FIG. 7.
The hrpP mutant secretes small amounts of the pilus subunit HrpA and fails to secrete AvrPto, HrpZ1, HrpW1, or HrpB-Cya and fails to translocate HrpB-Cya. (A) Wild-type DC3000, DC3000 ΔhrcQRSTU (CUCPB5113), and DC3000 ΔhrpP (CUCPB5453) were grown in HMM from an OD600 of 0.15 to 0.3. Cell pellet and supernatant fractions were separated by centrifugation. Proteins were separated by SDS-PAGE, transferred to the PVDF membrane, and subjected to immunoblot analysis using the indicated antibodies and secondary antibodies conjugated to alkaline phosphatase. CP, cell pellet; SN, supernatant. Bacteria in the bottom-most panel contained pCPP5688, which expresses HrpB-Cya from a hrp promoter. All other panels involved proteins expressed from native genes in the DC3000 genome. (B) Cya reporter translocation assay, performed as described in the legend for Fig. 3A, shows that HrpB-Cya translocation into N. benthamiana cells is impaired in DC3000 ΔhrpP. Values are the means and standard deviations from triplicate samples. The experiment was repeated three times with similar results.