Efficient infection of Nicotiana benthamiana by Tomato bushy stunt virus is facilitated by the coat protein and maintained by p19 through suppression of gene silencing

Tomato bushy stunt virus (TBSV) is one of few RNA plant viruses capable of moving systemically in some hosts in the absence of coat protein (CP). TBSV also encodes another protein (p19) that is not required for systemic movement but functions as a symptom determinant in Nicotiana benthamiana. Here, the role of both CP and p19 in the systemic spread has been reevaluated by utilizing transgenic N. benthamiana plants expressing the movement protein (MP) of Red clover necrotic mosaic virus and chimeric TBSV mutants that express CP of Turnip crinkle virus. Through careful examination of the infection phenotype of a series of mutants with changes in the CP and p19 genes, we demonstrate that both of these genes are required for efficient systemic invasion of TBSV in N. benthamiana. The CP likely enables efficient viral unloading from the vascular system in the form of assembled virions, whereas p19 enhances systemic infection by suppressing the virus-induced gene silencing.     

Identification of an ARGONAUTE for antiviral RNA silencing in Nicotiana benthamiana

ARGONAUTE proteins (AGOs) are known to be key components of the RNA silencing mechanism in eukaryotes that, among other functions, serves to protect against viral invaders. Higher plants encode at least 10 individual AGOs yet the role played by many in RNA silencing-related antiviral defense is largely unknown, except for reports that AGO1, AGO2, and AGO7 play an antiviral role in Arabidopsis (Arabidopsis thaliana). In the plant virus model host Nicotiana benthamiana, Tomato bushy stunt virus (TBSV) P19 suppressor mutants are very susceptible to RNA silencing. Here, we report that a N. benthamiana AGO (NbAGO) with similarity to Arabidopsis AGO2, is involved in antiviral defense against TBSV. The activity of this NbAGO2 is shown to be directly associated with anti-TBSV RNA silencing, while its inactivation does not influence silencing of transiently expressed transgenes. Thus, the role of NbAGO2 might be primarily for antiviral defense.     

The nuclear shuttle protein of Tomato leaf curl New Delhi virus is a pathogenicity determinant

The role of the movement protein (MP) and nuclear shuttle protein (NSP) in the pathogenicity of Tomato leaf curl New Delhi virus (ToLCNDV), a bipartite begomovirus, was studied. Both genes were expressed in Nicotiana benthamiana, Nicotiana tabacum, and Lycopersicon esculentum plants with the Potato virus X (PVX) expression vector or by stable transformation of gene constructs under the control of the 35S promoter in N. tabacum. No phenotypic changes were observed in any of the three species when the MP was expressed from the PVX vector or constitutively expressed in transgenic plants. Expression of the ToLCNDV NSP from the PVX vector in N. benthamiana resulted in leaf curling that is typical of the disease symptoms caused by ToLCNDV in this species. Expression of NSP from PVX in N. tabacum and L. esculentum resulted in a hypersensitive response (HR), demonstrating that the ToLCVDV NSP is a target of host defense responses in these hosts. The NSP, when expressed as a transgene under the control of the 35S promoter, resulted in necrotic lesions in expanded leaves that initiated from a point and then spread across the leaf. The necrotic response was systemic in all the transgenic plants. Deletion of 100 amino acids from the C terminus did not compromise the HR response, suggesting that this region has no role in HR. Deletion of 60 or 100 amino acids from the N terminus of NSP abolished the HR response, suggesting that these sequences are required for the HR response. These findings demonstrate that the ToLCNDV NSP is a pathogenicity determinant as well as a target of host defense responses.     

Complete sequencing of potato virus X new strain genome and construction of viral vector for production of target proteins in plants

The complete nucleotide sequence of the genome of a new potato virus X (PVX) strain Tula isolated by us has been determined. Based on comparison of the PVX Tula nucleotide sequence with the sequences of 12 other PVX strains, this strain was assigned to the European cluster of PVX strains. Phylogenetic analysis revealed the same phylogeny for both full genome sequences and nucleotide sequences of polymerase and coat protein genes, suggesting that the PVX evolution did not involve recombination between different strains. The full-size cDNA copy of the PVX Tula genome was cloned and the accumulation of the viral coat protein in infected Nicotiana benthamiana was shown to be about twofold higher than for the PVX strain UK3. Based on the PVX Tula genome, a new vector which contained the target gene instead of the removed triple transport gene block and the coat protein gene has been constructed for expression of target proteins in plants. The productivity of the new vector was about 1.5-2-fold higher than the productivity of the vector of the same structure based on the standard PVX strain genome. The new viral vector can be used for superproduction of recombinant proteins in plants.     

Phloem unloading of potato virus X movement proteins is regulated by virus and host factors

To determine the requirements for viral proteins exiting the phloem, transgenic plants expressing green fluorescent protein (GFP) fused to the Potato virus X (PVX) triple gene block (TGB)p1 and coat protein (CP) genes were prepared. The fused genes were transgenically expressed from the companion cell (CC)-specific Commelina yellow mottle virus (CoYMV) promoter. Transgenic plants were selected for evidence of GFP fluorescence in CC and sieve elements (SE) and proteins were determined to be phloem mobile based on their ability to translocate across a graft union into nontransgenic scions. Petioles and leaves were analyzed to determine the requirements for phloem unloading of the fluorescence proteins. In petioles, fluorescence spread throughout the photosynthetic vascular cells (chlorenchyma) but did not move into the cortex, indicating a specific barrier to proteins exiting the vasculature. In leaves, fluorescence was mainly restricted to the veins. However, in virus-infected plants or leaves treated with a cocktail of proteasome inhibitors, fluorescence spread into leaf mesophyll cells. These data indicate that PVX contributes factors which enable specific unloading of cognate viral proteins and that proteolysis may play a role in limiting proteins in the phloem and surrounding chlorenchyma.     

Sonchus Yellow Net Rhabdovirus Nuclear Viroplasms Contain Polymerase-Associated Proteins

We have initiated a study of the cytopathology of nucleorhabdoviruses by analyzing the subcellular localization of sonchus yellow net virus (SYNV) genomic and antigenomic RNAs and the encoded polymerase proteins. In situ hybridizations demonstrated that the minus-strand genomic RNA sequences are restricted to the nuclei of infected cells, while the complementary plus-strand antigenomic RNA sequences are present in both the nuclei and the cytoplasm. Immunofluorescence and immunogold labeling experiments also revealed that the nucleocapsid (N) protein and phosphoprotein (M2) are primarily localized to discrete regions within the nuclei and in virus particles that accumulate in perinuclear spaces. The N protein antiserum specifically labeled the nuclear viroplasms, whereas the M2 antiserum was more generally distributed throughout the nuclei. Antibody detection also indicated that the polymerase (L) protein is present in small amounts in the viroplasm. When the N and M2 proteins were expressed individually from the heterologous potato virus X (PVX) vector, both proteins preferentially accumulated in the nuclei. In addition, viroplasm-like inclusions formed in the nuclei of cells infected with the PVX vector containing the N gene. Fusions of the carboxy terminus of β-glucuronidase to N and M2 resulted in staining of the nuclei of infected cells following expression from the PVX vector. Deletion analyses suggested that multiple regions of the N protein contain signals that are important for nuclear localization.     

Cell-to-cell movement of Potato virus X: the role of p12 and p8 encoded by the second and third open reading frames of the triple gene block

Potato virus X (PVX) requires three proteins, p25, p12, and p8, encoded by the triple gene block plus the coat protein (CP) for cell-to-cell movement. When each of these proteins was co-expressed with a cytosolic green fluorescent protein (GFP) in the epidermal cells of Nicotiana benthamiana by the microprojectile bombardment-mediated gene delivery method, only p12 enhanced diffusion of co-expressed GFP, indicating an ability to alter plasmodesmal permeability. p25, p12, and CP, expressed transiently in the initially infected cells, transcomplemented the corresponding movement-defective mutants to spread through two or more cell boundaries. Thus, these proteins probably move from cell to cell with the genomic RNA. In contrast, p8 only functioned intracellularly and was not absolutely required for cell-to-cell movement. Since overexpression of p12 overcame the p8 deficiency, p8 appears to facilitate the functioning of p12, presumably by mediating its intracellular trafficking. Considering the likelihood that p12 and p8 are membrane proteins, it is suggested that intercellular as well as intracellular movement of PVX involves a membrane-mediated process.     

Transgenic Plants Expressing Potato Virus X ORF2 Protein (p24) Are Resistant to Tobacco Mosaic Virus and Ob Tobamoviruses

The p24 protein, one of the three proteins implicated in local movement of potato virus X (PVX), was expressed in transgenic tobacco plants (Nicotiana tabacum Xanthi D8 NN). Plants with the highest level of p24 accumulation exhibited a stunted and slightly chlorotic phenotype. These transgenic plants facilitate the cell-to-cell movement of a mutant of PVX that contained a frameshift mutation in p24. Upon inoculation with tobacco mosaic virus (TMV), the size of necrotic local lesions was significantly smaller in p24+ plants than in nontransgenic, control plants. Systemic resistance to tobamoviruses was also evidenced after inoculation of p24+ plants with Ob, a virus that evades the hypersensitive response provided by the N gene. In the latter case, no systemic symptoms were observed, and virus accumulation remained low or undetectable by Western immunoblot analysis and back-inoculation assays. In contrast, no differences were observed in virus accumulation after inoculation with PVX, although more severe symptoms were evident on p24-expressing plants than on control plants. Similarly, infection assays conducted with potato virus Y showed no differences between control and transgenic plants. On the other hand, a considerable delay in virus accumulation and symptom development was observed when transgenic tobacco plants containing the movement protein (MP) of TMV were inoculated with PVX. Finally, a movement defective mutant of TMV was inoculated on p24+ plants or in mixed infections with PVX on nontransgenic plants. Both types of assays failed to produce TMV infections, implying that TMV MP is not interchangeable with the PVX MPs.     

Proteome-wide overexpression of host proteins for identification of factors affecting tombusvirus RNA replication: an inhibitory role of protein kinase C

To identify host genes affecting replication of Tomato bushy stunt virus (TBSV), a small model positive-stranded RNA virus, we overexpressed 5,500 yeast proteins individually in Saccharomyces cerevisiae, which supports TBSV replication. In total, we identified 141 host proteins, and overexpression of 40 of those increased and the remainder decreased the accumulation of a TBSV replicon RNA. Interestingly, 36 yeast proteins were identified previously by various screens, greatly strengthening the relevance of these host proteins in TBSV replication. To validate the results from the screen, we studied the effect of protein kinase C1 (Pkc1), a conserved host kinase involved in many cellular processes, which inhibited TBSV replication when overexpressed. Using a temperature-sensitive mutant of Pkc1p revealed a high level of TBSV replication at a semipermissive temperature, further supporting the idea that Pkc1p is an inhibitor of TBSV RNA replication. A direct inhibitory effect of Pkc1p was shown in a cell-free yeast extract-based TBSV replication assay, in which Pkc1p likely phosphorylates viral replication proteins, decreasing their abilities to bind to the viral RNA. We also show that cercosporamide, a specific inhibitor of Pkc-like kinases, leads to increased TBSV replication in yeast, in plant single cells, and in whole plants, suggesting that Pkc-related pathways are potent inhibitors of TBSV in several hosts.     

利用病毒载体在烟草中瞬时表达融合HBsAg基因

利用马铃薯PVX病毒载体构建了外源人工融合乙肝表面抗原HBsAg基因的表达载体,在烟草中利用农杆菌介导进行瞬时表达,以快速鉴定外源基因瞬时表达的状况以及重组蛋白的免疫活性。利用PCR技术从含有人工融合HBsAg基因的表达载体中分别扩增出LP PreS1 PreS2 S、PreS1 PreS2 S、PreS2 S序列,将其分别与PVX病毒载体pgR106连接,构建成PVX-LP、PVX-S1和PVX-S2等3个转化载体,并将此载体导入农杆菌菌株GV3101中用于侵染烟草植株叶片。感染植株经RT-PCR、RNA Dot blotting和HBsAg蛋白的ELISA检测显示,3个人工融合的HBsAg基因均可在植物体内得到转录,翻译成具有活性的蛋白。结果表明,外源融合HB-sAg基因经过植物病毒载体瞬时表达系统可以在植物系统中正常转录和翻译。     

Functional complementation of nuclear targeting-defective mutants of simian virus 40 structural proteins.

Structural proteins of simian virus 40 (SV40), Vp2 and Vp3 (Vp2/3) and Vp1, carry individual nuclear targeting signals, Vp3(198-206) (Vp2(316-324) and Vp1(1-8), respectively, which are encoded in different reading frames of an overlapping region of the genome. How signals coordinate nuclear targeting during virion morphogenesis was examined by using SV40 variants in which there is only one structural gene for Vp1 or Vp2/3, nuclear targeting-defective mutants thereof, Vp2/3(202T) and Vp1 delta N5, or nonoverlapping SV40 variants in which the genes for Vp1 and Vp2/3 are separated, and mutant derivatives of the gene carrying either one or both mutations. Nuclear targeting was assessed immunocytochemically following nuclear microinjection of the variant DNAs. When Vp2/3 and Vp1 mutants with defects in the nuclear targeting signals were expressed individually, the mutant proteins localized mostly to the cytoplasm. However, when mutant Vp2/3(202T) was coexpressed in the same cell along with wild-type Vp1, the mutant protein was effectively targeted to the nucleus. Likewise, the Vp1 delta N5 mutant protein was transported into the nucleus when wild-type Vp2/3 was expressed in the same cells. These results suggest that while Vp1 and Vp2/3 have independent nuclear targeting signals, additional signals, such as those defining protein-protein interactions, play a concerted role in nuclear localization along with the nuclear targeting signals of the individual proteins.     

Cell-to-cell and phloem-mediated transport of potato virus X. The role of virions

Movement-deficient potato virus X (PVX) mutants tagged with the green fluorescent protein were used to investigate the role of the coat protein (CP) and triple gene block (TGB) proteins in virus movement. Mutants lacking either a functional CP or TGB were restricted to single epidermal cells. Microinjection of dextran probes into cells infected with the mutants showed that an increase in the plasmodesmal size exclusion limit was dependent on one or more of the TGB proteins and was independent of CP. Fluorescently labeled CP that was injected into epidermal cells was confined to the injected cells, showing that the CP lacks an intrinsic transport function. In additional experiments, transgenic plants expressing the PVX CP were used as rootstocks and grafted with nontransformed scions. Inoculation of the PVX CP mutants to the transgenic rootstocks resulted in cell-to-cell and systemic movement within the transgenic tissue. Translocation of the CP mutants into sink leaves of the nontransgenic scions was also observed, but infection was restricted to cells close to major veins. These results indicate that the PVX CP is transported through the phloem, unloads into the vascular tissue, and subsequently is transported between cells during the course of infection. Evidence is presented that PVX uses a novel strategy for cell-to-cell movement involving the transport of filamentous virions through plasmodesmata.     

Cell-to-cell movement of potato virus X revealed by micro-injection of a viral vector tagged with the β-glucuronidase gene

The replication and cell-to-cell movement of potato virus X (PVX) has been studied using a PVX vector construct which expressed the β-glucuronidase (GUS) reporter gene in infected cells. Nicotiana clevelandii leaf trichome cells were micro-injected with the PVX-GUS vector and histochemical staining was used to locate GUS activity. The distribution of GUS activity revealed that PVX had moved from the injected cell into other trichome cells and into the cells along the leaf margin. GUS activity was always restricted to the cells at the edge of the leaf suggesting that PVX was unable to move out of the marginal cells. At the infection front, scattered along the leaf margin, there were isolated groups of cells staining for GUS activity. The absence of GUS activity in the intervening cells suggests that PVX had moved through several cells without replicating within them. This latter observation is consistent with previously reported observations that viral movement proteins are capable of moving between cells.     

Cell-to-cell movement of the 25K protein of potato virus X is regulated by three other viral proteins

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.