Fig. 9.

NbBeclin1 targets RdRps of plant viruses distinct to potyviruses and inhibits viral infection. a, b Schematic representation of the full-length CGMMV RdRp (a) and PepMV RdRp (b). The positions of the first and last amino acid residues of each conserved domain are indicated. Met methyltransferase domain, Hel helicase domain, RdRp2 RdRp2 domain. c NbBeclin1 interacts with CGMMV (CG) and PepMV (PE) RdRps or their RdRp2 domains but not with their GDD mutants or other domains, i.e., Met and Hel in the Y2H assays. d Co-IP analysis of possible interactions of NbBeclin1 with different domains or the GDD mutant of CGMMV and PepMV RdRps in planta. N. benthamiana leaves were agroinfiltrated with the plasmids indicated. Leaf extracts were incubated with GFP-Trap®_MA magnetic agarose beads (ChromoTek). Samples before (Input) and after (IP) immunopurification were analyzed by immunoblotting using GFP or Myc antibody. Yellow asterisks indicate the expected band sizes. e BiFC assays for the interaction of NbBeclin1 with different domains of CGMMV RdRp or PepMV RdRp in H2B-RFP transgenic N. benthamiana leaves at 48 hpi. Bars = 50 μm. f Quantification of CGMMV or PepMV RNA levels by qRT-PCR. The plants were pre-inoculated with buffer (mock), TRV1+TRV2-GUS, or TRV1+TRV2-NbBeclin1 for 7 days. RNA was extracted from CGMMV or PepMV–inoculated or systemically infected leaves at 3 dpi and 14 dpi, respectively. The values are presented as means of fold change ±SD relative to mock-treated plants. Error bars represent SD. Three independent experiments, each consisting of three biological replicates, were carried out. Values from one representative result were used to plot a histogram and were normalized with NbActin as the internal reference. The data were analyzed using Student’s t-test and double asterisks denote significant differences compared to the CGMMV- or PepMV-infected NbBeclin1-silenced plants from control plants pretreated with mock (two-sided, **P < 0.01). g, h Symptoms of CGMMV (g) or PepMV (h) infected plants at 21 dpi. The plants were pre-inoculated with buffer (mock), TRV-GUS, or TRV-NbBeclin1 for 7 days. Mock inoculated with buffer, CGMMV inoculated with CGMMV, PepMV inoculated with PepMV