Phosphorylated status of ChvI regulates T6SS expression and Hcp secretion.

<p><i>A. tumefaciens</i> wild-type C58 strain containing the empty vector (V) or one of the plasmids expressing ChvG, ChvI, ChvI(D52E) or ChvI(D52A) grown in AB-MES (pH 7.0 or 5.5) at 25°C for 6 h was analyzed for T6SS expression and Hcp secretion. (<b>A</b>) Total proteins were analyzed by western blot analysis with antibodies against C-IcmF, Fha1 (filled arrow), Atu4343, ClpV, Hcp, Atu4349, and RpoA. (<b>B</b>) qRT-PCR analysis of mRNA levels of <i>fha1</i>, <i>atu4343</i>, <i>hcp</i>, and <i>atu4349</i>. Data are mean ± SD of 2 biological replicates, each of which contains 3 technical replicates. (<b>C</b>) Total (T) and secreted (S) proteins were analyzed by western blot analysis with antibodies against Hcp and RpoA. (<b>D</b>) Total and secreted (Sup) proteins were analyzed by western blot analysis with antibodies against C-IcmF, Fha1 (filled arrow), Atu4343, Hcp, and RpoA. RpoA was an internal control. The positions of molecular mass markers (in kDa) are indicated on the left.