Live-cell RNA imaging with the inactivated endonuclease Csy4 enables new insights into plant virus transport through plasmodesmata.

Plant-infecting viruses spread through their hosts by transporting their infectious genomes through intercellular nano-channels called plasmodesmata. This process is mediated by virus-encoded movement proteins. Whilst the sub-cellular localisations of movement proteins have been intensively studied, live-cell RNA imaging systems have so far not been able to detect viral genomes inside the plasmodesmata. Here, we describe a highly sensitive RNA live-cell reporter based on an enzymatically inactive form of the small bacterial endonuclease Csy4, which binds to its cognate stem-loop with picomolar affinity. This system allows imaging of plant viral RNA genomes inside plasmodesmata and shows that potato virus X RNA remains accessible within the channels and is therefore not fully encapsidated during movement. We also combine Csy4-based RNA-imaging with interspecies movement complementation to show that an unrelated movement protein from tobacco mosaic virus can recruit potato virus X replication complexes adjacent to plasmodesmata. Therefore, recruitment of potato virus X replicase is mediated non-specifically, likely by indirect coupling of movement proteins and viral replicase via the viral RNA or co-compartmentalisation, potentially contributing to transport specificity. Lastly, we show that a 'self-tracking' virus can express the Csy4-based reporter during the progress of infection. However, expression of the RNA-binding protein in cis interferes with viral movement by an unidentified mechanism when cognate stem-loops are present in the viral RNA.