1.8.56
MOLECULAR ANALYSIS OF TOBACCO MOSAIC VIRUS RESISTANCE GENE N

SP DINESH-KUMAR, S WHITHAM and B BAKER

Department of Plant Biology, University of California, Berkeley, and Plant Gene Expression Center, USDA-ARS, Albany, CA 94710, USA

Background and objectives
Plants are hosts to thousands of infectious diseases caused by a vast array of phytopathogenic fungi, bacteria, viruses and nematodes. Plants recognize and resist many invading phytopathogens by inducing hypersensitive response (HR, localized cell death) and systemic acquired resistance (SAR). HR and SAR depend upon interaction between a dominant or semi-dominant resistance (R) gene product in the plant and a corresponding dominant phytopathogen avirulence (Avr) gene product. Phytopathogen Avr products are predicted to function as ligands and host R products to function as receptors in an interaction leading to plant disease resistance.

Over the past 3 years, numerous R genes were cloned from several plant species. Although these genes confer resistance to diverse bacterial, fungal, viral and nematode pathogens, their products share striking structural similarities, suggesting that certain signalling events are held in common in plant defence [1]. To understand the molecular-genetic basis of disease resistance, we have isolated the resistance gene, N, of tobacco that mediates resistance to the viral pathogen tobacco mosaic virus (TMV) [2]. The deduced amino acid sequence of N comprises three functionally significant domains: a putative nucleotide binding site (NBS), a leucine-rich repeat (LRR) region, and an amino-terminal domain with similarity to cytoplasmic domains of the Drosophila developmental regulator Toll and the mammalian interleukin 1 receptor (IL-1R) (TIR, Toll-IL-1R homology region).

Results and conclusions
The presence of conserved LRR, NBS and TIR motifs between different R genes from distantly related plant species indicates that these motifs are structural and/or functional factors in determining resistance responses to diverse groups of plant pathogens. In order to understand the importance of the putative TIR, NBS and LRR motifs in N-mediated TMV resistance, we created an array of deletion and point mutations in the N gene. The results of our structure-function analysis indicate that the TIR, NBS and LRR domains are required for N function.

Sequence analysis of N cDNAs and genomic clones indicate that the N gene encodes both full-length (N) and truncated (Ntr) forms of proteins. The N gene contains five exons that are spliced together to form an open reading frame (ORF) of 3432 nt encoding a protein of 1144 amino acids. Ntr form results from alternative splicing of a 70-bp exon to form a 1956-nt ORF encoding a protein of 652 amino acids. Ntr is identical to the amino terminus of N except for the 36 additional amino acids at the carboxyl terminus. To understand functional significance of alternative splicing in N expression, we created number of deletions and transgenic reconstructions using N cDNAs and genomic clone. Results indicate that alternative splicing and genomic-3' untranslated region (g-3'UTR) are required for proper N function.

References
1. Baker B, Zambryski P, Staskawicz B, Dinesh-Kumar SP, 1997. Science 276, 726-733.
2. Whitham S, Dinesh-Kumar SP, Choi D et al., 1994. Cell 78, 1101-1115.