Plasmodesmata are intercellular organelles in plants that establish cytoplasmic continuity between neighboring cells. Microinjection studies showed that plasmodesmata facilitate the cell-to-cell transport of a plant-encoded transcription factor, KNOTTED1 (KN1). KN1 can also mediate the selective plasmodesmal trafficking of kn1 sense RNA. The emerging picture of plant development suggests that cell fate is determined at least in part by supracellular controls responding to cellular position as well as lineage. One of the mechanisms that enables the necessary intercellular communication appears to involve transfer of informational molecules (proteins and RNA) through plasmodesmata.

Contrary to a previous report, electron microscopic studies on the Fny strain of cucumber mosaic virus (CMV)-infected tobacco tissues revealed that plasmodesmata were not structurally modified during CMV infection, nor were virions ever observed in plasmodesmata connecting infected cells. To further explore the basis of CMV infection, experiments were performed on the CMV 3a ORF. The 3a protein of CMV was expressed in and purified from Escherichia coli. The purified protein was labeled with fluorescein isothiocyanate (FITC) and subsequently microinjected into mesophyll cells of mature leaves of Nicotiana tabacum cv. Turkish Samsun NN. Within a brief period (as little as 1 sec), the microinjected FITC-labeled CMV 3a protein moved into neighboring cells. Co-injection of unlabeled CMV 3a protein with 9.4-kDa fluorescein-conjugated dextran (F-dextran) resulted in extensive cell-to-cell movement (diffusion) of the F-dextran, indicating that the 3a protein can interact with and dilate plasmodesmata. Furthermore, co-injection of unlabeled 3a protein with fluorescently labeled infectious CMV RNA molecules resulted in rapid and extensive cell-to-cell transport. In contrast, a mutant form of the 3a protein was unable to traffic from cell to cell, to increase the size exclusion limit of plasmodesmata, or to potentiate cell-to-cell trafficking of CMV RNA molecules. Microinjection studies performed on transgenic tobacco plants expressing the CMV 3a protein indicated that fluorescently labeled CMV RNA moved out of the target cell into the surrounding mesophyll tissue. In addition, expression of the CMV 3a protein also potentiated the cell-to-cell movement of 9.4-kDa F-dextran. Collectively, these results provide direct experimental evidence that the CMV 3a protein functions as the movement protein of CMV. These findings are consistent with the hypothesis that CMV moves from cell-to-cell in the form of a ribonucleoprotein complex.

Transgenic tobacco (Nicotiana tabacum cv. Turkish Samsun NN) plants expressing a truncated replicase gene sequence from RNA-2 of strain Fny of cucumber mosaic virus (CMV) are resistant to systemic CMV disease. This is due to suppression of virus replication and cell-to-cell movement in the inoculated leaves of these plants. In this study, microinjection protocols were used to directly examine cellto-cell trafficking of CMV viral RNA in these resistant plants.CMV RNA fluorescently labeled with the nucleotide-specific TOTO-1 iodide dye, when coinjected with unlabeled CMV 3a movement protein (MP), moved rapidly into the surrounding mesophyll cells in mature tobacco leaves of vector control and untransformed plants. Such trafficking required the presence of functional CMV 3a MP. In contrast, coinjection of CMV 3a MP and CMV TOTO-RNA failed to move in transgenic resistant plants expressing the CMV truncated replicase gene. Furthermore, coinjection of 9.4-kDa fluorescein-conjugated dextran (F-dextran) along with unlabeled CMV 3a MP resulted in cell-to-cell movement of the F-dextran in control plants, but not in the transgenic plants. Similar results were obtained with viral RNA when the 30-kDa MP of tobacco mosaic virus (TMV) was coinjected with TMV TOTO-RNA into replicase-resistant transgenic tobacco expressing the 54-kDa gene sequence of TMV. However, in these transgenic plants, the TMV-MP was still capable of mediating cell-to-cell movement of itself and the 9.4-kDa F-dextran. These results indicate that an inhibition of cell-to-cell viral RNA trafficking is correlated with replicase-mediated resistance. This raises the possibility that the RNA-2 product is potentially involved in the regulation of cell-to-cell movement of viral infectious material during CMV replication.To systemically infect its host, a plant virus must be competent to replicate, move cell-to-cell via plasmodesmata, and both enter and egress the long-distance transport system of the phloem (1). Hence, strategies to develop transgenic plants resistant to viral infection have focused on reducing the efficacy of replication and/or cell-to-cell movement. Such strategies have included expression of viral coat protein (2, 3), or dysfunctional movement protein (MP; refs. 4 and 5), which result in either a delay or an inhibition of the establishment of systemic infection. Additionally, complete resistance for a number of virus diseases has been achieved through the expression of different forms of viral replicase genes (6-8). In this situation, viral replication within inoculated transgenic leaves or protoplasts is markedly reduced (9, 10). Resistance is thought to be achieved either by the establishment of a dysfunctional replicase complex that impairs protein synthesis (11)(12)(13)(14) or by gene silencing (15).The situation of strain-specific resistance reported for transgenic tobacco plants expressing the truncated CMV 2a (CMV, cucumber mosaic virus) replicase protein (10, 16) may be complex. Chlorotic spots or lesions were occasionally obser...