Structural Determination and Analysis of Proteins Involved in the Plant Innate Immune Syste

Plants and pathogens are locked in a never-ending evolutionary arms race for the advantage in fitness and proliferation. Plants employ protein receptors to monitor their extra- and intracellular environments for signs of microbial presence and initiate an innate immune response to protect the plant cell and prevent the spread of the threat. These responses are carefully controlled and regulated, as an increase in the immune related expression patterns leads to a decrease in plant growth and maturation. Pathogens use a system of small molecules and proteins to evade, inhibit, or dysregulate the plant response. Much research has been done on determining the molecular pathways that detect and then initiate the plant immune response and how pathogens attempt to block or disrupt these pathways, but so far very little information about the structures of the proteins and their mechanisms has been generated. A structural and mechanistic understanding of these proteins and pathways could lead to new disease prevention strategies or new targets for transgenic plants.;The protein BIK1 has been shown to be involved in regulating plant growth and immunity. BIK1 is a membrane localized kinase and associates with several membrane-bound receptors. When an associated receptor binds to its target, BIK1 is phosphorylated and initiates a response, either leading to plant growth or induction of the immune response, which are inversely regulated, but little is known about how BIK1 distinguishes between the two. The structure of BIK1 from the model organism Arabidopsis thaliana has been solved. The structural fold is that of a canonical Ser/Thr and Tyr kinases. BIK1 features a uniquely extended loop, that is missing in other plant kinases, and might provide a platform for protein-protein interactions. This loop is the location of residues Ser89 and Thr90 which were shown to be phosphorylated upon immune system stimulation by the bacterial elongation factor Tu (EF-Tu). Mutational analysis of these residues to create phospho-mimetic and phosphor-null variants showed that these residues are responsible for the regulation of jasmonic acid during an immune response.;On the microbe side, pathogenic organisms produce small molecules or proteins known as effectors. The highly studied effector, Avr4, from the tomato pathogen Cladosporium fulvum (CfAvr4) is an extracellular chitin binding protein that protects the fungus from plant-derived chitinases and sequesters chitin fragments from plant receptors. Chitin, a polysaccharide of N-acetylglucosamine, is the main structural component of the fungal cell wall and a potent inducer of the plant immune response. Orthologs of Avr4 are found in numerous fungal species including pathogens of plants ranging from banana to pine tree. Here we solved the structure of two Avr4 orthologs of tomato pathogens, Avr4 from Pseudocercospora fuligena (PfAvr4) and CfAvr4. The structures were the first Avr4 structure solved and some of the first structures from the carbohydrate binding module family 14 (CBM14) determined. CfAvr4 was solved bound to the chitin hexasaccharide, which provided the first structural information of the ligand binding mechanism of the CBM14 family. Both proteins had nearly identical cysteine-knot like folds and forms dimers in the crystal structure. In the CfAvr4 structure the ligand binds in a shallow cleft formed along the length of the protein and stacks against another ligand-protein pair with the ligands nearly fulling occluded from the solvent. Structure-guided mutational analysis, biochemical characterization, and plant based assays confirmed the ligand binding mechanism and showed that the residues necessary for binding are separate from those that lead to reception by a plant based receptor, Cf-4, that directly recognizes Avr4.;The structure of the UDP-GlcNAc 2-epimerase from Neisseria meningitidis Serogroup A (NmSacA) was recently solved bound to the substrate UDP-GlcNAc. The epimerase converts UDP-GlcNAc to the C2-epimer UDP-ManNAc which is used to form the bacterial capsular polysaccharides (CPSs). CPSs are organized structures surrounding the cell that protect the bacteria from the environment and the host immune system. While NmSacA is from a human pathogen, similar 2-epimerases are found in plant pathogens and information from this structure can be applied to them. The structure showed a similar active site architecture to previously published enzymes and provides a potential understanding as to why NmSacA lacks the allosteric regulation that is found in other UDP-GlcNAc 2-epimerases.