Figure 5.

Immunoblot analysis of AvrPto secretion by Pto DC3000 derivatives with deletions affecting the three major regions of the Hrp Pai. Bacteria were grown in Hrp-inducing minimal medium at pH 5.5 and 22°C to an OD₆₀₀ of 0.35 and then separated into cell-bound (C) and supernatant (S) fractions by centrifugation. Proteins were then resolved by SDS/PAGE, blotted, and immunostained with antibodies against AvrPto and β-lactamase as described (18), except that supernatant fractions were concentrated 3-fold relative to cell-bound fractions before loading. Pto DC3000, CUCPB5115 (CEL deletion), CUCPB5114 (hrp/hrc deletion), and CUCPB5110 (EEL deletion) all carried pCPP2318, which expresses β-lactamase without a signal peptide as a cytoplasmic marker.