Progress 10/01/06 to 09/30/11
Outputs
OUTPUTS: There were four major objectives for this project, and the first three were completed: (1) The signal molecules from one Medicago truncatula A17 compatible strain and one incompatible strain (Sinorhizobium meliloti NRG185 and S. meliloti NRG247, respectively) were characterized. We found that the difference in the signals was the degree of substitution with succinate. Those oligosaccharides that were fully substituted functioned with A17, and those that lacked succinate functioned in A20.In addition, the change in the production of these molecules in response to plant signals has also been performed. Incubation of the bacteria with plant flavonoids resulted in an attenuation of active oligosaccharide production, which would function to shut down that signal system after infection. (2) The genetic response of Medicago truncatula A17 to the compatible and incompatible strains has been analyzed and the specific genes identified from the genome bank. Many of the genes which are associated with compatible signal induction function in plant cell re-arrangement. (3) The cell surface structural changes associated with phase shift of Sinorhizobium sp. NGR234 during infection have been identified; however, the other strains were lost prior to this analysis, so only one strain was studied. All of this work will be published over the next two years. In the case of NGR234, the cell-surface chemistry changed from a hydrophilic glucan to a hydrophobic rhamnan, which would allow for close association of the plant and bacterial membranes in the symbiosome. Objective 4 was only partially completed prior to the loss of the strains, so very little work was done towards that objective. PARTICIPANTS: Senay Simsek worked on the project through the end of 2006. She is now an Assistant Professor in the Plant Science Department at North Dakota State University. Jae Wook Yoon worked on this project during the last reporting term. He graduated in July 2008, and has moved to a postdoctoral position in Korea. TARGET AUDIENCES: Plant and microbiology scientists. PROJECT MODIFICATIONS: There were no major changes to the project.
Impacts
The most significant impact of the work is the identification of the active structures in the compatible interactions. Second is the understanding of how signal molecule production by Sinorhizobium meliloti is altered and attenuated by the plant signals. This probably functions to turn off any further signaling by the bacteria, in order to prevent problems with the plant physiology and nodule development. Another impact is the identification of plant genes associated with the compatible interaction.
Publications
- 1. Reuhs,B.L., B. Relic, L.S. Forsberg, C. Marie, T. Ojanen-Reuhs, S.B. Stephens, C.H. Wong, S. Jabbouri, and W.J. Broughton. 2005. Structural characterization of a flavonoid-inducible Pseudomonas aeruginosa A-band-like O antigen of Rhizobium sp strain NGR234, required for the formation of nitrogen-fixing nodules. J. Bacteriol. 187:6479-6487.
- 2. Broughton, W.J., M. Hanin, B. Relic, J. Kopcinska, W. Golinowski, S. Simsek, T. Ojanen-Reuhs, B. Reuhs, H. Kobayashi, B. Bordogna, S. Jabbouri, R. Fellay, W.J. Deakin, X. Perret, and C. Marie. 2006. Flavonoid-inducible to Rhamnan O-antigens are necessary for Rhizobium sp. NGR234-Legume Interactios. J. Bacteriol. 188:3654-3663.
- 3. Simsek, S., T. Ojanen-Reuhs, S. B. Stephens, and B.L. Reuhs. 2007. Strain-ecotype specificity in Sinorhizobium meliloti-Medicago truncatula symbiosis is correlated to succinoglycan oligosaccharide structure. J Bacteriol. 189:7733-7740
- 4. Structural analysis of lipopolysaccharides from Sinorhizobium sp. NGR234 induced by apigenin. Jae Wook Yoon. 2008. PhD thesis, Purdue University.
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: There were four major objectives for this project, and the first three have been completed: (1) The signal molecules from one Medicago truncatula A17 compatible strain and one incompatible strain (Sinorhizobium meliloti NRG185 and S. meliloti NRG247, respectively) have been characterized. In addition, the change in the production of these molecules in response to plant signals has also been performed. (2) The genetic response of Medicago truncatula A17 to the compatible and incompatible strains has been analyzed and the specific genes identified from the genome bank. (3) The cell surface structural changes associated with phase shift of Sinorhizobium sp. NGR234 during infection have been identified; however, the other strains were lost prior to this analysis, so only one strain was studied. All of this work will be published over the next two years. Objective 4 was only partially completed prior to the loss of the strains, so very little work can be done towards that objective. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Plant microbiologists PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The most significant impact of the work will be the understanding of how signal molecule production by Sinorhizobium meliloti is altered and attenuated by the plant signals. This probably functions to turn off any further signaling by the bacteria, in order to prevent problems with the plant physiology and nodule development. The second impact is in the identification of plant genes associated with the compatible interaction.
Publications
- No publications reported this period
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: The completed objectives of the research include (1) the structural characterization of specific S. meliloti oligosaccharide signals required for infection and (2) analysis of changes in oligosaccharide production due to plant signals. These initial structural results have been published, and the more detailed mass spectrometry results will soon be published. The signal transduction results are also complete and will be submitted within three to six months. Other objectives still that have been completed include (3) a genetic analysis of the plant (Medicago truncatula) response to compatible and incompatible symbiotic infection by S. meliloti, (4) the in vitro determination of the structural changes associated with phase shift in Sinorhizobium sp. NGR234, S. fredii, and S. meliloti during the infection of host plants. The manuscripts will be written and submitted during 2010. One objective remains to be completed: (5) the structural characterization of novel symbiotic polysaccharides from each species that are expressed post infection by recovery of the cells from the infection sites. We continue to analyze the phase shift oligosaccharides that anchor the infection-related O antigen to the cell wall. As these products are present in low abundance, the last year was a continuation of the intense method development for the isolation and analysis of these molecules. A genetic analysis of the plant (Medicago truncatula) response to compatible and incompatible symbiotic infection by S. meliloti has been completed, and we have found an interesting mix of genes that are activated by both the compatible and incompatible strains. Three manuscripts will be submitted in the next period, and the final two manuscripts will be finished in 2011. PARTICIPANTS: I have primarily responsible for this research in the last year, but Anton Terekhov, my lab technician, has also participated. TARGET AUDIENCES: Plant and microbiology scientists PROJECT MODIFICATIONS: Nothing significant to report during this reporting period. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The major impact of the work over the last year is completing the method development for the determination of phase-shift structural changes in bacteria that have entered the infection state. This is important for moving forward with research into phase shift changes in Gram-negative bacteria, which can be very difficult to identify. It may lead to a better understanding of bacterial infection in general. The improved techniques include an electrophoresis purification of lipopolysaccharides and oligosaccharides for the purification of molecules that differ only slightly in structure. This can be applied to both in vitro-derived samples and those from the bacterial infection site in the plant. This will be described in detail in a methods manuscript to be submitted in the coming year. In addition, we have used the developed methods to show that the major phase shift antigen is anchored to a modified oligosaccharide in the infection state cells. This is a major development, as this sort of change has been suspected, but not proven, in other bacterial-host systems.
Publications
- No publications reported this period
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Progress 10/01/07 to 09/30/08
Outputs
OUTPUTS: The completed objectives of the research include (1) the structural characterization of specific S. meliloti oligosaccharide signals required for infection and (2) analysis of changes in oligosaccharide production due to plant signals. These results have been published or will soon be published. Other objectives still in progress include (3) a genetic analysis of the plant (Medicago truncatula) response to compatible and incompatible symbiotic infection by S. meliloti, (4) the in vitro determination of the structural changes associated with phase shift in Sinorhizobium sp. NGR234, S. fredii, and S. meliloti during the infection of host plants, and (5) the structural characterization of novel symbiotic polysaccharides from each species that are expressed post infection by recovery of the cells from the infection sites. During the last year, we pursued the in vitro determination of the structural changes associated with phase shift in Sinorhizobium sp. NGR234, S. fredii, and S. meliloti during infection. The major antigenic shifts in Sinorhizobium sp. NGR234 were previously characterized, but the related changes that accompany, and lead to, those shifts remain under investigation. We continue to analyze the oligosaccharides that anchor them to the cell wall. As these products are present in low abundance, the last year involved intense method development for the isolation and analysis of these molecules. The work focused on the degradation of previously intact phase shift products from the wild-type strain and the partial products from a mutant strain, which is unable to infect. A genetic analysis of the plant (Medicago truncatula) response to compatible and incompatible symbiotic infection by S. meliloti continues to be a focus of research, but it is still in the initial stages of study. We have also worked on method development for objective 5, the extraction of phase shift polysaccharides from infection sites. PARTICIPANTS: Jae Wook Yoon worked on this project during the last reporting term. He graduated in July 2008, and has moved to a postdoctoral position in Korea. TARGET AUDIENCES: Plant and microbiology scientists PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The major impact of the work over the last year is the development of methods for the determination of phase-shift structural changes in bacteria that have entered the infection state. This is important for moving forward with research into phase shift changes in Gram-negative bacteria, which can be very difficult to identify. It may lead to a better understanding of bacterial infection in general. The improved techniques include an electrophoresis purification of lipopolysaccharides and oligosaccharides for the purification of molecules that differ only slightly in structure. This can be applied to both in vitro-derived samples and those from the bacterial infection site in the plant. This will be described in detail in a methods manuscript to be submitted in the coming year. In addition, we have used the developed methods to show that the major phase shift antigen is anchored to a modified oligosaccharide in the infection state cells. This is a major development, as this sort of change has been suspected, but not proven, in other bacterial-host systems.
Publications
- Structural analysis of lipopolysaccharides from Sinorhizobium sp. NGR234 induced by apigenin. Jae Wook Yoon. 2008. PhD thesis, Purdue University.
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Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: During the last year, we completed many aspects of the project that relate to the Sinorhizobium meliloti oligosaccharide signal-molecules required for infection of the host plant. This includes the complete mass spectrometric analysis of the oligosaccharides produced in normally cultured cells, and after exposure of the bacterial cells to plant flavonoids. The active fraction has been isolated from the A17-compatible strain, and the final plant assays are all that remain for that part of the project. These analyses showed that unlike Nod factor biosynthesis, the biosynthesis of the active oligosaccharide is actually turned off in response to the plant. This is likely due to the numerous cellular changes in the plant that are induced by the oligosaccharide; if the bacteria continued to produce the signal, there would be developmental problems in the plant. The in vitro determination of the structural changes associated with phase shift in Sinorhizobium sp. NGR234, S. fredii,
and S. meliloti during infection is still at a midway point; i.e., the major antigenic shifts have been characterized, but the related changes that accompany, and lead to, those shifts remain under investigation. The major phase-shift antigen from Sinorhizobium sp. NGR234 was previously structurally characterized, and we continue to analyze the oligosaccharides that anchor them to the cell wall. A genetic analysis of the plant (Medicago truncatula) response to compatible and incompatible symbiotic infection by S. meliloti continues to be a focus of research, but it is still in the initial stages of study.
PARTICIPANTS: Senay Simsek worked on the project through the end of 2006. She is now an Assistant Professor in the Plant Science Department at North Dakota State University.
TARGET AUDIENCES: Plant and food scientists
Impacts
The major impact of the work is the determination that phase-shift structural changes in bacteria that have entered the infection state is common, maybe ubiquitous, in Sinorhizobium-legume symbiosis. It may lead to a better understanding of bacterial infection in general.
Publications
- Simsek, S., T. Ojanen-Reuhs, S. B. Stephens, and B.L. Reuhs. 2007. Strain-ecotype specificity in Sinorhizobium meliloti-Medicago truncatula symbiosis is correlated to succinoglycan oligosaccharide structure. J Bacteriol. 189:7733-7740
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Progress 10/01/05 to 09/30/06
Outputs
The structural characterization of specific Sinorhizobium meliloti oligosaccharide signal-molecules required for successful infection of the host plant, including oligosaccharide production after exposure of the bacterial cells to plant signals has been largely and successfully completed. The active fraction has been isolated from the A17-compatible strain, and the final structural analyses and plant assays are all that remain for that specific part of the project. We have also determined that the expression of these signal molecules is regulated by plant-derived flavonoids, and this continues to be investigated. The in vitro determination of the structural changes associated with phase shift in Sinorhizobium sp. NGR234, S. fredii, and S. meliloti during infection is at a midway point; i.e., the major antigenic shifts have been characterized, but the related changes that accompany, and lead to, those shifts remain under investigation. The major phase-shift antigen
from Sinorhizobium sp. NGR234 has been structurally characterized, and we are now analyzing the oligosaccharides that anchor them to the cell wall. A genetic analysis of the plant (Medicago truncatula) response to compatible and incompatible symbiotic infection by S. meliloti, and the structural characterization of novel symbiotic polysaccharides from each species that are expressed post infection by recovery of the cells from the infection sites have been addressed but are only in the initial stages of study.
Impacts
The identification of the differences in the signal molecules from each bacteria and the modification of these molecules in response to the plant will be of great interest to the scientific community. It may lead to a better basis for choosing the bacterial strains that will be used in the field.
Publications
- Reuhs,B.L., B. Relic, L.S. Forsberg, C. Marie, T. Ojanen-Reuhs, S.B. Stephens, C.H. Wong, S. Jabbouri, and W.J. Broughton. 2005. Structural characterization of a flavonoid-inducible Pseudomonas aeruginosa A-band-like O antigen of Rhizobium sp strain NGR234, required for the formation of nitrogen-fixing nodules. J. Bacteriol. 187:6479-6487.
- Broughton, W.J., M. Hanin, B. Relic, J. Kopcinska, W. Golinowski, S. Simsek, T. Ojanen-Reuhs, B. Reuhs, H. Kobayashi, B. Bordogna, S. Jabbouri, R. Fellay, W.J. Deakin, X. Perret, and C. Marie. 2006. Flavonoid-inducible to Rhamnan O-antigens are necessary for Rhizobium sp. NGR234-Legume Interactios. J. Bacteriol. 188:3654-3663.
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Progress 10/01/04 to 09/30/05
Outputs
This project focuses on ecotype-strain specificity in Medicago truncatula-Sinorhizobium meliloti interactions. Initially, we determined that infectivity is dependent on the bacterial extracellular polysaccharides (EPS), and we found a correlation between the EPS oligosaccharide structure and host specificity. The project consists of two primary objectives and two secondary objectives: 1) A determination of the structural requirements for compatible infection in M. truncatula ecotypes A17 versus A20; that is, the structures of the distinct oligosaccharides that are specifically recognized by a compatible host. 2) A study of the genetic basis for the difference in EPS oligosaccharide production among the strains. 3) An identification of modifications in oligosaccharide production in response to plant signals. 4) A study of infection thread development in the compatible and incompatible systems. We have made great progress on objectives 1 and 3, and we have started the
work on objectives 2 and 4. The progress has been such that three manuscripts are on the way to submission: the first will focus on the fact that strict strain-ecotype specificity exists in M. truncatula-S. meliloti interactions, and that it is due to differences in the secreted oligosaccharides. The second will present the oligosaccharide composition from each strain, and a construct with an extended host range. The third includes the results from induction experiments, in which the cells were grown in the presence of known bacterial gene regulators produced by the host plants. This work has shown that the bacteria may participate in limiting the number of infection sites. In the course of analyzing the EPS and K antigen mutants for the M. truncatula work, we also tested each on alfalfa, and found that the K antigens may substitute for EPS in most wild-type strains. In addition, Rhizobia undergo significant physiological changes during infection, including structural changes to the
cell surface. These studies addressed the changes in bacterial LPS and K antigen during infection and the genetic regulation of these changes.
Impacts
The identification of the differences in the signal molecules from each bacteria and the modification of these molecules in response to the plant will be of great interest to the scientific community. It may lead to a better basis for choosing the bacterial strains that will be used in the field.
Publications
- Marie, C., W. J. Deakin, T. Ojanen-Reuhs, E. Diallo, B. Reuhs, W. J. Broughton, and X. Perret. 2004 TtsI, a Key Regulator of Rhizobium Species NGR234, is Required for Type III-Dependent Protein Secretion and Synthesis of Rhamnose-Rich Polysaccharides. Mol. Plant-Microbe Interact. 17: 958-966.
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Progress 10/01/03 to 09/29/04
Outputs
The work has progressed very well over the last eight months; during this time the graduate students who work on the project have had less of a class load and more time in the lab. We have completed the analysis of polysaccharide mutants on alfalfa and M. truncatula and we have completed the first round of structural analysis on the active polysaccharides in Sinorhizobium-M. truncatula interactions. These two analyses will be published in the near future. The results have brought surprises: First, since most research on Sinorhizobium meliloti-Medicago sativa (alfalfa) interactions focused on S. meliloti Rm1021, in which production of the extracellular polysaccharides (EPS) is required for infection and the K antigen (capsular polysaccharide) is not active in promoting infection, it has been a commonly held belief the functional activity of the Rm41 K antigen was the exception. However, we constructed eight EPS mutants of wild-type S. meliloti strains and found that 6
of the eight were able to infect, presumably due to K antigen activity, and we have structurally characterized six of those polysaccharides. Second, using Rm1021 as the standard, a picture has emerged about the nature of the "structurally-conserved" EPS (i.e., structurally identical in all strains of S. meliloti); however, we have found that EPS structure varies among strains and that the differences in structure are correlated to strain-ecotype specificity in S. meliloti-M. truncatula interactions.
Impacts
M. truncatula is the subject of a worldwide genomic project for the study of plant-microbe interactions, and the identification of specific signal-molecules will be followed by an analysis of the plant response to compatible and incompatible symbiotic infection. This work may lead to a better understanding of plant development and symbiosis.
Publications
- No publications reported this period
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Progress 10/01/02 to 09/30/03
Outputs
Molecular signals are instrumental in the establishment of a symbiotic association between gram-negative, nitrogen-fixing bacteria and legumes. Bacterial extracellular polysaccharides (EPS) promote the infection process in Sinorhizobium meliloti-Medicago sativa (alfalfa) symbiosis, and purified EPS shows signal activity at 10-11 M. This project focuses on ecotype-strain specificity in Medicago truncatula-S. meliloti interactions. S. meliloti strains NRG185 and NRG34 infect M. truncatula ecotype A17 (compatible), but fail to establish nitrogen-fixing nodules on ecotype A20 at 28 days post infection (incompatible). Importantly, the phenotypes are reversed with S. meliloti strains NRG247 and Rm41. We have determined that infectivity is EPS-dependent, indicating that different ecotypes of M. truncatula have different structural requirements for oligosaccharide activity. And we have found a correlation between the EPS oligosaccharide structure and the host specificity of
the bacterial strain. The goal of the proposed research is to understand the biochemical and genetic basis for compatible symbiosis. Reseach conducted during the last year has involved the isolation of the specific signal molecules for future characterization.
Impacts
M. truncatula is the subject of a worldwide genomic project for the study of plant-microbe interactions, and the identification of specific signal-molecules will be followed by an analysis of the plant response to compatible and incompatible symbiotic infection. This work may lead to a better understanding of plant development and symbiosis.
Publications
- No publications reported this period
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