HOST-SPECIFICITY RESTRICTION BY BROMOVIRUS CELL-TO-CELL MOVEMENT PROTEIN OCCURS AFTER INITIAL CELL-TO-CELL SPREAD OF INFECTION IN NONHOST PLANTS

The nonstructural 3a protein of the positive-strand RNA bromoviruses i s required for infection spread in plants and is a crucial determinant of host specificity in systemic infection. To determine the paths of wild-type (wt) bromovirus infection spread, the step at which 3a mutan ts are arrested, and the nature of the host specificity associated wit h the 3a gene, we used in situ hybridization to examine infection spre ad by cowpea chlorotic mottle bromovirus (CCMV) and its derivatives at the level of individual cells in cowpea leaf epidermis. From 1 to 3 d ays post inoculation (dpi), wt CCMV spread from initially infected cel ls to adjacent cells, creating expanding infection foci whose radii gr ew by one additional epidermal cell diameter every 5 hr. By 3 to 4 dpi , vascular elements contacting such foci acted as conduits for further infection spread. By contrast, a 3a frameshift derivative multiplied in initially infected epidermal cells but failed to move into neighbor ing cells even by 4 dpi, showing that the 3a gene is essential for cel l-to-cell spread. Most interestingly, a CCMV derivative with the 3a ge ne replaced by that of a bromovirus not adapted to cowpea, brome mosai c virus (BMV), initially spread from cell to cell in cowpea plants, bu t stopped spreading between 1 and 2 dpi, when most infection foci enco mpassed 40-80 epidermal cells. Thus, the host-specificity restriction imposed by BMV 3a protein did not result from an inability to direct t he spread of infection out of initially infected cowpea cells, but fro m a much later block. The apparent absence of any preexisting anatomic al boundary at the limit of infection spread and localized tissue chan ges at the infection foci suggested that induced host responses might have contributed to this block. (C) 1995 Academic Press, Inc.