Jean-Luc Gallois

The recessive potyvirus resistance gene pot-1 is the tomato orthologue of the pepper pvr2-eIF4E gene

The translation initiation factor 4E (eIF4E) has been implicated in naturally occurring resistance to Potato virus Y (PVY) determined by the pvr2 locus in pepper (Capsicum annuum). Here, the molecular basis of the recessive resistance to PVY and Tobacco etch virus (TEV) controlled by the pot-1 locus in tomato (Lycopersicon esculentum; now Solanum lycopersicum) was investigated. On the basis of genetic mapping data that indicated that pot-1 and pvr2 are located in syntenic regions of the tomato and pepper genomes, the possible involvement of eIF4E in pot-1-mediated resistance was assessed. Genetic mapping of members of the eIF4E multigenic family in tomato introgression lines revealed that an eIF4E locus indeed maps in the same genomic region as pot-1. By comparing eIF4E coding sequences between resistant and susceptible Lycopersicon genotypes, a small number of polymorphisms that co-segregate with the pot-1 locus were identified, suggesting that this gene could be involved in resistance to potyviruses. Functional complementation experiments using Potato virus X-mediated transient expression of eIF4E from a susceptible genotype in a resistant pepper genotype confirmed that a small number of amino acid substitutions in the eIF4E protein indeed account for resistance/susceptibility to both the PVY and TEV, and consequently that pot-1 and pvr2 are orthologues. Taken together, these results support the role of this eIF4E gene as a key component of recessive resistance to potyviruses, and validate the comparative genomic approach for the molecular characterization of recessive resistance genes.

AtPRD1 is required for meiotic double strand break formation in Arabidopsis thaliana

The initiation of meiotic recombination by the formation of DNA double‐strand breaks (DSBs) catalysed by the Spo11 protein is strongly evolutionary conserved. In Saccharomyces cerevisiae, Spo11 requires nine other proteins for meiotic DSB formation, but, unlike Spo11, few of these proteins seem to be conserved across kingdoms. In order to investigate this recombination step in higher eukaryotes, we have isolated a new gene, AtPRD1, whose mutation affects meiosis in Arabidopsis thaliana. In Atprd1 mutants, meiotic recombination rates fall dramatically, early recombination markers (e.g., DMC1 foci) are absent, but meiosis progresses until achiasmatic univalents are formed. Besides, Atprd1 mutants suppress DSB repair defects of a large range of meiotic mutants, showing that AtPRD1 is involved in meiotic recombination and is required for meiotic DSB formation. Furthermore, we showed that AtPRD1 and AtSPO11‐1 interact in a yeast two‐hybrid assay, suggesting that AtPRD1 could be a partner of AtSPO11‐1. Moreover, our study reveals similarity between AtPRD1 and the mammalian protein Mei1, suggesting that AtPRD1 could be a Mei1 functional homologue.

The Arabidopsis Proteasome RPT5 Subunits Are Essential for Gametophyte Development and Show Accession-Dependent Redundancy

We investigated the role of the ubiquitin proteasome system (UPS), which allows proteins to be selectively degraded, during gametophyte development in Arabidopsis thaliana. Three mutant alleles altering the UPS were isolated in the Wassilewskija (Ws) accession: they affect the Regulatory Particle 5a (RPT5a) gene, which (along with RPT5b) encodes one of the six AAA-ATPases of the proteasome regulatory particle. In the heterozygous state, all three mutant alleles displayed 50% pollen lethality, suggesting that RPT5a is essential for male gametophyte development. However, a fourth mutant in the Columbia (Col) accession did not display such a phenotype because the RPT5b Col allele complements the rpt5a defect in the male gametophyte, whereas the RPT5b Ws allele does not. Double rpt5a rpt5b mutants showed a complete male and female gametophyte lethal phenotype in a Col background, indicating that RPT5 subunits are essential for both gametophytic phases. Mitotic divisions were affected in double mutant gametophytes correlating with an absence of the proteasome-dependent cyclinA3 degradation. Finally, we show that RPT5b expression is highly increased when proteasome functioning is defective, allowing complementation of the rpt5a mutation. In conclusion, RPT5 subunits are not only essential for both male and female gametophyte development but also display accession-dependent redundancy and are crucial in cell cycle progression.

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.

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.

Evolution of plant eukaryotic initiation factor 4E (eIF4E) and potyvirus genome-linked protein (VPg): A game of mirrors impacting resistance spectrum and durability

Polymorphism in the plant eukaryotic translation initiation factor 4E (eIF4E) and potyvirus genome-linked protein (VPg) determine, in many cases, the outcome of the confrontation between these two organisms: compatibility (i.e. infection of the plant by the virus) or incompatibility (i.e. resistance of the plant to the virus). The two interacting proteins eIF4E and VPg show strikingly similar evolution patterns. Most codon positions in their coding sequences are highly constrained for nonsynonymous substitutions but a small number shows evidence for positive selection. Several of these latter positions were shown to be functionally important, conferring resistance to the host or pathogenicity to the virus. Determining the mutational pathways involved in pepper eIF4E diversification revealed a link between an increase of the pepper resistance spectrum towards a panel of potyvirus species and an increase of durability of the resistance towards Potato virus Y. This relationship questions the interest of using more generally the spectrum of action of a plant resistance gene as a predictor of its durability potential.

eIF4E Resistance: Natural Variation Should Guide Gene Editing

eIF4E translation initiation factors have emerged as major susceptibility factors for RNA viruses. Natural eIF4E-based resistance alleles are found in many species and are mostly variants that maintain the translation function of the protein. eIF4E genes represent major targets for engineering viral resistance, and gene-editing technologies can be used to make up for the lack of natural resistance alleles in some crops, often by knocking out eIF4E susceptibility factors. However, we report here how redundancy among eIF4E genes can restrict the efficient use of knockout alleles in breeding. We therefore discuss how gene-editing technologies can be used to design de novo functional alleles, using knowledge about the natural evolution of eIF4E genes in different species, to drive resistance to viruses without affecting plant physiology.

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.

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

BackgroundIn 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.ResultsA 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.ConclusionWe 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.

Functional characterization of the plant ubiquitin regulatory X (UBX) domain-containing protein AtPUX7 in Arabidopsis thaliana

p97/CDC48 is a major AAA-ATPase that acts in many cellular events such as ubiquitin-dependent degradation and membrane fusion. Its specificity depends on a set of adaptor proteins, most of them containing the ubiquitin regulatory X (UBX) domain. Using a differential hybridization system, we isolated a UBX-containing protein that is expressed during the early phase of male gametophyte development in the crop Brassica napus and isolated and characterized its closest Arabidopsis thaliana homolog, AtPUX7. The AtPUX7 gene is expressed broadly in both the sporophyte and gametophyte due to regulation inferred by its first intron. The subcellular localization of AtPUX7 was assigned mainly to the nucleus in both the sporophyte and in pollen, mirroring the AAA-ATPase AtCDC48A localization. Furthermore, AtPUX7 interacts specifically with AtCDC48A in yeast as well as in planta in the nucleus. This interaction was mediated through the AtPUX7 UBX domain, which is located at the protein C-terminus, while an N-terminal UBA domain mediated its interaction with ubiquitin. Consistent with those results, a yeast-three hybrid analysis showed that AtPUX7 can act as a bridge between AtCDC48A and ubiquitin, suggesting a role in targeted protein degradation. It is likely that AtPUX7 acts redundantly with other members of the Arabidopsis PUX family because a null Atpux7-1 mutant does not display obvious developmental defects.