Evaluation of the RNA Silencing Suppression Activity of Three Cherry Virus F-Encoded Proteins.

Cherry virus F (CVF) is a newly emerged sweet cherry virus. CVF has been identified in a small number of countries and it has not been associated with discrete symptomatology. RNA silencing is a natural defense mechanism of plants against invaders that degrades viral RNA in a sequence-specific manner. As a counter-defense, plant viruses encode one or more RNA silencing suppressors (RSSs) interfering with the silencing pathway via several mechanisms. To identify putative RSSs, the three proteins (MP, CPL, CPS) encoded by the RNA2 of CVF were selected and separately cloned into the binary vector pART27. The clones were used for transient expression experiments in Nicotiana benthamiana leaves, using co-agroinfiltration with a GFP-expressing vector. In both CPL and CPS, a rapid decrease in fluorescence was recorded, comparable to the negative control, whereas the MP of CVF retained the GFP's fluorescence for a few days longer even though this was observed in a small number of infiltrated leaves. Further experiments have shown that the protein was not able to inhibit the cell-to-cell spread of the silencing signal; however, a putative interference with systemic silencing was recorded especially when the induction was carried out with double-stranded GFP RNA. Overall, our results indicate that the MP of CVF is putatively implicated in the suppression of RNA silencing, though further experimentation is needed to unveil the exact mode of action.

A novel grapevine badnavirus is associated with the Roditis leaf discoloration disease.

Roditis leaf discoloration (RLD), a graft-transmissible disease of grapevine, was first reported in Greece in the 1980s. Even though various native grapevine viruses were identified in the affected vines, the etiology of the disease remained unknown. In the present study, we used an NGS platform for sequencing siRNAs from a twenty-year old Roditis vine showing typical RLD symptoms. Analysis of the NGS data revealed the presence of various known grapevine viruses and viroids as well as a hitherto uncharacterized DNA virus. The circular genome of the new virus was fully reassembled. It is 6988 nts long and includes 4 open reading frames (ORFs). ORF1, ORF2 and ORF4 code for proteins with unknown functions while ORF3 encodes a polyprotein with motifs related to the replication, encapsidation and movement of the virus. Phylogenetic analysis classified the novel virus within the genus Badnavirus, with closest relationship to Fig badnavirus 1. Further studies showed that the new badnavirus is closely related with the RLD disease and the provisional name grapevine Roditis leaf discoloration-associated virus (GRLDaV) is proposed. Our findings extend the number of DNA viruses identified in grapevine, further drawing attention to the potential importance of this virus group on grapevine pathology.