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Osmotic water permeability (Pos) was measured in protoplasts isolated from different tissues of Phaseolus vulgaris twining shoot. Parenchyma protoplasts exhibited more frequently high Pos values than epidermis protoplasts did. Water channels could facilitate water movement between parenchyma cells whereas cell-to-cell water transport mostly occurs through plasmodesmata in epidermis.
Transroot osmotic water flux (Jos) and radial hydraulic conductivity (Lpr) in onion roots were greatly increased by three means; infiltration of roots by pressurization, repetition of osmosis and chilling at 5 degrees C. Jos was strongly reduced by the water channel inhibitor HgCl2 (91%) and the K+ channel inhibitor nonyltriethylammonium (C9, 75%), which actually made the membrane potential of root cells less sensitive to K+. C9 decreased the rate of turgor reduction induced by sorbitol solution to the same extent as HgCl2. Thus, C9 is assumed to decrease the hydraulic conductivity (Lp) of the plasma membrane by blocking water channels, although possible inhibition of the plasmodesmata of the root symplast by C9 cannot be excluded. Onion roots transported water from the tip to the base in the absence of the osmotic gradient. This non-osmotic water flux (Jnos) was equivalent to Jos induced by 0.029 M sorbitol. Jnos increased when Jos was increased by repetition of osmosis and decreased when Jos was decreased by either HgCl2 or by C9. The correlation between Jnos and Jos suggests that non-osmotic water transport occurs via the same pathways as those for osmotic water transport.
Sieve tubes are comprised of sieve elements, enucleated cells that are incapable of RNA and protein synthesis. The proteins in sieve elements are supplied from the neighboring companion cells through plasmodesmata. In rice plants, it was unclear whether or not all proteins produced in companion cells had the same distribution pattern in the sieve element-companion cell complex. In this study, the distribution pattern of four proteins, beta-glucuronidase (GUS), green fluorescent protein (GFP), thioredoxin h (TRXh) and glutathione S-transferase (GST) were analyzed. The foreign proteins GUS and GFP were expressed in transgenic rice plants under the control of the TRXh gene promoter (PTRXh), a companion cell-specific promoter. Analysis of leaf cross-sections of PTRXh-GUS and PTRXh-GFP plants indicated high accumulation of GUS and GFP, respectively, in companion cells rather than in sieve elements. GUS and GFP were also detected in phloem sap collected from leaf sheaths of the transgenic rice plants, suggesting these proteins could enter sieve elements. Relative amounts of GFP and endogenous phloem proteins, TRXh and GST, in phloem sap and total leaf extracts were compared. Compared to TRXh and GST, GFP content was higher in total leaf extracts, but lower in phloem sap, suggesting that GFP accumulated mainly in companion cells rather than in sieve elements. On the other hand, TRXh and GST appeared to accumulate in sieve elements rather than in companion cells. These results indicate the evidence for differential distribution of proteins between sieve elements and companion cells in rice plants.
The systemic movement of Cucumber mosaic virus (CMV) in cucumber plants was analyzed. The structure that is translocated and its putative interactions with phloem components were analyzed in phloem exudate (PE) samples, which reflect sieve tubes stream composition. Rate zonal centrifugation and electron-microscopy analyses of PE from CMV-infected plants showed that CMV moves through sieve tubes as virus particles. Gel overlay assays revealed that CMV particles interact with a PE protein, p48. The amino-acid sequence of several tryptic peptides of p48 was determined. Partial amino-acid sequence of p48 showed it was a cucumber homolog of phloem protein 1 (PP1) from pumpkin, with which p48 also shares several chemical properties. PP1 from pumpkin has plasmodesmata-gating ability and translocates in sieve tubes. Encapsidated CMV RNA in PE samples from infected plants was less accessible to digestion by RNase A than RNA in purified CMV particles, a property that was reconstituted by the in vitro interaction of purified CMV particles and protein p48. These results indicate that the interaction with p48 modifies CMV particle structure and suggest that CMV particles interact with the cucumber homolog of PP1 during translocation in the sieve tubes.
The 25K, 12K, and 8K proteins and coat protein (CP) of Potato virus X (PVX) are required for virus cell-to-cell movement. In this study, experiments were conducted to determine whether the PVX 25K protein moves cell to cell and to explore potential interactions between the PVX 25K, 12K, and 8K proteins and CP. The PVX 25K gene was fused to the green fluorescent protein (GFP) gene and inserted into plasmids adjacent to the cauliflower mosaic virus 35S promoter. These plasmids were introduced by biolistic bombardment to transgenic tobacco expressing the PVX 12K, 8K, and CP genes. The GFP:25K fused proteins moved cell to cell on nontransgenic tobacco and tobacco expressing either the 12K or 8K proteins. However, the GFP:25K proteins did not move on transgenic tobacco expressing the combined 12K/8K genes or the CP gene. Thus, movement of the PVX 25K protein through plasmodesmata may be regulated by interactions with other PVX proteins.
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.
The Tobacco mosaic virus (TMV) movement protein (MPTMV) mediates cell-to-cell viral trafficking by altering properties of the plasmodesmata (Pd) in infected cells. During the infection cycle, MPTMV becomes transiently associated with endomembranes, microfilaments, and microtubules (MT). It has been shown that the cell-to-cell spread of TMV is reduced in plants expressing the dysfunctional MP mutant MPNT-1. To expand our understanding of the MP function, we analyzed events occurring during the intracellular and intercellular targeting of MPTMV and MPNT-1 when expressed as a fusion protein to green fluorescent protein (GFP), either by biolistic bombardment in a viral-free system or from a recombinant virus. The accumulation of MPTMV:GFP, when expressed in a viral-free system, is similar to MPTMV:GFP in TMV-infected tissues. Pd localization and cell-to-cell spread are late events, occurring only after accumulation of MP:GFP in aggregate bodies and on MT in the target cell. MPNT-1:GFP localizes to MT but does not target to Pd nor does it move cell to cell. The spread of transiently expressed MPTMV:GFP in leaves of transgenic plants that produce MPNT-1 is reduced, and targeting of the MPTMV:GFP to the cytoskeleton is inhibited. Although MPTMV:GFP targets to the Pd in these plants, it is partially impaired for movement. It has been suggested that MPNT-1 interferes with host-dependent processes that occur during the intracellular targeting program that makes MP movement competent.
Incubation with cucumber phloem exudate in vitro results in a dramatic decrease in the electrophoretic mobility of Hop stunt viroid. UV cross-linking and a combination of size exclusion and ion exchange chromatography indicate that this phenomenon reflects a previously unsuspected ability of phloem protein 2, a dimeric lectin and the most abundant component of phloem exudate, to interact with RNA. In light of its demonstrated ability to move from cell to cell via plasmodesmata as well as long distances in the phloem, our results suggest that phloem protein 2 may facilitate the systemic movement of viroids and, possibly, other RNAs in vivo.
TGBp1, TGBp2, and TGBp3, three plant virus movement proteins encoded by the "triple gene block" (TGB), may act in concert to facilitate cell-to-cell transport of viral RNA genomes. Transient expression of Potato mop-top virus (genus Pomovirus) movement proteins was used as a model to reconstruct interactions between TGB proteins. In bombarded epidermal cells of Nicotiana benthamiana, green fluorescent protein (GFP)-TGBp1 was distributed uniformly. However, in the presence of TGBp2 and TGBp3, GFP-TGBp1 was directed to intermediate bodies at the cell periphery, and to cell wall-embedded punctate bodies. Moreover, GFP-TGBp1 migrated into cells immediately adjacent to the bombarded cell. These data suggest that TGBp2 and TGBp3 mediate transport of GFP-TGBp1 to and through plasmodesmata. Mutagenesis of TGBp1 suggested that the NTPase and helicase activities of TGBp1 were not required for its transport to intermediate bodies directed by TGBp2 and TGBp3, but these activities were essential for the protein association with cell wall-embedded punctate bodies and translocation of TGBpl to neighboring cells. The C-terminal region of TGBp1 was critical for trafficking mediated by TGBp2 and TGBp3. Mutation analysis also suggested an involvement of the TGBp2 C-terminal region in interactions with TGBp1.
We have reported previously that tobamoviruses enable the transport of red clover mottle comovirus (RCMV) in tobacco plants normally resistant to RCMV. Here we show that RCMV transport does not take place in transgenic tobacco plants (line To-4) producing the 30K transport protein of tobacco mosaic virus (TMV), whereas the transport of the TMV Ls1 mutant, the cell-to-cell movement of which is temperature sensitive, is complemented in these plants. However, RCMV transport is observed when these transgenic plants are infected with both RCMV and TMV Ls1 at the non-permissive temperature (33 degrees C). It is suggested that (i) the hypothetical modification of transgenic plant plasmodesmata by the TMV 30K transport protein can specifically mediate the cell-to-cell movement of the homologous virus (TMV), but is insufficient to mediate RCMV transport; (ii) the presence of the full-length TMV genome or a certain TMV-encoded product(s) besides the 30K protein is essential for complementation of the RCMV transport function. The possibility that line To-4 might provide enough 30K protein to complement TMV Ls1 but not RCMV cannot be ruled out. During double infection the mutant 30K protein may, in concert with the wild-type 30K protein, provide the transport function for RCMV.
During an infection with cowpea mosaic virus (CPMV) both virion assembly and formation of tubules associated with plasmodesmata are required for cell-to-cell movement. These functions are encoded by the M-RNA of CPMV. To study the mechanism of CPMV movement, mutant N123 was used in complementation studies with sunn-hemp mosaic virus (SHMV), a legume-infecting tobamovirus. Previous studies have shown that N123 fails to spread in cowpea plants because of mutation(s) in its M-RNA. However, the mutant was efficiently replicated in cowpea protoplasts, in which virions were formed and tubular transport structures were induced. After high-dose inoculation of cowpeas with N123, only a few infected protoplasts could be isolated, indicating that cell-to-cell transport of N123 was greatly impaired, if not completely abolished. Upon coinoculation with SHMV, mutant N123 infected cowpea plants systemically and accumulated to levels which were comparable to those of wild-type CPMV. In contrast, separate B-RNA of CPMV and a CPMV deletion mutant lacking the tubule-inducing function, were complemented by SHMV to only low levels. It is concluded that SHMV-facilitated spread of CPMV in the non-virion tobamovirus mode is inefficient and that spread of mutant N123 is probably in the CPMV mode, SHMV providing an as yet unidentified helper function.
The movement proteins (MP) of cowpea mosaic virus and cauliflower mosaic virus (CaMV) are associated with tubular structures in vivo which participate in the transmission of virus particles from cell to cell. Both proteins have been expressed in plant protoplasts and insect cells. In all cases, immunofluorescent histochemistry showed that the MPs accumulate intracellularly as tubular extensions projecting from the cell surface. Additionally, electron microscopy revealed intracellular MP aggregates in CaMV MP-expressing cells. The data presented establish common features for the tubule-forming MPs: no other virus gene products are required for tubule formation and unique plant components (e.g. plasmodesmata) are not essential for tubule synthesis.
The N-terminal P1 proteinase of potato virus Y (ordinary strain group isolate PVY-O) was expressed in E. coli. Antiserum was raised against the expressed protein and used to detect the viral proteins in infected tobacco leaf tissue by Western blotting and by electron microscopy with immunogold labelling. In the immunogold localization studies P1 protein was detected in association with the cytoplasmic inclusion bodies characteristic of PVY infections and in the cytoplasm of the infected plant cells. No significant P1 antibody binding with other plant cell organelles, or with the cell wall and plasmodesmata, was detected by immunogold labelling.
Brome mosaic bromovirus (BMV) 3a protein is required for cell-to-cell movement of the virus in host plants. The BMV 3a protein (B3a) was produced in Escherichia coli using an expression vector. Gel retardation analysis and UV cross-linking experiments demonstrated that B3a bound single-stranded RNA cooperatively without sequence specificity. Binding competition analysis showed that B3a bound to single-stranded nucleic acids more strongly than to double-stranded nucleic acids. Deletion mutagenesis located a nucleic acid-binding domain to amino acids 189-242. Western blot analysis of fractionated proteins of BMV-infected barley using monoclonal antibodies against B3a indicated that B3a may interact with membrane materials and form complexes in the cytoplasm. Immunogold labelling of thin sections of infected barley tissues revealed that B3a was associated with plasmodesmata and cytoplasmic inclusions.
An isolate of Strawberry mottle virus (SMoV) was transferred from Fragaria vesca to Nicotiana occidentalis and Chenopodium quinoa by mechanical inoculation. Electron micrographs of infected tissues showed the presence of isometric particles of approximately 28 nm in diameter. SMoV-associated tubular structures were also conspicuous, particularly in the plasmodesmata of C. quinoa. DsRNA extraction of SMoV-infected N. occidentalis yielded two bands of 6.3 and 7.8 kbp which were cloned and sequenced. Gaps in the sequence, including the 5' and 3' ends, were filled using RT-PCR and RACE. The genome of SMoV was found to consist of RNA1 and RNA2 of 7036 and 5619 nt, respectively, excluding a poly(A) tail. Each RNA encodes one polyprotein and has a 3' non-coding region of approximately 1150 nt. The polyprotein of RNA1 contains regions with identities to helicase, viral genome-linked protein, protease and polymerase (RdRp), and shares its closest similarity with RNA1 of the tentative nepovirus Satsuma dwarf virus (SDV). The polyprotein of RNA2 displayed some similarity to the large coat protein domain of SDV and related viruses. Phylogenetic analysis of the RdRp region showed that SMoV falls into a separate group containing SDV, Apple latent spherical virus, Naval orange infectious mottling virus and Rice tungro spherical virus. Given the size of RNA2 and the presence of a long 3' non-coding region, SMoV is more typical of a nepovirus, although atypically for a nepovirus it is aphid transmissible. We propose that SMoV is a tentative member of an SDV-like lineage of picorna-like viruses.
Many plant virus genera encode a 'triple gene block' (TGB), a specialized evolutionarily conserved gene module involved in the cell-to-cell and long-distance movement of viruses. The TGB-based transport system exploits the co-ordinated action of three polypeptides to deliver viral genomes to plasmodesmata and to accomplish virus entry into neighbouring cells. Although data obtained on both the TGB and well-studied single protein transport systems clearly demonstrate that plant viruses employ host cell pathways for intra- and intercellular trafficking of genomic nucleic acids and proteins, there is no integral picture of the details of molecular events during TGB-mediated virus movement. Undoubtedly, understanding the molecular basis of the concerted action of TGB-encoded proteins in transporting viral genomes from cell to cell should provide new insights into the general principles of movement protein function. This review describes the structure, phylogeny and expression of TGB proteins, their roles in virus cell-to-cell movement and potential influence on host antiviral defences.
Cowpea mosaic virus (CPMV) moves from cell to cell as virus particles which are translocated through a plasmodesmata-penetrating transport tubule made up of viral movement protein (MP) copies. To gain further insight into the roles of the viral MP and capsid proteins (CP) in virus movement, full-length and truncated forms of the MP were expressed in insect cells using the baculovirus expression system. Using ELISA and blot overlay assays, affinity purified MP was shown to bind specifically to intact CPMV virions and to the large CP, but not to the small CP. This binding was not observed with a C-terminal deletion mutant of the MP, although this mutant retained the capacity to bind to other MP molecules and to form tubules. These results suggest that the C-terminal 48 amino acids constitute the virion-binding domain of the MP.
The genomes of umbraviruses differ from those of most other viruses in that they do not encode a coat protein, and thus no virus particles are formed in infected plants. Protection of umbraviral RNA outside the host plant, during vector transmission, utilizes the coat protein of an assistor luteovirus, but this review focuses on the mechanisms that compensate for the lack of a coat protein in processes within the host plant. As well as an RNA-dependent RNA polymerase, umbravirus genomes encode two other proteins from almost completely overlapping open reading frames. One of these is a cell-to-cell movement protein that can mediate the transport of homologous and heterologous viral RNAs through plasmodesmata without the participation of a coat protein. The other, the ORF3 protein, binds to viral RNA to form filamentous ribonucleoprotein particles that have elements of helical structure. It serves to stabilize the RNA and facilitates its transport through the vascular system of the plant. It may also be involved in protection of the viral RNA from the plant's defensive RNA-silencing response, although it is not a suppressor of silencing. The ORF3 protein also enters the cell nucleus, specifically targeting the nucleolus. Although the function of this localization is unknown, the ORF3 protein may provide a valuable tool for investigating plant nucleolar function.