Identification and Characterization of the suppressors of RNAi encoded by Grapevine leafroll-associated virus 3

Abstract

Grapevine leafroll-associated virus 3 (GLRaV-3) infection results in grapevine leafroll disease and impairs viticulture worldwide. No natural resistance against GLRaV-3 is reported in grapevines therefore alternative protection is required. Since viral suppressors of RNAi (VSRs) are key factors in symptom induction this project sought to extend knowledge of the single VSR (p19.7) to characterise all GLRaV-3 VSRs. To identify even weak VSRs and to predict VSR-target(s) within plant RNAi pathways, five local and three systemic RNAi assays were used to screen nine Group I GLRaV- 3 proteins and to compare VSR activity among five sequence variants of p19.7. VSR activity of each GLRaV-3 protein co-infiltrated with either of three RNAi inducers (eGFP, mGFP or hpGFP) were assessed by GFP fluorescence and mRNA accumulation in two Nicotiana benthamiana plant variants (WT and 16c) using a negative control (GUS) or positive controls (Citrus tristeza virus) p23 (a local VSR) and p20 (a local and systemic VSR). Of GLRaV-3 p6, p5, p55, CP, p21, p19.6, p19.7, p4 and p7, only CP, p21, and p19.6 were identified as new-to-science VSRs. The order of local VSR strength was CP < p21 and p19.6 < p19.7. Only GLRaV-3 p19.7 suppressed RNA-directed DNA methylation and blocked development of peripheral red halos, hypothesised by binding small interfering RNAs. CP, p21 and p19.6 derepressed GFP in stomatal guard cells, whereas p19.7 additionally de-repressed GFP in mesophyll cells. The local VSR activities of CP, p21 or p19.6 were markedly reduced with p4 or p5, but minutely with p55. The order of systemic VSR strength was CP < p19.7< p21 and p19.6. The order of local VSR strength of p19.7 sequence variants was Group III < Groups I, II and VI < Group X but for systemic activity was Group VI and X < Group III < Groups I and II. Presence of conserved arginine-glycine and/or lysine-arginine DNA/dsRNA binding motifs may explain differences in putative RNA binding of p19.7. The presence of four GLRaV-3-encoded VSRs and p19.7’s inter-group VSR variation sheds some light on GLRaV-3’s ability to cause leafroll disease and may inform development of new plant protection strategies such as mild strain cross-protection.