Thèse Etude des Mécanismes Impliqués dans la Recombinaison et l'Exclusion de la Surinfection dans les Interactions Plante-Géminivirus-Aleurode Vecteur et la Transmission de Virus H/F - Doctorat.Gouv.Fr

Établissement : Institut Agro Montpellier École doctorale : GAIA - Biodiversité, Agriculture, Alimentation, Environnement, Terre, Eau Laboratoire de recherche : PHIM - Plant Health Institute Montpellier Direction de la thèse : MIKHAIL POOGGIN ORCID 000000032308393X Début de la thèse : 2026-10-01 Date limite de candidature : 2026-05-07T23:59:59 Les bégomovirus à ADN simple brin circulaire (genre Begomovirus, famille Geminiviridae) et leurs satellites sont transmis par l'aleurode Bemisia tabaci de manière persistante et circulante, causant de graves maladies dans les plantes cultivées dans le monde entier. La recombinaison et les réassortiments sont des facteurs d'évolution important chez les bégomovirus. Ils surviennent suite à la rencontre de différentes espèces dans les plantes coinfectées ou les aleurodes virulifères. Ces phénomènes génèrent des recombinants et réassortants qui peuvent avoir des propriétés différentes de leurs parents et devenir invasifs en échappant aux défenses de l'hôte ou à l'exclusion de la surinfection. L'émergence et les propriétés de recombinants hautement compétitifs du TYLCV (virus des feuilles jaunes en cuillère de la tomate) caractérisés par des échanges de courtes séquences dans la région d'origine de réplication, ont été étudiées en detail dans l'UMR PHIM, mais les mécanismes qui sous-tendent leur émergence sont encore largement mal compris. Nous utiliserons une combinaison de séquençage Illumina, séquençage Nanopore et l'approche VIGS (virus induced gene silencing) pour disséquer les mécanismes de recombinaison virale, d'évasion des défenses et d'exclusion de la surinfection dans un pathosystème tomate-bégomovirus-aleurode. Sur la base des connaissances mécanistiques obtenues, nous concevrons et testerons des variants de TYLCV capables de bloquer la surinfection par les recombinants invasifs. Les résultats attendus apporteront un nouvel éclairage sur les mécanismes sous-jacents aux interactions plante-virus-insecte vecteur dans les infections mixtes et sur l'évasion virale des défenses de l'hôte par recombinaison. Ils contribueront au développement de nouvelles stratégies pour contrôler la réplication et la transmission des recombinants géminivirus invasifs. Owing to high propensity for recombination, geminiviruses generate new recombinant strains of parental viruses with short swaps of viral genome sequences and new recombinant viral species with long swaps thereof. Successful recombinants can outcompete parental viruses in co-infected plants and be transmitted by insect vectors to susceptible host plants and, if capable of breaking resistance, to resistant variants of host plants and non-host plants (Lefeuvre and Moriones 2015). Cica Urbino (team Camepi) and Michel Peterschmitt (team DefensiRNA) have documented the emergence in Morocco and spread of a TYLCV recombinant IS76 that acquired from a related begomovirus TYLCSV a 76 nt sequence within the origin of replication/bidirectional promoter region (Belabess et al. 2015; Urbino et al. 2022). IS76 outcompetes the parental viruses in co-infections, infects plants carrying the begomovirus resistance gene Ty-1 (Belabess et al. 2016) and overcomes superinfection exclusion following challenge-inoculation of plants infected with parental viruses (Jammes et al. 2023). In collaboration with Mikhail Pooggin (DefensiRNA), Illumina-seq profiling of transcriptome and small RNAome have been undertaken to begin uncovering the mechanisms underlying IS76 competitiveness and ability to break Ty-1 resistance mediated by Ty-1 gene-encoded RNA-dependent RNA polymerase (RDR). IS76 recombination event was found to facilitate virus replication and promote expression of its silencing suppressor and coat protein genes, thereby allowing the recombinant virus to evade the negative impact of RDR-boosted production of virus-derived small interfering (si)RNAs directing transcriptional and posttranscriptional silencing of viral genes (Jammes et al. 2024). Interestingly, another TYLCV recombinant, IS141, with a 141 nt swap in the same region has emerged in Italy and shown by Cica Urbino and co-workers to outcompete the parental viruses in co-infections and to overcome Ty-1 resistance (Urbino et al. 2020).
Begomoviruses replicate via rolling-circle replication (RCR) and recombination-dependent replication (RDR), generating respectively the circular ssDNA encapsidated in virions and the linear concatemeric double-stranded (ds)DNA molecules (Preiss & Jeske 2003) implicated in both resurrection of circular ssDNA genomes and evasion of the antiviral RNA interference (RNAi) generating virus-derived siRNAs (Pooggin 2013). We will investigate involvement of RCR vs RDR in emergence of IS76 and IS76-like recombinants and in their ability to evade RDR resistance and superinfection exclusion.
Superinfection exclusion and related cross-protection phenomena are exploited in control of severe viral strains by pre-inoculating crop plants with mild strains of some RNA viruses such as citrus tristeza virus (CTV) (Ziebell & Carr 2010; Folimonova 2020). The mechanisms underlying these phenomena are still poorly understood (Perdoncini et al. 2022) and can potentially depend on antiviral RNAi and/or viral proteins. Mikhail Pooggin together with Folimonova's team in Florida (USA) found that mild and severe strains of CTV have similar profiles of viral siRNAs (Aknadibossian et al. 2023), consistent with viral protein-mediated cross-protection in the CTV pathosystem (Folimonova 2020). Cica Urbino and co-workers obtained evidence that cross-protection operates in the TYLCV pathosystem, but is readily overcome by IS76 in both susceptible and Ty-1 resistant plants via an unknown mechanism. We will investigate the mechanisms of superinfection exclusion and its evasion by IS76. In-depth knowledge of these mechanisms should help us to design mild cross-protective variants of TYLCV that block superinfection with IS76 and other invasive recombinants.
Begomoviruses are often associated with ssDNA betasatellites that depend on helper viruses for replication, encapsidation and transmission and encode a single protein suppressing RNAi. Cica Urbino, Michel Peterschmitt and co-workers have shown that coinfection of TYLCV with a betasatellite compromises Ty-1 resistance (Conflon et al. 2018). In an ongoing Indo-French CEFIPRA project in collaboration with Himalayan Institute of Bioresources and Technology (Palumpur, India), the PhD student Fani Gousi (team DefensiRNA) generated, under co-supervision of Mikhail Pooggin and Cica Urbino, a virus-induced gene silencing (VIGS) vector based on a disarmed betasatellite (lacking RNAi suppressor), which is capable of efficient transreplication with helper begomovirus TYLCV, transmission by whiteflies, and silencing of tomato genes such as PCNA encoding a DNA replication processivity factor. We will use this VIGS vector to validate involvement of candidate host genes in viral replication and recombination as well as in RDR-mediated resistance and superinfection exclusion and their evasion by IS76 and IS141. The candidate genes will be selected based on published research, further analysis of the transcriptome and sRNAome resources generated by a former PhD student in the team DefensiRNA (Margaux Jammes) for TYLCV and IS76 in susceptible and Ty-1 plants (Jammes et al. 2024) and new omics data to be generated in this project.
1. Investigate the involvement of rolling-circle and recombination-dependent replication mechanisms in emergence of invasive recombinants of tomato yellow leaf curl virus (TYLCV) and TYLCV-like geminiviruses and in their ability to evade host resistance and superinfection exclusion
2. Dissect the mechanisms of superinfection exclusion and its evasion by resistance-breaking recombinants of TYLCV and TYLCV-like geminiviruses
3. Design and test mild cross-protective variants of TYLCV that block superinfection with its invasive recombinants
Susceptible and Ty-1 resistant plants will be co-inoculated with TYLCV and TYLCSV or mock-inoculated and leaf samples will be collected at 30 and 120 days post-inoculation for DNA and RNA extraction. B. tabaci whiteflies will be placed at respective plants at both time points for virus acquisition, followed by DNA and RNA extraction. Viral circular ssDNA and linear dsDNA products of RCR and RDR, respectively, will be analyzed by rolling-circle amplification (RCA), followed by Illumina and Nanopore sequencing (RCR products), and by direct Nanopore sequencing (RDR products). By bioinformatic analysis we will characterize recombination breakpoints and frequencies in RCR vs RDR products (followed by validation by (q)PCR) to evaluate relative contributions of RDR vs RCR to IS76 and other recombination events. A former PhD student co-supervised by Cica Urbino and Michel Peterschmitt has documented with PCR the emergence of IS76 and IS141 recombinants following co-inoculation of susceptible and Ty-1 plants with TYLCV and TYLCSV (Belabess et al. 2018). Our preliminary Nanopore-seq analysis in single infections confirmed the linear concatemeric dsDNA structures of RDR products deduced by 2D gel blot hybridization (Preiss and Jeske 2003) and revealed new mechanistic details of RDR. Comparative analysis of the plant vs whitefly samples will be done to evaluate competitiveness of different recombinants in encapsidation and movement to and within phloem sieve tubes where whiteflies acquire virions. The plant RNA samples will be analyzed by Illumina mRNA and sRNA sequencing to identify differentially expressed host genes (DEGs) and profile viral transcriptome and sRNAome at different conditions as described by Jammes et al. (2024). Likewise, Illumina sequencing of whitefly RNA samples will be done to evaluate insect transcriptome responses to circulating viruses and their recombinants and to explore potential virus replication in insect cells that would trigger antiviral RNAi. Indeed, TYLCV was reported to replicate in the whitefly salivary glands (He et al. 2020). We will also explore if recombination can occur in whitefly cells co-replicating TYLCV and TYLCSV.
To investigate the mechanisms of superinfection exclusion and its evasion by TYLCV recombinants, susceptible and Ty-1 plants infected with TYLCV, TYLCSV and their combination will be superinfected with TYLCV, IS76 or their derivatives with point mutations in the cis-elements of the origin of replication/bidirectional promoter region identified previously by Urbino et al. (2022) and Jammes et al. (2024).
Involvement of candidate tomato genes in recombination (RAD51 recombinase, its paralogs, etc.), RNAi (Dicers, RDR, etc.) and superinfection exclusion (RNAi genes, other DEGs) will be evaluated using our disarmed betasatellite VIGS approach. In coinfection experiments, betasatellite VIGS constructs carrying inserts of tomato genes will be co-inoculated with TYLCV and TYLCSV, while in superinfection experiments, the VIGS constructs will be co-inoculated with the protective or the challenging virus (TYLCV or IS76). Analysis of DNA and RNA samples from selected plants will be done as described above to evaluate alterations in recombination profiles and frequencies and in viral gene expression and antiviral RNAi responses to co-infecting or superinfecting viruses.
Based on the obtained results we will design and test TYLCV derivatives capable of blocking superinfection with IS76 and IS141 in susceptible and Ty-1 plants.

Formation en Biologie Moléculaire, Bioinformatique; Phytopathologie ou Biologie Végétale