In vivo visualization of RNA localization and transport in plants

HEINLEIN, MANFRED; E.J. PEÑA; AGÜERO, MATÍAS

Berlin

Workshop; Intercellular communication in development and disease; 2017

Tobacco mosaic virus (TMV) infectious clones in which the coat protein gene has been removed and the movement protein (MP) is expressed as a fusion to fluorescent proteins (MP:FP) allow us to study the roles of MP during infection (A). The MP:FP accumulates transiently and reaches a maximum level after 2-3 days before it is degraded. Thus, infection foci produced by TMV-MP:FP are characterized by a ring-shaped fluorescence. (B, left-middle). At the leading front of infection, i.e. in recently infected cells in which the cell-to-cell movement of vRNA occurs, MP accumulates at plasmodesmata (PD) and in ER-associated mobile particles, which are proposed to represent early replication complexes (VRCs) (B, right). These particles are associated with the ability of MP to support virus movement 1 . Previously, we adapted the bacteriophage MS2-derived RNA-labeling system to study TMV-RNA transport. This method is based on the tagging of an RNA of interest with a protein-binding motif (stem loop, SL) and the co-expression of its corresponding fluorescently tagged RNA-binding protein (RBP). As the RBP is fused to an NLS and targeted to the nucleus, its retention to particles in the cytoplasm indicates the position of the labeled RNA-SL2 (Ci). Using this method we showed that TMV particles produced upon ectopic expression of MP contain MP mRNA (Cii) and that the mRNA accumulates together with MP at plasmodesmata3 (PD, Ciii), however the sensitivity of the method doe not allow us to study composition and trafficking of the particles. In this work we present an alternative method with higher sensitivity that not only allowed us to confirm previous results but also to visualize cell-to-cell RNA transport events. It also opens the possibility to address the compositions of the MP particles.