Identification of Ralstonia solanacearum exoproteins secreted by the type two secretion system using proteomic techniques

Ralstonia solanacearum, the causal agent of bacterial wilt, has a functional type II secretion system (T2SS), which uses a pore formed by SdpD1 to secret cell wall degrading enzymes and other proteins. Deletion of sdpD1 in R. solanacearum GMI1000 produced a mutant reduced in virulence, colonization and secretion of many proteins. However, some proteins are still secreted by the sdpD1 mutant. GMI1000 is predicted to have three incomplete T2SS that include SdpD-related proteins that could be partially functional. To identify proteins secreted via the principle T2SS and alternative T2SS, all sdpD genes were deleted using unmarked mutagenesis. Proteomic techniques such as two-dimensional gel electrophoresis and peptide labeling with isobaric tags followed by mass spectrometry allowed the identification of proteins secreted through the principle T2SS. Although candidates for the alternative T2SS were not identified, functionality of the SdpD-related proteins was not ruled out.