FIG. 1.

HrpJ is secreted in culture and translocated into plant cells by the DC3000 type III system. (A) DC3000 and the DC3000 hrcC mutant, both carrying pLN426, which encodes HrpJ fused to the hemagglutinin epitope (HrpJ-HA), were grown under conditions that induce type III secretion and separated into cell-bound and supernatant fractions as described in Materials and Methods. Samples were subjected to SDS-PAGE and immunoblot analysis using anti-HA or anti-NPTII antibodies. The NPTII protein is also encoded by pLN426 and is used here as a control for nonspecific cell lysis. HrpJ-HA was detected in supernatant fractions of DC3000 but not in supernatant fractions from the hrcC mutant, indicating that HrpJ is secreted by the DC3000 TTSS. (B) Adenylate cyclase (CyaA) assays with HrpJ-CyaA and AvrPto1-CyaA fusions were carried out by infiltration of Nicotiana benthamiana with DC3000 strains carrying construct pLN2234 or pLN1985, which produced either HrpJ-CyaA or AvrPto1-Cya (a type III effector known to be injected into plant cells), respectively. Plant tissue was harvested 10 h after infiltration, and cAMP levels were determined as described in Materials and Methods. Levels of cAMP are reported in picomoles of cAMP per microgram of protein, with standard errors. WT, wild type.