MOLECULAR AND BIOCHEMICAL ANALYSIS OF MECHANISMS INVOLVED IN THE MOVEMENT OF A BIPARTITE GEMINIVIRUS IN PLANTS

Amine O. NOUEIRY

Plant viruses infect plants by moving through plasmodesmata, but little is known of the mechanism(s) involved. The roles of two proteins involved in the movement, in plants, of a single-stranded DNA virus, bean dwarf mosaic geminivirus (BDMV), were investigated using in vivo microinjection and in vitro gel retardation assays. Using site­directed mutagenesis of infectious BDMV clones, it was shown that the BL1 and BR1 proteins of BDMV are not involved in replication but are both required for movement. Using functional proteins expressed in Escherichia coli and microinjected into plant cells, it was established that the BL1 protein moves extensively from cell-to-cell, increases mesophyll plasmodesmal size exclusion limit, and potentiates the cell-to-cell movement of double-stranded (ds-) DNA. The BR1 protein mediated in the export of single-stranded (ss-) and ds-DNA out of the nucleus. These results provide direct experimental evidence for intercellular macromolecular transport in plants, and establish that the BR1 and BL1 proteins coordinate the movement of viral DNA across nuclear and plasmodesmal boundaries, respectively.

Consistent with this hypothesis, the BR1 and BL1 proteins were shown to bind ds-DNA using an in vitro gel retardation assay. The binding of the BL1 and BR1 proteins to DNA was of a cooperative-type nature, but was not sequence-specific. Both proteins displayed size-selectivity in their ability to bind DNA in vitro, but in vivo microinjection experiments indicated that the BL1 and BRl proteins had the capacity to facilitate the cell-to-cell movement or nuclear export of small and large DNA molecules, respectively. These results are consistent with the hypothesis that the BLI and BRl proteins possess two DNA binding properties; one that mediates in cell-to-cell movement and nuclear export, respectively, and another involved in cooperative binding and size-­selectivity. Size-selective DNA binding by geminiviral movement proteins may provide an explanation for the maintenance of the geminiviral genome size during the systemic infection of plants.