Mechanisms of Plant Colonization by Ralstonia Solanacearum

NON-TECHNICAL SUMMARY: Ralstonia solanacearum, an important phytopathogenic bacterium, infects and kills many plants worldwide. The attributes and processes used by this pathogen to systemically colonize host plants need to be better understood before new control strategies can be devised. This project studies the role that multiple extracellular molecules produced by R. solanacearum have on its ability to invade and systemically colonize host tomato plants and to survive osmotic and matric stresses.

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APPROACH: 1. Mutants that express the green fluorescent protein (GFP) but lack extracellular polysaccharide, type four pili, or flagella are already available or will be constructed using standard methods. Both wounded-petiole and excised-stem inoculation methods will be evaluated for their utility in studying systemic colonization in more detail. Colonization will be determined over time by plating for viable cells and by epifluorescence. 2. Two putative lipopolysaccharide-negative mutants created previously will be further characterized genetically and phenotypically. The ability of the mutants to colonize tomato tissues will be studied as described above. The mutants also will be tested for their ability to induce resistance in tobacco and tomato tissues and for whether they induce expression of pathogenesis-related (PR) protein genes and other defense-response related genes. 3. A R. solanacearum mutant that cannot produce any of the known plant cell wall degrading exoenzymes will be created by site-specifically deleting their structural genes using a `classic' two-step recombination, sacB counterselection method that introduces no foreign DNA. This mutant will be tested for its ability to colonize tomato plants and two cause wilt symptoms using both wounded-petiole and soil-drench inoculation methods. Annotation of the genomic sequence of strain GMI1000 revealed a number of other genes that might encode structures or activities that are important for systemic colonization. Such loci will be inactivated using a standard antibiotic-resistance marked gene replacement strategy and tested for virulence and colonization ability. 4. The ability of wild-type and mutant R. solanacearum strains to survive osmotic and matric stresses in culture will be studied by exposure to various concentrations of NaCl and polyethylene glycol, respectively. The ability of these same strains to survive in soils from the Piedmont and coastal plain regions of Georgia will be studied in microcosms subjected to varying levels of desiccation. The ability of R. solanacearum to enter the `viable but nonculturable' state in water and in soil will be assessed using standard procedures to evaluate culturability versus viability
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PROGRESS: 2003/04 TO 2008/03<BR>
OUTPUTS: Obj 1.The role of type IV pili and flagella play during colonization of tomato was examined. Pili were required for efficient colonization regardless of the inoculation method, but flagella were important only for invasion of unwounded roots. A double mutant lacking both appendages sometimes reduced colonization synergistically, but movement still occurred, suggesting that additional unknown pathogen or plant factors contribute to bacterial movement within tomato plants. Obj 2. The lipopolysaccharide (LPS) O-antigen moiety was eliminated by mutating the waaL gene. This mutant was moderately more sensitive to carbenicillin and novobiocin, but was comparable to the wild type in EPS1 production, growth rate in culture, short-term survival in potting soil, natural transformation, auto-aggregation, biofilm formation, and elicitation of the hypersensitive reaction when infiltrated into tobacco leaves. However, both twitching motility and swimming motility were less than normal. These defects could contribute to the reduced virulence of this mutant, so we could not conclude that the lack of LPS is solely responsible for the reduced virulence of the waaL mutant. Obj 3. The role of six plant cell wall degrading enzymes made by R solanacearum was studied using 15 GMI1000 mutants lacking all combinations of these enzymes. Mutants lacking only pectolytic enzymes were equally virulent or more virulent than the wild type, but cellulolytic mutants were significantly less virulent than either the wild type or a mutant lacking all six enzymes. All enzyme mutants were significantly more virulent than a sdpD1 mutant lacking its primary type II protein secretion system. Therefore, both cellulolytic enzymes and unknown other protein(s) secreted by the type II system contribute to colonization of tomato by R. solanacearum. Inactivation of any one or all of three of the "incomplete" type II secretion systems encoded in the GMI1000 genome showed that few if any proteins, and none important for pathogenesis, transit these potential alternative systems. Inactivation of a tolC ortholog (RSc0695) in GMI1000 had no effect on protein secretion or virulence. Obj 4. A R. solanacearum pilA mutant, a type II secretion system mutant, a phcA mutant, and an EPS-negative mutant survived fluctuating wet and dry conditions in soil microcosms as well as wild type strain AW1. This and other wild type strains from the SE USA survived best in 4 C water, followed by race 3 biovar 2 strains, and tropical biovar 1 strains survived least well. All strains survived better if cells entered a starvation mode prior to the cold treatment, but RpoS, a stationary phase sigma factor, was not important for survival. Low concentrations of various inorganic ions, buffers and organic media components reduced the survival at 4 C, but compounds in some river water apparently can counteract the toxic effect of inorganic ions. <BR> PARTICIPANTS: Dr. Timothy Denny (PI) conceptualized the project, designed the experiments, and analyzed the results. Ms. Marla Popov (Research Professional I) conducted experiments. Ms. Nicole Lynch, Ms. Vaishali Bhakta, Mr. Erik Hansen, and Ms. C. Daniele Gates, UGA undergraduates, received training as part of a directed study lab course. Ms. Maria Carolina Zuleta a UGA graduate student in Plant Pathology received training during her M.S. degree program. Mr. David Samuels and Ms. Erica Miller, both UGA graduated students, received training while on rotations in Dr. Denny's lab during their first semester's study. <BR> TARGET AUDIENCES: Target audiences were undergraduate students, graduate students, post-doctoral associates, and professional scientists. Efforts included classroom and laboratory instruction, practicum experiences, and presentations at professional meetings.
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IMPACT: 2003/04 TO 2008/03<BR>
The results advanced basic understanding of how this plant pathogenic bacterium can systematically colonize tomato plants and survive in the absence of a plant host.