Plants can detect thousands of different pathogens and actively respond. This detection system employs two major classes of receptors: transmembrane receptors on the cell surface that detect conserved pathogen associated molecular patterns (PAMPs), and intracellular receptors that detect specialized pathogen effector proteins. These intracellular receptors share a common structure consisting of a nucleotide binding (NB) domain and leucine rich repeats (LRR). NB-LRR genes are also found in humans, and mutations in several of these are associated with various autoinflammatory diseases, including Crohn's disease and Familial Cold-Induced Autoinflammatory Syndrome. Two human NB-LRR proteins, NOD1 and NOD2, have been shown to activate immune responses upon detection of bacterial cell wall fragments, thus NB-LRR proteins in plants and animals fulfill similar roles. However, the mechanisms by which NB-LRR proteins detect pathogen molecules and activate immune responses are poorly understood. This proposal focuses on the molecular mechanisms of NB-LRR activation, the role of subcellular localization of NB-LRR proteins, and the identification of downstream proteins engaged by activated NB-LRR proteins in plants. Specifically, this proposal focuses on the RPS5 protein from Arabidopsis. RPS5 mediates recognition of the AvrPphB protein secreted by the bacterial pathogen Pseudomonas syringae. A specific goal of the proposed research is to identify the mechanisms by which RPS5 detects AvrPphB and activates defense responses. Past work has shown that AvrPphB is a cysteine protease that targets the Arabidopsis PBS1 kinase. Recent work has demonstrated that PBS1 forms a complex with RPS5 and that cleavage of PBS1 is required for activation of RPS5. Activation appears to be dependent on ATP binding as mutations in the P- loop of the RPS5 NB domain block activation, while mutations in the Walker B motif that impair ATP hydrolysis cause RPS5 to be constitutively activated. Based on these and other data, it is hypothesized that PBS1 cleavage is detected by the LRR domain of RPS5, which causes a conformational change in RPS5 that enables exchange of ADP for ATP. Binding of ATP then releases the N-terminal coiled-coil domain of RPS5 from autoinhibition, freeing it to engage downstream signaling proteins. This model will be tested by pursuing three specific aims. First, an in vitro system containing soluble RPS5, PBS1 and AvrPphB proteins will be used to assess whether ATP binding, ATP hydrolysis and/or ADP/ATP exchange are regulated by PBS1 cleavage. Second, immunomicroscopy and live-cell imaging will be used to assess whether activation of downstream signaling events by RPS5 requires its subcellular relocalization, particularly to the nucleus. Finally, proteins that associate with activated forms of RPS5 will be identified using both yeast two-hybrid approaches and by purification of protein complexes from plant extracts. These experiments will provide key insights into the innate immune systems of both plants and humans.
The immune systems of plants and animals employ similar proteins to detect disease-causing organisms. Mutations in the genes encoding these proteins are associated with debilitating autoimmune diseases in humans, and susceptibility to disease in plants. By studying at a molecular level, how these proteins function, this research will lead to new treatments for autoimmune diseases, and new approaches to breeding disease resistant crops.
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