Figure 4

Effect of ectopically expressed GPRP on ClYVV virulence in infected broad bean. (A) Each of the recombinant ClYVV vectors possessing GFP, VfGPRP, or VfGPmycRP between P1 and HC-Pro cistrons was biolistically inoculated into broad bean leaves of four plants. (B) The reactions of broad beans inoculated with the recombinant ClYVV vectors, pClYVV/C3-S65T (GFP), pClYVV/VfGPRP (VfGPRP), or pClYVV/VfGPmycRP (VfGPmycRP), were evaluated at 13 and 19 days post-inoculation (dpi). The vertical axis indicates the number of plants showing the indexed symptoms. (C) The coat protein (CP) of ClYVV was detected in extract from a third upper leaf from the inoculated leaf by Western blotting with anti-CP polyclonal antibodies at 13 and 19 dpi. Since an extract from ClYVV/C3-S65T was no longer available at 19 dpi because of lethal necrosis, the extract from 13 dpi was applied for comparison of CP accumulation. VfGPmycRP protein was also detected using anti-Myc monoclonal antibody (Sigma-Aldrich, St Louis, MO, USA). Lane H is extract from a healthy broad bean leaf. CBB stained gels are shown as loading controls. (D) Viral genome cDNA including that of transgenes was detected in RNA extracts from the same leaves as used for Western blotting by reverse transcription-coupled PCR (RT-PCR) using primers 5'-GATGTACACGTGTGTCCAATGTCTTTTGG-3' and 5'-CTTATGGCATGCACATAATTGTTAACC-3', whose positions on the viral genome are shown in A. Lane P is PCR products amplified from the parental ClYVV vectors using the same primer pair. Lane H is the RT-PCR product with RNA extract from a healthy broad bean leaf. Lane M is a 100-bp ladder. (E) Photographs of tips of plants infected with recombinant ClYVVs.