variant with overrepresentation of the resistance allele has been selected.

[0485] A panel of 7 different constructs were tested for eIF4E1:

[0486] Zm00001d041682_eIF4E1_Natural (Optimized) Zm00001d041682_eIF4E1_Var1 A63D / A64D Zm00001d041682_eIF4E1_Var2 D99G Zm00001d041682_eIF4E1_Var3 A63D / A64D / D99G Zm00001d041682_eIF4E1_Var4 V96D / G97R Zm00001d041682_eIF4E1_Var5 A63D / A64D / V96D / G97R Zm00001d041682_eIF4E1_Var6 A63D / A64D / V96D / G97R / D99G / G211L Zm00001d041682_eIF4E1_Var7 G211L

[0487] For each construct, 3 independent events were selected, and for each event, 5 plants were infected with MDMV. Plants were grown on a cultivation platform with a growth condition of 24°C during the day / 19°C at night and a photoperiod of 16 hours during the day / 8 hours at night. 13 days after sowing, fresh MDMV-infected leaves ground in phosphate buffer containing abrasives were used to mechanically infect the 2nd or 3rd leaf, and the plants were artificially infected. Scoring was performed 21 days after infection, with a score range of 0 (resistant) to 5 (susceptible).

[0488] Expression of variants carrying the mutation of interest identified by Y2H showed a significant reduction in MDMV infection rates compared to J1A and plants overexpressing the native allele (see e.g. Figure 4 ). These results demonstrate virus resistance of these variants of interest in maize.

[0489] Example 5 : Generation of eIF4E resistance variants resistant to SCMV & MDMV (Potyviridae) and MCMV (Tomato Bushy Viridae) in maize by a lead editing approach

[0490] Susceptible plants containing the eif4e gene were converted to resistant plants by genome editing. Amino acid modifications at the positions listed in the table below were achieved by the lead editing approach (Anzalone et al., 2019) using epegRNA (enhanced lead editing guide RNA) (Nelson et al., 2021). These epegRNAs have a 3'tevoprecQ1 pseudoknot sequence designed to stabilize the pegRNA (Nelson et al., 2021). The spacer, primer binding site (PBS) and reverse transcriptase template region (RTT) of the epegRNA (Table 5) were designed with the help of the PlantPegDesigner tool. The epegRNA combination expressed from the maize U6 promoter (SEQ ID NO: 24) alone was cloned together with the ZmUbiquitin promoter-PE2 lead editor (SEQ ID NO: 25) into a plant binary vector containing a rice actin promoter-:BAR selection marker cassette. The binary vector was transformed into an Agrobacterium strain and maize transformation of susceptible J1A varieties was performed essentially as described by Ishida et al., (2007). Edited plants with the desired nucleotide changes (alone or in combination) were identified by next generation sequencing of PCR amplicons covering the lead editing target site.

[0491]

[0492] Table 5: epegRNA design

[0493]

[0494]

[0495]

[0496] Example 6 :Phenotypic analysis of GE (gene-edited) plants after SCMV, MDMV and / or MCMV virus infection

[0497] GE (gene edited) plants were phenotyped in the greenhouse to evaluate resistance to 3 different viruses: SCMV & MDMV (Potavirus family) and MCMV (Tomato Bushy Viridae family), which mimicked maize lethal necrosis disease alone or by combining MCMV and Potatovirus Y. For each virus infection method (5 in total), plants were grown on separate culture tables to avoid unnecessary virus cross-contamination. Growth conditions were 24°C day / 19°C night with a photoperiod of 16 h day / 8 h night. Plants were artificially infected 3 days after sowing by mechanical infection of the 2nd or 3rd leaf using freshly infected leaves ground in phosphate buffer containing abrasives.

[0498] Example 7 : ELISA test to monitor the presence / absence of the virus

[0499] Leaf sampling completed at 15 DPI was tested with ELISA to monitor and quantify the viruses used for infection (SCMV, MDMV and MCMV). ELISA can measure the extent of virus accumulation in plants using anti-SCMV / MDMV / MCMV antibodies. Virus levels were compared to wild-type corn plants in GE / GM corn plants with eIF4E protein variants.

Claims

1. An eIF(iso)4E protein variant capable of conferring resistance to at least one corn virus, wherein the protein variant comprises at least one mutation compared to the corresponding native corn eIF(iso)4E.

2. The eIF(iso)4E protein variant according to claim 1, wherein the eIF(iso)4E protein variant is an eIF4E1 protein variant, an eIF4E2 protein variant, an eIFiso4E1 protein variant or an eIFiso4E2 protein variant.

3. The eIF(iso)4E protein variant according to claim 1 or 2, wherein the native corn eIF(iso)4E protein is the protein eIF4E1 of sequence SEQ ID NO: 1, the protein eIF4E2 of sequence SEQ ID NO: 2, the protein eIFiso4E1 of sequence SEQ ID NO: 3 or the protein eIFiso4E2 of sequence SEQ ID NO:

4.

4. The eIF(iso)4E protein variant according to any one of claims 1 to 3, wherein the corn virus is potato virus Y or maize chlorotic mottle virus.

5. The eIF(iso)4E protein variant according to any one of claims 1 to 4, wherein the at least one mutation disrupts the interaction between the protein variant and at least one virus selected from the group consisting of MCMV (maize chlorotic mottle virus), SCMV (sugarcane mosaic virus), MDMV (maize dwarf mosaic virus) and WSMV (wheat streak mosaic virus).

6. The eIF(iso)4E protein variant according to any one of claims 1 to 5, wherein the at least one mutation is selected from: - the group consisting of V96D, G97R, T7S, A19V, L33I, N55D / K, P56S / T, A63D, A64D, S67A, D99G, S159K, Q164K and G211L / D / S, with reference to the native zein sequence of SEQ ID NO: 1, - the group consisting of D101G, A65D, A66D, N57D / K, P58S / T, S69A, G98D, G99R, S161K, Q166K and G213L / D / S, with reference to the native zein sequence of SEQ ID NO: 2, - the group consisting of A60D, A61D, N94R, W108L, S157K and R161E / L, with reference to the native zein sequence of SEQ ID NO: 3, or - The group consisting of A54D, A55D, T88R, W102L, S151K and R155E / L, with reference to the native zein sequence of SEQ ID NO:

4.

7. The eIF(iso)4E protein variant according to any one of claims 1 to 6, wherein the protein variant comprises at least two mutations compared to the corresponding native maize eIF(iso)4E protein.

8. The eIF(iso)4E protein variant according to any one of claims 1 to 7, wherein the protein variant comprises: - the mutations (i) A63D and A64D, (ii) V96D and G97R, (iii) D99G and / or (iv) G211L / D / S, with reference to the native zein of sequence SEQ ID NO: 1, - the mutations (i) A65D and A66D, (ii) G98D and G99R, (iii) D101G and / or (iv) G213L / D / S, with reference to the native zein of SEQ ID NO: 2, - a native zein having the mutations (i) A60D and A61D, (ii) N94R, (iii) W108L and R161E / L and / or (iv) S157K, with reference to the sequence SEQ ID NO: 3, or - A native zein having the mutations (i) A54D, A55D, (ii) T88R, (iii) W102L and R155E / L and / or (iv) S151K, with reference to the sequence SEQ ID NO:

4.

9. A nucleic acid encoding at least one eIF(iso)4E protein variant according to any one of claims 1 to 8.

10. A vector comprising at least one nucleic acid encoding at least one eIF(iso)4E protein variant according to any one of claims 1 to 8.

11. A genetically modified or edited corn plant that is resistant to at least one corn virus, or a genetically modified or edited corn seed that is resistant to at least one corn virus, wherein the plant or seed comprises at least one eIF(iso)4E protein variant according to any one of claims 1 to 8, and wherein the plant is not obtained by means of a substantially biological process.

12. Progeny of the genetically modified or edited plant according to claim 11.

13. A method of obtaining a genetically modified or edited corn plant that is resistant to at least one corn virus, wherein the method comprises: - transforming a maize cell or maize tissue using at least one vector according to claim 10 to obtain a transformed maize cell or a transformed maize tissue, and - Regenerating a maize plant from said transformed maize cell or transformed maize tissue.

14. A method for obtaining an edited modified corn plant resistant to at least one corn virus, wherein the method comprises introducing at least one mutation into an endogenous corn gene encoding eIF(iso)4E protein to obtain a mutant gene encoding an eIF(iso)4E protein variant according to any one of claims 1-8.

15. Use of at least one eIF(iso)4E protein variant according to any one of claims 1 to 8 for preventing at least one viral disease of corn.

16. A method for identifying a corn plant that is resistant to at least one corn virus, wherein the method comprises detecting the expression of the eIF(iso)4E protein variant according to any one of claims 1 to 8 and / or the presence of a nucleic acid encoding the eIF(iso)4E protein variant according to any one of claims 1 to 8 in cells of the corn plant.

Images