Cell-to-Cell Trafficking of Macromolecules through Plasmodesmata Potentiated by the Red Clover Necrotic Mosaic Virus Movement Protein

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...

Section: Methodsmentioning

confidence: 99%

Viral RNA trafficking is inhibited in replicase-mediated resistant transgenic tobacco plants.

Nguyen

Lucas

Ding

et al. 1996

“…However, based on pioneering studies performed by using viral-encoded movement proteins (MPs; refs. [5][6][7][8][9], and more recently with plant proteins (10,11), it is now clear that higher plant plasmodesmata have evolved the capacity to engage in the transport of macromolecules (proteins and nucleic acids). Direct microinjection experiments, performed with viral MPs and plant proteins, have established that cell-to-cell trafficking of macromolecules involves (i) proteinmediated increase in plasmodesmal size exclusion limit (SEL), (ii) protein transport, and (iii) protein-mediated trafficking of RNA͞ DNA (9,12).…”

mentioning

confidence: 99%

Phloem sap proteins from Cucurbita maxima and Ricinus communis have the capacity to traffic cell to cell through plasmodesmata

Balachandran

Schobert

et al. 1997

Self Cite

214

126

In angiosperms, the functional enucleate sieve tube system of the phloem appears to be maintained by the surrounding companion cells. In this study, we tested the hypothesis that polypeptides present within the phloem sap traffic cell to cell from the companion cells, where they are synthesized, into the sieve tube via plasmodesmata. Coinjection of f luorescently labeled dextrans along with sizefractionated Cucurbita maxima phloem proteins, ranging in size from 10 to 200 kDa, as well as injection of individual f luorescently labeled phloem proteins, provided unambiguous evidence that these proteins have the capacity to interact with mesophyll plasmodesmata in cucurbit cotyledons to induce an increase in size exclusion limit and traffic cell to cell. Plasmodesmal size exclusion limit increased to greater than 20 kDa, but less than 40 kDa, irrespective of the size of the injected protein, indicating that partial protein unfolding may be a requirement for transport. A threshold concentration in the 20-100 nM range was required for cell-to-cell transport indicating that phloem proteins have a high affinity for the mesophyll plasmodesmal binding site(s). Parallel experiments with glutaredoxin and cystatin, phloem sap proteins from Ricinus communis, established that these proteins can also traffic through cucurbit mesophyll plasmodesmata. These results are discussed in terms of the requirements for regulated protein trafficking between companion cells and the sieve tube system. As the threshold value for plasmodesmal transport of phloem sap proteins falls within the same range as many plant hormones, the possibility is discussed that some of these proteins may act as long-distance signaling molecules.

“…These plant viruses each encode a single, functionally homologous MP with conserved amino acid sequences (14,17), and neither virus requires coat protein for cell-to-cell movement (16,18). These MPs have been shown conclusively to bind RNA in vitro and to increase the size exclusion limit of plasmodesmata in mesophyl cells to allow cell-to-cell movement (19). Moreover, both TMV MP and RCNMV MP have been shown to complement the transport of other plant viruses belonging to different taxonomic groups, such as barley stripe mosaic virus (20,21), potato virus X (22), and brome mosaic virus (23).…”

mentioning

confidence: 99%

Systemic spread of an RNA insect virus in plants expressing plant viral movement protein genes

Dasgupta

Garcia

Goodman

2001

nodavirus V iruses whose host range spans two kingdoms are few. Most are insect-vectored plant viruses that multiply in a narrow insect host range (1). Flock house virus (FHV), a member of the insect͞animal virus family called Nodaviridae (for reviews, see refs. 2-4), is a unique example of a virus that crosses the kingdom barrier. FHV was originally isolated from the New Zealand grass grub Costelytra zealandica (5). The range of insect hosts for FHV is not well studied. In the laboratory, FHV replicates in the larvae of the wax moth Galleria mellonella and cultured Drosophila melanogaster cells (4). In 1990, FHV was shown to replicate and produce infectious virions in barley protoplasts and the inoculated leaves of several other monocotyledon and dicotyledon plant species without producing symptoms (6). FHV thus replicates in both insects (2) and plants (6), although it is not transmitted to plants by its insect hosts (2).The genome of FHV consists of two small messenger sense RNAs: RNA1 (3.1 kb; ref. Broad host range, simple genome organization, and asymptomatic growth in plants make FHV a powerful tool for the study of fundamental questions of virus-host interactions and the modification of gene expression in plants. A drawback to its use is that FHV does not encode a protein that enables it to move inside the plant. In all plants previously tested, FHV accumulated only in inoculated tissues or cells (6).Here we report the results of experiments designed to determine whether FHV could be mobilized in plants with the use of plant viral movement proteins (MPs). We used genes encoding the 30-kDa protein of tobacco mosaic virus (TMV) and the 35-kDa protein of red clover necrotic mosaic virus (RCNMV) (dianthovirus), two of the most well-studied and widely used plant viral MPs, both expressed as transgenes in N. benthamiana plants (11-16). These plant viruses each encode a single, functionally homologous MP with conserved amino acid sequences (14, 17), and neither virus requires coat protein for cell-to-cell movement (16, 18). These MPs have been shown conclusively to bind RNA in vitro and to increase the size exclusion limit of plasmodesmata in mesophyl cells to allow cell-to-cell movement (19). Moreover, both TMV MP and RCNMV MP have been shown to complement the transport of other plant viruses belonging to different taxonomic groups, such as barley stripe mosaic virus (20, 21), potato virus X (22), and brome mosaic virus (23).Our results show that MPs of both TMV and RCNMV initiate local as well as long-distance movement of FHV in MP transgenic plants. RCNMV MP is more effective in mobilizing movement of FHV to noninoculated leaves. We also show that FHV replicates in inoculated leaves of six more plant species, in addition to those reported previously (6). Newly discovered experimental hosts of FHV include alfalfa, Arabidopsis, Brassica, cucumber, rice, and sweet corn. Plants were grown from seeds planted in Scotts Redi-earth Plug and Seedling Mix and placed in growth chambers maintained at 24°C, with 12 h of l...