Plants employ a branched immune system that prevents disease in the vast majority of plant-microbe interactions. One branch recognizes pathogen associated molecular patterns (PAMPs) that are contained within conserved microbial molecules. A second branch recognizes effector proteins, which are pathogen-encoded virulence factors. These two branches utilize common signaling pathways and induce common outputs, yet little is known about the mechanistic basis of intersections within the network they comprise. The RIN4 protein of the model plant Arabidopsis thaliana is a hub protein with key roles in regulating both branches, thus a detailed understanding of RIN4 will provide fundamental insight into the function of the plant immune system. The work will address three aspects of RIN4 function. Changes in the sub-cellular localization of RIN4, which are induced by bacterial effector proteins, have distinct effects on the activity of the two branches of the plant immune system. The proposed work will determine how sub-cellular localization of RIN4 affects its defense regulatory function. Preliminary data also indicate that RIN4 interacts with components of the plant exocyst, which is a multi-protein complex that regulates vesicle trafficking and membrane fusion events that play important roles in the function of the plant immune system. The proposed work will determine how interaction of the exocyst with RIN4 contributes to plant immune function. Finally, RIN4 functions in guard cells to regulate stomatal aperture in response to bacterial infection. The ability of plants to prevent invasion by epiphytically growing bacterial pathogens depends on their ability to close and keep closed their stomata. Control of guard cell function by RIN4 and bacterial effector proteins that target RIN4 will be investigated. The research aims will be addressed using plant pathology, molecular, biochemical, and cell biological methods.
Broader Impacts: The trend to use agriculture to fuel as well as feed the world places great demand on optimizing crop productivity. To most effectively breed and/or engineer disease resistance in food and biofuel crops while minimizing deleterious side effects, such as reduced yield, requires fundamental knowledge about connections within the network comprising the plant immune system. Though the work will be done in the Arabidopsis thaliana model system, the findings will be applicable to agriculturally important plants, as RIN4 homologs have been identified in all land plants examined and demonstrated to play important roles in immune function of several crop plants. Findings will be presented at meetings and published in peer-reviewed journals. The PI has a proven record of working with underserved groups at the undergraduate, graduate, and post-doctoral levels, including the recent graduation of an Hispanic Ph.D. student. Funds from the project will also support participation of students from small liberal arts colleges in the Summer Undergraduate Research Experience program at The Ohio State University. Finally, the PI will participate in the organization and instruction of a module focusing on the plant immune system during the 2012 Summer Practical Workshop in Functional Genomics at Ohio State.
Diseases of crop plants are a major limiting factor in efforts to produce sufficient food, fiber, fuel, and other bioproducts to meet the demands of an increasing world population. The most ecologically friendly means of combatting plant diseases is through the deployment of plant varieties that are resistant to infection. The innate immune system of plants is a major component of resistance that functions through a battery of responses including plant cell fortification and deployment of antimicrobial activities. Defense responses elicited by the plant immune system are quite effective when activated, but can also be quite detrimental to plant health if inappropriately activated. Thus, the ability of plants to recognize the presence of threatening entities, such as microbial or fungal pathogens, and thus turn on defense responses at the appropriate time and location is critical to their overall health and production. Recognition and response to potential pathogens is mediated by two distinct branches of the plant immune system. The first is by recognition of conserved and abundant components of a microbe, called pathogen associated molecular patterns or PAMPs. PAMPs are recognized by receptors on the surface of plant cells resulting in the activation of intracellular signaling to elicit defense responses. Pathogens actively deploy virulence factors, including pathogen-encoded proteins called effector proteins, to suppress PAMP-triggered immunity. The second mechanism for pathogen recognition is based on perception of these pathogen-derived effector proteins. The effectors are typically recognized by intracellular plant proteins called resistance or R-proteins. Our project focused on a protein of the model plant, Arabidopsis thaliana, called RIN4. Bacterial effector proteins target RIN4 to suppress PAMP-triggered immunity. However, RIN4 interacts with several Arabidopsis R-proteins and the perturbation of RIN4 by the bacterial effectors is the signal that activates the R-proteins. This unique role of RIN4 in linking the PAMP- and effector-triggered immune responses makes its study very informative for the function of plant immune systems in general. Our research revealed two key findings about RIN4. First, its role in mediating PAMP-triggered immunity, and the activity that is inhibited when RIN4 is targeted by bacterial effectors, involves interaction with a protein that mediates secretion from the plant cell. Targeting of RIN4 by bacterial effectors causes mis-localization of the secretion protein and disrupts the secretion of an antimicrobial protein. Our second key finding is that perturbation of RIN4 by bacterial effector proteins can affect its activity by altering its sub-cellular localization. Specifically, one bacterial effector protein cleaves RIN4 to produce a fragment that moves away from the plasma membrane. This non-membrane tethered fragment of RIN4 potently suppresses PAMP-triggered immunity. However, the R-protein that recognizes this bacterial effector does so by recognizing the non-membrane tethered fragment of RIN4 and activating effector-triggered immunity. Thus, our work has revealed novel insights into the molecular function of RIN4 and, more generally, into the mechanisms linking the two main branches of the plant immune system.
