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

Abstract

AbstractPlant-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, which bind nucleic acids and target and dilate plasmodesmata. 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 new, 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 nanomolar affinity. This system allows imaging of plant viral RNA genomes inside plasmodesmata and shows thatPotato virus XRNA 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 fromTobacco mosaic viruscan recruitPotato virus Xreplication complexes to plasmodesmata entrances. Therefore, recruitment of replication complexes is mediated non-specifically, likely by indirect coupling of movement proteins and viral replicaseviathe 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 proteinin cisinterferes with viral movement by an unidentified mechanism when cognate stem-loops are present in the viral RNA.