In vivo recombination of cauliflower mosaic virus DNA

Ligation and recombination of the DNA of cauliflower mosaic virus (CaMV) is demonstrated by the following experiments: (i) Ligation: Different noninfectious fragments of the CaMV genome (obtained after insertion into plasmid pBR322 followed by enzymatic excision) regained infectivity when mixtures of them were used to inoculate their host. The symptom appearance was delayed by comparison with a typical CaMV infection, and only the newly formed leaves were affected. (ii) Recombination: Pairs of noninfectious recombinant full-length CaMV genomes (integrated into pBR322 at different restriction endonuclease sites) regained infectivity upon simultaneous inoculation of a sensitive host. The symptomatology of the resulting infection was indistinguishable from that of a typical CaMV infection. We show that progeny DNA had the same characteristics (size, structure, restriction endonuclease digestion pattern) as bona fide CaMV DNA, and that the vector pBR322 had been completely eliminated. A cloned tandem dimer of CaMV DNA with a partial deletion similarly was infectious in the plant assays. This system should be useful to study the expression of mutant genomes, thus allowing characterization of the CaMV genes.     

HSP70 homolog functions in cell-to-cell movement of a plant virus

Plant closteroviruses encode a homolog of the HSP70 (heat shock protein, 70 kDa) family of cellular proteins. To facilitate studies of the function of HSP70 homolog (HSP70h) in viral infection, the beet yellows closterovirus (BYV) was modified to express green fluorescent protein. This tagged virus was competent in cell-to-cell movement, producing multicellular infection foci similar to those formed by the wild-type BYV. Inactivation of the HSP70h gene by replacement of the start codon or by deletion of 493 codons resulted in complete arrest of BYV translocation from cell to cell. Identical movement-deficient phenotypes were observed in BYV variants possessing HSP70h that lacked the computer-predicted ATPase domain or the C-terminal domain, or that harbored point mutations in the putative catalytic site of the ATPase. These results demonstrate that the virus-specific member of the HSP70 family of molecular chaperones functions in intercellular translocation and represents an additional type of a plant viral-movement protein.     

Arabidopsis synaptotagmin SYTA regulates endocytosis and virus movement protein cell-to-cell transport

Synaptotagmins are calcium sensors that regulate synaptic vesicle exo/endocytosis. Thought to be exclusive to animals, they have recently been characterized in plants. We show that Arabidopsis synaptotagmin SYTA regulates endosome recycling and movement protein (MP)-mediated trafficking of plant virus genomes through plasmodesmata. SYTA localizes to endosomes in plant cells and directly binds the distinct Cabbage leaf curl virus (CaLCuV) and Tobacco mosaic virus (TMV) cell-to-cell movement proteins. In a SYTA knockdown line, CaLCuV systemic infection is delayed, and cell-to-cell spread of TMV and CaLCuV movement proteins is inhibited. A dominant-negative SYTA mutant causes depletion of plasma membrane-derived endosomes, produces large intracellular vesicles attached to plasma membrane, and inhibits cell-to-cell trafficking of TMV and CaLCuV movement proteins, when tested in an Agrobacterium-based leaf expression assay. Our studies show that SYTA regulates endocytosis, and suggest that distinct virus movement proteins transport their cargos to plasmodesmata for cell-to-cell spread via an endocytic recycling pathway.Synaptotagmins (Syts) are a large family of Ca²⁺/lipid binding proteins widely studied in animals due to their role in neurotransmitter release. They are also found in Drosophila and Caenorhabditis elegans and were recently described in plants (1, 2). Syts have a conserved domain structure: a short uncleaved N-terminal signal peptide that overlaps a transmembrane (TM) domain, followed by a cytosolic variable region and two C-terminal C₂ domains, C₂A and C₂B. Whereas C₂A and C₂B each bind phospholipids in a Ca²⁺-dependent manner, fold independently and act synergistically, C₂B is essential for activity (1). SytI, the best studied Syt, is proposed to act as a Ca²⁺ sensor to regulate rapid and synchronous synaptic vesicle exocytosis (1). Whether it regulates SNARE complex formation in a temporal and spatial manner, or is itself fusogenic, is unclear. Studies in PC12 cells, and of mouse and Drosophila sytI mutants, suggest that the SNARE complex VAMP1/SNAP25/syntaxin-1 targets the synaptic vesicle to the plasma membrane to create a metastable fusion intermediate. SytI on the vesicle membrane, and perhaps a distinct partner Syt on the plasma membrane, would then interact with phospholipids and the SNARE complex to accelerate SNARE-mediated fusion pore dilation. Liposome studies suggest a direct fusogenic role for SytI, in which shallow insertion of the C₂ region into target membranes induces curvature to destabilize the lipid bilayer and form the fusion pore opening (1, 3). Studies in mice, Drosophila, and C. elegans show that SytI also regulates the kinetics of endocytosis at nerve terminals, apparently in a clathrin-mediated manner (1, 4).Plant virus movement proteins (MPs) mediate the transport of progeny genomes across the cell wall for local and systemic infection. Despite diverse strategies for cell-to-cell movement, two common features have emerged: movement proteins alter plasmodesmata (PD), transwall pores that connect adjacent plant cells; and protein localization and interaction studies implicate the endoplasmic reticulum (ER) and membrane trafficking in this process (5 –8). This is typified by the Begomoviruses Cabbage leaf curl virus (CaLCuV) and Squash leaf curl virus (SqLCV) and Tobamovirus Tobacco mosaic virus (TMV), with their respective single strand DNA (ssDNA) or RNA (ssRNA) genomes. CaLCuV and SqLCV encode two movement proteins: the nuclear shuttle protein NSP and the cell-to-cell movement protein MP. NSP binds replicated viral ssDNA in the nucleus and shuttles it to the cytoplasm, where MP traps these complexes to direct them to and across the cell wall via ER-derived transwall tubules. NSP then targets the viral genome to the nucleus for new cycles of replication (7). The ER-derived tubules are proposed to be the analog of the desmotubule, the PD axial membrane component that is first derived from cortical ER “trapped” by the wall during cell division (6). TMV genomes replicate at ER-derived membrane sites in the cytosol. TMV encodes a single 30-kDa movement protein (30K), which binds and targets progeny genomes to cortical ER sites and PD. The 30K protein increases PD size exclusion limits to allow viral ssRNA to move cell to cell (5, 7). Mutational and antisense suppression studies show that interaction of 30K with a cell wall pectin methylesterase (PME) is required for TMV cell-to-cell movement and infection. Hence, PME may direct 30K, complexed with TMV genomes, to PD and/or act to alter PD gating (9). These studies, and those of other movement proteins, link vesicular traffic to virus movement and lead to speculation that viral genomes and other macromolecules may target to and through PD by “grabbing” a receptor or exo/endocytosis (10).We report here the functional analysis of a plant synaptotagmin, Arabidopsis SYTA, which we identified in a yeast interactive screen using CaLCuV MP (MP_CaLCuV) as bait. SYTA directly binds to MP_CaLCuV in vitro, and to the related SqLCV MP (MP_SqLCV) and the distinct TMV 30K. We found that SYTA localizes to endosomes, using FM4-64 and compartment-specific markers. To establish the functions of SYTA in virus movement and in plant cells, we showed that CaLCuV infection is delayed, and TMV 30K and MP_CaLCuV cell-to-cell spread are inhibited, in an SYTA knockdown line; and a dominant-negative form of SYTA inhibited endocytosis and the recycling of an endosome marker at the plasma membrane, and the cell-to-cell trafficking of TMV 30K and MP_CaLCuV in an Agrobacterium tumefaciens-based leaf transient expression assay. We conclude that SYTA regulates both endosome recycling and the activities of the diverse MP_CaLCuV and TMV 30K in virus cell-to-cell movement, and suggest that distinct virus movement proteins transport their cargos to PD for cell-to-cell spread via an endocytic recycling pathway.     

Recombinant tobacco mosaic virus movement protein is an RNA-binding, alpha-helical membrane protein

The 30-kDa movement protein (MP) is essential for cell-cell spread of tobacco mosaic virus in planta. To explore the structural properties of MP, the full-length recombinant MP gene was expressed in Escherichia coli, and one-step purification from solubilized inclusion bodies was accomplished by using anion exchange chromatography. Soluble MP was maintained at >4 mg/ml without aggregation and displayed approximately 70% alpha-helical conformation in the presence of urea and SDS. A trypsin-resistant core domain of the MP had tightly folded tertiary structure, whereas 18 aa at the C terminus of the monomer were rapidly removed by trypsin. Two hydrophobic regions within the core were highly resistant to proteolysis. Based on results of CD spectroscopy, trypsin treatment, and MS, we propose a topological model in which MP has two putative alpha-helical transmembrane domains and a protease-sensitive carboxyl terminus.     

Role of microtubules in the intracellular distribution of tobacco mosaic virus movement protein

Despite its central role in virus infection, little is known about the mechanisms of intracellular trafficking of virus components within infected cells. In this study, we followed the dynamics of tobacco mosaic virus movement protein (MP) distribution in living protoplasts after disruption of microtubules (MTs) by cold treatment and subsequent rewarming to 29 degrees C. At early stages of infection, cold treatment (4 degrees C) caused the accumulation of MP fused to green fluorescent protein (GFP) in large virus replication bodies that localized in perinuclear positions, whereas at midstages of infection, the association of MP:GFP with MTs was disrupted. Rewarming the protoplasts to 29 degrees C reestablished the association of MTs with the replication bodies that subsequently spread throughout the cytoplasm and to the periphery of the cell. The role of MTs in the intracellular distribution of the MP also was analyzed by examining the distribution pattern of a nonfunctional mutant of MP (TAD5). Like MP:GFP, TAD5:GFP interacted with the endoplasmic reticulum membranes and colocalized with its viral RNA but did not colocalize with MTs. The involvement of MTs in the intracellular distribution of tobacco mosaic virus MP is discussed.     

Intracellular distribution of viral gene products regulates a complex mechanism of cauliflower mosaic virus acquisition by its aphid vector

Interactions between Cauliflower mosaic virus (CaMV) and its aphid vector are regulated by the viral protein P2, which binds to the aphid stylets, and protein P3, which bridges P2 and virions. By using baculovirus expression of P2 and P3, electron microscopy, surface plasmon resonance, affinity chromatography, and transmission assays, we demonstrate that P3 must be previously bound to virions in order that attachment to P2 will allow aphid transmission of CaMV. We also show that a P2:P3 complex exists in the absence of virions but is nonfunctional in transmission. Hence, unlike P2, P3 and virions cannot be sequentially acquired by the vector. Immunogold labeling revealed the predominance of spatially separated P2:P3 and P3:virion complexes in infected plant cells. This specific distribution indicates that the transmissible complex, P2:P3:virion, does not form primarily in infected plants but in aphids. A model, describing the regulating role of P3 in the formation of the transmissible CaMV complex in planta and during acquisition by aphids, is presented, and its consequences are discussed.     

Interaction between the aphid transmission factor and virus particles is a part of the molecular mechanism of cauliflower mosaic virus aphid transmission.

Cauliflower mosaic virus (CaMV) aphid transmission factor (ATF or P18) is presumed to interact with both virus particles and vector mouthparts, thereby mediating virus aphid transmission. We developed a protein-protein binding assay and our results clearly show that virus particles bind strongly and specifically to P18 whether P18 was obtained from plants, a baculovirus expression system, or the pGEX-3X Escherichia coli expression system. We overproduced, using the pGEX-3X expression system, various fragments of P18 and thereby demonstrated that the C-terminal 31 amino acid residues are responsible for the interaction. Using PCR-based mutagenesis, 2 amino acid residues essential for interaction were identified. Point substitutions (amino acids 157 from Ile to Asn or 159 from Gly to Ser) were sufficient to abolish the interaction, whereas another mutation (amino acid 158 from Ile to Ser) had no effect on P18 virus binding. We evaluated whether there was a correlation between the ability of P18 to interact with CaMV particles and its biological activity. Aphid transmission assays were carried out and we demonstrated that the loss of the virus binding capacity had a dramatic effect on the ability of P18 to mediate aphid transmission. Thus, our results suggest that binding between P18 and virus particles is likely to be one of the molecular mechanisms involved in CaMV aphid transmission.     

Genomic position affects the expression of tobacco mosaic virus movement and coat protein genes.

Alterations in the genomic position of the tobacco mosaic virus (TMV) genes encoding the 30-kDa cell-to-cell movement protein or the coat protein greatly affected their expression. Higher production of 30-kDa protein was correlated with increased proximity of the gene to the viral 3' terminus. A mutant placing the 30-kDa open reading frame 207 nucleotides nearer the 3' terminus produced at least 4 times the wild-type TMV 30-kDa protein level, while a mutant placing the 30-kDa open reading frame 470 nucleotides closer to the 3' terminus produced at least 8 times the wild-type TMV 30-kDa protein level. Increases in 30-kDa protein production were not correlated with the subgenomic mRNA promoter (SGP) controlling the 30-kDa gene, since mutants with either the native 30-kDa SGP or the coat protein SGP in front of the 30-kDa gene produced similar levels of 30-kDa protein. Lack of coat protein did not affect 30-kDa protein expression, since a mutant with the coat protein start codon removed did not produce increased amounts of 30-kDa protein. Effects of gene positioning on coat protein expression were examined by using a mutant containing two different tandemly positioned tobamovirus (TMV and Odontoglossum ringspot virus) coat protein genes. Only coat protein expressed from the gene positioned nearest the 3' viral terminus was detected. Analysis of 30-kDa and coat protein subgenomic mRNAs revealed no proportional increase in the levels of mRNA relative to the observed levels of 30-kDa and coat proteins. This suggests that a translational mechanism is primarily responsible for the observed effect of genomic position on expression of 30-kDa movement and coat protein genes.     

Direct functional assay for tobacco mosaic virus cell-to-cell movement protein and identification of a domain involved in increasing plasmodesmal permeability.

Plasmodesmata are cytoplasmic bridges betweenplant cells thought to generally allow only the passage of small molecules andmetabolites. However, large structures such as plant viruses also move from cellto cell via plasmodesmata. In tobacco mosaic virus (TMV) infection a viralmovement protein (TMV-MP) mediates viral spread. Here, a microinjection assay isused to monitor the dynamics of TMV-MP function directly in wild-type plants.The results indicate that TMV-MP interacts with an endogenous plant pathwayincreasing plasmodesmal size exclusion limit to permit passage of 20-kDadextrans. Furthermore, TMV-MP influences plasmodesmal size exclusion limitseveral cells distant from the injection site, indicating either that TMV-MPitself crosses plasmodesmata or that TMV-MP induces a diffusable signal capableof dilating microchannels of plasmodesmata. The region of TMV-MP responsible forincreasing plasmodesmal size exclusion limit was mapped to the carboxyl-terminalpart of the 268-amino acid residue protein between amino acid residues 126 and224.     

Identification of C-terminal amino acid residues of cauliflower mosaic virus open reading frame III protein responsible for its DNA binding activity.

We cloned in Escherichia coli truncated versions of the protein p15 encoded by open reading frame III of cauliflower mosaic virus. We then compared the ability of the wild-type p15 (129 amino acids) and the deleted p15 to bind viral double-stranded DNA genome. Deletions of > 11 amino acids in the C-terminal proline-rich region resulted in loss of DNA binding activity of wild-type p15. Moreover, a point mutation of the proline at position 118 sharply reduced the interaction between the viral protein and DNA. These results suggest that cauliflower mosaic virus p15 belongs to the family of DNA binding proteins having a proline-rich motif involved in interaction with double-stranded DNA.     

The aphid transmission factor of cauliflower mosaic virus forms a stable complex with microtubules in both insect and plant cells

We analyzed the distribution of the cauliflower mosaic virus (CaMV) aphid transmission factor (ATF), produced via a baculovirus recombinant, within Sf9 insect cells. Immunogold labeling revealed that the ATF colocalizes with an atypical cytoskeletal network. Detailed observation by electron microscopy demonstrated that this network was composed of microtubules decorated with paracrystalline formations, characteristic of the CaMV ATF. A derivative mutant of the ATF, unable to self-assemble into paracrystals, was also analyzed. This mutant formed a net-like structure, with a mesh of four nanometers, tightly sheathing microtubules. Both the ATF– and the derivative mutant–microtubule complexes were highly stable. They resisted dilution-, cold-, and calcium-induced microtubule disassembly as well as a combination of all three for over 6 hr. CaMV ATF cosedimented with microtubules and, surprisingly, it bound to Taxol-stabilized microtubules at high ionic strength, thus suggesting an atypical interaction when compared with that usually described for microtubule-binding proteins. Using immunofluorescence double labeling we also demonstrated that the CaMV ATF colocalizes with the microtubule network when expressed in plant cells.     

Molecular characterization and biological function of the movement protein of tobacco mosaic virus in transgenic plants.

We previously demonstrated, in transgenic tobacco plants, that the role of the movement protein (MP) of tobacco mosaic virus is to facilitate the cell-to-cell spread of viral progeny during infection. An analysis of different tissues of these transgenic plants indicated that the MP accumulated in leaf, stem, and root tissue. The highest levels were detected in older leaves. The relative levels of MP in leaf tissue from transgenic plants were equivalent to, or higher than, the levels of MP in tobacco mosaic virus-infected leaf tissue. Results of subcellular fractionation of homogenates of transgenic leaf tissue showed that the MP was most abundant in the cell wall fraction of older leaves and that the protein remained at high levels in the cell wall fraction as the leaves continued to age. Significant levels of the MP were detected in a crude membrane/organelle fraction and a soluble fraction in younger leaves but decreased to low levels in older leaves. These results suggest that the MP accumulates and is stable in cell walls. We have previously shown that the MP modifies the molecular exclusion limit of plasmodesmata, which is consistent with the hypothesis that plant viruses move from cell to cell through altered plasmodesmata. We show here that the ability of the tobacco mosaic virus MP to modify the molecular exclusion limit of plasmodesmata in tobacco depends on the developmental stage of the leaf. The implications of these findings on understanding virus movement and how plasmodesmata function are discussed.     

Expression of alfalfa mosaic virus coat protein in tobacco mosaic virus (TMV) deficient in the production of its native coat protein supports long-distance movement of a chimeric TMV

Alfalfa mosaic virus (AlMV) coat protein is involved in systemic infection of host plants, and a specific mutation in this gene prevents the virus from moving into the upper uninoculated leaves. The coat protein also is required for different viral functions during early and late infection. To study the role of the coat protein in long-distance movement of AlMV independent of other vital functions during virus infection, we cloned the gene encoding the coat protein of AlMV into a tobacco mosaic virus (TMV)-based vector Av. This vector is deficient in long-distance movement and is limited to locally inoculated leaves because of the lack of native TMV coat protein. Expression of AlMV coat protein, directed by the subgenomic promoter of TMV coat protein in Av, supported systemic infection with the chimeric virus in Nicotiana benthamiana, Nicotiana tabacum MD609, and Spinacia oleracea. The host range of TMV was extended to include spinach as a permissive host. Here we report the alteration of a host range by incorporating genetic determinants from another virus.     

Invasion of minor veins of tobacco leaves inoculated with tobacco mosaic virus mutants defective in phloem-dependent movement.

To fully understand vascular transport of plant viruses, the viral and host proteins, their structures and functions, and the specific vascular cells in which these factors function must be determined. We report here on the ability of various cDNA-derived coat protein (CP) mutants of tobacco mosaic virus (TMV) to invade vascular cells in minor veins of Nicotiana tabacum L. cv. Xanthi nn. The mutant viruses we studied, TMV CP-O, U1mCP15-17, and SNC015, respectively, encode a CP from a different tobamovirus (i.e., from odontoglossum ringspot virus) resulting in the formation of non-native capsids, a mutant CP that accumulates in aggregates but does not encapsidate the viral RNA, or no CP. TMV CP-O is impaired in phloem-dependent movement, whereas U1mCP15-17 and SNC015 do not accumulate by phloem-dependent movement. In developmentally-defined studies using immunocytochemical analyses we determined that all of these mutants invaded vascular parenchyma cells within minor veins in inoculated leaves. In addition, we determined that the CPs of TMV CP-O and U1mCP15-17 were present in companion (C) cells of minor veins in inoculated leaves, although more rarely than CP of wild-type virus. These results indicate that the movement of TMV into minor veins does not require the CP, and an encapsidation-competent CP is not required for, but may increase the efficiency of, movement into the conducting complex of the phloem (i.e., the C cell/sieve element complex). Also, a host factor(s) functions at or beyond the C cell/sieve element interface with other cells to allow efficient phloem-dependent accumulation of TMV CP-O.