M CHU, J-W PARK, S GARCIA, J KUECKER, B DESVOYES, B WHITEHEAD and HB SCHOLTHOF

Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX 77843, USA

Background and objectives
A critical host-range determinant for plant-virus interactions is compatibility for long-distance movement. An infectious cDNA clone of the 4800-nt single-stranded positive-sense RNA genome of tomato bushy stunt virus (TBSV) provides a suitable genetic system for studies on virus spread because this virus contains two 3' proximal overlapping movement genes, p22 and p19, that are expressed from one subgenomic RNA. The membrane-bound p22 protein is expressed at relatively low levels and is required for cell-to-cell movement. Although the start codon for p19 is located downstream of the AUG for p22, the soluble p19 protein is abundantly expressed and promotes long-distance systemic invasion in some hosts. These features of p19 provide an ideal model system for detailed investigations on interactions that govern long-distance virus spread [1].

Results and conclusions
To determine whether the high level of p19 expression is controlled by leaky scanning of ribosomes past the AUG of p22, changes were introduced to improve the context sequences of the p22 start codon. Subsequent in vitro translation assays and in vivo experiments in protoplasts and whole plants revealed that improvement of the context for p22 caused an increase in protein accumulation and a concomitant decline in p19 protein production. The biological consequence of this reduction in p19 protein levels was ineffective long-distance movement in spinach plants. These results illustrate the biological relevance of dosage control of protein accumulation, and therefore this may present another mode of regulation for gene function.

In related genetic studies on the involvement of host-specific interactions with the p19 protein, we mutated codons for clustered charged amino acids that were considered candidates to be exposed on the protein surface for specific interactions with host proteins. Bio-assays with several mutants revealed that amino-acid residues located towards the central domain of the p19 protein were required for TBSV to systemically invade spinach plants. Further analyses revealed that replication and cell-to-cell movement were not affected by these mutations, but the mutants were defective for long-distance spread. These results suggest that the central domain of the p19 protein may interact with specific host factors to stimulate a systemic invasion.

During time-course studies in spinach it became obvious that a systemic infection proceeded from the inoculated leaves downward to the roots prior to infection of upper leaves, and the p19 protein was the first virus protein to be detected in roots. Subsequent experiments revealed that a p19 mutant that previously was shown to be debilitated in its ability to invade spinach plants was defective for movement from inoculated leaves down into the roots.

The combined results of the above studies lead us to speculate that a functional p19 protein is required in abundance for interaction with host factors, to permit spread of virus out of the inoculated leaves into the vasculature and to permit transport to the roots and subsequently to the upper parts.

References
1. Scholthof HB, Scholthof K-BG, Kikkert M, Jackson AO, 1995. Virology 213, 425-438.