Cathy Dayan-Glick, Ludmila Maslenin, Yifat Iddan and Munir Mawassi Characterization of the grapevine virus A (GVA) AlkB motif and its involvement in plant viral infection Cathy Dayan-Glick, Ludmila Maslenin, Yifat Iddan and Munir Mawassi The S. Tolkowsky Laboratory, Department of Plant Pathology, The Virology Unit, Agricultural Research Organization, Volcani Center, Israel. Cathy Dayan-Glick kathy@volcani.agri.gov.il Introduction: The grapevine virus A (GVA), genus Vitivirus, family Betaflexiviridae, is closely associated with Rugose wood (RW) complex diseases. RW disease characterized by modifications of infected grape woody cylinder (fig. 1). Infected grapes show less vigorous growth and delayed bud opening, and may decline and die within a few years. GVA’s positive single-stranded RNA genome consists of five open reading frames (ORFs). ORF1 encodes replication-associated proteins, and an AlkB-like protein (fig. 2a). AlkB proteins have homologues in multicellular organisms, and involved in nucleic acids de-methylation mechanism. Their specific role in viruses is unknown. In this research, we have utilized GVA- and AlkB-mutated derived GVA clones (fig. 2b) to examine the role of viral AlkB motif in plant infection. Figure 1: Infected grapes by Rugose wood complex diseases. (a) Field-grown vine affected by rugose wood; (b) Stem grooving induced by GVA. (Van den Born et al., 2008) Figure 2: GVA point mutations in AlkB Sequence. (a) GVA schematic genome organization. (b) Viral AlkB sequence alignment. Blue arrows pointing towards conserved amino acids in all organisms. Red arrows pointing towards conserved amino acids only in viruses. Black arrow pointing towards a non-conserved amino acid in GVA-AlkB sequence (aa E). Each of the 7 amino acids was modified to Alanin. a b Figure 3: GVA viral symptoms appearance in N. benthamiana plants. Each average calculated using data from 7 plants. P<0.001 Figure 4: GVA replication in grapevine plantlets. Figure 5: GVA replication dynamics in N. benthamiana plants. Positive control (pJG4-5 + pSH17-4) Negative control (pJG4-5 + pRFHM1) p10 + AlkB p10 + p10 Figure 6: Protein:protein interactions using the Yeast Two-Hybrid system. Aim of research The role of AlkB in viral infection and plant RNA silencing suppression Methods 1. N. benthamiana plants were inoculated using agro-infiltration. 2. Grapevine plantlets were inoculated using agro-drenching. 3. Viral RNA replication was measured using Real-Time PCR. 4. Virus titer was determined using western-blot. 5. Protein-protein interaction was examined by a Yeast Two-Hybrid system. 6. Suppression of RNA silencing was examined by Confocal microscopy (using GFP as a reporter protein). GFP + p10 + AlkB GFP + p10 GFP GFP + p10 + R2 GFP + p10 + H1 GFP + p10 + E Figure 7: Examination of RSS activity of AlkB and p10 using Confocal microscopy. Results Figure 3: The mutations H1 and R2 inhibit symptoms appearance (6-10 days after WT-GVA) in N. benthamiana plants. Figure 4+5: The mutations H1, R2 and E, decrease viral RNA levels in N. benthamiana plants, as well as in grapevine plantlets. Figure 6: P10 exhibits self interaction and capable to interact with AlkB protein. Figure 7+8+9: Co expression of AlkB with p10 enhanced the RSS activity. The mutations H1, R2 inhibit the RSS activity of AlkB and p10. # The mutations H1, R2 and E decrease accumulation of the viral coat protein in N. benthamiana plants (data not shown). Figure 8: MICA image analysis of GFP levels (RSS activity). Figure 9: (a) GFP siRNA accumulation in N. benthamiana leaves, which were agro-infiltrated with a binary vector expressing: (1) Negative control; (2) GFP; (3) GFP+p10; (4) GFP+p10+AlkB; (5) GFP+p10+H1; (6) GFP+p10+R2; (7) GFP+p10+E. (b) EtBr stained RNA gel as an equal loading control. a b 1 2 3 4 5 6 7 21-24 bp Conclusions 1. In this research, we have found that co-expression of AlkB with p10 strongly suppresses the plant RNA silencing mechanism, and as a result enables GVA to accumulate in the host plant. 2. We have identified two amino acids (H1 and R2) at the GVA AlkB motif sequence, which affect RSS activity and GVA accumulation.