Figure 3. Expression of Avr3b^P6497 in N. benthamiana increased Phytophthora susceptibility and suppressed ROS accumulation.

(A) Western blot assay of Avr3b^P6497 and GFP protein levels in N. benthamiana leaves following Agrobacterium-mediated transient expression. Anti-FLAG monoclonal antibody was used to detect levels of FLAG-tagged Avr3b^P6497 protein. Anti-GFP monoclonal antibody was used to detect GFP protein levels. (B) Real-time PCR measurement of pathogen and plant DNA ratios was used to determine P. capsici biomass in infected plant tissues following Agrobacterium-mediated transient expression of FLAG-Avr3b or GFP. Nicotiana benthamiana leaves infiltrated with Agrobacterium carrying different constructs were grown in a greenhouse for 48 hours then inoculated with agar plugs containing P. capsici mycelium. DNA from P. capsici infected regions was isolated at 36 hpi. Real-time PCR employed primers specific for the N. benthamiana and P. capsici actin genes. Statistical analysis was performed by the Wilcoxon rank sum test (asterisk indicates p<0.01). Bars represent standard errors from 6 replicates (two technical replicates each from three biological replicates). (C) Photomicrographs from P. capsici infected regions. Leaf regions transiently expressing Avr3b^P6497 or GFP, and inoculated with P. capsici for 16 hours, were stained with trypan blue. Bar  = 2.5 µm. IS: inoculation sites, SP: sporangia. (D) ROS generation during P. capsici infection following Avr3b^P6497 or GFP transient expression in N. benthamiana leaves at 12 hpi (upper panel) and 36 hpi (lower panel). Leaves were stained with DAB. Bar  = 5 mm. Typical results are shown from 4 replicates.