Progress 04/01/03 to 03/31/08
Outputs
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. 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. 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. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
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.
Publications
- Young, J. M., C. Allen, T. Coutinho, T. Denny, J. Elphinstone, M. Fegan, M. Gillings, T. R. Gottwald, J. H. Graham, N. S. Iacobellis, J. D. Janse, M. A. Jacques, M. M. Lopez, C. E. Morris, N. Parkinson, P. Prior, O. Pruvost, J. R. Neto, M. Scortichini, Y. Takikawa, and C. D. Upper. 2008. Plant-pathogenic bacteria as biological weapons - Real threats Phytopathology 98:1060-1065.
- Bull, C. T., S. H. De Boer, T. P. Denny, G. Firrao, M. Fischer-Le Saux, G. S. Saddler, M. Scortichini, D. E. Stead, and Y. Takikawa. 2008. Demystifying nomenclature of bacterial plant pathogens. J. Plant Pathol. 90:403-417.
|
Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: Obj. 1. Additional preliminary colonizations assays were run to refine the experimental methods necessary to examine movement of R. solanacearum within the rachis of excised tomato leaves. The wild-type strain moved up to 8 cm in 4 days, whereas three motility-defective mutants (lacking either or both twitching and swimming motility) generally moved less quickly. However, since the motility mutants did move within the tissue, and in some cases apparently hop-scotched long distances, we speculate that there are unknown pathogen or plant factors that contribute to bacterial movement. Obj. 2. Re-examination of the waaL mutant that lacks the LPS O-antigen revealed that, contrary to our initial conclusions, it is somewhat reduced in production of EPS, and in both twitching and swimming motility in culture. These defects, either alone or in combination, could contribute to the reduced virulence of this mutant. Therefore, we were incorrect to conclude that the lack of LPS is solely
responsible for the reduced virulence of the waaL mutant. Obj. 3. To investigate whether the R. solanacearum Type I secretion system (T1SS) contributes to colonization, we tried to inactivate this protein secretion system. The putative tolC gene (RSc0695) in strain GMI1000 was site-specifically inactivated by introduction of an unmarked deletion. The mutant did not exhibit an increased sensitivity to selected detergents or antibiotics that is typical of tolC mutants of enteric bacteria. In addition, 2-dimensional gel electrophoresis of extracellular proteins produced by the putative tolC mutant were indistinguishable from those made by wild-type R. solanacearum, and the mutant exhibited normal virulence on tomato plants. However, he protein encoded by R. solanacearum locus RSc0695 was not tested for its ability to complement an E. coli tolC mutant, so we are uncertain if it is functionally a TolC-like outer membrane pore. Two new graduate students in the UGA Microbiology Department
performed this work during rotations in my laboratory. Obj. 4. The ability of R. solanacearum to survive exposure to 4 C while suspended in water was further explored. Allowing the cells to enter a starvation mode by incubation at room temperature for 3 to 7 days prior to beginning the cold treatment substantially improved survival of all strains tested. Additional Race 3 Biovar 2 strains and Biovar 1 strains isolated from either the southeastern United States or tropical locations were tested and preliminary patterns held true: tropical Bv 1 strains were the most sensitive to cold, R3Bv2 strains exhibited intermediate sensitivity, and Bv 1 strains from the US were the least sensitive. Two samples of river water from Athens, GA reduced cold survival whereas two other samples improved survival in comparison to pure water. Therefore, it appears that other compounds in river water can counteract the toxic effect of inorganic ions, which were previously observed to reduce cold survival of
R. solanacearum in pure water. Ms. Maria Carolina Zuleta graduated from my program with her M.S. degree in Plant Pathology. Dr. Denny served on a USDA NRI grant review panel for functional genomics.
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 and Ms. Vaishali Bhakta , both a 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.
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.
Impacts
The results advanced basic understanding of how a plant pathogenic bacterium can systemically colonize and kill a host plant.
Publications
- Liu, H. and Denny, T. P. 2007. Proteomics analysis indicates that Ralstonia solanacearum has a distinctive type II secretion system. Phytopathology 97:S66-S67.
- Denny, T. P., Milling, A. S., Bhakta, V. G., and Allen, C. 2007. Ralstonia solanacearum race 3 biovar 2 strains are not uniquely cold tolerant in vitro. Phytopathology 97:S28.
|
Progress 01/01/06 to 12/31/06
Outputs
Objective 3. We found previously that the type II protein secretion system (T2SS) is essential for efficient colonization of host tomato plants by R. solanacearum. This pathogen secretes at least 36 proteins from the periplasm through a pore in the outer membrane formed by the SdpD1 secretin. Strain GMI1000 is predicted to have three SdpD-related proteins, SdpD2, SdpD3 and SdpD4, which are part of three gene clusters that appear to encode incomplete T2SS, and might form alternative pores that partially compensate for the loss of the major SdpD1 secretin. To determine if any proteins are secreted via the SdpD-related proteins, we created a mutants lacking each individual sdpD gene, all three sdpD-related genes, or all four sdpD genes. Two-dimensional gel analysis of the extracellular proteins of the four-fold mutant compared with that of the SdpD1-secretin mutant, revealed only subtle differences for proteins between 25 and 250 kDa and 3 to 11 pH range. In addition,
the triple mutant remained fully virulent. Thus, few proteins, and none important for pathogenesis, transit the alternative SdpD secretins. Objective 4. Unlike most strains in the R. solanacearum species complex, the race 3 biovar 2 (r3b2) subgroup can survive and cause potato brown rot in cool climates. R3b2 is not present in North America and APHIS listed it as a Select Agent because its supposed cold tolerance potentially threatens the potato industry. We found that R. solanacearum strains vary in their ability to survive exposure to 4 degrees C in pure water. Contrary to expectations, strains AW1 and K60, which are native to the southeastern United States, remained culturable for more than 10 weeks and were the most cold tolerant. GMI1000 from French Guiana was among the least tolerant, whereas r3b2 strains UW551 and IPO1609 exhibited intermediate tolerance (these tests were performed by C. Allen, who has APHIS permits to work with r3b2). GMI1000 survived as well as AW1 in water
at 10 to 30 degrees C. Low concentrations of various inorganic ions, buffers and organic media components reduced the survival of both GMI1000 and AW1. Survival of AW1 in water was not strongly dependent on RpoS, a stationary phase sigma factor. Viability assays revealed that a high proportion of cells had an intact plasma membrane despite being non-culturable (they appear to have entered a "viable but nonculturable" state). Although these results must still be confirmed using more natural assays, they challenge the assumption that the cold tolerance of r3b2 strains makes them a greater threat to the US potato industry than native R. solanacearum strains.
Impacts
The results advanced basic understanding of how a plant pathogenic bacterium can systemically colonize and kill a host plant.
Publications
- Zuleta, M.C., Liu, H.,Schell, M.A., and Denny, T.P. 2006. Identification of Ralstonia solanacearum proteins secreted through alternative type II secretion systems. Phytopathology 96:S131.
|
Progress 01/01/05 to 12/31/05
Outputs
Objective 1. R. solanacearum reportedly requires swimming motility to infect plants efficiently via unwounded roots. To test the role of swimming motility in systemic movement of bacteria from roots, wild type, a pilA mutant (non-piliated, twitching motility negative), a fliC mutant (non-flagellated, swimming negative) and a pilA fliC double mutant were tested for virulence on tomato plants inoculated by soil drench with or without intentionally wounding the roots. The fliC mutant was as virulent as the wild type only when the roots had been wounded, indicating that swimming motility is not required for vertical movement from roots to the rest of the plant. The pilA mutant was consistently less virulent than wild type regardless of whether roots had been wounded. The pilA fliC double mutant was consistently less virulent than the fliC mutant; sometimes it was as virulent as the pilA mutant, but other times it was less virulent than the pilA mutant. Similar results
were observed for the double mutant inoculated via a wounded petiole. Objective 3. Bioinformatics identified 15 genes in R. solanacearum that are conserved only in xylem-colonizing bacteria. Each of these genes was site-specifically inactivated and each mutant (missing a single gene each) was tested for virulence on tomato plants with unwounded roots. None of the mutants was significantly less virulent than the wild type. Objective 4. Survival in soil of a pilA mutant, a type II secretion mutant, and a phcA mutant (which is a global regulator virulence and other genes) was assayed. These mutants survived as well as the wild type during the 3 month test.
Impacts
The results advanced basic understanding of how a plant pathogenic bacterium can systemically colonize and kill a host plant.
Publications
- Liu H., Zhang S., Schell M.A., Denny T.P. 2005. Pyramiding unmarked mutations in Ralstonia solanacearum shows that secreted proteins in addition to plant cell wall degrading enzymes contribute to virulence. Mol. Plant-Microbe Interact. 18(12):1296-1305.
- Genin S., Brito B., Denny T.P., Boucher C. 2005. Control of the Ralstonia solanacearum type III secretion system (Hrp) genes by the global virulence regulator PhcA. FEBS Lett. 579(10):2077-2081.
- Gabriel D.W., Allen C., Schell M.A., Denny T.P., Greenberg J.T., Duan Y.P., Flores-Cruz Z., Huang Q., Clifford J.M., Presting G., Gonzalez E.T., Reddy J., Elphinstone J., Swanson J., Yao J., Mulholland V., Liu L., Farmerie W., Patnaikuni M., Balogh B., Norman D., Alvarez A., Castillo J.A., Jones J., Saddler G., Walunas T., Zhukov A., Mikhailova N. 2006. Identification of open reading frames unique to a Select Agent: Ralstonia solanacearum race 3 biovar 2. Mol. Plant-Microbe Interact. 19(1):69-79.
- Aves-ILagan Y., Lavina W.A., Denny T.P., Raymundo A.K. 2003. Development of a polymerase chain reaction-based technique for the detection of Philippine banana strains of Ralstonia solanacearum (Smith) Yabuuchi et al. Phil. Agric. Scientist 86:385-393.
|
Progress 01/01/04 to 12/31/04
Outputs
Objective 2. The lipopolysaccharide (LPS) O-antigen moiety was eliminated without altering the remaining LPS molecule or the extracellular polysaccharide (EPS1) by site-specifically inactivating in R. solanacearum strain GMI1000 putative genes encoding either an undecaprenyl phosphate N-acetylglucosaminyl transferase (wecA; RS01594) or a lipid A core:O-antigen ligase (waaL; RS01395). Extensive characterization of these mutants was completed. Gel electrophoresis and composition analysis of purified LPS indicated that the wecA mutant produces at most a single O-antigen oligosaccharide repeat unit, whereas the waaL mutant completely lacks the O-antigen. Both of the mutants were resistant to an LPS-specific phage. The wecA mutant was substantially more sensitive to the antibiotics carbenicillin, novobiocin, nalidixic acid and bacitracin, whereas the waaL mutant was moderately more sensitive only to carbenicillin and novobiocin. Both mutants were 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. The wecA mutant lacked twitching motility and exhibited reduced swimming motility, but the waaL mutant was normal. Both mutants were dramatically less virulent than the wild type on tomato plants when inoculated via wounded petioles or unwounded roots. For the waaL mutant, the loss of virulence can be attributed solely to the loss of O-antigen production. Objective 3. The virulence of 15 mutants that lack one to six plant cell wall degrading enzymes was tested extensively on tomato plants. Mutants lacking any combination of the three polygalacturonases and/or the pectin methyl esterase were as virulent as the wild-type parent strain. Elimination of either the single known endoglucanase or the single putative exoglucanase significantly reduced virulence, and
elimination of both enzymes had an additive effect. The mutant lacking all six enzymes was more virulent than that lacking both the endo- and exoglucanases. However, all 15 mutants were significantly more virulent than a mutant lacking its type II protein secretion system. These data strongly suggest that proteins in addition to the endo- and exoglucanases contribute to colonization of tomato by R. solanacearum.
Impacts
The results advanced basic understanding of how a plant pathogenic bacterium can systemically colonize and kill a host plant.
Publications
- Bhatt, G. and T. P. Denny. 2004. Ralstonia solanacearum iron scavenging by the siderophore staphyloferrin B is controlled by PhcA, the global virulence regulator. J. Bacteriol. 186:7896-7904.
- Denny, T. P., Liu, H., Wolff, J. J., Amster, I. J., and Schell, M. A. 2004 Extracellular space: the final frontier for Ralstonia solanacearum proteins required for colonization. Phytopathology 94:S121-S122.
- Kang, Y., Liu, H., Carlson, R. W., and Denny, T. P. 2004. Lipopolysaccharide O-antigen of Ralstonia solanacearum contributes to virulence on tomato plants. Phytopathology 94:S49. 2004.
- Liu, H., Zhang, S., Rodriguez, C., Schell, M. A., and Denny, T. P. Pyramiding unmarked mutations in Ralstonia solanacearum to assess the role of extracellular enzymes in pathogenesis. Phytopathology 94:S62. 2004.
- Robertson, A. E., W. P. Wechter, T. P. Denny, B. A. Fortnum, and D. A. Kluepfel. 2004. Relationship between avirulence gene (avrA) diversity in Ralstonia solanacearum and bacterial wilt incidence. Mol. Plant-Microbe Interact. 17:1376-1384.
|
Progress 01/01/03 to 12/31/03
Outputs
Objective 1. Preliminary petiole-inoculation assays showed that a GFP+ wild-type strain spread both up and down the stem in the major vessel bundle subtending the inoculated petiole and move laterally to infect other major vessel bundles. In contrast, a GFP+ pilA (non-piliated, twitching negative) mutant was remained confined mostly to the one major vessel bundle subtending the inculcated petiole. The necessary GFP+, non-flagellate mutants of R. solanacearum were created by site specifically introducing a fliC::Gm allele into existing wild-type and pilA-mutant strains. Objective 2. Two LPS O-antigen mutants were thoroughly characterized in vitro and tested for several traits in vivo. One mutant was indistinguishable from the wild-type parent in culture except for the LPS defect, and this mutant exhibited reduced virulence on tomato. Objective 3. A set of 15 mutants that have up to all six genes for known plant cell wall degrading enzymes deleted was created using a
mutagenesis procedure that leaves only a few nucleotides of foreign DNA in place of the targeted gene. Preliminary virulence assays showed that the 6-fold mutant largely retained virulence, which contrasts with the very poor colonization exhibited by a mutant lacking its type II protein secretion pathway. Thus, other proteins that transit the type II pathway are important for colonization of tomato plants. A R. solanacearum mutant lacking the ability to produce an iron-binding siderophore was created and characterized. The mutant was not affected in its ability to wilt tomato plants, and growth of bacteria in xylem sap suggested that iron is not limiting in that niche. It was reported previously that R. solanacearum produces the siderophore schizokinen, but we determine that it actually makes a different siderophore, which is probably staphyloferrin B. Objective 4. A method for examining survival of R. solanacearum in natural soil was established. A 5-month long test showed that a
wild-type strain and an EPS-negative strain survived equally well in soil that was constantly moist, constantly dry, or alternately moist and dry. Both strains survived least well in the soil with fluctuating moisture.
Impacts
The results advanced basid understanding of how a plant pathogenic bacterium can systemically colonize and kill a host plant.
Publications
- No publications reported this period
|
|