The goal of the proposed research is to characterize proteins that interact with type III effectors, using the Arabidopsis- P. syringae interaction as a model pathosystem. One type III effector, the Pseudomonas syringae AvrPpiB protein, has no known or predicated biochemical function. However, preliminary data have shown that this factor increases bacterial growth by 10-fold under mild water deficit conditions when introduced into a bacterial strain with reduced virulence. AvrPpiB also causes systemic chlorosis when expressed in older leaves of Arabidopsis. These observations suggest that AvrPpiB plays a physiological role in microbes and plants. Several objectives are proposed to understand the precise function of AvrPpiB. First, ecotype differences in AvrPpiB-dependent host responses will be mapped as a prelude to positional cloning of the responsible genes. Second, a mutant screen will be undertaken to identify host (Arabidopsis) genes that are required for AvrPpiB-dependent host responses. The mutants will be characterized using physiological, biochemical, and "-omics" technologies, and potentially informative genes will be cloned. Third, changes in transcription profiles will be assessed in Arabidopsis that either respond or fail to respond to AvrPpiB. This should provide insight into signaling pathways that give rise to the response. Fourth, proteins that interact with AvrPpiB will be identified in vitro using yeast expression assays, and in vivo by expressing TAP-tagged effector protein fusions in Arabidopsis and purifying tagged protein complexes.
Broader Impacts: This project will add to our understanding of host components that interact or mediate plant response to bacterial pathogens. This has implications for the design of resistant crop species. The proposed research will also involve undergraduates, graduate students, and postdoctoral fellows, including minorities. Outreach activities will be undertaken that relate to educating the public on issues about genetically modified foods.
PI: Sarah R Grant, Department of Biology, University of North Carolina at Chapel Hill Intellectual Merit: Type III effectors of Gram-negative bacteria are injected directly into host cells, where they inactivate host pathogen defenses by interacting with host proteins. HopAM1 is a typical type III effector from Pseudomonas syringae: it can suppress plant defense responses and render a weakly virulent P. syringae strain more virulent. Because they suppress plant defenses by interacting with and modifying host proteins, type III effectors like HopAM1 are useful tools to identify essential components to the complex plant defense response. What makes HopAM1 unique among the many type III effectors being studied by researchers around the world is a clearly visible HopAM1-dependent phenotype that we have directly related to its virulence function using host genetics. We used this phenotype to isolate mutants in the model plant Arabidopsis that have lost the ability to respond to HopAM1. These mutants will have genetic defects in the genes for plant defense proteins that are affected by HopAM1. In year 2 of this project, we demonstrated that HopAM1 makes Arabidopsis plants more susceptible to bacterial infection and also resistant to drought. We found that transgenic plants expressing HopAM1 protein were unusually sensitive to the plant drought response hormone Abscisic Acid (ABA). Work published in 2008 (Goel et al., The Pseudomonas syringae Type III Effector HopAM1 Enhances Virulence on Water-Stressed Plants. Molecular Plant Microbe Interactions. Volume 21, 361-370.) demonstrates that transgenic plants expressing HopAM1 are hypersensitive to ABA in germination responses and stomatal closure. Increasing evidence from several research groups indicates that ABA inhibits plant defense responses and our data shows that HopAM1affects ABA responses but not production. We predict that HopAM1 affects plant cell biology at steps that overlap with ABA responses resulting in defense response suppression as well as stimulation of ABA responses. We have been granted renewed funding from NSF (grant number IOB-1022286) to continue our studies of HopAM1 function in which we proposed to extend our genetic analyses of HopAM1 responses in order to identify plant proteins that are affected by HopAM1.The mutants we have isolated in this project will reveal genes involved in plant defense. Broader Impacts Four undergraduate students, one doctoral student and two postdoctoral fellows participated in this project, four women and three men. The postdoctoral fellows are now researchers at Dupont India and at the Agriculture Ministry in Japan. The doctoral student is now a lecturer in the Biology Department at Johns Hopkins University. The undergraduate students are doing graduate research in biology at the University of North Carolina, studying dentistry at UNC or studying education Wake Forest University. I work through the UNC Botanical Garden and the UNC Morehead Science Center to discuss controversial research developments with the public and make presentations of genetics for the general public. I have also used aspects of the research in this grant to develop laboratory exercises for my undergraduate Laboratories in Genetics class.
