Direct functional assay for tobacco mosaic viruscell-to-cell movement protein and identification of a domain involved inincreasing plasmodesmal permeability.

Abstract

Plasmodesmata are cytoplasmic bridges between plant cells thought to generally allow only the passage of small molecules and metabolites. However, large structures such as plant viruses also move from cell to cell via plasmodesmata. In tobacco mosaic virus (TMV) infection a viral movement protein (TMV-MP) mediates viral spread. Here, a microNuection assay is used to monitor the dynamics of TMV-MP function directiy in wild-type plants. The results indicate that TMV-MP interacts with an endogenous plant pathway increasing plasmodesmal size exclusion limit to permit passage of 20-kDa dextrans. Furthermore, TMV-MP influences plasmodesmal size exclusion limit several cells distant from the ijection site, indicating either that TMV-MP itself crosses plasmodesmata or that TMV-MP induces a diffusable signal capable of dilating microchannels of plasmodesmata. The region of TMV-MP responsible for increasing plasmodesmal size exclusion limit was mapped to the carboxyl-terminal part of the 268-amino acid residue protein between amino acid residues 126 and 224.In plants, cell communication and molecular trafficking are largely dependent on cytoplasmic bridges between plant cells, the plasmodesmata (PD). These plasma membranelined pores link neighboring cells and thereby generate a three-dimensional cellular network (1). Each PD contains about 10 microchannels (diameter, 2.5 nm) thought to function as transport pathways (for more details see ref.2). In mesophyll cells, only small molecules, such as metabolites or dextrans with a molecular mass up to 1 kDa (corresponding to a Stokes radius of 0.75 nm), can pass through these channels freely (3). Due to their structural complexity, it seems possible that PD might also function in transport of endogenous macromolecules (4). In fact, PD are the major route for plant viruses moving from infected to adjacent healthy cells. Compared to the size of PD microchannels, plant viral dimensions are enormous-e.g., tobacco mosaic virus (TMV) particles are 300 x 18 nm and even free-folded TMV RNA has an average diameter of 10 nm (5). How can these large structures fit through so small a passageway?Several lines of evidence suggest that viral encoded proteins are involved in the movement process. For TMV, a 30-kDa protein, which is expressed only transiently during infection (6), has been implicated in movement (7). Clues about the function ofTMV movement protein (TMV-MP) are derived from in vitro experiments with purified TMV-MP and from in vivo experiments with transgenic tobacco plants expressing TMV-MP. Two functions have been identified: (i) TMV-MP cooperatively binds single-stranded (ss) nucleic acids (8) and (ii) transgenic tobacco plants expressing TMV-MP show an elevated PD size exclusion limit (SEL) of >10 kDa for dextrans (Stokes radius of 2.4 nm to 3.1 nm) (3). These transgenic plants also facilitate intercellular movement of unencapsidated viral RNA, thereby indicating that formation of viral particles is not necessary for TMV-MP-mediated movement (9).A model for TMV-MP-me...

Domains of the TMV movement protein involved in subcellular localization

“…Based on experiments in which purified MP mutants were co-injected with fluorescently labeled dextrans into plant cells, the region of the protein from amino acids 126-224 has been assigned the ability to modify the size exclusion limit of plasmodesmata (Waigmann et al, 1994). A protein fragment comprising amino acids 116-268 could modify plasmodesmata, although in a way that was different from the wt protein.…”

Tobacco mosaic virus: a pioneer to cell–to–cell movement

“…Unlike wild-type tobacco mesophyll plasmodesmata, which can tra¤c only dextrans up to 0.75^1.0 kDa, the P30-transgenic plants exhibited a plasmodesmatal size exclusion limit of almost 10 kDa (Wolf et al 1989 tissue represents a steady state of P30 accumulation and activity, it remained unclear whether the increase in plasmodesmatal size exclusion limit was due to activation of an endogenous plasmodesmatal transport pathway or induction of a speci¢c host response to this viral protein. Direct microinjection of puri¢ed P30 into wild-type tobacco mesophyll cells resulted in a relatively fast (3^5 min) increase in the size exclusion limit up to 20 kDa, indicating that P30 functions via the existing plasmodesmatal transport machinery (Waigmann et al 1994). The increased size exclusion limit of 10^20 kDa (Wolf et al 1989;Waigmann et al 1994) corresponds to a dilated channel diameter of 59 nm, potentially allowing passage of 2.0^2.5 nm-wide P30^TMV RNA complexes (Citovsky et al 1992a).…”

“…Direct microinjection of puri¢ed P30 into wild-type tobacco mesophyll cells resulted in a relatively fast (3^5 min) increase in the size exclusion limit up to 20 kDa, indicating that P30 functions via the existing plasmodesmatal transport machinery (Waigmann et al 1994). The increased size exclusion limit of 10^20 kDa (Wolf et al 1989;Waigmann et al 1994) corresponds to a dilated channel diameter of 59 nm, potentially allowing passage of 2.0^2.5 nm-wide P30^TMV RNA complexes (Citovsky et al 1992a).…”

“…It was noted that large £uorescent dextrans moved not only into the cells adjacent to the microinjected cell, but also that they travelled as far as 20^50 cells away from the site of injection (Waigmann et al 1994). These observations indicated that P30 itself must have moved through plasmodesmata to induce the increase in size exclusion limit in the distant mesophyll cells, providing the ¢rst evidence that plasmodesmata can allow the passage of protein molecules.…”

Cell-to-cell movement of three genera (+) ss RNA plant viruses

“…MP TMV was the first movement protein that showed binding with single-stranded (ss) RNA or DNA (Citovsky et al 1990). Complexes of MP TMV-nucleic acid are estimated to measure 1.5-3.5 nm in diameter, which is compatible with 3.2-4.3 nm SEL-broadened PD (Waigmann et al 1994;Kiseylova et al 2001). The MP TMV mutation analysis indicated that activity of nucleic acid single strand is present in domains between amino acid radical 112-185 and 186-268 movement protein (Citovsky et al 1992).…”