Use of GFP-Tagged Viruses in Plasmodesmal Research

Abstract

The insertion of the gene for the green fluorescent protein (GFP) from the jellyfish Aequorea victoria into the genome of a number of plant viruses is proving to be a powerful tool in the study of virus-plasmodesmata interactions. Initially, the gfp gene was introduced as a separate open-reading frame into the genome of potato virus X (PVX), allowing the expression of ‘free’ GFP in the cytosol of virus-infected cells (Baulcombe et al. 1995). This initial study of GFP, expressed from a virus vector, demonstrated that GFP could be used as a non-invasive marker of viral infections, allowing the detection of infected cells at both the whole-plant and single-cell level. However, not all viruses are amenable to this technology and some have proved problematical due to either a lack of full-length infectious clones (see Oparka et al. 1996a), or because of constraints on genome function or virus assembly due to the insertion of the additionalgfp gene. For example, in the case of the spherical viruses cucumber mosaic virus (CMV; Canto et al. 1997), and brome mosaic virus (BMV; Rao 1997), ‘free’ GFP can be expressed from the complete viral genome by omitting one of the existing viral genes and:replacing it with the gfp gene. However, this may result in loss of virus movement unless a complementation strategy is used (e.g. Canto et al., 1997). Free GFP has also been expressed from the tubule-forming virus cowpea mosaic virus (CPMV) by replacing; either the coat protein gene or movement protein gene with gfp (Wellink et al. 1997). Rodshaped plant RNA viruses appear to offer greater potential over spherical viruses, as insertion of an additional gene in the former does not appear to prevent particle assembly or virus movement (Santa Cruz et al. 1996). To date, GFP has been expressed successfully from the rod-shaped viruses PVX (Baulcombe et al. 1995; Oparka et al. 1995,1996a, b; Santa Cruz et al. 1996), tobacco mosaic virus (TMV; Heinlein et al. 1995; Padgett et al. 1996; Chen et al. 1997) and tobacco etch virus (TEV; Schaad et al. 1997)