resistance in transgenic crop plants; influence of transport protein genes
on viral host range, symptom expression on resistance of transgenic plants
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.
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
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
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.
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.
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
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
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.
August 1994 December 1998
Biologische Bundesanstalt für Land- und Forstwirtschaft
Moscow State University (RU)
Institute of Molecular Biology and Biotechnology