PAPAYA WITH NOVEL TRAITS AND METHODS FOR PRODUCING SUCH PAPAYA PLANTS

Abstract

The present application relates to papaya plants having increased resistance to the papaya ringspot virus as compared to a wild type plant due to a mutation in the eukaryotic translation initiation factor 4e and/or eukaryotic translation initiation factor iso4e gene leading to non-functional eukaryotic translation initiation factor 4e and/or eukaryotic translation initiation factor iso4e proteins. Methods of producing such papaya plants having increased resistance to the papaya ringspot virus are described.

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Claims

1. An isolated mutated eukaryotic translation initiation factor 4e (eIF4e) and/or eukaryotic translation initiation factor iso4e gene (eIF(iso)4e) gene comprising a Guanine to Adenine substitution at position 2112 as compared to the wild type eIF4e and/or eIF(iso)4e gene.

2. The gene according to claim 1, wherein the substitution encodes a non-functional eIF4e and/or eIF(iso)4e protein.

3. The gene according to claim 1, wherein the mutated eIF4e gene comprises the nucleic acid set forth in SEQ ID NO:2.

4. The gene according to claim 1, wherein the mRNA sequence of the mutated eIF4e gene comprises the nucleic acid set forth in SEQ ID NO: 5.

5. The gene according to claim 1, wherein the mutated eIF4e gene encodes the amino acid set forth in SEQ ID NO:7.

6. A plant or plant part comprising the mutated eIFe4 gene according to claim 1.

7. The plant or plant part according to claim 6, wherein the plant part is selected from the group consisting of cell, tissue, calli, flowers, seeds, roots, and stems.

8. The plant or plant part according to claim 5, wherein the plant is papaya plant.

9. The plant or plant part according to claim 5, wherein the plant part is a seed.

10. A plant grown from the seed of claim 9.

11. The plant according to claim 9, wherein the plant comprises mutated eIF4e gene eIF(iso)4e gene comprising a Guanine to Adenine substitution at position 2112 as compared to the wild type eIF4e and/or eIF(iso)4e gene.

12. The plant according to claim 9, wherein the substitution encodes a non-functional eIF4e and/or eIF(iso)4e protein.

13. The plant according to claim 9, wherein the mutated eIF4e gene comprises the nucleic acid set forth in SEQ ID NO:2.

14. The plant according to claim 9, wherein the mRNA sequence of the mutated eIF4e gene comprises the nucleic acid set forth in SEQ ID NO:5.

15. The plant according to claim 9, wherein the mutated eIF4e gene encodes the amino acid set forth in SEQ ID NO:7.

16. A seed produced by the plant grown from the plant of claim 10.

17. The seed according to claim 16, wherein the seed comprises a mutated eIF4e gene eIF(iso)4e gene comprising a Guanine to Adenine substitution at position 2112 as compared to the wild type eIF4e and/or eIF(iso)4e gene.

18. The seed according to claim 17, wherein the mutated eIF4e gene comprises the nucleic acid set forth in SEQ ID NO:2.

19. The seed according to claim 17, wherein the mRNA sequence of the mutated eIF4e gene comprises the nucleic acid set forth in SEQ ID NO:5.

20. The seed according to claim 17, wherein the mutated eIF4e gene encodes the amino acid set forth in SEQ ID NO:7.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0022] FIG. 1 shows disease severity rating scale of papaya ringspot virus infection.

[0023] FIG. 2 shows Papaya M2(0.3)-1709 mutant seedlings demonstrating resistance to papaya ringspot virus along with infection ratings; S-check is the susceptible check i.e. a positive control.

BRIEF DESCRIPTION OF SEQUENCES

[0024] SEQ ID NO: 1 pertains to wild type eIF4e gene nucleotide sequence in papaya (Annotated from sequencing report and NCBI sequence: ABIM01000000, ABIM01005433.1, LG8 contig 5440);

[0025] SEQ ID NO: 2 pertains to mutated eIF4e nucleotide sequence in papaya (papaya mutant line number, M2(0.3)-1709 G2112A (UGG->UGA) W140*);

[0026] SEQ ID NO: 3 pertains to mutated eIF4e nucleotide sequence in papaya (papaya mutant line number M2(0.3)-1220 G2301A (GGA->GAA) G173E);

[0027] SEQ ID NO: 4 pertains to wild type eIF4e gene (mRNA) sequence in papaya (GenBank: FJ644949.1);

[0028] SEQ ID NO: 5 pertains to mutated eIF4e gene (mRNA) sequence in papaya (papaya mutant line number M2(0.3)-1709);

[0029] SEQ ID NO: 6 pertains to wild type eIF4e amino acid sequence in papaya (protein_id=ACN38307.1);

[0030] SEQ ID NO: 7 pertains to mutated eIF4e amino acid sequence in papaya (papaya mutant line number M2(0.3)-1709);

[0031] SEQ ID NOs: 8, 10, 12, 14, 16, and 18 pertain to papaya eIF4e forward primer sequences;

[0032] SEQ ID NOs: 9, 11, 13, 15, 17, and 19 pertain to papaya eIF4e reverse primer sequences;

[0033] SEQ ID NO: 20 pertains to eIF(iso)4e gene nucleotide sequence in wild type papaya (gb|ABIM01010894.1:25000-29500, Carica papaya chromosome LG6 contig_10909, whole genome shotgun sequence);

[0034] SEQ ID NO: 21 pertains to eIF(iso)4e mutant nucleotide sequence in papaya (papaya mutant line number, M2(0.3)-552 G2046A (GGG->GAG) G105E);

[0035] SEQ ID NO: 22 pertains to wild type eIF(iso)4e gene (mRNA) sequence in papaya (GenBank: FJ595992.1);

[0036] SEQ ID NO: 23 pertains to mutated eIF(iso)4e gene (mRNA) sequence in papaya (papaya mutant line number M2(0.3)-552);

[0037] SEQ ID NO: 24 pertains to wild type eIF(iso)4e amino acid sequence in papaya; SEQ ID NO: 25 pertains to mutated eIF(iso)4e amino acid sequence in papaya (papaya line number M2(0.3)-552);

[0038] SEQ ID NOs: 26- 37 pertain to papaya eIF(iso)4e forward primer sequences; and SEQ ID NOs: 38-49 pertain to papaya eIF(iso)4e reverse primer sequences.

DETAILED DESCRIPTION OF THE INVENTION

[0039] As used herein, the term plant includes the whole plant or any parts or derivatives thereof, such as plant organs (e.g., harvested or non-harvested fruits, flowers, leaves, stem, roots etc.), plant cells, plant protoplasts, plant cell or tissue cultures from which whole plants can be regenerated, regenerable or non-regenerable plant cells, plant calli, plant cell clumps, and plant cells that are intact in plants, or parts of plants, such as embryos, pollen, ovules, ovaries, fruits flowers, leaves, seeds, tubers, clonally propagated plants, roots, stems, cotyledons, hypocotyls, root tips and the like. Also, any developmental stage is included, such as seedlings, immature and mature, etc.

[0040] An M1 population is a plurality of mutagenized seeds/plants of a certain plant line or cultivar. M2, M3, M4, etc. refers to the consecutive generations obtained following selfing of a first mutagenized seed/plant (M1).

[0041] The term selfing means self-pollination, e.g., when the pollen self-pollinates the ovule of the same plant.

[0042] A mutation in a nucleic acid molecule coding for a protein is a change of one or more nucleotides compared to the wild type sequence, e.g. by replacement, deletion or insertion of one or more nucleotides. A point mutation is the replacement of a single nucleotide, or the insertion or deletion of a single nucleotide.

[0043] As used herein, disease resistance refers to the acquired (i.e. inherited) ability of a plant to survive (e.g. grow and optionally reproduce) and/or restrict the multiplication of the pathogen following exposure to a pathogen that can cause disease in wild type plants. In other words, the plant has phenotypically changed compared to the wild type plant such that it is less affected and lacks disease symptoms. In a plant, disease resistance may be naturally occurring or induced by such techniques as genetic engineering or selection of variants produced by tissue culture or mutagenesis. A disease resistant plant is therefore a plant that is resistant to disease at a level that would normally kill, or inhibit the normal growth of, a wild type plant of the same species. An increased resistant plant may be defined as a plant which when compared to wild type plant has an increased ability to survive (e.g. grow and optionally reproduce, lack disease symptoms) following exposure to a disease that would normally affect the wild type plant.

[0044] A loss-of-function protein refers to a mutant eIF4e or eIF(iso)4e protein that may be a truncated protein that will not encode a functional eukaryotic RNA translation initiation factor. Non-functional and loss-of-function may be used interchangeably.

[0045] The present invention may provide methods to generate papaya plants that have increased resistance as compared to the wild type papaya plant to papaya ringspot virus due to a mutation in at least one of its eIF4e and/or eIF(iso)4e genes, leading to non-functional proteins without the inclusion of foreign nucleic acids in the papaya genomes

[0046] The present invention may also provide a series of independent mutations in the eIF4e and/or eIF(iso)4e gene; papaya plants having these mutations in at least one of its eIF4e and/or eIF(iso)4egenes; and a method of creating and identifying similar and/or additional mutations in the eIF4e and/or eIF(iso)4e gene of papaya.

[0047] The inventors in the present invention used a method called TILLING (Targeting Induced Local Lesions in Genomes) (McCallum et al., 2000; Till et al. 2003, Triques et al., 2007). TILLING, relies on random mutations in the genome caused by treatment of the seeds using chemical or physical mutagens which causes single nucleotide polymorphism mutations in the target genome. DNA or RNA from samples from the resulting mutagenized plants are pooled and subjected to screening for mutations in the gene of interest. Once a mutation is identified in the gene of interest, the seeds of the plant carrying that mutation are grown and genotyped for heterozygous and homozygous mutants using the gene marker. M2 plants from a selected family may then be subjected to artificial inoculation with the virus and ELISA test is performed to check for young plant resistance and also grown in hotspots (a place where the incidence of target disease infection is very high) to check for adult plant resistance. The M2 plants are further grown into adults and M3 seeds are harvested. The M3 plants are screened again in disease hotspots and screened for the disease resistance phenotypic characteristics associated with the gene of interest.

[0048] The single homozygous mutants in the eIF4e or eIF(iso)4e gene exhibiting a disease resistant phenotype may be combined with another homozygous eIF4e or eIF(iso)4e mutant line to generate a PRSV resistant hybrid papaya variety. The single gene eIF4e mutants may also be crossed with mutants in the eIF(iso)4e gene to generate a hybrid carrying resistance derived from combination of single mutants in eIF4e and eIF(iso)4e by stacking leading to resistance for a longer period (durable resistance).

[0049] Using the process of TILLING, the inventors in the present invention have induced independent mutations in the eIF4e and/or eIF(iso)4e genes in papaya, causing single nucleotide polymorphisms that resulted in non-functional proteins thereby leading to increased resistance to the papaya ringspot virus when compared to wild type papaya plant. The eIF4e gene encodes a eukaryotic RNA translation initiation factor. It is known that an isoform of eukaryotic translation initiation factor eIF4e from Arabidopsis thaliana interacts with the viral protein VPg of the Turnip Mosaic Virus (TuMV). Several natural resistance genes against potyviruses, from distinct crops, were shown to encode defective forms of eIF4e and eIF(iso)4e, for example, pvr2 for pepper resistance against Potato Virus Y (PVY), Tobacco Etch Virus (TEV), mol for resistance in lettuce to Lettuce Mosaic Virus (LMV), sbm1 for resistance in pea to Pea Seed-Borne Mosaic Virus (PSbMV) and pot-1 for resistance in tomato to Potato Virus Y (PVY) and Tobacco Etch Virus (TEV) (Caranta et al, 1996). Although these genes control diverse resistance phenotypes, resistance in all cases results from a small number of amino acid changes in the proteins encoded by the recessive resistance alleles that harbour point mutations. Melon Necrotic Spot Virus (MNSV) resistant melon plants have been generated by TILLING of the eIF4e gene in melon (Nieto et al., 2007). As PRSV is a potyvirus it is hypothesised that TILLING orthologs of Papaya eIF4e and eIF(iso)4e genes has a good potential of generating PRSV resistant lines. US919677 B2 (CARANTA et al. 2004) demonstrated that there exists a co-relation between the eIF4e and VPg gene of the potyvirus and mutations in eIF4e results in resistance in Solanaceae, Cucurbitaceae, Cruciferae and Compositae plants. However, no prior art exists that demonstrates the use of this method for inducing potyvirus resistance in papaya.

[0050] Thus, an aspect of the present invention may provide, a papaya plant with increased disease resistance to papaya ring spot virus as compared to wild type papaya plant due to mutation(s) in at least one of its eIF4e and/or eIF(iso)4e genes.

[0051] Based on the type of mutation(s), the increase in disease resistance may be 60% as compared to wild type, the increase in disease resistance may be 70% as compared to wild type, the increase in disease resistance may be 80% as compared to wild type, the increase in disease resistance may be 90% as compared to wild type, the increase in disease resistance may be 100% as compared to wild type, the increase in disease resistance may range from 60%-100% as compared to wild type, the increase in disease resistance may range from 60%-90% as compared to wild type, the increase in disease resistance may range from 70%-100% as compared to wild type, the increase in disease resistance may range from 80%-100% as compared to wild type, the increase in disease resistance may range from 90%-100% as compared to wild type, the increase in disease resistance may range from 70%-90% as compared to wild type, the increase in disease resistance may range from 70%-80% as compared to wild type, the increase in disease resistance may range from 80%-90% as compared to wild type.

[0052] In an embodiment, the papaya plant may comprise plant organs (e.g., harvested or non-harvested fruits, flowers, leaves, etc.), plant cells, plant protoplasts, plant cell or tissue cultures from which whole plants can be regenerated, regenerable or non-regenerable plant cells, plant calli, plant cell clumps, and plant cells that are intact in plants, or parts of plants, such as embryos, pollen, ovules, ovaries, fruits, (e.g., harvested tissues or organs, such as harvested papaya or parts thereof), flowers, leaves, seeds, tubers, clonally propagated plants, roots, stems, cotyledons, hypocotyls, root tips and the like. Also, any developmental stage is included, such as seedlings, immature and mature, etc.

[0053] In an embodiment, the mutation is induced using the mutagens that may be chemical or physical mutagens.

[0054] In an embodiment, the chemical mutagen used may be selected from but not limited to ethyl methanesulfonate (EMS), methylmethane sulfonate (MMS), N-ethyl-N-nitrosurea (ENU), triethylmelamine (TEM), N-methyl-N-nitrosourea (MNU), procarbazine, chlorambucil, cyclophosphamide, diethyl sulfate, acrylamide monomer, melphalan, nitrogen mustard, vincristine, dimethylnitosamine, N-methyl-N-nitro-Nitrosoguanidine (MNNG), nitrosoguanidine, 2-aminopurine, 7,12 dimethyl-benz(a)anthracene (DMBA), ethylene oxide, hexamethylphosphoramide, bisulfan, diepoxyalkanes (diepoxyoctane (DEO), diepoxybutane (BEB), and the like), 2-methoxy-6-chloro-9 [3(ethyl -2-chloro-ethyl)aminopropylamino] acridine dihydrochloride (ICR- 1 70), and formaldehyde.

[0055] In an embodiment, the mutation is induced using physical mutagens such as fast neutrons or electromagnetic radiation such as X-rays or gamma rays.

[0056] In an embodiment, methods by which mutations like deletion and insertions may be induced may include but are not limited to new breeding techniques (NBTs) that include targeted gene editing using, TALEN, Zinc Finger Nucleases (ZFN), Oligonucleotide-directed mutagenesis (ODM) and CRISPR-Cas9. Gene editing techniques may be used to induce targeted mutations, insertions or deletions in the papaya genes eIF4e and/or eIF(iso)4e and other members of this gene family which are involved in the cascade of molecular mechanisms such as host protein-viral protein interaction which is required for PRSV genes replication within papaya cells leading to PRSV resistance in papaya.

[0057] Thus, in this embodiment, papaya seeds from various strains may be cleaned and treated using 1M KNO3 solution for improving the germination as per methods known in the art. After which the seeds may be washed and treated with a mutagen (M1 seed) such that the mutagen selected causes point mutations. The M1 seeds are then grown to maturity and M2 seeds are harvested individually from each plant. The M2 seeds are then sown and the plants may then be prepared for DNA extraction using any suitable method of plant DNA preparation to prepare the papaya plant DNA for eIF4e and/or eIF(iso)4e mutation screening such as using commercial DNA extraction kits following manufacturer's protocol, for example, QiagenDneasy plant mini kit or similar known DNA kits. Papaya M2 DNA samples may then be pooled to expedite screening for mutations in eIF4e and/or eIF(iso)4e gene from the entire population of M2 plant families originating from the mutagenized plant tissue. The size of the pooled group may depend on the sensitivity of the screening method used. In accordance with one aspect of an exemplary embodiment of the invention, groups of four or more individual papaya plants per M2 family are pooled.

[0058] In an embodiment, after the DNA samples are pooled, the pools are subjected to eIF4e and/or eIF(iso)4e sequence-specific amplification techniques, such as Polymerase Chain Reaction (PCR). Any primer specific to the eIF4e and/or eIF(iso)4e locus or the sequences immediately adjacent to the eIF4e and/or eIF(iso)4e locus may be utilized to amplify the eIF4e and/or eIF(iso)4e sequences within the pooled DNA sample. Preferably, the primer is designed to amplify the regions of the eIF4e and/or eIF(iso)4e locus where useful mutations are most likely to arise. Most preferably, the primer is designed to detect mutations in the coding region of the eIF4e and/or eIF(iso)4e gene. Preferably, the primer used may be such that it avoids known polymorphic sites to ease screening for point mutations. The present invention may therefore provide in an embodiment, suitable primers for this purpose (SEQ ID NOs: 8-19 and 26-49).

[0059] In an embodiment, DNA and/or RNA from the mutagenized papaya plant or subsequent generations may be sequenced by various sequencing methods such as using the Illumina/PacBio/BioNano/Nanopore or other next generation sequencing (NGS) platforms.

[0060] To facilitate detection of PCR products on a gel, the PCR primer may be labeled using Infra-Red dye (IRdye) or any conventional labeling method. In an alternative embodiment, other methods of amplification and screening may be used such as denaturing high pressure liquid chromatography (dHPLC), constant denaturant capillary electrophoresis (CDCE), temperature gradient capillary electrophoresis (TGCE) (see Li et al., Electrophoresis 23(10):1499-1511, 2002), or by fragmentation using enzymatic cleavage, such as used in the high throughput method described by Colbert et al. (2001), Triques et al. (2007), and in combination with next generation sequencing platforms as described above (Kumar et al., 2017). In an alternate embodiment, Targeted Genotyping by sequencing (GBS) may be used to detect the mutation in the eIF4e and/or eIF(iso)4e gene

[0061] The PCR amplification products may be incubated with an endonuclease that preferentially cleaves mismatches in heteroduplexes between wild type and mutant sequences which recognize and cleave at mismatches in a heteroduplex. Suitable endonucleases may include but are not limited to resolvases, RNases, bacteriophage T4 endonuclease VII, bacteriophage T7, endonuclease I, Saccharomyces cerevisiae endonuclease XI, Saccharomyces cerevisiae endonuclease X2, Saccharomyces cerevisiae endonuclease X3, SI nuclease, CEL I, PI nuclease, ENDO I nuclease or mung bean nuclease. In an embodiment, cleavage products are electrophoresed using an automated sequencing gel apparatus such as Licor (LICOR Inc.), and gel images are analyzed with the aid of a standard commercial image-processing program as per manufacturer's instructions.

[0062] The present inventors have determined that to achieve increased resistance in papaya as compared to wild type plants, mutations that alter and completely abolish eIF4e and/or eIF(iso)4e gene function are desirable. Preferred mutations include missense, nonsense and splice junction changes, including mutations that prematurely truncate the translation of the eIF4eand/or eIF(iso)4e protein from messenger RNA or make non-functional protein, such as those mutations that create a stop codon or codon change within the coding regions or splice junctions of the eIF4e and/or eIF(iso)4e gene. Such mutations include insertions, repeat sequences, modified open reading frames (ORFs), deletions, intron-exon splice junction point mutations and, most preferably, single nucleotide polymorphisms that lead to a loss of functional protein.

[0063] Thus, in one embodiment the present invention relates to a nucleic acid sequence (or gene) encoding a protein whose amino acid sequence is represented herein or encoding a protein derived from this (eIF4e and/or eIF(iso)4e) protein by substitution, deletion or addition of one or several amino acids, such as 2, 3, 4, 5, 6, 7, 8, 9 amino acids, or more amino acids, such as 10 or more than 10 amino acids in the amino acid sequence of the parent protein and losing or partially losing the activity of the parent protein.

[0064] In an embodiment once an M2 plant having a mutated eIF4e and/or eIF(iso)4e sequence is identified, the mutations may be analyzed to determine its effect on the expression, translation, and/or activity of the protein. Thus, in an embodiment, the PCR fragment containing the mutation may be sequenced, using standard sequencing techniques, to determine the exact location of the mutation in relation to the overall eIF4e and/or eIF(iso)4e sequence. Each mutation is evaluated to predict its impact on protein function (i.e., completely tolerated to loss-of-function), molecular changes in bonding pattern of altered amino acid and altered nucleotide interaction in the new gene using in-silico bioinformatics tools such as SIFT, PARSESNP, CODDLE, CLUSTAL-W, BLASTP, BLASTN, PDB or other tools known in the art.

[0065] In an embodiment, if the initial assessment of a mutation in an M2 plant indicates it to be of a desirable nature and in a useful position within the eIF4e and/or eIF(iso)4e gene, then phenotypic analysis of the selected papaya plant containing that mutation maybe pursued using artificial inoculation with the PRSV strain and ELISA tests as well as growing the mutant plants in a disease hot spots where the incidence of PRSV infection is very high. Various methods may be used to obtain the line of papayas according to the present invention, first the selected M2 plant is backcrossed to the original parent at least twice or more using marker assisted selection (MAS) to create a backcross 1 (BC1) progeny plant to eliminate background mutations. The backcrossed BC1 plant may be self-pollinated to create a BC1F2 plant that maybe homozygous for the eIF4e and/or eIF(iso)4e mutation. Several phenotypic characteristics of these homozygous eIF4e and eIF(iso)4e mutant plants may be assessed to determine if the mutation results in a useful and desirable genotypic and phenotypic changes in the papaya plant.

[0066] In an aspect, the present invention may provide novel mutations in the eIF4e and/or eIF(iso)4e gene of the papaya genome, such mutations may be created and identified according to various embodiments of the present invention. There is no previously reported mutation in the eIF4e gene (SEQ ID NO: 1,) and protein in papaya (SEQ ID NO: 6) beginning at nucleotide position 2112 (G2112A) (SEQ ID NO: 2) where a single nucleotide polymorphism (SNP) conversion of Guanine (G) to Adenine (A) in the eIF4e gene results in the formation of stop codon from Tryptophan (W) at the amino acid position of 140 leading to complete loss of protein and function (SEQ ID NO: 7). The STOP mutation (G2112A, W140*) in papaya eIF4e gene shows the stop of further translation beyond the protein sequence from amino acid position at 140. The mutation of the present invention results in a defective eIF4e gene leading to loss of complete transcription of the gene and translation into functional protein, thereby leading to the non-availability of the eIF4e translational initiation factor protein that is required for the multiplication of papaya ring spot viral RNA thereby leading to lack of PRSV symptoms and hence resistance. It has been shown that for successful PRSV infection, the cap protein of the PRSV physically interacts with the eIF4e and/or eIF(iso)4e protein for multiplication (Caranta et al., 1996, Piron et al., 2010).

[0067] In an embodiment, the present invention may also provide other mutations in eIF4e, which also leads to similar types of resistance towards PRSV in papaya as shown in Table 1.

[0068] Thus, an aspect of the present invention may provide a papaya plant, having a mutation in the eIF4e gene at the nucleotide position 2112 resulting in a stop codon on the 140-amino acid position of the SEQ ID NO: 7, resulting in a non-functional protein leading to increased resistance to the papaya ringspot virus, when compared to a wild type papaya plant.

[0069] As will be demonstrated in the examples, several physical characteristics of these homozygous eIF4e and/or eIF(iso)4e mutant plants may be assessed to determine if the mutation results in increased resistance to the papaya ringspot virus in the papaya plants. Mutant papaya plants are evaluated for degree of resistance as compared to normal (e. g., wild type) papaya plants. Such evaluation may include but not be limited to disease resistance screening bio-assays as well as phenotypic evaluations.

[0070] The PRSV screening technique was utilized to observe the resistance of the mutants towards the PRSV infection. The mutant seeds identified by TILLING and confirmed by sequencing were soaked in water and incubated in damped cloth for three days. Further the mutant seeds are sown in papaya media (coir and sand in 3:1 ratio). PRSV strains for inoculation were prepared by collecting the PRSV symptomatic papaya leaves, which are homogenized by crushing the collected leaves in mortar and pestle with inoculation buffer. The homogenized PRSV inoculum was subsequently inoculated on the leaves of wild type and mutant plants and observations were taken after 7, 14 and 21 days after inoculation (DAI). Various rating of infection was observed and severity was denoted by rating 0 for no infection and rating 4 for severe leaf mottling and stunting of plants. The level of resistance was determined based on the difference in rating of severity of virus infection between mutant plants and wild type plants. The value of the viral load was calculated using ELISA test and co-related to percentage (%) increase in mutation based on the severity rating. Thus a 0 rating may correlate to 100% increase in resistance as compared to wild type papaya plant.

[0071] Thus, in an embodiment, the increase in disease resistance in the papaya mutant of the present invention may range from 70%-100% in some cases, based on the type of mutation.

[0072] Thus, in an embodiment, the present invention may provide a papaya plant, having a mutation in the eIF4e and/or eIF(iso)4e gene such that the papaya has an increased resistance of up to 70%-100% as compared to a wild type papaya plant.

[0073] In an aspect of the present invention may provide eIF4e gene from papaya comprising a G2112A mutation; wherein the G2112A mutation comprises a nucleotide change within the eIF4e gene; and wherein the nucleotide change is identified according to SEQ ID NO: 2.

[0074] In an aspect of the present invention may provide novel mutations in the eIF(iso)4e gene, such mutations may be created and identified according to various embodiments of the present invention. There are no previously reported mutation in the eIF(iso)4e gene (SEQ ID NO: 20) and protein (SEQ ID NO: 24) in papaya beginning at nucleotide position 2046 (G2046A) (SEQ ID NO: 21) where a single nucleotide polymorphism (SNP) conversion of Guanine (G) to Adenine (A) in the eIF(iso)4e gene results in the formation of missense codon from Glycine (G) at the amino acid position of 105 to E leading to non-functional protein (G105E) (SEQ ID NO: 25). The non-tolerated missense mutation (G2046A) in papaya eIF(iso)4e gene shows the formation of non-functional protein due to change in protein sequence at the amino acid position 105 (G105E). The mutation of the present invention results in defective eIF(iso)4e gene leading to expression of the gene with a non-functional protein, leading to the non-availability of the eIF(iso)4e translational initiation factor protein for the multiplication of papaya ring spot viral RNA thereby leading to lack of PRSV symptoms and hence resistance. In an embodiment, the present invention may present other mutations in eIF(iso)4e which also leads to non-functional proteins leading to similar types of resistance towards PRSV in papaya.

[0075] In an alternate embodiment, the present invention may provide a papaya plant having increased disease resistance to the papaya ringspot virus as compared to a wild type papaya plant, comprising integrated in its genome (a) the nucleotide sequence set forth in SEQ ID NO: 2; (b) the nucleotide sequence set forth in SEQ ID NO:3, (c) a nucleotide sequence that encodes the polypeptide set forth in SEQ ID NO: 7; (d) a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 7, wherein the polypeptide comprises at least one mutation described herein; (e) a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to the nucleotide sequence set forth in SEQ ID NO: 2 and encodes a polypeptide for translation initiation factor eIF4e as described herein; (f) a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to the nucleotide sequence set forth in SEQ ID NO: 3 and encodes a polypeptide for translation initiation factor eIF4e as described herein; or (g) a nucleotide sequence fully complementary to any one of (a) to (f).

[0076] In an alternate embodiment, the present invention may provide a papaya plant having increased disease resistance to the papaya ringspot virus infection as compared to a wild type papaya, comprising integrated in its genome (a) the nucleotide sequence set forth in SEQ ID NO: 21, (b) a nucleotide sequence that encodes the polypeptide set forth in SEQ ID NO: 25; (c) a nucleotide sequence that encodes a polypeptide having at least 50%, 55%, 60, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity to amino acid sequence SEQ ID NO: 25, wherein the polypeptide comprises at least one mutation described herein; (d) a nucleotide sequence having at least 50%, 55%, 60,65%, 70%, 75%, 80%, 85%, 90%, or 95% identity to the nucleotide sequence set forth in SEQ ID NO: 21 and encodes a polypeptide for translation initiation factor eIF(iso)4e as described herein; (e) a nucleotide sequence fully complementary to any one of (a) to (d).

[0077] In an aspect, the present invention may provide a papaya plant having increased resistance as compared to a wild type papaya plant to the papaya ringspot virus, comprising a mutation in the eIF4e gene, such that it introduced a stop codon in the corresponding nucleotide sequence such that it results in a non-functional polypeptide.

[0078] In an aspect, the present invention may provide a papaya plant having increased resistance as compared to a wild type papaya plant to the papaya ringspot virus, comprising a mutation in the eIF(iso)4e gene such that it results in a non-functional polypeptide.

[0079] Thus, an aspect of the present invention may provide methods of producing a papaya plant with increased resistance to papaya ringspot virus created by the steps comprising: [0080] a. treating papaya seeds with a mutagen and growing such plants; [0081] b. extracting DNA and/or RNA and analyzing progeny papaya plants to detect at least one mutation in at least one copy of a eIF4e and/or eIF(iso)4e gene/RNA transcript leading to non-functional eIF4e and/or eIF(iso)4e proteins; [0082] c. selecting progeny papaya plants carrying the mutation and that have increased resistance to papaya ringspot virus phenotypically; [0083] d. selecting directly for increased disease resistance by growing the progeny papaya plants in the presence of the disease. [0084] e. repeating the cycle of growing and culturing the progeny papaya plants to produce additional plants with increased resistance to papaya ringspot virus; and [0085] f. combining two lines both homozygous for the mutation in eIF4e gene present in the two parents to generate a hybrid; or combining the homozygous mutants in eIF(iso)4e gene present in the two parents to generate a hybrid; or combining the homozygous mutants in eIF4e and/or eIF(iso)4e genes present in two different parent plants to generate a hybrid by gene stacking.

[0086] Another aspect of the present invention may provide food and food products incorporating papaya fruits with increased resistance to the papaya ringspot virus caused by a mutation in the eIF4e and/or eIF(iso)4e gene leading to non-functional eIF4e and/or eIF(iso)4e proteins.

[0087] The present invention provides several advantages over the prior art. The process of TILLING results in the generation of stable mutant papaya plants that demonstrate an increased disease resistance to the papaya ringspot virus through novel mutations in the papaya genome. The method used in the present invention is both novel and inventive and cost efficient in comparison to other methods such as the transgenic methods and crossing with wild species with great difficulty described in the prior art. The papaya plants of the present invention results in the decrease in the use of agrochemicals for the control of the papaya ringspot virus vectors. The present invention results in a non-transgenic plant which is more acceptable by the consumers worldwide. These and other advantages will be further demonstrated in the examples below. The following examples are offered by way of illustration only, and not limitation thereof. It is to be understood that the mutations below are merely exemplary and that similar mutations leading to non-functional protein are also contemplated.

EXAMPLES

Example 1

Mutagenesis and DNA Extraction

[0088] Papaya seeds (10,000 seeds, called MO) of the breeding line (BVC#13243-2398) were washed with water and treated with 1M KNO3 and then placed on a shaker (100 rpm) for overnight incubation. The mutagen ethyl methanesulfonate (EMS) was added under a fume hood to the imbibing seeds to final concentrations ranging from about 0.1% to about 1.6% (v/v). The treated seeds (called M1) were then incubated for a period of 6 to 12 hours, and the seeds were then rinsed with running water for approximately one hour. The mutagenized seeds were planted in potted trays and allowed to germinate in the nursery. Plants that were four to six weeks old were transferred to the field to grow to fully mature M1 plants. The flowers on the mature M1 plants were hermaphrodite and self-pollinated and then M2 seeds were collected from individual plants.

[0089] DNA extractions of M2 were performed using Qiagen plant DNA extraction kit following manufacturers instruction. Ten seeds per M2 families were sown in nursery pots inside a greenhouse and leaves were collected from four leaf stage M2 plants. The leaf samples were collected in 96-well collection plates with two 4 mm steel beads per well. The samples were frozen in liquid nitrogen and stored at 80 C. prior to DNA extraction. The protocol used for DNA extraction was from DNeasy 96-plant kit (Qiagen-Hilden, Germany). The samples were ground to powder with the help of the custom made vibrator shaker (Abraham et al., 2009). The extracted genomic DNA was run on 0.8% w/v agarose gel (Invitrogen) to check the quality. The quantity of M2 DNA was quantified with Nanodrop (Implen). The M2 DNA was normalized to 50 ng/l and 8-fold pooling plates were made for TILLING.

Example 2

Mutation Detection

[0090] Nested PCR was carried out by using target gene specific primers on 5ng of papaya M2 genomic DNA. The first N1 PCR amplified product (1 l) was used by diluting 1:10, which served as a template for the nested PCR, using 5end infra-red dyes (IRD) labeled combinations of IRD700 and IRD800 M13 universal primers along with unlabeled N2 primers with M13 sequence and the section of N1 primer sequence

Example 3

Bioinformatic Analysis

[0091] CODDLE (Codons Optimized to Discover Deleterious Lesions,) was used to ascertain regions of the target gene in which G/C to A/T transitions, which are most likely to result in deleterious effects on the protein, had occurred. PARSESNP (Project Aligned Related Sequences and Evaluate SNPs,) was used to demonstrate the distribution of mutations within the target gene, and to indicate the nature of each single mutation. To predict the impact of the mutation on the target protein sequence, SIFT (Sorting Intolerant from Tolerant,) analysis and PPOPEN software analysis was done. ClustalW was utilized for Multiple Sequence Alignment of the gene of interest. Primers were designed according to the M13 TILLING system for each gene (Triques et al., 2007). TILLING primers for eIF4e (SEQ ID NOs: 8-19) and eIF(iso)4e (SEQ ID NOs: 26-49) were designed by Primer 3 software and further TILLING were carried out.

Example 4

Identification and Evaluation Of Mutations (Tilling Screening)

[0092] M13 TILLING methodology from INRA-URGV (Triques et al., 2008; Triques et al., 2007), France was utilized to screen the Papaya mutant population. The workflow for M13 TILLING starts by designing sequence specific primers for the gene which it is intended for mutation screening. The PCR condition for the sequence specific reaction is an initial denaturation of 94 C. for 5 min, 35 cycles of denaturation at 94 C. for 10 sec, annealing at 55 C. for 15 sec, and extension at 72 C. for lmin 30 sec, and a final extension of 72 C. for 5 min and cool down to 4 C. The Infrared labeled M13 universal primers were multiplexed by using the PCR product of the gene specific reaction as template. The PCR condition for the nested labeled reaction were as follows, initial denaturation of 94 C. for 5 min and 10 cycles of denaturation at 94 C. for 15 sec, annealing at 60 C. for 30 sec, and extension at 72 C. for 1 min 30 sec (specific for the gene sequence), and 25 cycles of 94 C. for 15 sec, 50 C. for 30 sec and extension at 72 C. for 1 min 30 sec (specific for M13 universal primers) and a final extension of 72 C. for 5 min and cool down to 4 C. The labeled samples were then loaded into agarose gel for observing and obtaining an idea of how much PCR product should be taken for ENDO-I digestion. The samples were then passed through a RAMP reaction in which the samples were denatured at 94 C. and gradually cooled to 8 C. at the rate of 0.1 C. per second for enhanced formation of heteroduplex. The PCR product was then used for ENDO-I digestion at 45 C. for 20 minutes and 5 l of 0.15 M EDTA (pH-8.0) was added to stop the reaction. The samples were then passed through the sephadex (GE, G-50, medium) for purification of the DNA samples. The sephadex were prepared by mixing equal quantities into the wells of Millipore filtration plate 94 well (Multiscreen-HV, MAHVN4550) with the help of sephadex column loader 45 l. After transferring the sephadex onto filtration plate, about 325 l of sterile distilled water was added to make the sephadex to swell which requires an incubation of at least one hour. Excess water is centrifuged out by spinning at 500 G for 2 min. Sample plate is fit onto the filtration plate with 5 formamide loading dye. After stopping the ENDO-I digestion the samples are transferred into the Sephadex G-50 column and then centrifuged again to obtain the purified sample onto the formamide plates. The filtrate is further dried to 5 l at 65 C. in a vacuum centrifuge concentrator for about an hour. Furthermore, the samples are denatured just before loading onto the Genetic Analyzer (Licor, 4300).

Acrylamide Gels on Licor 4300 System

[0093] TILLING gel plates were cleaned thoroughly and the gel plates and rails were paired respectively by taking the front and back plates placed on a flat surface. The plates were sandwiched with 0.25 mm spacers. 17.5 ml of KB.sup.+LICOR 6.5% gel matrix was taken and 196 1 of 10% Ammonium persulfate and 19.6 1 added and mixed gently. The mix was taken up in a 20 ml syringe and the gel was squeezed into the back plate by gentle tapping on the front plate. The plastic comb (0.25 mm) was inserted in between the plates and left to polymerize for about 90 min. After the polymerization the casting combs were removed and the glass plates were cleaned thoroughly with Milli-Q water to avoid the persistence of acrylamide gel on the outer surface. The cleaned plates were placed on to the Licor machine and the upper and lower tank was filled with 1 Tris Borate EDTA (TBE) (Licor) buffer. The notch was cleaned with a syringe and needle pre-run was started which goes on for 20 min at the following condition i.e. 1500V, 40 W, 40 mA and 45 C. The denatured samples along with size-markers were denatured at 93 C. for 3 min and both were placed on ice. Using an 8-channel pipette 1.0 l of the samples were loaded on to the cold sample-loading tray, which should be kept on ice while loading. The samples were absorbed by inserting the membrane comb in the tray for several seconds until complete absorption the comb was left to remain for 2 min at room temperature for drying. The notch was cleaned up with 1 TBE buffer and the comb inserted rapidly but gently at 45-degree angle with sample one on the left. The system was closed with the tank lids and wire. The run was started with the same conditions as the one done for pre-run for initially for four minutes and then the combs removed and the notch cleaned up with 1 TBE and the run restarted. TILLING images were detected by red and green spots for IRD700 and IRD800 respectively, which are represented by black and white picture for each fluorescent image using software provided with Licor 4300 TILLING was done on eIF4e and eIF(iso)4e gene on all the seven super pools (SP). And further deconvolution was done on probable mutants. The TILLING mutants were confirmed by sequencing and positions of mutations were confirmed. Genotyping by TILLING and sequencing was done to find whether the M2 seeds are Homozygous/Heterozygous/ Wild type for the obtained mutations.

[0094] Table 1 below lists various mutations obtained after screening using TILLING method in eIF4e gene.

TABLE-US-00001 TABLE 1 Effect of Mutation Mutant Line Codon Amino acid SIFT SIFT No. No. Code Nature Exon Change Change Analysis Score 1 M2(0.3)-1709 2398 (A) G2112A Exon UGG W140* NA NA UGA 2 M2(0.3)-1220 2654 (A) G2301A Exon GGA G173E Not 0 GAA Tolerated 3 M2(0.4)-578 882 (B) C2539T Intronic NA NA NA NA 4 M2(0.4)-713 1114 (B) A3393G Intronic NA NA NA NA 5 M2(0.4)-638 974 (B) A3553G Intronic NA NA NA NA

[0095] Table 2 below lists various mutations obtained after screening using TILLING method in eIF(iso)4e gene.

TABLE-US-00002 TABLE 2 Effect of Mutation Mutant Line Codon Amino acid SIFT SIFT No. No. Code Nature Exon Change Change Analysis Score 1 M2(0.4)-740 1170 (B) C1912T Intronic NA NA NA NA 2 M2(0.4)-882 1465 (B) A1941G Exon UAC Y70C Not 0 UGC Tolerated 3 M2(0.3)-1159 2503 (A) A2117G Intronic NA NA NA NA 4 M2(0.3)-1159 2503 (A) A2147T Intronic NA NA NA NA 5 M2(0.3)-1159 2503 (A) C2118G Intronic NA NA NA NA 6 M2(0.3)-1159 2503 (A) C2391T Intronic NA NA NA NA 7 M2(0.3)-1159 2503 (A) G2239A Exon UUG L125= NA NA UUA 8 M2(0.3)-1159 2503 (A) A3576G Intronic NA NA NA NA 9 M2(0.3)-1159 2503 (A) C3408G Intronic NA NA NA NA 10 M2(0.3)-49 117 (A) A2040G Exon AAU N103S Tolerated 0.17 AGU 11 M2(0.3)-181 387 (A) A1998G Exon AAA K89R Not 0 AGA Tolerated 12 M2(0.3)-704 1322 (A) T1940C Exon UAC Y70H Tolerated 0.06 CAC 13 M2(0.3)-87 189 (A) T2098C Exon CUU L122= NA NA CUC 14 M2(0.4)-1379 2460 (B) G2095A Exon UGG W121* NA NA UGA 15 M2(0.4)-1278 2286 (B) C938T Intronic NA NA NA NA 16 M2(0.3)-459 883 (A) G1868A Intronic NA NA NA NA 17 M2(0.3)-552 1038 (A) G2046A Exon GGG G105E Not 0 GAG Tolerated

Example 5

Phenotypic Analysis of the Mutants by Artificial Inoculation

[0096] To prepare the test plants, pre-germination was done by soaking the seeds in water for 3 days and then transferred in a layer of damped cloth. Seeds were incubated for 3 days at room temperature. After pre-germination, the seeds were sown in individual polyethylene bags with papaya media (3:1-coir dust: sand). Seedlings were drenched with calcium nitrate once a week and with fungicide (promocarb hydrochloride/metalaxyl) were applied to prevent damping-off problems. Papaya mutant plants are grown to 8-leaf stage and the inoculum is prepared from PRSV infected symptomatic leaves. To be able to determine the suitable variety that will serve as good propagation host for PRSV, susceptible papaya controls were inoculated and their disease severity were assessed and compared. The suitable inoculum concentration was determined by comparing two inoculum dilutions (1:5 and 1:10). Virus titers of the first and second fully expanded leaf were compared using their ELISA values, leaf samples were collected at different days after inoculation (DAI), (7 DAI, 14 DAI and 21DAI). Young, symptomatic leaves from PRSV infected seedlings were collected (preferably 14-21 days after inoculation) and homogenized in chilled inoculation buffer using a blender or a mortar and pestle in a 1:5 dilution (2 g of infected papaya leaves in 10 ml of inoculation buffer). Then virus homogenate was filtered through a layer of cheesecloth and 1:10 dilution was prepared from the 1:5 dilution. Thirty-days old seedlings were inoculated with ice-cold inoculum containing Celite (5g/liter) on the upper leaf surface using sponge and washed with tap water after 5 minutes to remove the Celite and plant debris on the leaf surface (FIG. 1). The inoculation was repeated day after the first inoculation. Symptom development, disease severity and incidence were assessed at different times after inoculation (7, 14 and 21 DAI). Inoculated seedlings were rated based on disease severity 21 DAI using 0-4 scale below (Table 3):

TABLE-US-00003 TABLE 3 Rating Description 0 No symptom 1 Vein yellowing and/or mosaic 2 Mosaic + mottling 3 Mottling + leaf deformation 4 Severe mottling + shoe-stinging and/or stunting

[0097] The value of the viral load was calculated and co-related to percentage (%) increase in mutation based on the severity rating. Thus a 0 rating may correlate to 99-100% increase in disease resistance as compared to wild type papaya plant. M2(0.3)-1709 is the eIF4e mutant papaya according to the present invention. M2(0.4)-740 is the eIF(iso)4e intronic mutation. M2(0.4)-882 is a eIF4e(iso) missense mutant with amino acid changes Y at position 70 to C (Y70C). The table below demonstrates observations (Table 4).

TABLE-US-00004 TABLE 4 Phenotypic observation of the mutant versus wildtype papaya plants for PRSV resistance. Accession Accession Bench Bio Severity OD ELISA Code Name coordinates rating value Result PH-02691 EW 2398 (A) M2(0.3)-1709 0 0.069 Negative M2(0.3)-1709 0 0.074 Negative M2(0.3)-1709 0 0.07 Negative M2(0.3)-1709 1 0.074 Negative M2(0.3)-1709 1 0.08 Negative M2(0.3)-1709 0 0.08 Negative PH-02692 EW 2654 (A) WILD TYPE 4 0.332 Positive PH-02693 EW 1170 (B) M2(0.4)-740 4 0.922 Positive PH-02694 EW 1465 (B) M2(0.4)-882 4 0.935 Positive PH-02695 EW 2503 (A) WILD TYPE 4 0.787 Positive PH-1301 S-check WILD TYPE 4 1.023 Positive Healthy check 1 0.079 Healthy check 2 0.08 Healthy check 3 0.08 Buffer 0.068 Positive value 0.199* Positive check 0.554 *Average of healthy checks X 2.5

[0098] The above table clearly demonstrates, eIF4e mutants with increased resistance compared to wild type and other mutants (M2(0.4)-740, 882) which served as positive control plants. It is clearly observed that all resistant plants have low OD values of viral load at 0.069, translating to 100% increased resistance over wild type susceptible plant which showed high OD values of viral load at 0.787. When compared with positive controls, some mutant plants showed a 90% increase in resistance. All the homozygous mutants confirmed by TILLING and by genotyping showed no symptoms after the inoculation, whereas the positive control (susceptible check) showed severe mottling and stunting effect in the papaya plants typical of PRSV infection (FIG. 2). In doing standard ELISA, negative controls have to be included, these negative controls refer to the healthy checks. Three healthy checks were taken from seedlings which are not challenged by PRSV. To calculate the threshold value in which we can declare that the sample is infected by PRSV, we take the average readings of the 3 healthy checks and multiply it by 2.5. And this calculated value (=positive value) is the threshold. Anything that is higher or equal to the positive value means it is infected with PRSV. If the value is lower than the threshold, then it is declared not infected by PRSV. The positive check is the positive controlleaves coming from PRSV-infected plants.