FIG. 5.

N- and C-terminal truncations of HrpP reveal an atypical T3SS translocation signal and a C terminus that is important for function in promoting DC3000 secretion of AvrPto-Cya and elicitation of the HR in tobacco leaves. For the immunoblot data at the top of the figure, DC3000 ΔhrpP (CUCPB5453) carrying plasmids expressing full-length and truncated versions of HrpP (as described in the legend to Fig. 4) was grown in HMM from an OD₆₀₀ of 0.3 to 0.5. Cell pellets and supernatant fractions were separated by centrifugation. Supernatant proteins were separated by SDS-PAGE, transferred to the PVDF membrane, and subjected to immunoblot analysis using an anti-AvrPto antibody and secondary antibody conjugated to alkaline phosphatase. For the HR tests in the middle of the figure, the same DC3000 ΔhrpP derivatives were infiltrated into N. tabacum at 5 × 10⁸ CFU/ml. Leaves were scored for visual collapse at 48 hpi. +, HR; −, no HR. For the Hrp-Cya translocation tests at the bottom of the figure, full-length and truncated versions of hrpP were fused to Cya, in pCPP5371, which provides expression from the AvrPto promoter, and introduced into DC3000 or DC3000 ΔhrcQRSTU as indicated. Strains were infiltrated into N. benthamiana at 3 × 10⁸ CFU/ml. Leaf discs were taken at 7 hpi and assayed for cAMP (Assay Designs direct cyclic AMP kit). Total protein was measured by the Bradford method. Values are the means and standard deviation from triplicate samples. The experiment was repeated three times with similar results.