The use of inter-specific complementation approach to understand the role of localised replication in plant virus cell-to-cell transport through plasmodesmata

The only way for plant viruses to spread through their hosts is transport of viral genomes through plasmodesmata (PD) with the aid of virus-encoded movement proteins (MPs), which can target and open PD and bind nucleic acids. This transport typically happens early during infection, when viral genomes are not yet abundant, in order to escape non-cell-autonomous defence signalling and mobile RNA silencing. However, none of the MPs characterised to date show any sequence specificity, raising the question whether transport is selective and how this occurs. The RNA virus, Potato virus X targets some of its replication sites to the entrances of plasmodesmata (PD), where it may insert newly synthesized viral RNA directly into the channels (co-replicational movement). Complementation of movement by ectopic MPs is widespread amongst plant viruses and provides a way to dissect the mechanism and significance of localised replication. In this study, PVX-based plasmid vector is constructed by modifying it to express the 30K MP of Tobacco mosaic virus, instead of its cognate triple gene block (TGB) proteins, which are responsible for cell to cell transport of PVX. Using a new, highly sensitive RNA imaging system based on the high-affinity RNA binding protein Csy4 which can track viral genomes within PD, it is shown that PVX with in cis movement complmentation, nonetheless, is still capable of forming PD-associated replication sites, suggesting that MPs may recruit viral replicases non-specifically through mutual interactions with the same RNA molecule(s) or host proteins. Key Words: PVX, Confocal microscopy, Complementation, Cell-to-cell movement