DISSECTION OF PLANT VIRUS RESISTANCE SIGNALING PATHWAYS

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Plants exhibit natural resistance to diseases through systems of gene-for-gene interactions. Pathogens express genes called avirulence genes (Avr) that interact with plant encoded resistance genes (R). One example of such an interaction is between the tobacco N gene that encodes resistance to tobacco mosaic virus (TMV). Another is the RCY1 gene of Arabidopsis, which encodes resistance to cucumber mosaic virus-Y (CMV-Y). In fact, many avirulence and resistance genes have been identified in recent years, yet little is known about the mechanism of resistance. Pathologically, resistance is characterized by a hypersensitive response that results in massive cell death, but restricts propagation of the virus. The structure of many of resistance proteins suggests that the responses may activate signaling pathways. This proposal will use an Arabidopsis thaliana genetic system to study both the N/TMV and Rcy1/CMV-Y systems. Expression in Arabidopsis of the Avr protein of TMV (p50 helicase) in N- expressing cells or CMV-Y coat protein (CP) in Rcy1-expressing cells during germination of seedlings causes rapid death of the developing plant. This provides a genetic selection for plant mutants that fail to respond to the Avr protein, potentially identifying proteins downstream of the resistance genes. The proposal has 7 Specific Aims. The first three address the identity of cellular components that make up the response system to the two pathogens. Aim 1 will attempt to identify mutations that affect components of the pathway, both loss and gain-of- function mutations. In addition, yeast two and three-hybrid approaches and biochemical approaches (co-purification, affinity chromatography or interaction cloning) will also be used to identify proteins that interact with the resistance proteins. Aim 2 will identify genes whose expression is altered during response to pathogens and Aim 3 will test whether known genes affecting other gene-for-gene responses also affect the two being studied here. The last four Specific Aims concern the biochemical characterization of resistance genes. Aim 4 will determine whether the Rcy1 gene product resembles the N protein by isolating and sequencing the RCY1 gene. Aim 5 will further characterize the elicitor of the Rcy1 response, the CMV-Y coat protein. Aim 6 will test the function of a putative nucleotide binding site found in the N protein. Binding of nucleotides and nucleotide analogs to the protein will be done to determine the specificity of the interaction. Dr. Dinesh-Kumar will determine if the N protein is an NTPase or kinase, including testing if it is phosphorylated by itself or other cytosolic proteins. Aim 7 will determine whether N protein directly or indirectly interacts with its elicitor, TMV replicase.