Bromovirus movement protein conditions for the host specificity of virus movement through the vascular system and...

Previously, we reported that CCMV(B3a), a hybrid of bromovirus Cowpea chlorotic mottle virus (CCMV) with the 3a cell-to-cell movement protein (MP) gene replaced by that of cowpea-nonadapted bromovirus Brome mosaic virus (BMV), can form small infection foci in inoculated cowpea leaves, but that expansion of the foci stops between 1 and 2 days postinoculation. To determine whether the lack of systemic movement of CCMV(B3a) is due to restriction of local spread at specific leaf tissue interfaces, we conducted more detailed analyses of infection in inoculated leaves. Tissue-printing and leaf press-blotting analyses revealed that CCMV(B3a) was confined to the inoculated cowpea leaves and exhibited constrained movement into leaf veins. Immunocytochemical analyses to examine the infected cell types in inoculated leaves indicated that CCMV(B3a) was able to reach the bundle sheath cells through the mesophyll cells and successfully infected the phloem cells of 50% of the examined veins. Thus, these data demonstrate that the lack of long-distance movement of CCMV(B3a) is not due to an inability to reach the vasculature, but results from failure of the virus to move through the vascular system of cowpea plants. Further, a previously identified 3a coding change (A776C), which is required for CCMV(B3a) systemic infection of cowpea plants, suppressed formation of reddish spots, mediated faster spread of infection, and enabled the virus to move into the veins of inoculated cowpea leaves. From these data, and the fact that CCMV(B3a) directs systemic infection in Nicotiana benthamiana, a permissive systemic host for both BMV and CCMV, we conclude that the bromovirus 3a MP engages in multiple activities that contribute substantially to host-specific long-distance movement through the phloem.