Le beet necrotic yellow vein virus (BNYVV) est un phytovirus qui possède un génome segmenté à ARN de polarité positive. L’ARN3 viral renferme le domaine « core » qui contient une séquence de 20 nucléotides appelée « coremin », indispensable au mouvement systémique du virus chez Beta macrocarpa. L’ARN3 subit un processus de dégradation qui conduit à la formation d’un ARN non codant (ncRNA3) correspondant à son extrémité 3’. Ce dernier est stabilisé par la séquence « coremin » à son extrémité 5’. Grâce à l’outil génétique levure, l’exoribonucléase Xrn1 puis l’exoribonucléase XRN4 de plante ont été identifiées comme étant responsable de l’accumulation du ncRNA3 à partir d’ARN3. Nous avons démontré in vitro que l’accumulation de ncRNA3 est liée au blocage de Xrn1 par « coremin ». La protéine virale p14, un suppresseur du RNA silencing codée par l’ARN2, est aussi nécessaire au mouvement systémique du virus et interagit avec la séquence « coremin ». Nos travaux confirment que l’ARN3 est capable de complémenter partiellement un mutant allélique de p14 dans l’infection locale et systémique. Nos résultats mettent en évidence un effet de la protéine p14 sur la systémie du RNA silencing et sur une éventuelle cible cellulaire RDR6. / The beet necrotic yellow vein virus (BNYVV) is a multipartite positive-stranded RNA phytovirus. The RNA3 contains a « core » sequence in which resides the « coremin » motif of 20 nucleotides absolutely required for the viral systemic movement in Beta macrocarpa. The RNA3 undergoes a process that produces a noncoding RNA3 (ncRNA3), stabilized by « coremin » at its 5’ end. Using a yeast genetic approach, the exoribonuclease Xrn1 and plant XRN4 have been identified as being responsible for the ncRNA3 accumulation from RNA3 processing. In vitro, we showed that the ncRNA3 accumulation is due to the stalling of Xrn1 by “coremin”. The viral p14 protein, an RNA silencing suppressor encoded by the RNA2, is also required for the systemic movement and interacts with the “coremin” sequence. Our studies demonstrated the ability of RNA3 to partially complement an allelic p14 mutant in local and systemic infections. Our data highlighted an effect of the p14 protein on the RNA silencing movement and on the potential cellular target RDR6.

Rhizomania, caused by beet necrotic yellow vein virus (BNYVV), has been considered as an economically important disease around the world because of the extreme reduction in sugar beet yield and sugar content in affected plants. The spread of rhizomania all over the world, including the emergence of resistance- breaking virus isolates, have been become a major concern for the plant pathologists and plant breeders aiming at improving sugar beet resistance to   BNYVV as well as better understanding sugar beet-virus interactions during disease development. The main focus of this project is to elucidate the role of P25-interacting partner, the VIP1 transcription factor, in activation of transcription.  The isolation of the gene-of-interest (VIP1) was performed by RT-PCR on total RNA preparations extracted from root tissue of sugar beet (Beta vulgaris ssp. vulgaris).  The isolated gene of interest was cloned using gateway system into a binary expression vector and the obtained construct was then transformed into Agrobacterium tumefaciens for analysing transient expression in the experimental host (Nicotinana benthamiana).  Dual-luciferase promoter activity assay was performed on isolated leaf discs co-expressing P25 and VIP1 and compared to appropriate controls.  Six promoter constructs were tested. However, we observed an increase in luciferase activity (1.8-4.2-fold) upon co-expression of P25 and VIP1 only for two constructs tested, although the increase was not supported by Student’s t-test at 0.05 significance level. Nevertheless, the luciferase activity assay data for these two constructs were consistent with RNA-seq and RT-qPCR data obtained previously showing upregulation of the expression of these two specific sugar beet genes during BNYVV infection in sugar beets.     Thus, the results support our hypothesis that the interaction of the virus virulence factor P25 with VIP1 transcription factor is needed to activate transcription of certain genes in the nucleus for the virus benefit.

No description available.