Resistance to subterranean clover mottle virus in subterranean clover results from restricted cell-to-cell movement

Abstract

Subterranean clover mottle sobemovirus (SCMoV) is an important pathogen of subterranean clover (Trifolium subterraneum) pastures in Australia. Cultivars of subterranean clover are susceptible (e.g. Dalkeith, Woogenellup), moderately susceptible (e.g. Green Range), partially resistant (e.g. Goulburn) or highly resistant (e.g. Meteora, Trikkala) to SCMoV. The resistant class does not become systemically infected on sap inoculation, while only a proportion of plants develop systemic infection with the moderately susceptible and partially resistant classes (Wroth and Jones 1992b; Ferris and Jones 1994). These different classes were used to examine the basis of SCMoV resistance using cell biological approaches. Virus levels were quantified at the tissue level by ELISA and at the cell level by counting protoplasts which fluoresced after antibody staining. Three aspects were investigated: (1) initial cell infection, (2) frequencies of cell infection in sap-inoculated leaves and in systemically infected leaves from graft-inoculated plants, and (3, ability of the virus to replicate in isolated protoplasts. Fewer and smaller starch lesions formed in inoculated leaves of a highly resistant cultivar than in those of moderately susceptible and susceptible cultivars. In protoplasts isolated from epidermal cell strips from inoculated leaves, initially the numbers of fluorescing protoplasts were the same for all four classes of cultivars (5-7%). Subsequently, however, the numbers remained unchanged for the two highly resistant cultivars, but increased considerably in the susceptible one used, and to a lesser extent in the moderately susceptible and partially resistant ones. For protoplasts isolated from whole inoculated leaves, the percentage of protoplasts that fluoresced was greatest in the susceptible cultivars used, lower in the moderately susceptible and partially resistant cultivars, and least in the highly resistant ones. All classes became systemically infected when graft-inoculated, but with the systemically infected leaves the proportions of protoplasts that fluoresced were least for the highly resistant and greatest for the susceptible classes. When protoplasts were inoculated with SCMoV in vitro, the proportions which fluoresced were the same (53-56%) for highly resistant, moderately susceptible and susceptible cultivars, showing that resistance was not expressed at the single cell level as an inhibition of virus replication. Because movement of SCMoV was restricted in inoculated leaves of the highly resistant class (starch lesion, epidermal strip and whole leaf data) and in graft-induced systemically infected leaves, it is concluded that the basis of resistance to SCMoV in highly resistant cultivars of subterranean clover is restriction of cell-to-cell movement of the virus.