Fig. 5.

Overexpression of NbBeclin1 promotes autophagy-dependent degradation of NIb and inhibits TuMV replication. a, b Immunoblotting of total protein extracts from the N. benthamiana leaves agroinfiltrated with buffer (−) or the plasmids indicated. The membrane was probed with GFP (@GFP), or Myc antibodies (@Myc). c Immunoblotting analysis of total protein extracts from leaves infiltrated with buffer (−) or TuMV-CFP-NIb together with an empty vector (Vec) or Myc-NbBeclin1 with antibodies against GFP or Myc. d Quantification of TuMV RNA levels by qRT-PCR. RNA was extracted from leaves agroinfiltrated with TuMV-CFP-NIb together with Vec or Myc-NbBeclin1 at 60 hpi. Values represent means ± SD relative to plants infiltrated with TuMV-CFP-NIb and Vec (n = 3 biological replicates). The data were analyzed using Student’s t-test and asterisks denote significant differences between the two treatments (two-sided, **P < 0.01). e Immunoblotting analysis of total protein extracts from leaves co-infiltrated with 6K2-GFP and buffer (−), empty vector (Vec), or Myc-NbBeclin1. Antibodies against GFP or Myc were used as a primary antibody. f, g The effect of the autophagy inhibitor 3-MA on the NbBeclin1-mediated degradation of NIb-YFP or TuMV-CFP-NIb. Total proteins were isolated from plant leaves agroinfiltrated with NIb-YFP alone or with Myc-NbBeclin1 (f) or TuMV-CFP-NIb alone or with Myc-NbBeclin1 (g) followed by DMSO or 3-MA treatment. h, i The effect of silencing of NbATG8a on the NbBeclin1-mediated degradation of NIb-YFP or CFP-NIb. Plants inoculated with TRV-GUS or TRV-NbATG8a at 14 dpi were agroinfiltrated with NIb-YFP alone or with Myc-NbBeclin1 (h) or TuMV-CFP-NIb alone or with Myc-NbBeclin1 (i). Total protein was extracted from infiltrated leaves at 3 dpi. Immunoblotting was performed using GFP or Myc antibodies. All immunoblotting assays in this figure were repeated at least three times, and one representative blot was shown. CBB staining of Rubisco large subunit serves as a loading control