Virus Evolution Faced to Multiple Host Targets: The Potyvirus-Pepper Case Study.

The wealth of variability amongst genes controlling immunity against potyviruses in pepper (Capsicum spp.) has been instrumental in understanding plant-virus co-evolution and major determinants of plant resistance durability. Characterization of the eukaryotic initiation factor 4E1 (eIF4E1), involved in mRNA translation, as the basis of potyvirus resistance in pepper initiated a large body of work that showed that recessive resistance to potyviruses and other single-stranded positive-sense RNA viruses resulted from mutations in eukaryotic initiation factors in many plant crop species. Combining mutations in different eIF4Es in the same pepper genotype had complex effects on the breadth of the resistance spectrum and on resistance durability, revealing a trade-off between these two traits. In addition, combining eIF4E1 mutations with a quantitatively resistant genetic background had a strong positive effect on resistance durability. Analysing the evolutionary forces imposed by pepper genotypes onto virus populations allowed identifying three key factors improving plant resistance durability: the complexity of mutational pathways involved in virus adaptation to the plant resistance, the decrease of competitivity induced by these mutations on the virus and the intensity of genetic drift imposed by plant genotypes on the virus during its infection cycle.

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.

Cloning of the Arabidopsis rwm1 gene for resistance to Watermelon mosaic virus points to a new function for natural virus resistance genes.

Arabidopsis thaliana represents a valuable and efficient model to understand mechanisms underlying plant susceptibility to viral diseases. Here, we describe the identification and molecular cloning of a new gene responsible for recessive resistance to several isolates of Watermelon mosaic virus (WMV, genus Potyvirus) in the Arabidopsis Cvi-0 accession. rwm1 acts at an early stage of infection by impairing viral accumulation in initially infected leaf tissues. Map-based cloning delimited rwm1 on chromosome 1 in a 114-kb region containing 30 annotated genes. Positional and functional candidate gene analysis suggested that rwm1 encodes cPGK2 (At1g56190), an evolutionary conserved nucleus-encoded chloroplast phosphoglycerate kinase with a key role in cell metabolism. Comparative sequence analysis indicates that a single amino acid substitution (S78G) in the N-terminal domain of cPGK2 is involved in rwm1-mediated resistance. This mutation may have functional consequences because it targets a highly conserved residue, affects a putative phosphorylation site and occurs within a predicted nuclear localization signal. Transgenic complementation in Arabidopsis together with virus-induced gene silencing in Nicotiana benthamiana confirmed that cPGK2 corresponds to rwm1 and that the protein is required for efficient WMV infection. This work uncovers new insight into natural plant resistance mechanisms that may provide interesting opportunities for the genetic control of plant virus diseases.

Viral factor TAV recruits TOR/S6K1 signalling to activate reinitiation after long ORF translation.

The protein kinase TOR (target-of-rapamycin) upregulates translation initiation in eukaryotes, but initiation restart after long ORF translation is restricted by largely unknown pathways. The plant viral reinitiation factor transactivator-viroplasmin (TAV) exceptionally promotes reinitiation through a mechanism involving retention on 80S and reuse of eIF3 and the host factor reinitiation-supporting protein (RISP) to regenerate reinitiation-competent ribosomal complexes. Here, we show that TAV function in reinitiation depends on physical association with TOR, with TAV-TOR binding being critical for both translation reinitiation and viral fitness. Consistently, TOR-deficient plants are resistant to viral infection. TAV triggers TOR hyperactivation and S6K1 phosphorylation in planta. When activated, TOR binds polyribosomes concomitantly with polysomal accumulation of eIF3 and RISP--a novel and specific target of TOR/S6K1--in a TAV-dependent manner, with RISP being phosphorylated. TAV mutants defective in TOR binding fail to recruit TOR, thereby abolishing RISP phosphorylation in polysomes and reinitiation. Thus, activation of reinitiation after long ORF translation is more complex than previously appreciated, with TOR/S6K1 upregulation being the key event in the formation of reinitiation-competent ribosomal complexes.

Potyviruses differ in their requirement for TOR signalling.

Potyviruses are important plant pathogens that rely on many plant cellular processes for successful infection. TOR (target of rapamycin) signalling is a key eukaryotic energy-signalling pathway controlling many cellular processes such as translation and autophagy. The dependence of potyviruses on active TOR signalling was examined. Arabidopsis lines downregulated for TOR by RNAi were challenged with the potyviruses watermelon mosaic virus (WMV) and turnip mosaic virus (TuMV). WMV accumulation was found to be severely altered while TuMV accumulation was only slightly delayed. In another approach, using AZD-8055, an active site inhibitor of the TOR kinase, WMV infection was found to be strongly affected. Moreover, AZD-8055 application can cure WMV infection. In contrast, TuMV infection was not affected by AZD-8055. This suggests that potyviruses have different cellular requirements for active plant TOR signalling.

Specific requirement for translation initiation factor 4E or its isoform drives plant host susceptibility to Tobacco etch virus.

BACKGROUND: In plants, eIF4E translation initiation factors and their eIFiso4E isoforms are essential susceptibility factors for many RNA viruses, including potyviruses. Mutations altering these factors are a major source of resistance to the viruses. The eIF4E allelic series is associated with specific resistance spectra in crops such as Capsicum annum. Genetic evidence shows that potyviruses have a specific requirement for a given 4E isoform that depends on the host plant. For example, Tobacco etch virus (TEV) uses eIF4E1 to infect Capsicum annuum but uses eIFiso4E to infect Arabidopsis thaliana. Here, we investigated how TEV exploits different translation initiation factor isoforms to infect these two plant species. RESULTS: A complementation system was set up in Arabidopsis to test the restoration of systemic infection by TEV. Using this system, Arabidopsis susceptibility to TEV was complemented with a susceptible pepper eIF4E1 allele but not with a resistant allele. Therefore, in Arabidopsis, TEV can use the pepper eIF4E1 instead of the endogenous eIFiso4E isoform so is able to switch between translation initiation factor 4E isoform to infect the same host. Moreover, we show that overexpressing the pepper eIF4E1 alleles is sufficient to make Arabidopsis susceptible to an otherwise incompatible TEV strain. Lastly, we show that the resistant eIF4E1 allele is similarly overcome by a resistance-breaking TEV strain as in pepper, confirming that this Arabidopsis TEV-susceptibility complementation system is allele-specific. CONCLUSION: We report here a complementation system in Arabidopsis that makes it possible to assess the role of pepper pvr2-eIF4E alleles in susceptibility to TEV. Heterologous complementation experiments showed that the idiosyncratic properties of the 4E and iso4E proteins create a major checkpoint for viral infection of different hosts. This system could be used to screen natural or induced eIF4E alleles to find and study alleles of interest for plant breeding.

Can genome editing help transitioning to agroecology?

Meeting the challenges of agroecological transition in a context of climate change requires the use of various strategies such as biological regulations, adapted animal and plant genotypes, diversified production systems, and digital technologies. Seeds and plants, through plant breeding, play a crucial role in driving these changes. The emergence of genome editing presents a new opportunity in plant breeding practices. However, like any technological revolution involving living organisms, it is essential to assess its potential contributions, limits, risks, socio-economic implications, and the associated controversies. This article aims to provide a comprehensive review of scientific knowledge on genome editing for agroecological transition, drawing on multidisciplinary approaches encompassing biological, agronomic, economic, and social sciences.

Single amino acid changes in the turnip mosaic virus viral genome-linked protein (VPg) confer virulence towards Arabidopsis thaliana mutants knocked out for eukaryotic initiation factors eIF(iso)4E and eIF(iso)4G.

Previous resistance analyses of Arabidopsis thaliana mutants knocked out for eukaryotic translation initiation factors showed that disruption of the At-eIF(iso)4E or both the At-eIF(iso)4G1 and At-eIF(iso)4G2 genes resulted in resistance against turnip mosaic virus (TuMV). This study selected TuMV virulent variants that overcame this resistance and showed that two independent mutations in the region coding for the viral genome-linked protein (VPg) were sufficient to restore TuMV virulence in At-eIF(iso)4E and At-eIF(iso)4G1xAt-eIF(iso)4G2 knockout plants. As a VPg-eIF(iso)4E interaction has been shown previously to be critical for TuMV infection, a systematic analysis of the interactions between A. thaliana eIF4Es and VPgs of virulent and avirulent TuMVs was performed. The results suggest that virulent TuMV variants may use an eIF4F-independent pathway.

Development and evaluation of robust molecular markers linked to disease resistance in tomato for distinctness, uniformity and stability testing.

Molecular markers linked to phenotypically important traits are of great interest especially when traits are difficult and/or costly to be observed. In tomato where a strong focus on resistance breeding has led to the introgression of several resistance genes, resistance traits have become important characteristics in distinctness, uniformity and stability (DUS) testing for Plant Breeders Rights (PBR) applications. Evaluation of disease traits in biological assays is not always straightforward because assays are often influenced by environmental factors, and difficulties in scoring exist. In this study, we describe the development and/or evaluation of molecular marker assays for the Verticillium genes Ve1 and Ve2, the tomato mosaic virus Tm1 (linked marker), the tomato mosaic virus Tm2 and Tm2 ( 2 ) genes, the Meloidogyne incognita Mi1-2 gene, the Fusarium I (linked marker) and I2 loci, which are obligatory traits in PBR testing. The marker assays were evaluated for their robustness in a ring test and then evaluated in a set of varieties. Although in general, results between biological assays and marker assays gave highly correlated results, marker assays showed an advantage over biological tests in that the results were clearer, i.e., homozygote/heterozygote presence of the resistance gene can be detected and heterogeneity in seed lots can be identified readily. Within the UPOV framework for granting of PBR, the markers have the potential to fulfil the requirements needed for implementation in DUS testing of candidate varieties and could complement or may be an alternative to the pathogenesis tests that are carried out at present.

Natural variation and functional analyses provide evidence for co-evolution between plant eIF4E and potyviral VPg.

Amino acid substitutions in the eukaryotic translation initiation factor 4E (eIF4E) result in recessive resistance to potyviruses in a range of plant species, including Capsicum spp. Correspondingly, amino acid changes in the central part of the viral genome-linked protein (VPg) are responsible for the potyvirus's ability to overcome eIF4E-mediated resistance. A key observation was that physical interaction between eIF4E and the VPg is required for viral infection, and eIF4E mutations that cause resistance prevent VPg binding and inhibit the viral cycle. In this study, polymorphism analysis of the pvr2-eIF4E coding sequence in a worldwide sample of 25 C. annuum accessions identified 10 allelic variants with exclusively non-synonymous variations clustered in two surface loops of eIF4E. Resistance and genetic complementation assays demonstrated that pvr2 variants, each with signature amino acid changes, corresponded to potyvirus resistance alleles. Systematic analysis of the interactions between eIF4E proteins encoded by the 10 pvr2 alleles and VPgs of virulent and avirulent potato virus Y (PVY) and tobacco etch virus (TEV) strains demonstrated that resistance phenotypes arose from disruption of the interaction between eIF4E and VPg, and that viral adaptation to eIF4E-mediated resistance resulted from restored interaction with the resistance protein. Complementation of an eIF4E knockout yeast strain by C. annuum eIF4E proteins further shows that amino acid changes did not impede essential eIF4E functions. Altogether, these results argue in favour of a co-evolutionary 'arms race' between Capsicum eIF4E and potyviral VPg.

Allele mining in the pepper gene pool provided new complementation effects between pvr2-eIF4E and pvr6-eIF(iso)4E alleles for resistance to pepper veinal mottle virus.

Molecular cloning of recessive resistance genes to potyviruses in a large range of host species identified the eukaryotic translation initiation factor 4E (eIF4E) as an essential determinant in the outcome of potyvirus infection. Resistance results from a few amino acid changes in the eIF4E protein encoded by the recessive resistance allele that disrupt the direct interaction with the potyviral protein VPg. In plants, several loci encode two protein subfamilies, eIF4E and eIF(iso)4E. While most eIF4E-mediated resistance to potyviruses depends on mutations in a single eIF4E protein, simultaneous mutations in eIF4E (corresponding to the pvr2 locus) and eIF(iso)4E (corresponding to the pvr6 locus) are required to prevent pepper veinal mottle virus (PVMV) infection in pepper. We used this model to look for additional alleles at the pvr2-eIF4E locus that result in resistance when combined with the pvr6-eIF(iso)4E resistant allele. Among the 12 pvr2-eIF4E resistance alleles sequenced in the pepper gene pool, three were shown to have a complementary effect with pvr6-eIF(iso)4E for resistance. Two amino acid changes were exclusively shared by these three alleles and were systematically associated with a second amino acid change, suggesting that these substitutions are associated with resistance expression. The availability of new resistant allele combinations increases the possibility for the durable deployment of resistance against this pepper virus which is prevalent in Africa.

[The struggle for translation: how RNA phytoviruses use eukaryotic Initiation Factor 4E]. / La lutte pour la traduction: utilisation des facteurs d'initiation de la traduction eIF4E par les phytovirus à ARN.

Potyvirus are one of the largest groups of phytopathogenic virus and are responsible for significant agronomic loss. Host proteins belonging to the eukaryotic translation initiation complex, and particularly eIF4E (eukaryotic Initiation Factor 4E, which binds to the mRNA cap), play an important role in the success of a productive potyvirus infection. Plant eIF4E interacts with the viral VPg protein, which binds to the 5' end of the viral genome. In several plant species eIF4E isoforms have amino acid changes that prevent the interaction with VPg, leading to recessive resistance to potyviruses. Analysis of the natural variability of eIF4E and VPg proteins suggests that their diversity is structured by coevolution between the host and the pathogen. The role of the eIF4E and VPg interaction in the viral life cycle remains largely unknown: It could be involved in translation, replication or cell to cell trafficking of the viral genome. Genetic analysis shows that, besides eIF4E, other proteins of the translation initiation complex are likely to be involved in viral production. Furthermore, other groups of RNA virus require proteins of the translation initiation complex for completing their life cycle. This pinpoints the importance of translation initiation, a key target used by viruses to subvert the cell's machinery.

Translation initiation factors: a weak link in plant RNA virus infection.

Recessive resistance genes against plant viruses have been recognized for a long time but their molecular nature has only recently been linked to components of the eukaryotic translation initiation complex. Translation initiation factors, and particularly the eIF4E and eIF4G protein families, were found to be essential determinants in the outcome of RNA virus infections. Viruses affected by these genes belong mainly to potyviruses; natural viral resistance mechanisms as well as mutagenesis analysis in Arabidopsis all converged to identify the same set of translation initiation factors. Their role in plant resistance against RNA viruses remains to be elucidated. Although the interaction with the protein synthesis machinery is probably a key element for successful RNA virus infection, other possible mechanisms will also be discussed.

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.

Different mutations in the genome-linked protein VPg of potato virus Y confer virulence on the pvr2(3) resistance in pepper.

Five different amino acid substitutions in the VPg of Potato virus Y were shown to be independently responsible for virulence toward pvr2(3) resistance gene of pepper. A consequence of these multiple mutations toward virulence involving single nucleotide substitutions is a particularly high frequency of resistance breaking (37% of inoculated plants from the first inoculation) and suggests a potentially low durability of pvr2(3) resistance. These five mutants were observed with significantly different frequencies, one of them being overrepresented. Genetic drift alone could not explain the observed distribution of virulent mutants. More plausible scenarios were obtained by taking into account either the relative substitution rates, the relative fitness of the mutants in pvr2(3) pepper plants, or both.

Serological, Molecular, and Pathotype Diversity of Pepper veinal mottle virus and Chili veinal mottle virus.

ABSTRACT Variability within the pepper-infecting potyviruses Pepper veinal mottle virus (PVMV) and Chili veinal mottle virus (ChiVMV) in Africa and Asia was investigated. Coat protein (CP) gene sequence diversity revealed three clades that corresponded to three geographic locations and there was no evidence of presence of the ChiVMV/Asian group in western or central Africa. These clades included closely related isolates that potentially belong to two viral species, which is consistent with current nomenclature. These clades could not be unambiguously identified with polyclonal antisera; however, reverse transcription-polymerase chain reactions allowed differentiation of the isolates into two species based on a large indel in the CP gene. PVMV and ChiVMV isolates were classified into three and two pathotypes, respectively, in relation to pepper genotypes carrying different resistance factors. Specificity of resistance only partially corresponded to molecular diversity of the isolates. Only one isolate of PVMV could infect pepper genotypes carrying the two recessive genes pvr6 and pvr2 (2); however, these genotypes were not infected by PVMV in field trials in Senegal, despite a high prevalence of PVMV in the surrounding pepper plants.

Mutations in potato virus Y genome-linked protein determine virulence toward recessive resistances in Capsicum annuum and Lycopersicon hirsutum.

The recessive resistance genes pot-1 and pvr2 in Lycopersicon hirsutum and Capsicum annuum, respectively, control Potato virus Y (PVY) accumulation in the inoculated leaves. Infectious cDNA molecules from two PVY isolates differing in their virulence toward these resistances were obtained using two different strategies. Chimeras constructed with these cDNA clones showed that a single nucleotide change corresponding to an amino acid substitution (Arg119His) in the central part of the viral protein genome-linked (VPg) was involved in virulence toward the pot-1 resistance. On the other hand, 15 nucleotide changes corresponding to five putative amino acid differences in the same region of the VPg affected virulence toward the pvr2(1) and pvr2(2) resistances. Substitution models identified six and five codons within the central and C terminal parts of the VPg for PVY and for the related potyvirus Potato virus A, respectively, which undergo positive selection. This suggests that the role of the VPg-encoding region is determined by the protein and not by the viral RNA apart from its protein-encoding capacity.

Structural analysis of the eukaryotic initiation factor 4E gene controlling potyvirus resistance in pepper: exploitation of a BAC library.

The pvr2 locus in pepper codes for a eukaryotic translation initiation factor 4E (eIF4E) gene that confers resistance to viruses belonging to the potyvirus genus. In this work, we describe the isolation and characterisation of the genomic sequence carrying the pvr2 locus. A Bacterial Artificial Chromosome (BAC) library that consisted of 239,232 clones with an average insert size of 123 kilobases (kb) was constructed from a Capsicum annuum line with the pvr2(+) allele for susceptibility to potato virus Y (PVY) and tobacco etch virus (TEV). Based on a polymerase chain reaction (PCR) screen with single-copy markers, three to seven positive BAC clones per markers were identified, indicating that the BAC library is suitable for pepper genome analysis. To determine the genomic organization of the pepper eIF4E gene, the library was screened with primers designed from the cDNA sequence and four positive BAC clones carrying the pvr2 locus were identified. A 7-kb DNA fragment containing the complete eIF4E gene was sub-cloned from the positive BAC clones and analysed. The eIF4E gene is organised into five exons and four introns and showed a strictly conserved exon/intron structure with eIF4E genes from Arabidopsis thaliana and rice. Moreover, the splice sites between plant exons 1/2 and 2/3 are conserved among eukaryotes including human, Drosophila and yeast. Several potential binding sites for MADS box transcription factors within the 5' flanking region of eIF4E genes from the three plant species were also predicted.

The Am Gene Controlling Resistance to Alfalfa mosaic virus in Tomato Is Located in the Cluster of Dominant Resistance Genes on Chromosome 6.

ABSTRACT The dominant gene Am from Lycopersicon hirsutum f. sp. glabratum PI134417 confers resistance to most strains of Alfalfa mosaic virus, including the recently identified necrotic strains. The phenotypic response includes a lack of symptom development following mechanical inoculation of leaves. To study the resistance mechanism controlled by Am, biological (back-inoculation to susceptible hosts), serological (double-antibody sandwich, enzyme-linked immunosorbent assay), and molecular (reverse transcription-polymerase chain reaction and hybridization with specific riboprobes) methods of virus detection have been conducted on mechanically inoculated PI134417 leaves. The virus was never recovered, indicating that Am acts by an inhibition of viral accumulation during the early events of the virus life cycle. Am has been mapped genetically to the short arm of tomato chromosome 6 in the resistance hotspot, which includes the R-genes Mi and Cf-2/Cf-5 and the quantitative resistance factors Ty-1, Ol-1, and Bw-5.

Exploitation of natural genetic diversity to study plant-virus interactions: what can we learn from Arabidopsis thaliana?

The development and use of cultivars that are genetically resistant to viruses is an efficient strategy to tackle the problems of virus diseases. Over the past two decades, the model plant Arabidopsis thaliana has been documented as a host for a broad range of viral species, providing access to a large panel of resources and tools for the study of viral infection processes and resistance mechanisms. Exploration of its natural genetic diversity has revealed a wide range of genes conferring virus resistance. The molecular characterization of some of these genes has unveiled resistance mechanisms distinct from those described in crops. In these respects, Arabidopsis represents a rich and largely untapped source of new genes and mechanisms involved in virus resistance. Here, we review the current status of our knowledge concerning natural virus resistance in Arabidopsis. We also address the impact of environmental conditions on Arabidopsis-virus interactions and resistance mechanisms, and discuss the potential of applying the knowledge gained from the study of Arabidopsis natural diversity for crop improvement.