Figure 6
Overexpression of Hsc70-2 alleviated the inhibitory effect of CP on BBSV replication. (a) Schematic representation of plasmids used in Agrobacterium tumefaciens-mediated infection assay. Rectangles show ORFs in the BBSV genome. A stop codon read-through (RT) between p23 and p82 is illustrated by a vertical dashed line. Rz represents a cis-acting ribozyme. For these BBSV variants, the substitutions of these residues in the wild-type BBSV are linked by dashed lines across the expanded regions to indicate their sequence positions. The names of the mutants are indicated on the left. (b) Northern blot analysis of viral RNAs accumulation in N. benthamiana leaves agroinfiltrated with different BBSV-derived mutants. (c) Analysis of the BBSVmMP accumulation in leaves transiently expressing GFP (OD600 = 1.0), CP-Flag (OD600 = 0.1) plus GFP (OD600 = 1.0) or CP-Flag (OD600 = 0.1) plus Flag-Hsc70-2 (OD600 = 1.0). The replication-deficient mutant, BBSVmGDD, served as the negative control. Bands corresponding to gRNA and sgRNAs as well as the antibodies used for Western blot analysis in figures b and c are indicated on the right. Methylene blue-stained rRNAs and CBB-stained rbcL were used as RNA and protein loading controls, respectively. Open arrow and solid arrowheads indicate bands corresponding to wild-type CP and CP-Flag, respectively. Numbers below rRNA loading panel represent relative abundance of BBSV genomic RNA in each treatment group normalized to that of the GFP group (n = 3 per group).
