Using the Ralstonia solanacearum Tat Secretome To Identify Bacterial Wilt Virulence Factors

Abstract

To identify secreted virulence factors involved in bacterial wilt disease caused by the phytopathogen Ralstonia solanacearum, we mutated tatC, a key component of the twin-arginine translocation (Tat) secretion system. The R. solanacearum tatC mutation was pleiotropic; its phenotypes included defects in cell division, nitrate utilization, polygalacturonase activity, membrane stability, and growth in plant tissue. Bioinformatic analysis of the R. solanacearum strain GMI1000 genome predicted that this pathogen secretes 70 proteins via the Tat system. The R. solanacearum tatC strain was severely attenuated in its ability to cause disease, killing just over 50% of tomato plants in a naturalistic soil soak assay where the wild-type parent killed 100% of the plants. This result suggested that elements of the Tat secretome may be novel bacterial wilt virulence factors. To identify contributors to R. solanacearum virulence, we cloned and mutated three genes whose products are predicted to be secreted by the Tat system: RSp1521, encoding a predicted AcvB-like protein, and two genes, RSc1651 and RSp1575, that were identified as upregulated in planta by an in vivo expression technology screen. The RSc1651 mutant had wild-type virulence on tomato plants. However, mutants lacking either RSp1521, which appears to be involved in acid tolerance, or RSp1575, which encodes a possible amino acid binding protein, were significantly reduced in virulence on tomato plants. Additional bacterial wilt virulence factors may be found in the Tat secretome.

The Twin Arginine Translocation System Is Essential for Aerobic Growth and Full Virulence of Burkholderia thailandensis

“…In E. coli O157:H7 (25), Pseudomonas aeruginosa (26), Agrobacterium tumefaciens (27), and Ralstonia solanacearum (28), the Tat system is required for anaerobic growth. Conversely, in Rhizobium leguminosarum (29), Myxococcus xanthus (42), M. smegmatis (41), and Shewanella oneidensis (33), Tat mutants show reduced growth under aerobic conditions.…”

“…Proteins exported by the Tat system have also been shown to be important for virulence (21,24). In many bacterial species, proteins exported by the Tat system are associated with anaerobic growth, and consequently, many Tat system mutants show defects in their abilities to grow under these conditions (25)(26)(27)(28). Conversely, in some species, there is evidence that the Tat system plays a role in aerobic growth.…”

Transcriptomic and Phenotypic Analysis Reveals New Functions for the Tat Pathway in Yersinia pseudotuberculosis

“…Several studies indicate that a functional Tat system is required for many pathogenic bacteria, such as Pseudomonas syringae pv. tomato DC3000, S. enterica, Campylobacter jejuni, and Ralstonia solanacearum (5,(48)(49)(50)(51), to efficiently re- spond to diverse forms of physicochemical stresses. Indeed, our transcriptomic analysis of the Y. pseudotuberculosis ⌬tatC mutant revealed that a number of prominent stress-responsive genes were induced at both temperatures, and this number was higher during stationary-phase growth.…”

Ralstonia solanacearum Dps Contributes to Oxidative Stress Tolerance and to Colonization of and Virulence on Tomato Plants

“…solanacearum virulence is quantitative and complex, with many contributing factors such as type II-secreted proteins, type III-secreted effectors, extracellular polysaccharide, and several plant cell wall-degrading enzymes (16,17,36,38).…”