1.12.2S
INCREASING THE EFFICACY OF COAT PROTEIN- AND MOVEMENT PROTEIN-MEDIATED RESISTANCE REQUIRES AN UNDERSTANDING OF MECHANISMS OF RESISTANCE

RN BEACHY, M BENDAHMANE, C-O LIM and C REICHEL

The Scripps Research Institute, 10550 No. Torrey Pines Rd, La Jolla, CA 92037, USA

Background and objectives
Following our initial reports of coat protein-mediated resistance (CP-MR) to tobacco mosaic virus (TMV), and subsequently, resistance due to expression of non-functional mutants of the TMV movement protein (dMP-MR), we investigated the biochemical and structural basis of resistance. The ultimate purpose of these studies is to increase the efficacy of resistance to virus diseases based on knowledge of the mechanisms of resistance. Most of our studies have involved TMV and transgenic tobacco and tomato plants.

Materials and methods
Mutants of the TMV CP were constructed, based on the known 3-dimensional structures of the molecule as well as the virus particle, to alter the intra- and intermolecular protein-protein interactions. Mutant CPs were then expressed as transgenes in transgenic plants and the effect of mutation on the CP-MR was determined. For studies of the MP, for which 3-D structure is not yet determined, we constructed a series of non-functional mutants of the protein, and determined the effect of the mutation on subcellular distribution of the protein: for these studies we fused the MP with the green fluorescent protein and determined the sites of accumulation of the fusion protein using fluorescence microscopy. Mutant proteins that accumulate in cells in a manner similar, or dissimilar to that of the wild-type MP were produced in transgenic plants, and the plants were tested for resistance to TMV and other viruses.

Results and conclusions
In previous work we presented data that supported the hypothesis that CP-MR against TMV results because the CP restricts disassembly of challenge virus, and proposed that protein-protein interactions between transgenic CP and challenge virus were essential for the reaction. To further define the role of protein structure and sequence in these interactions plant lines that accumulated equivalently high levels of wild-type or mutant CP were developed. Sucrose density gradient centrifugation and electron microscopy were used to determine whether or not the mutant CP molecules assembled to form virus-like particles, or other states of aggregation or assembly. R1 progeny of selected plant lines were challenged by inoculation with TMV and the degree of resistance was correlated with the ability of the CP to assemble. Plant lines that contained CP that was capable of self-assembly provided CP-MP, while CP that did not self-assemble did not. Interestingly, certain CP mutants, in particular CP molecules that form highly stable aggregates and virus-like particles, provided greater levels of resistance than did wild-type CP. A molecular model that predicts the factors that contribute to resistance will be presented.

We have demonstrated that during TMV infection the MP accumulates on the endoplasmic reticulum, in association with microtubules, and plasmodesmata and in punctate structures at or near the plasmalemma. We are currently characterizing what role, if any, the MP in each of these subcellular compartments plays in virus infection and replication. We previously reported that transgenic plants that accumulate a certain non-functional mutant of the movement protein conferred resistance to TMV infection while other mutants did not. A goal of our research is to construct mutant MPs that do not function in virus movement but act as dominant negative mutants to interfere with the virus infection in transgenic plants. Our progress in identifying such mutants will be described.

References
1. Bendahmane M, Fitchen JH, Zhang G, Beachy RN, 1997. Journal of Virology 71, 7942-7950.
2. Heinlein M, Padgett HS, Gens S, Pickard B, Casper SJ, Epel BL, Beachy RN, 1998. Plant Cell (in press).