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A 50-KILODALTON DOMAIN OF THE TOBACCO MOSAIC VIRUS REPLICASE PROTEIN ELICITS THE N-MEDIATED HYPERSENSITIVE RESPONSE

A 50-KILODALTON DOMAIN OF THE TOBACCO MOSAIC VIRUS REPLICASE PROTEIN ELICITS THE N-MEDIATED HYPERSENSITIVE RESPONSE
FL ERICKSON1, SP HOLZBERG2, AA CALDERON-URREA1,3, VANESSA HANDLEY1, C CORR1, BJ BAKER1
1Dept. of Plant and Microbial Biology, Plant Gene Expression Center, University of California, Berkeley, and United States Department of Agriculture, 2Dept. of Environmental Science, Policy, and Management, University of California, Berkeley, CA 94710 USA; 3Dept. of Biology, California State University, Fresno, CA 93740 USA

Background and objectives
The interaction between tobacco bearing the N gene and tobacco mosaic virus (TMV) is a classic model system for studying gene-for-gene disease resistance. N encodes a protein belonging to the TIR/NBS/LRR family of plant disease resistance proteins. The TMV replicase protein has been implicated as the avirulence factor that triggers the N-mediated hypersensitive response (HR) [1]. Using a vector based on the non-eliciting tobamovirus, Ob, Padgett demonstrated that expression of a fragment of the 126 kD replicase elicits the HR [2]. Because a tobamovirus was used as a vector system in these studies, uncertainty remains whether viral replication, movement, and/or other viral factors are also required for triggering N-mediated HR. Identifying and characterizing the minimal viral component(s) required for triggering N-mediated defense responses are important steps toward understanding the biochemical mechanisms of recognition and the subsequent responses to viral infection.


Results and conclusions
We employed Agrobacterium-mediated transient and stable transgenic expression strategies to express portions of the TMV genome in SR1 and SR1:NN tobacco. Importantly, these methods express viral proteins in the absence of viral replication and movement. Both methods demonstrated that expression of the C-terminal 50 kD region of the 126 kD replicase is sufficient to elicit N-mediated HR. The thermosensitivity of the N-mediated response to TMV is retained when induced by expression of this 50 kD domain. Deletion and site-directed mutagenesis of the elicitor domain is in progress. Preliminary results indicate that small truncations from either the N- or C- terminus of the 50 kD domain abolishes elicitor activity. However, immunoblot analysis indicates that the failure of these smaller polypeptides to induce the HR may be due to reduced protein expression. Biochemical characterization of the elicitor domain is in progress using recombinant proteins purified from E. coli and insect cells. These genetic and biochemical analyses should further define the molecular determinants involved in TMV recognition by N-containing tobacco.


References
1. Padgett HS, and Beachy RN 1993. Plant Cell 5, 577-86. 2. Padgett HS, Watanabe Y, and Beachy RN 1997. Molecular Plant-Microbe Interactions 10, 709-15.