The triple gene block proteins (TGBp1-3) and coat protein (CP) of potexviruses are required for cell-to-cell movement. Separate models have been proposed for intercellular movement of two of these viruses, transport of intact virions, or a ribonucleoprotein complex (RNP) comprising genomic RNA, TGBp1, and the CP. At issue therefore, is the form(s) in which RNA transport occurs and the roles of TGBp1-3 and the CP in movement. Evidence is presented that, based on microprojectile bombardment studies, TGBp1 and the CP, but not TGBp2 or TGBp3, are co-translocated between cells with viral RNA. In addition, cell-to-cell movement and encapsidation functions of the CP were shown to be separable, and the rate-limiting factor of potexvirus movement was shown not to be virion accumulation, but rather, the presence of TGBp1-3 and the CP in the infected cell. These findings are consistent with a common mode of transport for potexviruses, involving a non-virion RNP, and show that TGBp1 is the movement protein, whereas TGBp2 and TGBp3 are either involved in intracellular transport or interact with the cellular machinery/docking sites at the plasmodesmata.

Phloem-mobile endogenous RNA is trafficked selectively into the shoot apex. In contrast, most viruses and long-distance post-transcriptional gene silencing (PTGS) signals are excluded from the shoot apex. These observations suggest the operation of an underlying regulatory mechanism. To examine this possibility, a potexvirus movement protein, known to modify cell-to-cell trafficking and PTGS, was expressed ectopically in transgenic plants. These plants were found to be compromised in their capacity to exclude both viral RNA and silencing signals from the shoot apex. The transgenic plants also displayed various degrees of abnormal leaf polarity depending on transgene expression level. Normal patterns of organ development were restored by either virus- or Agrobacterium tumefaciens-mediated induction of PTGS. This revealed the presence of an RNA signal surveillance system that acts to allow the selective entry of RNA into the shoot apex. We propose that this surveillance system regulates signaling and protects the shoot apex, in particular the cells that give rise to reproductive structures, from viral invasion.

Cell-to-cell movement of potexviruses requires cognate recognition between the viral RNA, the triple gene block proteins (TGBp1-3) and the coat protein (CP). cis-acting motifs required for recognition and translocation of viral RNA were identified using an artificial potexvirus defective RNA encoding a green fluorescent protein (GFP) reporter transcriptionally fused to the terminal viral sequences. Analysis of GFP fluorescence produced in vivo from these defective RNA constructs, referred to as chimeric RNA reporters, was used to identify viral cis-acting motifs required for RNA trafficking. Mapping experiments localized the cis-acting element to nucleotides 1-107 of the Potato virus X (PVX) genome. This sequence forms an RNA secondary structural element that has also been implicated in viral plus-strand accumulation [Miller, E.D., Plante, C.A., Kim, K.-H., Brown, J.W. and Hemenway, C. (1998) J. Mol. Biol. 284, 591-608]. While replication and movement functions associated with this region have not been separated, these results are consistent with sequence-specific recognition of RNA by the viral movement protein(s). This situation is unusual among viral movement proteins that typically function to translocate RNA between cells in a non-sequence-specific manner. These data support the concept of cis-acting elements specifying intercellular potexvirus RNA movement and thus provide a basis for dissection of RNA-mediated intercellular communication in plants.

The triple gene block proteins (TGBp1-3) and coat protein (CP) of potexviruses are required for cell-to-cell movement. Both cell-to-cell and long-distance movement of White clover mosaic virus in which individual, combinations, or all movement functions were mutated could be rescued by transgenic Nicotiana benthamiana expressing complementary viral products. To address the importance of TGB functions in vascular transport, we used an experimental system based on grafted plants and trans-complementation, to define co-translocated viral products and the minimal requirements for viral exit from the plant vasculature. Evidence is presented that TGBp1 is co-translocated with viral RNA and CP and that, once viral RNA is loaded into the phloem translocation stream, it can exit in sink tissues and replicate in the absence of TGBp2-3. These results are discussed in the context of the recent finding that TGBp1 can mediate the suppression of signaling involved in systemic gene silencing.

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