FIG 1.

PPV HCPro expressed in trans complements the infectivity deficiency of PPV-P1P1b progeny. (A) Schematic representation of the viral cDNA of the PPV-P1P1b clone. (B) Experimental approach followed for testing the effect of HCPro added in trans on PPV-P1P1b infectivity. (1) N. benthamiana plants were infected with PPV-P1P1b by biolistic inoculation; (2) 2 weeks later, systemically infected leaves were infiltrated with cultures of A. tumefaciens strains carrying different binary vectors (the expression of the indicated proteins supplemented in trans by agroinfiltration was confirmed by carrying out an RNA-silencing suppression test of these constructs in parallel); (3) agroinfiltrated patches were harvested 5 days after infiltration; and (4) extracts prepared from these patches were used for manual inoculation of N. clevelandii plants. (C) Representative photographs taken under an epifluorescence microscope of N. clevelandii leaves from two plants inoculated with extracts from N. benthamiana plants infected with PPV-P1P1b and supplemented by agroinfiltration with the indicated proteins. —, empty vector. (D) Western blot analysis of the native extracts from the infected and agroinoculated N. benthamiana plants (two plants per sample) used to inoculate the N. clevelandii plants shown in panel C. A polyclonal serum specific for PPV CP and a monoclonal antibody raised against GFP were utilized for viral accumulation assessment. Protein sizes indicated on the right (in kilodaltons) were estimated from the positions of prestained molecular mass markers (New England BioLabs) run in the same gel.