Replication and trafficking of a plant virus are coupled at the entrances of plasmodesmata.

Plant viruses use movement proteins (MPs) to modify intercellular pores called plasmodesmata (PD) to cross the plant cell wall. Many viruses encode a conserved set of three MPs, known as the triple gene block (TGB), typified by Potato virus X (PVX). In this paper, using live-cell imaging of viral RNA (vRNA) and virus-encoded proteins, we show that the TGB proteins have distinct functions during movement. TGB2 and TGB3 established endoplasmic reticulum-derived membranous caps at PD orifices. These caps harbored the PVX replicase and nonencapsidated vRNA and represented PD-anchored viral replication sites. TGB1 mediated insertion of the viral coat protein into PD, probably by its interaction with the 5' end of nascent virions, and was recruited to PD by the TGB2/3 complex. We propose a new model of plant virus movement, which we term coreplicational insertion, in which MPs function to compartmentalize replication complexes at PD for localized RNA synthesis and directional trafficking of the virus between cells.

The ORF3-encoded proteins of vitiviruses GVA and GVB induce tubule-like and punctate structures during virus infection and localize to the plasmodesmata.

The genomic RNA of vitiviruses contains 5 open reading frames (ORF). ORF3 encodes a protein to which the function of a movement protein (MP) was assigned, based on sequence homology with other viral proteins. The aim of the research described in this paper was to gain further insight in distribution profile of the ORF3 product encoded by the vitiviruses Grapevine virus A (GVA) and Grapevine virus B (GVB). Expression of the GVA MP-GFP fusion protein via the virus genome in Nicotiana benthamiana leaves resulted in the formation of irregular spots and fibrous network structures on the outermost periphery of epidermal cells. Expression of GVA MP-GFP and GVB MP-GFP was involved in the formation of the tubule-like and punctate structures on the periphery of N. benthamiana and Vitis vinifera protoplasts. Co-expression of the GVA MP-GFP and GVA MP-RFP in protoplasts resulted in co-localization of these proteins into the same punctate structures, indicating that the MP is not accumulated randomly onto the cell surface, but targeted to particular sites at the cell periphery, where punctate and tubule-like structures are likely formed. With the use of cytoskeleton and secretory pathway inhibitors, we showed that the cytoskeletal elements are not likely to be involved in targeting of the MP-GFP to the punctate cellular structures. In addition to MP, a functional coat protein was found to be essential for virus spread within inoculated leaves.

Expression dynamics and ultrastructural localization of epitope-tagged Abutilon mosaic virus nuclear shuttle and movement proteins in Nicotiana benthamiana cells.

The geminivirus Abutilon mosaic virus (AbMV) encodes two proteins which are essential for viral spread within plants. The nuclear shuttle protein (NSP) transfers viral DNA between the nucleus and cytoplasm, whereas the movement protein (MP) facilitates transport between cells through plasmodesmata and long-distance via phloem. An inducible overexpression system for epitope-tagged NSP and MP in plants yielded unprecedented amounts of both proteins. Western blots revealed extensive posttranslational modification and truncation for MP, but not for NSP. Ultrastructural examination of Nicotiana benthamiana tissues showed characteristic nucleopathic alterations, including fibrillar rings, when epitope-tagged NSP and MP were simultaneously expressed in leaves locally infected with an AbMV DNA A in which the coat protein gene was replaced by a green fluorescent protein encoding gene. Immunogold labelling localized NSP in the nucleoplasm and in the fibrillar rings. MP appeared at the cell periphery, probably the plasma membrane, and plasmodesmata.

Evidence for unidirectional flow through plasmodesmata.

The leaf trichome of tobacco (Nicotiana tabacum) represents a unique secretory structure in which the basal trichome cell is connected to the epidermis by numerous plasmodesmata (PD). Small fluorescent probes microinjected into the basal trichome cell moved apically into distal trichome cells but not into the subtending epidermal cell. In marked contrast, the same probes moved apically into trichome cells when injected into the epidermal cell. Noninvasive methods of dye loading, including ester loading into the apical secretory cell by trichome "capping" and by infiltration of caged fluorescein, produced the same result. In transgenic tobacco plants constitutively expressing photoactivatable green fluorescent protein (PAGFP), activation of PAGFP above the epidermal/trichome (e/t) boundary resulted in movement of protein apically into the distal trichome cells but not across the e/t boundary, while PAGFP activated in the epidermal cell moved apically across the e/t boundary. Experiments with apoplastic tracers also revealed the presence of a distinct apoplastic barrier to solute movement at the e/t interface. These data point to unidirectional transport of solutes through PD. PAGFP activated in individual cells equidistant between the basal cell and the apical cell moved bidirectionally from these cells, suggesting that mass flow was not the driving force for unidirectional transport. We found that unidirectional transport across the e/t boundary was not affected by virus infection or by addition of the actin inhibitor latrunculin but could be dissipated completely by addition of sodium azide. Collectively, our data suggest that active, unidirectional transport of molecules may occur through PD located at unique interfaces in the plant.