News and Publications

Transcriptional Regulation of Gene Expression

R.N. Beachy, S. Dai, C. Fauquet,* I. Ordiz, S. Petruccelli, B. Verdaguer, Y. Yin

* ORSTOM, Paris, France

Regulated expression of gene transcription involves complex interactions between transcriptional regulatory proteins, target DNA sequences, and RNA polymerase complexes. We are studying the regulated expression of the promoters from 2 plant pararetroviruses that cause significant diseases in crop plants. Our goal is to develop strategies that limit gene expression and reduce the impact of infection.

RF2a, a b-ZIP protein involved in the expression of genes in phloem cells in rice leaves, is important for expression of the major promoter of rice tungro bacilliform virus. RF2a, which binds to a short oligonucleotide proximal to the TATA element, contains 3 putative regulatory domains, one of which binds the TATA-binding protein. In in vitro transcription assays, RF2a specifically activates transcription of the rice tungro bacilliform virus promoter, whereas mutants with deletions in selected domains do not.

We developed transgenic rice and tobacco plants that contain mutants of RF2a with deletions in one or more of the regulatory domains and determined the effect of the mutations on expression of the RF2a promoter. We found that certain mutants restrict expression from the promoter. Our goals include determining the nature of the protein-DNA and protein-protein interactions that control expression of the promoter and limit replication of rice tungro bacilliform virus in transgenic rice plants.

Role of P30 Movement Protein of Tobacco Mosaic Virus in Pathogenesis and Cell-Cell Spread of Infection

R.N. Beachy, L. Brill, M. Heinlein, T. Kahn, C.O. Lim, P. Mas, H.S. Padgett, C. Reichel, J. Szecsi

The P30 movement protein is required for local, cell-cell movement of tobacco mosaic virus in infected plants and therefore plays an important role in viral pathogenicity. Our goals are to determine the structural and cellular functions of the movement protein and to use the information to develop strategies that can be used to interfere with viral infection in transgenic plants.

To follow the subcellular locations in which movement protein accumulates during viral infection, we fused the protein with green fluorescent protein and used fluorescent and confocal microscopy to examine infected protoplasts and leaves. We found that the fusion protein was present, in a temporally regulated manner, in microtubules, the endoplasmic reticulum, plasmodesmata, and cortical punctate bodies that are probably sites of adhesion of the endoplasmic reticulum with the plasma membrane. Biochemical fractionation of infected cells confirmed that the C-terminus of movement protein is exposed on the cytoplasmic side of the endoplasmic reticulum. Similar studies are in progress to determine the nature of the adhesion sites on the endoplasmic reticulum and the plasma membrane, determine the components of plasmodesmata, and characterize the association of movement protein with these sites.

Internal deletions of 3 amino acids in movement protein resulted in changes in subcellular distribution, phosphorylation, and function of the protein in cell-to-cell spread of infection. This information will be used in conjunction with ongoing studies to determine the 3-dimensional structure of movement protein and to assign function to selected structures of the protein.

Role of Protein Structure in Coat Protein--Mediated Resistance

R.N. Beachy, M. Bendahmane, A. Voloudakis

Resistance to viral infection in transgenic plants caused by expression of transgenes that produce viral coat protein is termed coat protein--mediated resistance. Our studies of such resistance for tobacco mosaic virus and potyviruses include determining the structural and cellular nature of the resistance and developing mutants of coat protein that confer higher levels of resistance than does wild-type coat protein.

Mutants of the coat protein of tobacco mosaic virus that increased interactions between coat protein molecules conferred higher levels of resistance than did mutants that reduced such interactions. Previous studies showed that coat protein reduced disassembly of challenge virus, thereby limiting infection of transgenic plants. Like the wild-type, certain mutants of coat protein reduced the number of sites of infection in transgenic plants, whereas other mutants had little effect on infection per se but limited viral replication or disease. Ongoing studies include structural analyses of selected mutants of coat protein to clarify the intramolecular and intermolecular protein interactions that limit disassembly of challenge virus and other aspects of coat protein--mediated resistance.

Unlike the situation with tobacco mosaic virus, coat protein--mediated resistance to potyviruses, including tobacco etch potyvirus, is poorly understood, although examples of such resistance in transgenic plants are well established. We recently discovered that resistance to tobacco etch potyvirus is conferred equally by coat protein that does or does not assemble to form viruslike particles. We concluded that coat protein--mediated resistance against this virus probably does not involve blocking disassembly, and we proposed that the coat protein of tobacco etch potyvirus may interfere with viral replication or other steps not yet determined.

PUBLICATIONS

Ceriani, M.F., Marcos, J.F., Hopp, H.E., Beachy, R.N. Simultaneous accumulation of multiple viral coat proteins from a TEV-NIa based expression vector. Plant Mol. Biol. 36:239, 1997.

Heinlein, M., Padgett, H.S., Gens, S., Pickard, B., Casper, S.J., Epel, B.L., Beachy, R.N. Changing patterns of localization of TMV movement protein and replicase to endoplasmic reticulum and microtubules during infection. Plant Cell 10:1107, 1998.

Kahn, T.W., Lapidot, M., Heinlein, M., Reichel, C., Cooper, B., Gafny, R., Beachy, R.N. Domains of the TMV movement protein involved in subcellular localization. Plant J. 14:106, 1998.

Lewis, J.K., Bendahmane, M., Smith, T.J., Beachy, R.N., Siuzdak, G. Identification of viral mutants by mass spectrometry. Proc. Natl. Acad. Sci. U.S.A. 95:8596, 1998.

Verdaguer, B., de Kochko, A., Fux, C.I., Beachy, R.N., Fauquet, C. Functional organization of the cassava vein mosaic virus (CsVMV) promoter. Plant Mol. Biol. 37:1005, 1998.

Yin, Y., Zhu, Q., Dai, S., Lamb, C., Beachy, R.N. RF2a, a bZIP transcriptional activator of the phloem-specific rice tungro bacilliform virus promoter, functions in vascular dvelopment. EMBO J. 16:5247, 1998.