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EC-sponsored Research on Safety of Genetically Modified Organisms - A Review of Results
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image Virus resistance in transgenic crop plants; influence of transport protein genes on viral host range, symptom expression on resistance of transgenic plants

Background and objectives

The improvement of virus resistance in crop plants by means of genetic engineering is a very promising approach. To extend the availability of safe virus resistance genes, extensive basic research on viral infection is necessary. For cell-to-cell translocation of viral genomes many (if not all) plant viruses express so-called “movement proteins” which are responsible for the spread of virus to other plant tissues. Different viruses use different strategies for coding their transport function. Interfering with the functioning of transport proteins is a promising strategy for use in generating transgenic virus-resistant plants. We therefore studied the influence of such transport protein genes on viral host range, symptom expression and resistance of transgenic plants. The main aim of the research was to provide a functional analysis of several poorly studied genetic systems of intercellular transport of viral infection.

image image Microscopic fluorescent images of the plant leaf cells expressing GFP-tagged
movement proteins of poa semilatent virus.
A., B., and C. GFP-TGBp2 protein; D. the same optical section as in C. with
membrane structures staind by a rhodamine dye; E., overlap of the images in C.
and D.; F., GFP-TGBp3 protein; G., GFP-TGBp2 protein co-expressed with
non-tagged TGBp3 protein.



Approach and methodology

Microprojectile bombardment has been established as a very efficient tool for complementation analysis. This has firstly been demonstrated by complementation of a potato virus X (PVX) mutant with cloned viral movement protein genes. Microprojectile bombardment was used to examine the transport function of the 25 kDa movement protein (MP) encoded in the triple gene block (TGB) of PVX. An MP-defective full-length cloned PVX genome carrying a ß-glucuronidase (GUS) reporter gene was cobombarded with 35S promoter constructs containing either the MP gene of wild-type PVX, the MP gene of either of two tobamoviruses (tomato mosaic virus or crucifer tobamovirus), red clover necrotic mosaic dianthovirus (RCNMV) or brome mosaic bromovirus (BMV).


Main findings and outcome

When inoculated alone, the MP-defective PVX was unable to move out of the inoculated cell, as visualized by in situ staining for GUS activity. However, cell-to-cell movement of the mutant PVX genome was restored by coinoculation with 35S constructs containing the MP cDNA of PVX, either tobamovirus or RCNMV. These results demonstrate clearly that cobombardment of cDNA of an MP-defective virus with plasmids designed to express MP of other viruses could be used as a fast and simple method for trans-complementation experiments.

Studies on the movement of hordeivirus hybrids have been extended. The barley stripe mosaic virus (BSMV) TGB was replaced with the respective TGB genes from two other hordeiviruses, poa semilatent virus (PSLV) or lychnis ringspot virus (LRSV). The BSMV/LRSV recombinant did not exhibit infectivity on the plants tested, whereas the infection rate and host range of the BSMV/PSLV hybrid were similar to those of BSMV. Assuming that the PSLV TGB was functional in the BSMV genome context, a further series of recombinants was constructed, in which smaller portions of the BSMV TGB were replaced by the corresponding PSLV sequences. Examination of the infectivity of the hybrid viruses suggested that the TGB-coded proteins could interact in a host-dependent manner to mediate cell-to-cell movement. Analysis of recombinants with hybrid sequences of the first gene in the TGB (ßb gene) indicated that sequence-independent binding of ßb to viral RNAs could occur during formation of ßb-RNA complexes in vivo, and that the ßb MP is involved in virus long-distance movement.


Conclusions

Our experiments have studied the protein components that influence movement and targeting of infectious viral particles. The results of co-expression experiments with mutant forms of movement proteins argue against involvement of a direct interaction between the small TGB proteins in the targeting of related components to the cell peripheral compartments. These results do provide evidence for an interaction of the TGBp3 movement protein with cellular components and gives an excellent new basis for studying these very important interactions which influence virus movement and infection. We believe this research will facilitate the development of environmentally safe, virus-resistant crop plants.

 

Major publications

Morozov S.Yu., Fedorkin O.N., Jüttner G., Schiemann J., Baulcombe D.C., Atabekov J.G., “Complementation of a potato virus X mutant mediated by bombardment of plant tissues with cloned viral movement protein genes”.
J. Gen. Virol., 78, 1997, pp. 2077-2083.

Agranovsky A.A., Folimonov A.S., Folimonova S.Yu., Morozov S.Yu., Schiemann J., Lesemann D.-E., Atabekov J.G., “Beet yellows closterovirus HSP70-like protein mediates the cell-to-cell movement of a potexvirus transport-deficient mutant and a hordeivirus-based chimeric virus”.
J. Gen. Virol., 79, 1998, pp. 889-895.

Solovyev A.G., Savenkov E.I., Grdzelishvili V.Z., Kalinina N.O., Morozov S.Yu., Schiemann J., Atabekov J.G., “Movement of hordeivirus hybrids with exchanges in the triple gene block”.
Virology, 253, 1999, pp. 278-287.

Morozov S.Yu., Solovyev A.G., Kalinina N.O., Fedorkin O.N., Samuilova O.V., Schiemann J., Atabekov J.G., “Evidence for two nonoverlapping functional domains in the potato virus X 25K movement protein”.
Virology, 260, 1999, pp. 55-63.

Solovyev A.G., Stroganova T.A., Fedorkin O.N., Schiemann J., Morozov S.Yu., “Subcellular sorting of small membrane-associated triple gene block proteins: TGBp3-assisted targeting of TGBp2”.
Virology, 269, 2000, pp. 113-127.
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imageResearch project
 

Contract number
MA2B910029

Period
August 1994 – December 1998

Coordinator
R. Casper
Biologische Bundesanstalt für Land- und Forstwirtschaft
Braunschweig (DE)

 

 
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Partners


J.G. Atabekov
Moscow State University (RU)

M. Tsagris
Institute of Molecular Biology and Biotechnology
Heraklion (GR)

 
 
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