The main objectives are:
to determine how many apparently different mechanisms of cell to cell movement of viruses there are;
to study the basic interactions between the virus, the movement protein and plasmodesmata;
to use viral movement proteins to examine the physical structure of plasmodesmata and how plasmodesmata function;
to analyse the infection units of viruses which move through plasmodesmata;
to determine if the interactions between movement proteins and plasmodesmata can be interfered with and thus confer resistance.
Most of the effort in this first year has been directed at developing systems, solving problems and obtaining experimental materials from which the main cooperative aspects of the project can be built. Important results are reported.
The movement protein of alfalfa mosaic virus (AIMV) expressed in yeast and E coli has the same mobility as that from plants which suggests that none of the proteins is postranslationally modified. A series of 8 contiguous in frame deletions covering the whole sequence of the AIMV movement protein gene have been made and are being expressed in E coli. The cytomegalovirus (CMV) movement protein has been expressed using the T7 ribonucleic acid (RNA) polymerase system in E coli by cloning into pT7-7. Expression levels were higher in E coli strain K38 than in BL21 (DE3) at 2h post-induction but at 7h post induction they were higher in BL21 than in K38.
Genomic location of movement protein genes in different viruses. Beet necrotic yellow vein virus (BNYVV) RNA 1 is infectious to protoplasts which shows that it can autoreplicate but RNA 2 is required for infection of a local lesion host such as Chenopodium quinoa. This indicates that RNA 2 which has 6 open reading frames carries functions for cell to cell movement. Disabling mutations have been introduced into each of the open reading frames in RNA 2 and the ability of transcripts carrying each mutation to replicate when coinoculated with RNA 1 was tested both in protoplasts and on leaves. Three adjacent genes encoding proteins of 42, 13 and 15 kDa (the triple block) were found to be nonessential for virus multiplication in protoplasts but required for cell to cell movement.
A reliable method has been developed for the preparation of clean cell wall fragments containing intact plasmodesmata. This method involved grinding plasmolysed tobacco leaves or maize roots in liquid N22 followed by extrusion through a French Press and then repeated washing with extraction buffer. This produced c ell wall fragments ranging in size from 1 mu to several mu which, on embedding and sectioning, were shown by electron microscopy to contain seemingly intact plasmodesmata. After negative staining these preparations have proved suitable for high-resolution conventional electron microscopy with a tilting stage to give 3-D imaging. In initial experiments using uranyl acetate or methylamine tungstate as negative stains, substructure was revealed in the plasmodesmatal neck.
The cell-to-cell movement of plant viruses is via plasmodesmata which have to be modified to enable the passage of the infectious units. Many viruses encode movement protein(s) (MP) which gate plasmodesmata. This project has three main aims. 1. The Mps of six viruses from different groups - alfalfa mosaic, beet necrotic yellow vein (BNYVV), cauliflower mosaic (crucifer and tobacco strains), cucumber mosaic (CMV), grapevine fanleaf (GFLV) and tobacco mosaic viruses - Will be isolated and characterized using various approaches including in-vitro expression, biochemical analysis, mutagenesis and plant transformation. 2. The structure and function of plasmodesmata will be investigated. The host proteins to which MPs bind will be isolated and characterized. The structure of plasmodesmata will be examined by high resolution electron microscopy and by immuno-probing of plant tissues with antisera against MPs and MP-binding host proteins. Dye injection procedures Will be used to assess the gating capacity of plasmodesmata in Virus-infected and transgenic plants and the effects on plasmodesmata of co-injecting macromolecules, eg. MPs or host protein antisera. 3. The possibilities of inhibiting cell-to-cell movement of viruses Will be studies by transforming sugar beet, zucchini and grapevine with engineered forms of the Mps of BNYVV, CMV and GFLV respectively. Information from the studies of the functions of MPs and their interactions With
plasmodesmata Will be used in designing these novel MPs.
Funding SchemeCSC - Cost-sharing contracts