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. 2009 Mar 6;191(9):3120–3131. doi: 10.1128/JB.01623-08

FIG. 6.

FIG. 6.

An HrpP-GFP fusion cannot be translocated but can still restore to the DC3000 ΔhrpP mutant the ability to elicit HR in tobacco and to secrete AvrPto in culture. (A) DC3000 and DC3000 ΔhrcQRSTU (CUCPB5113) expressing HrpP-GFP-Cya (pCPP5909) or HrpP-Cya (pCPP5697) from the vector-provided hrpP promoter were infiltrated into N. benthamiana at 3 × 108 CFU/ml. Leaf discs were excised 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. (B) DC3000 ΔhrpP expressing HrpP-GFP-Cya or HrpP-GFP-HA (pCPP5911) were grown overnight in HMM to an OD600 of 1.0. Cells were collected by centrifugation and lysed, and cellular proteins were separated by SDS-PAGE, transferred to the PVDF membrane, and subjected to immunoblot analysis using an anti-GFP antibody and secondary antibodies conjugated to alkaline phosphatase. (C) DC3000, DC3000 ΔhrcQRSTU (CUCPB5113), and DC3000 ΔhrpP (CUCPB5453) and DC3000 ΔhrpP expressing HrpP-GFP-Cya (pCPP5909), HrpP-Cya (pCPP5697), HrpP-GFP-HA (pCPP5911), or HrpP-HA (pCPP5653) were infiltrated into N. tabacum at 5 × 108 CFU/ml. Leaves were scored for visual collapse at 48 hpi. One representative leaf is shown. (D) Immunoblot analysis, performed as described in the legend for Fig. 5, indicates that AvrPto is secreted in culture by DC3000 ΔhrpP expressing HrpP-HA and HrpP-GFP-HA.