Transgenic Brassica rapa plants over‐expressing eIF(iso)4E variants show broad‐spectrum Turnip mosaic virus (TuMV) resistance

The protein–protein interaction between VPg (viral protein genome‐linked) of potyviruses and eIF4E (eukaryotic initiation factor 4E) or eIF(iso)4E of their host plants is a critical step in determining viral virulence. In this study, we evaluated the approach of engineering broad‐spectrum resistance in Chinese cabbage (Brassica rapa) to Turnip mosaic virus (TuMV), which is one of the most important potyviruses, by a systematic knowledge‐based approach to interrupt the interaction between TuMV VPg and B. rapa eIF(iso)4E. The seven amino acids in the cap‐binding pocket of eIF(iso)4E were selected on the basis of other previous results and comparison of protein models of cap‐binding pockets, and mutated. Yeast two‐hybrid assay and co‐immunoprecipitation analysis demonstrated that W95L, K150L and W95L/K150E amino acid mutations of B. rapa eIF(iso)4E interrupted its interaction with TuMV VPg. All eIF(iso)4E mutants were able to complement an eIF4E‐knockout yeast strain, indicating that the mutated eIF(iso)4E proteins retained their function as a translational initiation factor. To determine whether these mutations could confer resistance, eIF(iso)4E W95L, W95L/K150E and eIF(iso)4E wild‐type were over‐expressed in a susceptible Chinese cabbage cultivar. Evaluation of the TuMV resistance of T₁ and T₂ transformants demonstrated that the over‐expression of the eIF(iso)4E mutant forms can confer resistance to multiple TuMV strains. These data demonstrate the utility of knowledge‐based approaches for the engineering of broad‐spectrum resistance in Chinese cabbage.     

A TILLING approach to generate broad‐spectrum resistance to potyviruses in tomato is hampered by eIF4E gene redundancy

Genetic resistance to pathogens is important for sustainable maintenance of crop yields. Recent biotechnologies offer alternative approaches to generate resistant plants by compensating for the lack of natural resistance. Tomato (Solanum lycopersicum) and related species offer a model in which natural and TILLING‐induced potyvirus resistance alleles may be compared. For resistance based on translation initiation factor eIF4E1, we confirm that the natural allele Sh–eIF4E1^PI24–pot1, isolated from the wild tomato species Solanum habrochaites, is associated with a wide spectrum of resistance to both potato virus Y and tobacco etch virus isolates. In contrast, a null allele of the same gene, isolated through a TILLING strategy in cultivated tomato S. lycopersicum, is associated with a much narrower resistance spectrum. Introgressing the null allele into S. habrochaites did not extend its resistance spectrum, indicating that the genetic background is not responsible for the broad resistance. Instead, the different types of eIF4E1 mutations affect the levels of eIF4E2 differently, suggesting that eIF4E2 is also involved in potyvirus resistance. Indeed, combining two null mutations affecting eIF4E1 and eIF4E2 re‐establishes a wide resistance spectrum in cultivated tomato, but to the detriment of plant development. These results highlight redundancy effects within the eIF4E gene family, where regulation of expression alters susceptibility or resistance to potyviruses. For crop improvement, using loss‐of‐function alleles to generate resistance may be counter‐productive if they narrow the resistance spectrum and limit growth. It may be more effective to use alleles encoding functional variants similar to those found in natural diversity.     

Engineering virus resistance using a modified potato gene

Natural mutations in translation initiation factor eIF4E confer resistance to potyviruses in many plant species. Potato is a staple food crop plagued by several potyviruses, yet to date no known eIF4E-mediated resistance genes have been identified. In this study, we demonstrate that transgenic expression of the pvr1(2) gene from pepper confers resistance to Potato virus Y (PVY) in potato. We then use this information to convert the susceptible potato ortholog of this allele into a de novo allele for resistance to PVY using site-directed mutagenesis. Potato plants overexpressing the mutated potato allele are resistant to virus infection. Resistant lines expressed high levels of eIF4E mRNA and protein. The resistant plants showed growth similar to untransformed controls and produced phenotypically similar tubers. This technique disrupts a key step in the viral infection process and may potentially be used to engineer virus resistance in a number of economically important plant-viral pathosystems. Furthermore, the general public may be more amenable to the 'intragenic' nature of this approach because the transferred coding region is modified from a gene in the target crop rather than from a distant species.     

Engineering of CRISPR/Cas9‐mediated potyvirus resistance in transgene‐free Arabidopsis plants

Members of the eukaryotic translation initiation factor (eIF) gene family, including eIF4E and its paralogue eIF(iso)4E, have previously been identified as recessive resistance alleles against various potyviruses in a range of different hosts. However, the identification and introgression of these alleles into important crop species is often limited. In this study, we utilise CRISPR/Cas9 technology to introduce sequence‐specific deleterious point mutations at the eIF(iso)4E locus in Arabidopsis thaliana to successfully engineer complete resistance to Turnip mosaic virus (TuMV), a major pathogen in field‐grown vegetable crops. By segregating the induced mutation from the CRISPR/Cas9 transgene, we outline a framework for the production of heritable, homozygous mutations in the transgene‐free T₂ generation in self‐pollinating species. Analysis of dry weights and flowering times for four independent T₃ lines revealed no differences from wild‐type plants under standard growth conditions, suggesting that homozygous mutations in eIF(iso)4E do not affect plant vigour. Thus, the established CRISPR/Cas9 technology provides a new approach for the generation of Potyvirus resistance alleles in important crops without the use of persistent transgenes.     

Coordinated and selective recruitment of eIF4E and eIF4G factors for potyvirus infection in Arabidopsis thaliana

The translation initiation factors eIF4E and eIF(iso)4E play a key role during virus infection in plants. During mRNA translation, eIF4E provides the cap-binding function and is associated with the protein eIF4G to form the eIF4F complex. Susceptibility analyses of Arabidopsis mutants knocked-out for At-eIF4G genes showed that eIF4G factors are indispensable for potyvirus infection. The colonization pattern by a viral recombinant carrying GFP indicated that eIF4G is involved at a very early infection step. Like eIF4E, eIF4G isoforms are selectively recruited for infection. Moreover, the eIF4G selective involvement parallels eIF4E recruitment. This is the first report of a coordinated and selective recruitment of eIF4E and eIF4G factors, suggesting the whole eIF4F recruitment.     

A natural recessive resistance gene against potato virus Y in pepper corresponds to the eukaryotic initiation factor 4E (eIF4E)

We show here that the pvr2 locus in pepper, conferring recessive resistance against strains of potato virus Y (PVY), corresponds to a eukaryotic initiation factor 4E (eIF4E) gene. RFLP analysis on the PVY-susceptible and resistant pepper cultivars, using an eIF4E cDNA from tobacco as probe, revealed perfect map co-segregation between a polymorphism in the eIF4E gene and the pvr2 alleles, pvr2(1) (resistant to PVY-0) and pvr2(2) (resistant to PVY-0 and 1). The cloned pepper eIF4E cDNA encoded a 228 amino acid polypeptide with 70-86% nucleotide sequence identity with other plant eIF4Es. The sequences of eIF4E protein from two PVY-susceptible cultivars were identical and differed from the eIF4E sequences of the two PVY-resistant cultivars Yolo Y (YY) (pvr2(1)) and FloridaVR2 (F) (pvr2(2)) at two amino acids, a mutation common to both resistant genotypes and a second mutation specific to each. Complementation experiments were used to show that the eIF4E gene corresponds to pvr2. Thus, potato virus X-mediated transient expression of eIF4E from susceptible cultivar Yolo Wonder (YW) in the resistant genotype YY resulted in loss of resistance to subsequent PVY-0 inoculation and transient expression of eIF4E from YY (resistant to PVY-0; susceptible to PVY-1) rendered genotype F susceptible to PVY-1. Several lines of evidence indicate that interaction between the potyvirus genome-linked protein (VPg) and eIF4E are important for virus infectivity, suggesting that the recessive resistance could be due to incompatibility between the VPg and eIF4E in the resistant genotype.     

Ectopic expression of a recessive resistance gene generates dominant potyvirus resistance in plants

Despite long-standing plant breeding investments and early successes in genetic engineering, plant viral pathogens still cause major losses in agriculture worldwide. Early transgenic approaches involved the expression of pathogen-derived sequences that provided limited protection against relatively narrow ranges of viral pathotypes. In contrast, this study demonstrates that the ectopic expression of pvr1 , a recessive gene from Capsicum chinense , results in dominant broad-spectrum potyvirus resistance in transgenic tomato plants ( Solanum lycopersicum ). The pvr1 locus in pepper encodes the eukaryotic translation initiation factor eIF4E. Naturally occurring point mutations at this locus result in monogenic recessive broad-spectrum potyvirus resistance that has been globally deployed via plant breeding programmes for more than 50 years. Transgenic tomato progenies that over-expressed the Capsicum pvr1 allele showed dominant resistance to several tobacco etch virus strains and other potyviruses, including pepper mottle virus, a range of protection similar to that observed in pepper homozygous for the pvr1 allele.     

Multiple copies of eukaryotic translation initiation factors in Brassica rapa facilitate redundancy,enabling diversification through variation in splicing and broad‐spectrum virus resistance

Recessive strain‐specific resistance to a number of plant viruses in the Potyvirus genus has been found to be based on mutations in the eukaryotic translation initiation factor 4E (eIF4E) and its isoform, eIF(iso)4E. We identified three copies of eIF(iso)4E in a number of Brassica rapa lines. Here we report broad‐spectrum resistance to the potyvirus Turnip mosaic virus (TuMV) due to a natural mechanism based on the mis‐splicing of the eIF(iso)4E allele in some TuMV‐resistant B. rapa var. pekinensis lines. Of the splice variants, the most common results in a stop codon in intron 1 and a much truncated, non‐functional protein. The existence of multiple copies has enabled redundancy in the host plant's translational machinery, resulting in diversification and emergence of the resistance. Deployment of the resistance is complicated by the presence of multiple copies of the gene. Our data suggest that in the B. rapa subspecies trilocularis, TuMV appears to be able to use copies of eIF(iso)4E at two loci. Transformation of different copies of eIF(iso)4E from a resistant B. rapa line into an eIF(iso)4E knockout line of Arabidopsis thaliana proved misleading because it showed that, when expressed ectopically, TuMV could use multiple copies which was not the case in the resistant B. rapa line. The inability of TuMV to access multiple copies of eIF(iso)4E in B. rapa and the broad spectrum of the resistance suggest it may be durable.     

An iterative gene-editing strategy broadens eIF4E1 genetic diversity in Solanum lycopersicum and generates resistance to multiple potyvirus isolates

Resistance to potyviruses in plants has been largely provided by the selection of natural variant alleles of eukaryotic translation initiation factors (eIF) 4E in many crops. However, the sources of such variability for breeding can be limited for certain crop species, while new virus isolates continue to emerge. Different methods of mutagenesis have been applied to inactivate the eIF4E genes to generate virus resistance, but with limited success due to the physiological importance of translation factors and their redundancy. Here, we employed genome editing approaches at the base level to induce non-synonymous mutations in the eIF4E1 gene and create genetic diversity in cherry tomato (Solanum lycopersicum var. cerasiforme). We sequentially edited the genomic sequences coding for two regions of eIF4E1 protein, located around the cap-binding pocket and known to be important for susceptibility to potyviruses. We show that the editing of only one of the two regions, by gene knock-in and base editing, respectively, is not sufficient to provide resistance. However, combining amino acid mutations in both regions resulted in resistance to multiple potyviruses without affecting the functionality in translation initiation. Meanwhile, we report that extensive base editing in exonic region can alter RNA splicing pattern, resulting in gene knockout. Altogether our work demonstrates that precision editing allows to design plant factors based on the knowledge on evolutionarily selected alleles and enlarge the gene pool to potentially provide advantageous phenotypes such as pathogen resistance.     

Mimicking natural polymorphism in eIF4E by CRISPR‐Cas9 base editing is associated with resistance to potyviruses

In many crop species, natural variation in eIF4E proteins confers resistance to potyviruses. Gene editing offers new opportunities to transfer genetic resistance to crops that seem to lack natural eIF4E alleles. However, because eIF4E are physiologically important proteins, any introduced modification for virus resistance must not bring adverse phenotype effects. In this study, we assessed the role of amino acid substitutions encoded by a Pisum sativum eIF4E virus‐resistance allele (W69L, T80D S81D, S84A, G114R and N176K) by introducing them independently into the Arabidopsis thaliana eIF4E1 gene, a susceptibility factor to the Clover yellow vein virus (ClYVV). Results show that most mutations were sufficient to prevent ClYVV accumulation in plants without affecting plant growth. In addition, two of these engineered resistance alleles can be combined with a loss‐of‐function eIFiso4E to expand the resistance spectrum to other potyviruses. Finally, we use CRISPR‐nCas9‐cytidine deaminase technology to convert the Arabidopsis eIF4E1 susceptibility allele into a resistance allele by introducing the N176K mutation with a single‐point mutation through C‐to‐G base editing to generate resistant plants. This study shows how combining knowledge on pathogen susceptibility factors with precise genome‐editing technologies offers a feasible solution for engineering transgene‐free genetic resistance in plants, even across species barriers.     

Development of broad virus resistance in non‐transgenic cucumber using CRISPR/Cas9 technology

Genome editing in plants has been boosted tremendously by the development of CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats) technology. This powerful tool allows substantial improvement in plant traits in addition to those provided by classical breeding. Here, we demonstrate the development of virus resistance in cucumber (Cucumis sativus L.) using Cas9/subgenomic RNA (sgRNA) technology to disrupt the function of the recessive eIF4E (eukaryotic translation initiation factor 4E) gene. Cas9/sgRNA constructs were targeted to the N′ and C′ termini of the eIF4E gene. Small deletions and single nucleotide polymorphisms (SNPs) were observed in the eIF4E gene targeted sites of transformed T1 generation cucumber plants, but not in putative off‐target sites. Non‐transgenic heterozygous eif4e mutant plants were selected for the production of non‐transgenic homozygous T3 generation plants. Homozygous T3 progeny following Cas9/sgRNA that had been targeted to both eif4e sites exhibited immunity to Cucumber vein yellowing virus (Ipomovirus) infection and resistance to the potyviruses Zucchini yellow mosaic virus and Papaya ring spot mosaic virus‐W. In contrast, heterozygous mutant and non‐mutant plants were highly susceptible to these viruses. For the first time, virus resistance has been developed in cucumber, non‐transgenically, not visibly affecting plant development and without long‐term backcrossing, via a new technology that can be expected to be applicable to a wide range of crop plants.     

Simultaneous CRISPR/Cas9‐mediated editing of cassava eIF4E isoforms nCBP‐1 and nCBP‐2 reduces cassava brown streak disease symptom severity and incidence

Cassava brown streak disease (CBSD) is a major constraint on cassava yields in East and Central Africa and threatens production in West Africa. CBSD is caused by two species of positive‐sense RNA viruses belonging to the family Potyviridae, genus Ipomovirus: Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV). Diseases caused by the family Potyviridae require the interaction of viral genome‐linked protein (VPg) and host eukaryotic translation initiation factor 4E (eIF4E) isoforms. Cassava encodes five eIF4E proteins: eIF4E, eIF(iso)4E‐1, eIF(iso)4E‐2, novel cap‐binding protein‐1 (nCBP‐1), and nCBP‐2. Protein–protein interaction experiments consistently found that VPg proteins associate with cassava nCBPs. CRISPR/Cas9‐mediated genome editing was employed to generate ncbp‐1, ncbp‐2, and ncbp‐1/ncbp‐2 mutants in cassava cultivar 60444. Challenge with CBSV showed that ncbp‐1/ncbp‐2 mutants displayed delayed and attenuated CBSD aerial symptoms, as well as reduced severity and incidence of storage root necrosis. Suppressed disease symptoms were correlated with reduced virus titre in storage roots relative to wild‐type controls. Our results demonstrate the ability to modify multiple genes simultaneously in cassava to achieve tolerance to CBSD. Future studies will investigate the contribution of remaining eIF4E isoforms on CBSD and translate this knowledge into an optimized strategy for protecting cassava from disease.     

The potyvirus recessive resistance gene, sbm1, identifies a novel role for translation initiation factor eIF4E in cell-to-cell trafficking

From the characterization of the recessive resistance gene, sbm1, in pea we have identified the eukaryotic translation initiation factor, eIF4E, as a susceptibility factor required for infection with the Potyvirus, Pea seed-borne mosaic virus. A functional analysis of the mode of action of the product of the dominant allele revealed a novel function for eIF4E in its support for virus movement from cell-to-cell, in addition to its probable support for viral RNA translation, and hence replication. Different resistance specificities in two independent pea lines were explained by different mutations in eIF4E. On the modelled structure of eIF4E the coding changes were in both cases lying in and around the structural pocket involved in binding the 5'-m7G cap of eukaryotic mRNAs. Protein expression and cap-binding analysis showed that eIF4E encoded by a resistant plant could not bind to m7G-Sepharose, a result which may point to functional redundancy between eIF4E and the paralogous eIF(iso)4E in resistant peas. These observations, together with related findings for other potyvirus recessive resistances, provide a more complete picture of the potyvirus life cycle.     

High resistance to plum pox virus (PPV) in transgenic plants containing modified and truncated forms of PPV coat protein gene

Two modified plum pox virus (PPV) coat protein (CP) gene constructs, designed to reduce putative biological risks associated with heteroen capsidation, were integrated into Nicotiana benthamiana plants. The first one contained a deletion of the nucleotides encoding for the DAG amino acid triplet involved in virus aphid-transmission. In the second one, the first 420 nucleotides of the PPV CP gene were removed. We present here the analysis and the selection throughout the generations of PPV-resistant transgenic lines containing these constructs. In most of the lines, a recovery phenotype was observed and was associated with a down-regulation of the transgene products (RNA or protein). We also describe two lines that were highly resistant to PPV. This immunity was correlated with a high number of transgene copies (at least three) and with low or undetectable transgene RNA levels. No heterologous protection was observed against other potyviruses. These characteristics indicate that the described resistance against PPV was RNA-mediated and can be classified as a 'sense suppression' or homology-dependent resistance. Moreover, the production of a highly resistant line containing the PPV CP gene with one third of its 5 end deleted indicated that this region is not necessary to trigger the plant resistance mechanism(s)     

Double mutations in eIF4E and eIFiso4E confer recessive resistance to <Emphasis Type="Italic">Chilli veinal mottle virus</Emphasis> in pepper

To evaluate the involvement of translation initiation factors eIF4E and eIFiso4E in Chilli veinai mottle virus (ChiVMV) infection in pepper, we conducted a genetic analysis using a segregating population derived from a cross between Capsicum annuum ‘Dempsey’ containing an eIF4E mutation (pvr1 ² ) and C. annuum ‘Perennial’ containing an eIFiso4E mutation (pvr6). C. annuum ‘Dempsey’ was susceptible and C. annuum ‘Perennial’ was resistant to ChiVMV. All F₁ plants showed resistance, and F₂ individuals segregated in a resistant-susceptible ratio of 166:21, indicating that many resistance loci were involved. Seventy-five F₂ and 329 F₃ plants of 17 families were genotyped with pvr1 ² and pvr6 allele-specific markers, and the genotype data were compared with observed resistance to viral infection. All plants containing homozygous genotypes of both pvr1 ² and pvr6 were resistant to ChiVMV, demonstrating that simultaneous mutations in eIF4E and eIFiso4E confer resistance to ChiVMV in pepper. Genotype analysis of F2 plants revealed that all plants containing homozygous genotypes of both pvr1 ² and pvr6 showed resistance to ChiVMV. In protein-protein interaction experiments, ChiVMV viral genome-linked protein (VPg) interacted with both eIF4E and eIFiso4E. Silencing of eIF4E and eIFiso4E in the VIGS experiment showed reduction in ChiVMV accumulation. These results demonstrated that ChiVMV can use both eIF4E and eIFiso4E for replication, making simultaneous mutations in eIF4E and eIFiso4E necessary to prevent ChiVMV infection in pepper. These authors contributed equally to this work.     

Nicotiana benthamiana resistance to non-adapted Melon necrotic spot virus results from an incompatible interaction between virus RNA and translation initiation factor 4E

Nicotiana benthamiana has been described as non-host for Melon necrotic spot virus (MNSV). We investigated the basis of this resistance using the unique opportunity provided by strain MNSV-264, a recombinant virus that is able to overcome the resistance. Analysis of chimeric MNSV mutants showed that virulence in N. benthamiana is conferred by a 49 nucleotide section of the MNSV-264 3'-UTR, which acts in this host as a cap-independent translational enhancer (3'-CITE). Although the 3'-CITE of non-adapted MNSV-Mα5 is active in susceptible melon, it does not promote efficient translation in N. benthamiana, thus preventing expression of proteins required for virus replication. However, MNSV-Mα5 gains the ability to multiply in N. benthamiana cells if eIF4E from a susceptible melon variety (Cm-eIF4E-S) is supplied in trans. These data show that N. benthamiana resistance to MNSV-Mα5 results from incompatibility between the MNSV-Mα5 3'-CITE and N. benthamiana eIF4E in initiating efficient translation of the viral genome. Therefore, non-host resistance conferred by the inability of a host susceptibility factor to support viral multiplication may be a possible mechanism for this type of resistance to viruses.     

Allelic variation for broad‐spectrum resistance and susceptibility to bacterial pathogens identified in a rice MAGIC population

Quantitative trait loci (QTL) that confer broad‐spectrum resistance (BSR), or resistance that is effective against multiple and diverse plant pathogens, have been elusive targets of crop breeding programmes. Multiparent advanced generation intercross (MAGIC) populations, with their diverse genetic composition and high levels of recombination, are potential resources for the identification of QTL for BSR. In this study, a rice MAGIC population was used to map QTL conferring BSR to two major rice diseases, bacterial leaf streak (BLS) and bacterial blight (BB), caused by Xanthomonas oryzae pathovars (pv.) oryzicola (Xoc) and oryzae (Xoo), respectively. Controlling these diseases is particularly important in sub‐Saharan Africa, where no sources of BSR are currently available in deployed varieties. The MAGIC founders and lines were genotyped by sequencing and phenotyped in the greenhouse and field by inoculation with multiple strains of Xoc and Xoo. A combination of genomewide association studies (GWAS) and interval mapping analyses revealed 11 BSR QTL, effective against both diseases, and three pathovar‐specific QTL. The most promising BSR QTL (qXO‐2‐1, qXO‐4‐1 and qXO‐11‐2) conferred resistance to more than nine Xoc and Xoo strains. GWAS detected 369 significant SNP markers with distinguishable phenotypic effects, allowing the identification of alleles conferring disease resistance and susceptibility. The BSR and susceptibility QTL will improve our understanding of the mechanisms of both resistance and susceptibility in the long term and will be immediately useful resources for rice breeding programmes.