Pyramiding Unmarked Deletions in Ralstonia solanacearum Shows That Secreted Proteins in Addition to Plant Cell-Wall-Degrading Enzymes Contribute to Virulence

Abstract

Ralstonia solanacearum, like many phytopathogenic bacteria, makes multiple extracellular plant cell-wall-degrading enzymes (CWDE), some of which contribute to its ability to cause wilt disease. CWDE and many other proteins are secreted to the milieu via the highly conserved type II protein secretion system (T2SS). R. solanacearum with a defective T2SS is weakly virulent, but it is not known whether this is due to absence of all the CWDE or the loss of other secreted proteins that contribute to disease. These alternatives were investigated by creating mutants of wild-type strain GMI1000 lacking either the T2SS or up to six CWDE and comparing them for virulence on tomato plants. To create unmarked deletions, genomic regions flanking the target gene were polymerase chain reaction (PCR)-amplified, were fused using splice overlap extension PCR, were cloned into a suicide plasmid harboring the sacB counter-selectable marker, and then, were site-specifically introduced into the genome. Various combinations of five deletions (delta pehA, delta pehB, delta B, PehC, and Pme) was not statistically different from GMI1000, but all the mutants lacking one or both cellulolytic enzymes (Egl or CbhA) wilted plants significantly more slowly than did the wild type. The GMI-6 mutant that lacks all six CWDE was more virulent than the mutant lacking only its two cellulolytic enzymes, and both were significantly more virulent than the T2SS mutant (GMI-D). Very similar results were observed in wounded-petiole inoculation assays, so GMI-6 and GMI-D appear to be less capable of colonizing tomato tissues after invasion. Because the T2SS mutant was much less virulent than the sixfold CWDE mutant, we conclude that other secreted proteins contribute substantially to the ability of R. solanacearum GMI1000 to systemically colonize tomato plants.

Ralstonia solanacearum RSc0411 (lptC) is a determinant for full virulence and has a strain-specific novel function in the T3SS activity

“…Finally, the crystal-violet-bound biofilm was dissolved in 1 % SDS and quantified by measuring OD 570 . Swimming motility assay was performed as described previously (Liu et al, 2005). Because of the variable swimming activity of R. solanacearum wild-type strains (our unpublished data), swimming motility of strains in Pss4 background was assayed in 0.3 % agar medium, while that in Pss190 background was tested in 0.22 % agar medium.…”

Global regulation of pathogenicity mechanism of Ralstonia solanacearum

“…The bacteria reportedly produce one b-1,4-endoglucanase (Egl), one endopolygalacturonase (PehA), two exopolygalacuturonases (PehB and PehC), one b-1,4-cellobiohydrolase (CbhA) and a pectin methyl esterase (Pme). Genetic inactivation of single genes has shown that Egl, PehA and PehB each contribute to its virulence (Liu et al 2005). Furthermore, Liu et al showed that a mutant lacking the six genes encoding these six CWDEs wilted plants significantly more slowly that the wildtype.…”

Elucidation of the Regulon and cis -Acting Regulatory Element of HrpB, the AraC-Type Regulator of a Plant Pathogen-Like Type III Secretion System in Burkholderia pseudomallei

“…Moreover, an attempt to demonstrate phytopathogenicity of B. thailandensis on Oryza sativa (rice) plants was similarly unsuccessful (34). These results, coupled with observation that, unlike those of R. solanacearum and Xanthomonas spp., the genomes of B. thailandensis and B. pseudomallei encode no plant cell wall-degrading enzymes (e.g., pectate lyases, polygalacturonases, or endoglucanases) (37,70), call into question any suggestion that B. thailandensis and, by extension, B. pseudomallei are typical plant pathogens. In fact, of the Ͼ30 described Burkholderia spp., only 6 are reported to cause plant disease (17,40), and none of these has a T3SS orthologous to T3SS2 bp .…”