FIG. 3.

HrpP-Cya is translocated into N. benthamiana cells by DC3000 at levels comparable to those of type III effectors when expressed from its native hrp promoter, but it is not secreted in culture. (A) pCPP5297 (HrpP-Cya), pCPP5716 (HopA1-Cya), pCPP5312 (AvrPto-Cya), and pCPP5898 (HrpW1-Cya) were introduced into wild-type DC3000 or the T3SS⁻ derivative DC3000 ΔhrcQRSTU (CUCPB5113). Strains were infiltrated into N. benthamiana at 3 × 10⁸ CFU/ml. Leaf discs were excised at 7 h postinfiltration and assayed for cAMP (using the 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. (B) DC3000 and DC3000 ΔhrcQRSTU (CUCPB5113) carrying pCPP5614 (expressing HrpP-HA from the nptII promoter) 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 polyvinylidene difluoride (PVDF) membrane, and subjected to immunoblot analysis using the indicated antibodies (indicated by α) and secondary antibodies conjugated to alkaline phosphatase. CP, cell pellet; SN, supernatant.