FIG. 7.
Translocation of AvrA::CyaA′ fusion proteins into plant cells through the TTSS. (A) Immunoblot analysis of AvrA1-99::CyaA′ protein fusions in supernatants and cellular lysates. Bacterial strains were cultivated in MMG supplemented with Congo red. Concentrated supernatant preparations (CSP-CR) (OUT) and concentrated lysate preparations (CLP-CR) (IN) were prepared from these cultures, and their protein contents were separated by SDS-PAGE and visualized by immunoblotting with a monoclonal antiserum raised against adenylate cyclase for the following strains: (lane 1) GMI1666 popF1 popF2 carrying pSC163 (pLAFR6::AvrA1-99::CyaA′), (lane 2) GMI1663popF1/pSC163, (lane 3) GMI1000/pSC163, (lane 4) GMI1000, (lane 5) GMI1664popF2/pSC163, and (lane 6) GMI1402hrcS/pSC163. Control experiments with an anti-LacI antibody that detected the intracellular LacI protein demonstrated that cell lysis could not account for the signals detected with anti-CyaA′ antibody (data not shown). (B) Cya translocation assays showing adenylate cyclase activity of the AvrA1-99::CyaA′ fusion protein. Measurements of cAMP detected in bottom special tobacco leaf cells 7 h postinoculation with derivatives of the wild-type strain GMI1000 and hrp and popF mutants. For a particular strain, the means and standard deviations were calculated from measurements of three independent samples. Bacterial strains were the following: (lane 1) GMI1666 carrying pSC163 (pLAFR6::AvrA1-99::CyaA′), (lane 2) GMI1663popF1/pSC163, (lane 3) GMI1000/pSC163, (lane 4) GMI1000, (lane 5) GMI1664popF2/pSC163, and (lane 6) GMI1402hrcS/pSC163.