The movement protein BC1 promotes redirection of the nuclear shuttle protein BV1 of Abutilon mosaic geminivirus to the plasma membrane in fission yeast.

In order to monitor their interaction and cellular localisation, the movement protein (MP; syn. BC1) and the nuclear shuttle protein (NSP; syn. BV1) of the geminivirus Abutilon mosaic virus (AbMV) were ectopically expressed in Schizosaccharomyces pombe cells, either alone or together under the control of an inducible promoter. For highest resolution, electron microscopy using freeze-fracture immunolabelling served to detect these proteins in situ. As expected from previous in planta and yeast experiments, NSP accumulated within the nuclei, whereas MP was targetted to the protoplasmic face of plasma membranes when expressed alone. Upon coexpression, NSP was localised at the plasma membranes, where it was strongly attached. These results support a model in which NSP transports viral DNA to the cell periphery to facilitate cell-to-cell movement of viral DNA within plants. In contrast to AbMV MP, no plant-specific protein seems to be necessary for the translocation of NSP to the plasma membrane.

Yeast two-hybrid systems confirm the membrane- association and oligomerization of BC1 but do not detect an interaction of the movement proteins BC1 and BV1 of Abutilon mosaic geminivirus.

Most of the plant begomoviruses use two proteins to transport their DNA from cell to cell, BV1 to shuttle it between nucleus and cytoplasm and BC1 to facilitate movement across plasmodesmata. In order to analyse their interaction for Abutilon mosaic geminivirus (AbMV) in yeast ( Saccharomyces cerevisiae), BC1 and BV1 genes were cloned into various plasmid vectors suitable for two-hybrid analysis. BC1 was fused to the binding domain (GBD) and BV1 to the activation domain (GAD) of the GAL4 transcription factor to check for interactions in the nucleus. Additionally, BC1 as well as BV1 were integrated into pMyr or pSos vectors to analyze protein binding at the plasma membrane using the CytoTraptrade mark system. Using freeze-fracture immuno-labelling (FreeFI), singly-expressed GBD:BC1 was localized at the plasma membrane although it was fused to a nuclear localization signal provided by the construct. GAD:BV1 was found in the nucleus of transformed cells as expected. Upon co-transformation of both constructs, cells grew poorly and exhibited symptoms of autolysis without any detectable level of GBD:BC1 or GAD:BV1, as revealed by FreeFI. In conclusion, both fusion proteins did not meet in the same compartment and appeared to be harmful to yeast if constitutively co-expressed. When expressed from pSos vector, BC1 induced the CytoTrap detection signal in the absence of pMyr indicating that BC1 protein alone is able to target the effector protein to the inner face of the plasma membrane. A mutated form of BC1 (DeltaBC1) lacking the previously identified membrane-binding domain was no longer able to auto-induce the CytoTrap signal cascade. Using DeltaBC1, an N-terminal, or a C-terminal third of BC1 revealed a homo-oligomerization of the C-terminal region of BC1 in two-hybrid analysis, but no interaction of BC1 with BV1.

Localizing the movement proteins of Abutilon mosaic geminivirus in yeast by subcellular fractionation and freeze-fracture immuno-labelling.

The movement proteins BC1 and BV1 of Abutilon mosaic geminivirus fused to glutathion-S-transferase (GST) and Flag-peptide were expressed in fission yeast (Schizosaccharomyces pombe) cells to analyse the fundamental intracellular distribution of these proteins in an eukaryotic cell in the absence of plant-specific factors. Most of BC1 protein sedimented rapidly after cell lysis and differential centrifugation. Using freeze-fracture immuno-labelling, the protein was detected in situ predominantly at plasma membranes and to a lower extent at cytoplasmic vesicles but not in the cytoplasm, the nuclei, or the mitochondria. Anti-BC1, anti-GST, and anti-Flag antibodies tagged smooth flecks only at the protoplasmic faces of the plasma membrane. The consequences of the BC1 behaviour for its use in two-hybrid analysis in yeast are discussed. In contrast, BV1 was detected mainly in the nucleus and partially in the cytoplasm but never associated with membranes.

Transcriptional analysis of the virion-sense genes of the geminivirus beet curly top virus.

The genome of the geminivirus beet curly top virus (BCTV) consists of a single circular DNA containing overlapping open reading frames (ORFs) located on both the virion-sense and complementary-sense DNA strands. To investigate the expression of these ORFs, RNA extracted from infected Nicotiana benthamiana and Beta vulgaris has been examined for the presence of viral transcripts. An abundant 1.1-kb virion-sense polyadenylated RNA and four complementary-sense polyadenylated RNAs of 1.7, 1.5, 1.3, and 0.7 kb have been identified by northern blot hybridization, confirming the bidirectional transcription strategy implied by the arrangement of ORFs. We previously demonstrated that two overlapping virion-sense ORFs are involved in coat protein synthesis (ORF V1) and viral single-stranded DNA accumulation (ORF V2). Mutants of a third virion-sense ORF (ORF V3), located upstream and overlapping ORFs V1 and V2, retain the ability to replicate efficiently in N. benthamiana leaf discs but produce an asymptomatic infection in N. benthamiana and B. vulgaris at low frequency, associated with reduced levels of viral DNA compared to wild-type infection. Our data support the recent suggestion that ORF V3 participates in virus movement. The 1.1 kb virion-sense RNA comprises a population of overlapping transcripts with 5' termini suitably positioned for the expression of ORFs V1, V2, and V3. The overlapping arrangement of the transcripts and juxtaposition of putative regulatory elements could provide a means for the temporal control of virion-sense gene expression.

Transcript mapping of Abutilon mosaic virus, a geminivirus.

The transcripts of the DNA of Abutilon mosaic virus, a geminivirus with a bipartite genome (DNA A and DNA B), were characterized by Northern blot hybridization, S1 nuclease assay, primer extension analysis, and sequencing of the 3' termini of cDNA clones. It was shown that transcription is bidirectional and that the transcripts are polyadenylated. Two overlapping transcripts of 1.6 and 0.7 kb were mapped to the complementary strand of DNA A and two of 1.3 and 1.2 kb to the complementary strand of DNA B. One transcript of 0.9 kb was mapped to the viral sense in DNA A and one of 1.0 kb to the viral sense in DNA B. The ends of complementary and viral mRNA overlapped in both genome parts in regions rich in polyadenylation signals.