Functional analyses of NPR1 in plant defense

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Innate immune mechanisms are conserved in evolution. The NPR1 protein of Arabidopsis is a master regulator of gene expression in plant defense. Similar to the mammalian immuno- regulator NF-kB, the NPR1 protein is nuclear translocated upon induction. In the resting state, NPR1 is present in the cytoplasm as a complex with the organelle translation elongation factor EF-Tu through disulfide bonds. An increase in salicylic acid upon pathogen challenge results in a biphasic redox change and the release of NPR1 monomer to enter the nucleus. The NPR1 protein levels oscillate through proteasome-mediated degradation in the nucleus. This dynamic change is required for NPR1 function and is facilitated by phosphorylation of DSXXXS ("IkB site") found in NPR1. NPR1 controls gene expression as a cofactor for the TGA transcription factors. Genomic and genetic studies led to the identification of HSF4 and several DNA damage repair proteins such as BRCA2A and RAD51D as additional nuclear factors involved in NPR1-mediated gene expression. In aim 1, we will reveal the biological significance of the NPR1-EF-Tu complex found in the cytoplasm. The NPR1-EF-Tu interaction will be disrupted through mutagenesis to determine whether EF-Tu is only part of the NPR1 oligomer or also links defense with organelle activities. Since EF-Tu is a known elicitor of plant defense, we will also investingate the possibility that Pseudomonas EF-Tu interferes with NPR1 oligomer formation during infection. In aim 2, we will study the oscillation of NPR1 monomer. The IkB site mutants will be further studied to understand the regulatory role of phosphorylation in NPR1 function. The corresponding protein kinase will be identified. In aim 3, we will isolate and characterize the NPR1 nuclear complex using TAP-tagged NPR1. Physical interactions between NPR1, transcription factors and chromatin remodeling proteins will be examined and characterized in a temporal fashion. The significance of the project to human health is two-fold: First, understanding the interplay between defense and organelle activity, the oscillation in transcription regulation, and a possible link between defense-associated transcription and DNA recombination are highly novel research areas relevant to all eukaryotic organisms. Second, human health is directly impacted by environmental health. Using plant innate immunity to control diseases will help reduce pesticide pollution, increase food nutrition, and save natural and human resources. The significance of the project to human health is two-fold: First, studying the crosstalk between cytoplasmic/nuclear function and organelle activity, understanding the biological significance of periodicity in transcription regulation, and establishing a link between pathogen-induced gene expression and DNA repair machinery may lead to exciting new discoveries in basic biology. Second, human health is directly impacted by environmental health. Using plant innate immunity to control disease in crop plants will help reduce pesticide pollution, increase food nutrition, and save natural and human resources.