Progress 09/01/07 to 08/31/11
Outputs
OUTPUTS: Our first sub-objective of Aim 1 was to determine which type III-secreted proteins were not secreted in the P. syringae hrpJ mutant. Interestingly, the only proteins that were not secreted were the harpin group of proteins (HrpZ1, HrpW, HrpAK1) and HrpK1 putative translocator. The harpins are also putative translocators so it looks like HrpJ is required for translocators to be secreted. Our next sub-objective of Aim 1 was to determine whether HrpJ could complement the secretion of the harpins and HrpK1 even if it remained cell-bound. HrpJ is normally a type-III secreted protein. Interestingly, when we made constructs that produced a HrpJ protein without its type III secretion signal or a construct that fused GST to HrpJ's N-terminus we found that these HrpJ constructs were still able to complement the hrpJ mutant allowing HrpK1 and the harpins to be secreted in culture via the type III system. Our third sub-objective of Aim 1 was to identify domains within HrpZ1 and HrpW1 that were required for their dependence on HrpJ. The idea behind this experiment is that by expressing deletions of HrpZ1 and HrpW1 we might find derivatives that could be secreted from P. syringae hrpJ mutants. Interestingly, we did find a C-terminal HrpZ1 deletion that allowed the HrpZ1 derivative to be secreted from the hrpJ mutant. This suggests that the C-terminal portion of HrpZ1 makes contact with HrpJ either directly or indirectly. The last sub-objective is to identify interacting proteins with HrpJ and we have been unsuccessful up to this point to in identifying HrpJ interactors even though we have tried extensively. Aim 2 of our USDA grant is focused on the HrpK1 protein, which is a type III secreted protein that is suspected to be a translocator protein - a protein that makes pores in host cells and allows other proteins, the type III effector proteins, to be injected into eukaryotic cells by the type III system. The first sub-objective was to determine the extent that many of the type III effectors are injected into plant cells from a P. syringae hrpK1 mutant. All of the type III effectors are reduced in their translocation when delivered by a P. syringae hrpK1 mutant. In the second sub-objective of Aim 2 we wanted to determine whether HrpK1 can form pores in membranes, a characteristic shared by translocators of animal pathogens. We had a lot of problems purifying HrpK1 because it aggregates. However, we were able to purify it in denaturing conditions and then renature it. We tested these HrpK1 preparations and found that they were able to form pores in a liposome assay, in collaboration with Dr. Bill Picking of the Oklahoma State University. We also tested whether HrpK1 interacted with any type III apparatus proteins and any type III-secreted substrates and were unable to identify any HrpK1 interactors. The last sub-objectives of Aim 2 - a microscopy approach to visualize real-time secretion of HrpK1 and other type III-secreted proteins was not successful.We have also investigated aspects of HrpJ and HrpK1 in experiments that were not described in our proposal. 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 discovery that HrpJ controls the secretion of a subset of proteins secreted by the P. syringae type III secretion system should be a major conceptually advance for researchers studying the type III secretion system. It is the first protein found in plant pathogens to clearly affect secretion hierarchy needed for type III secretion. The HrpK1 research establishes the HrpK1 is a translocator for the P. syringae type III secretion system. This also represents a major conceptually advance.
Publications
- Karpisek, A., E. Crabill, and J.R. Alfano. 2011. The Pseudomonas syringae HrpJ protein controls the secretion of translocator proteins. Manuscript in preparation.
- Crabill, E., W.L. Picking, W.D. Picking, and J.R. Alfano. 2011. The HrpK1 protein forms pores in liposomes as acts as the major translocator for the Pseudomonas syringae type III secretion system. Manuscript in preparation.
- Block, A., M. Guo, G. Li, C. Elowsky, T. E. Clemente, and J.R. Alfano. 2010. The Pseudomonas syringae type III effector HopG1 targets mitochondria, alters plant development, and suppresses plant innate immunity. Cell. Microbiol. 12:318-330.
- Walton, J.D., T.J. Avis, J.R. Alfano, M. Gijzen, P. Spanu, K. Hammond-Kosack, F. Sanchez. 2009. Effectors, effectors et encore des effectors: The XIV International Congress on Molecular-Plant Microbe Interactions, Quebec. Mol. Plant-Microbe Interact. 22: 1479-1483.
- Alfano, J.R. 2009. Roadmap for future research on plant pathogen effectors. Mol. Plant. Pathol. 10: 805-813.
- Guo, M., F. Tian, Y. Wamboldt, and J.R. Alfano. 2009. The majority of the type III effector inventory of Pseudomonas syrinage pv. tomato DC3000 can suppress plant immunity. Mol. Plant Microbe Interact. 22: 1069-1080.
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Progress 09/01/08 to 08/31/09
Outputs
OUTPUTS: We have conducted many experiments in the past year that constitute activity outputs. These experiments include many of the assays we have up and running in my laboratory. One of the graduate students working on this project, Emerson Crabill, learned an assay from Dr. Bill Picking research group. In this assay, Emerson purified several secreted accessory proteins including HrpK1 and HrpZ1 and these were tested whether they were able to lyse liposomes containing a fluorescent dye. These experiments clearly showed that both HrpK1 and HrpZ1 can disrupt liposomes supporting that they both act as translocators for type III effectors. That is, they both assist in the transfer of type III effectors across the plant cell wall and plasma membrane into the plant cell. The results in the past year have been disseminated in national and international talks at meetings and university seminars. I gave two international talks in the past year - one at the 22nd New Phytologist Symposium: Effectors in plant-microbe interactions held at the INRA Versailles Research Centre, Paris, France (Sep 2009) and the other at a meeting called The Bacterial Cell Envelope: Structure, function, and infection interface held in Blaubeuren, Germany (Oct 2009). This grant also partially funded my attendance and that of two graduate students, which each presented posters, at the Congress of the International Society of Molecular Plant-Microbe Interactions held in Quebec City, Canada in July 2009. PARTICIPANTS: The first two years of this award I supported a technician, Andrew Karpisek, partially on this award. He has left the position to go to graduate school at a different university. Emerson Crabill continues to be supported on this award and another graduate student, Tanja Toruno has been partially supported by this award. TARGET AUDIENCES: The main target audience for this research is molecular microbiologists, in particular, those that are interested in bacterial pathogens. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
We have made several significant advances this past year and we are now ready to write-up two manuscripts. The first paper will be on Pseudomonas syringae HrpJ protein. This protein was known to be a type III-secreted protein that was needed for type III effectors to be translocated into plant cells and HrpZ1 to be secreted in culture. We found in the past two years that HrpJ was needed for HrpZ1, HrpW1, HrpAK1, and HrpK1 all to be secreted from P. syringae in culture. Thus, HrpJ is acting as a control protein to regulate which proteins are secreted by the type III apparatus. Interestingly, all of these proteins are putative translocators, which makes sense since a hrpJ mutant was earlier shown not to be able to inject type III effectors. We found that the secretion of these translocators can be complemented by hrpJ constructs that encode secretion defective HrpJ derivatives. That is, HrpJ needs to be within the bacterial cell to complement the translocator secretion defect. We also found that if a 50 amino acid C-terminal region of HrpZ1 was deleted then the protein could be secreted in the absence of HrpJ. This suggests that this HrpZ1 region interacts with HrpJ, however, we have been unable to show that these proteins interact. The second paper that we are writing-up is focused mostly on the HrpK1 protein. Prior to this award we had indirect evidence that suggested HrpK1 was a translocator for the type III apparatus. P. syringae mutants defective in HrpK1 could secrete proteins in culture but did not inject them into plant cells based on a great reduction in the ability of this mutant to elicit a hypersensitive response (HR). The bacterial-induced HR is due to the injection of type III effectors into plant cells. Over the past two years we have more specifically determined the hrpK1 mutant translocation defect by assaying the ability of the hrpK1 mutant to translocate type III effector-adenylate cyclase (CyaA) fusions into plant cells. The hrpK1 mutant is greatly reduced in its ability to inject type III effector-CyaA fusions into plant cells. However, certain type III effector-CyaA fusions are injected at a higher level than other fusions. This year we also determined that HrpK1 and HrpZ1 could disrupt liposomes containing a fluorescent dye consistent with both acting as translocators. Finally, to try and determine the contribution of HrpZ1 and other related proteins to HrpK1 we have tested the ability to inject proteins in an array of P. syringae mutants lacking different combinations of these proteins. In general, we find that HrpK1 contributes the most to translocation. However, the HrpZ1 protein and other related proteins do appear to also subtly contribute.
Publications
- Block, A., M. Guo, G. Li, C. Elowsky, T. E. Clemente, and J.R. Alfano. 2009. The Pseudomonas syringae type III effector HopG1 targets mitochondria, alters plant development, and suppresses plant innate immunity. Cell. Microbiol. doi:10.1111/j.1462-5822.2009.01396.x
- Walton, J.D., T.J. Avis, J.R. Alfano, M. Gijzen, P. Spanu, K. Hammond-Kosack, F. Sanchez. 2009. Effectors, effectors et encore des effectors: the XIV International Congress on Molecular-Plant Microbe Interactions, Quebec. Mol. Plant-Microbe Interact. 22: 1479-1483.
- Alfano, J.R. 2009. Roadmap for future research on plant pathogen effectors. Mol. Plant. Pathol. 10: 805-813.
- Jeong, B-r., K. van Dijk, and J.R. Alfano. 2009. Pseudomonas syringae type III-secreted proteins and their activities and effects on plant innate immunity. Annu. Plant Reviews. 34: 48-76.
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Progress 09/01/07 to 08/31/08
Outputs
OUTPUTS: We have conducted many experiments that constitute activity outputs that have resulted in advances in our project during the past year. These experiments include many that are well established in the lab such as protein secretion assays, plant bioassays, and expression studies. A graduate student (Emerson Crabill) in my lab working on experiments that are part of this visited the lab of Bill Pickings at the University of Kansas to learn an assay important to our project. This assay determines whether a test protein can form a pore in a membrane. A goal of a subset of experiments on HrpK1 is to determine whether it can form pores in membranes, which would be predicted if it is acting a translocator protein. The other main output to report is that this grant supported two graduate students in my lab working on this project to attend the American Phytopathological Society meeting held in Minneapolis, Minnesota in summer 2008. The grant also has partially supported 4 undergraduate researchers in my lab the past year. Results from our project were disseminated in the form of presented posters at the APS meeting this past summer and in talks that I gave nationally and internationally the past year. PARTICIPANTS: Emerson Crabill, a PhD student in the lab, has been supported by this grant and is working on experiments of HrpK1. Andrew Karpisek, a research technician, has been working on the experiments with HrpJ and he is being supported off of this grant. TARGET AUDIENCES: The main target audience of our research is molecular microbiologists, in particular those that are interested in bacterial pathogens. PROJECT MODIFICATIONS: No major experimental approach changes.
Impacts
We have made several interesting observations over the past year that have resulted in a change of knowledge outcome that should be reported. Prior to this grant being awarded, we discovered that Pseudomonas syringae hrpJ mutants were unable to secreted HrpZ1 into culture supernatants via the type III protein secretion system (T3SS). HrpJ was also known to be a type III secreted protein so the implication was that HrpJ someone controlled which proteins were secreted via the T3SS. One of the main goals of Aim 1 was to determine whether other proteins required HrpJ for their secretion via the T3SS. Interestingly, we found that HrpW1, HrpK1, and HopAK1 all required HrpJ these proteins to be secreted via the T3SS. This is interesting because all of these proteins are predicted to participate in the translocation process, the step in type III secretion which transfers type III secreted proteins across the plant plasma membrane. Thus, it seems that HrpJ is required for any of these proteins to be secreted and, therefore, it seems to control whether translocation occurs which we find to be fascinating and relevant. Another important goals of Aim 1 was to determine whether a cell-bound unsecreted derivative of HrpJ could restore the secretion of the translocator proteins to determine whether HrpJ could complement when it was in the cell or whether it had to be secreted before the other proteins could be secreted. Interestingly, we found that a HrpJ derivative lacking its T3SS could complement the secretion of HrpZ1 indicating that HrpJ needed to be in the cytoplasm to allow for HrpZ1 to be secreted. Our current hypothesis is that HrpJ interacts with the translocator proteins near the type III secretion pore allowing these proteins to be secreted. We have also made progress on our understanding of the HrpK1 protein. Prior to the award we had indirect evidence that suggested HrpK1 acted as a translocator for the T3SS. HrpK1 was known to be secreted via the T3SS. P. syringae hrpK1 mutants retain the ability to secrete proteins but are greatly reduced in their ability to elicit the HR, which is an indication of translocation. We have now measured more specifically the ability of the hrpK1 mutants to inject specific effector proteins into plant cells using adenylate cyclase reporter fusions. In general, the hrpK1 mutants are reduced in the ability to inject proteins. Interestingly, some effector proteins are more dramatically reduced and our current experiments are focused on trying to determine why it appears this way. Additionally, we now have preliminary data indicating HrpK1 can form pores in membranes. We will work on this in the next year. This is critical if we are to determine that HrpK1 is a translocator.
Publications
- No publications reported this period
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