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Ningnanmycin (NNM) belongs to microbial pesticides that display comprehensive antiviral activity against plant viruses. NNM treatment has been shown to efficiently delay or suppress the disease symptoms caused by tobacco mosaic virus (TMV) infection in local-inoculated or systemic-uninoculated tobacco leaves, respectively. However, the underlying molecular mechanism of NNM-mediated antiviral activity remains to be further elucidated. In this study, 414 differentially expressed genes (DEGs), including 383 which were up-regulated and 31 down-regulated, caused by NNM treatment in TMV-infected BY-2 protoplasts, were discovered by RNA-seq. In addition, KEGG analysis indicated significant enrichment of DEGs in the plant-pathogen interaction and MAPK signaling pathway. The up-regulated expression of crucial DEGs, including defense-responsive genes, such as the receptor-like kinase FLS2 , RLK1, and the mitogen-activated protein kinase kinase kinase MAPKKK , calcium signaling genes, such as the calcium-binding protein CML19 , as well as phytohormone responsive genes, such as the WRKY transcription factors WRKY40 and WRKY70 , were confirmed by RT-qPCR. These findings provided valuable insights into the antiviral mechanisms of NNM, which indicated that the agent induces tobacco systemic resistance against TMV via activating multiple plant defense signaling pathways.

Cytosinpeptidemycin (CytPM) is a microbial pesticide that displayed broad-spectrum antiviral activity against various plant viruses. However, the molecular mechanism underlying antiviral activity of CytPM is poorly understood. In this study, the results demonstrated that CytPM could effectively delay the systemic infection of tobacco mosaic virus (TMV) in Nicotiana benthamiana and significantly inhibit the viral accumulation in tobacco BY-2 protoplasts. Results of RNA-seq indicated that 210 and 120 differential expressed genes (DEGs) were significantly up- and down-regulated after CytPM treatment in BY-2 protoplasts, respectively. In addition, KEGG analysis indicated that various DEGs were involved in endoplasmic reticulum (ER) protein processing, suggesting a possible correlation between ER homeostasis and virus resistance. RT-qPCR was performed to validate the gene expression of crucial DEGs related with defense, stress responses, signaling transduction, and phytohormone, which were consistent with results of RNA-seq. Our works provided valuable insights into the antiviral mechanism of CytPM that induced host resistance to viral infection.

A predicted stem-loop structure of 25 nucleotides, located in the coat protein (CP) gene and 3'-UTR sequences of Tobacco mosaic virus (TMV), was validated previously (Guo et al., 2015). In this study, both disrupted stem-loop and nucleotide deletion mutants of TMV replicated more rapidly in Nicotiana benthamiana protoplasts. The TMV mutant with a complete mirrored stem-loop structure showed similar level of viral RNA accumulation as TMV. Recovering the stem-loop structure also resulted in a similar replication level as TMV. All these mutants induced necrosis in N. benthamiana and assembled into typical rigid rod-shaped virions. TMV mutant without the stem-loop structure induced more local lesions in Chenopodium quinoa. When the putative stem-loop structure in Tomato mosaic virus (ToMV) was disrupted, the mutant also showed an enhanced virus replication. This suggests that the stem-loop structure of TMV is a new cis-acting element with a role in virus replication., (Copyright © 2018 Elsevier Inc. All rights reserved.)

The upstream pseudoknots domain (UPD) of Tobacco mosaic virus (TMV) is located at the 3'-untranslated region (UTR). It plays an important role in virus replication and translation. To determine the importance of UPD and 3'-UTR, and the effects of introduced RNA elements in TMV 3'-UTR, a series of TMV mutants with internal poly(A) tract upstream of UPD was constructed for structural analysis by selective 2'-hydroxyl acylation analyzed by primer extension (SHAPE). TMV(24A+UPD) and TMV(42A+UPD) formed a similar structure as that of TMV 3'-UTR, but TMV(62A+UPD) structures altered by the introduced poly(A) tract. In addition, TMV(24A+UPD) had a higher viral RNAs accumulation than TMV in N. benthamiana protoplasts, and induced lethal symptoms in the infected plants. TMV(62A+UPD) showed a drastically reduced accumulation, its coat protein was undetectable in protoplasts, and the inoculated plants remained symptomless. This study analyzed the structures of 3'-UTR of TMV and found that the longer poly(A) tract introduced upstream of UPD reduced viral RNAs accumulation and induced milder symptoms in N. benthamiana. In conclusion, different lengths of the internal poly(A) tract introduced into the TMV 3'UTR lead to structural variations that affect virus accumulation and symptom expression.

One striking feature of viruses with RNA genomes is the modification of the host membrane structure during early infection. This process requires both virus- and host-encoded proteins; however, the host factors involved and their role in this process remain largely unknown. On infection with Tobacco mosaic virus (TMV), a positive-strand RNA virus, the filamentous and tubular endoplasmic reticulum (ER) converts to aggregations at the early stage and returns to filamentous at the late infectious stage, termed the ER transition. Also, membrane- or vesicle-packaged viral replication complexes (VRCs) are induced early during infection. We used microarray assays to screen the Arabidopsis thaliana gene(s) responding to infection with TMV in the initial infection stage and identified an Arabidopsis gene, PAP85 (annotated as a vicilin-like seed storage protein), with upregulated expression during 0.5 to 6 h of TMV infection. TMV accumulation was reduced in pap85-RNA interference (RNAi) Arabidopsis and restored to wild-type levels when PAP85 was overexpressed in pap85-RNAi Arabidopsis. We did not observe the ER transition in TMV-infected PAP85-knockdown Arabidopsis protoplasts. In addition, TMV accumulation was reduced in PAP85-knockdown protoplasts. VRC accumulation was reduced, but not significantly (P = 0.06), in PAP85-knockdown protoplasts. Coexpression of PAP85 and the TMV main replicase (P126), but not their expression alone in Arabidopsis protoplasts, could induce ER aggregations.

The multiplicity of infection (MOI), i.e., the number of virus genomes that infect a cell, is a key parameter in virus evolution, as it determines processes such as genetic exchange among genomes, selection intensity on viral genes, epistatic interactions, and the evolution of multipartite viruses. In fact, the MOI level is equivalent to the virus ploidy during genome expression. Nevertheless, there are few experimental estimates of MOI, particularly for viruses with eukaryotic hosts. Here we estimate the MOI of Tobacco mosaic virus (TMV) in its systemic host, Nicotiana benthamiana. The progress of infection of two TMV genotypes, differently tagged with the green or red fluorescent proteins GFP and RFP, was monitored by determining the number of leaf cell protoplasts that showed GFP, RFP, or GFP and RFP fluorescence at different times postinoculation. This approach allowed the quantitative analysis of the kinetics of infection and estimation of the generation time and the number of infection cycles required for leaf colonization. MOI levels were estimated from the frequency of cells infected by only TMV-GFP or TMV-RFP. The MOI was high, but it changed during the infection process, decreasing from an initial level of about 6 to a final one of 1 to 2, with most infection cycles occurring at the higher MOI levels. The decreasing MOI can be explained by mechanisms limiting superinfection and/or by genotype competition within double-infected cells, which was shown to occur in coinfected tobacco protoplasts. To our knowledge, this is the first estimate of MOI during virus colonization of a eukaryotic host.

Expression of tobacco mosaic virus (TMV) coat protein (CP) restricts virus disassembly and alters the accumulation of the movement protein (MP). To characterize the role of structure of transgenic CP in regulating virus disassembly and production of MP, we generated CPs with mutations at residues Glu50 and Asp77, located in the interface between juxtaposed CP subunits. In transgenic Nicotiana tabacum and BY-2 cells, three categories of coat protein-mediated resistance (CP-MR) levels were identified: wild-type CP-MR; elevated CP-MR; and no CP-MR. Mutant CPs that interfered with the accumulation of virus replication complexes conferred very high levels of protection to TMV, except by CP(E50D) which provided no protection in the systemic host (Xanthi-nn) but high CP-MR in the local lesion host (Xanthi-NN). In transgenic BY-2 cells CP(E50D) strongly reduced accumulation of MP:GFP. In general, there was a strong correlation between the capacity for CP to assemble to pseudovirions and CP-MR, while there was not strong correlation with packaging viral RNA and CP-MR. The data demonstrate that interference with one or more steps in virus infection and replication by wild type and mutant CP determine the degree of CP-MR.

The replication of eukaryotic positive-strand RNA virus genomes occurs in the membrane-bound RNA replication complexes. Previously, we found that the extract of evacuolated tobacco BY-2 protoplasts (BYL) is capable of supporting the translation and subsequent replication of the genomic RNAs of plant positive-strand RNA viruses, including Tomato mosaic virus (ToMV). Here, to dissect the process that precedes the formation of ToMV RNA replication complexes, we prepared membrane-depleted BYL (mdBYL), in which the membranes were removed by centrifugation. In mdBYL, ToMV RNA was translated to produce the 130-kDa and 180-kDa replication proteins, but the synthesis of any ToMV-related RNAs did not occur. When BYL membranes were added back to the ToMV RNA-translated mdBYL after the termination of translation with puromycin, ToMV RNA was replicated. Using a replication-competent ToMV derivative that encodes the FLAG-tagged 180-kDa replication protein, it was shown by affinity purification that a complex that contained the 130-kDa and 180-kDa proteins and ToMV genomic RNA was formed after translation in mdBYL. When the complex was mixed with BYL membranes, ToMV RNA was replicated, which suggests that this ribonucleoprotein complex is an intermediate of ToMV RNA replication complex formation. We have named this ribonucleoprotein complex the "pre-membrane-targeting complex." Our data suggest that the formation of the pre-membrane-targeting complex is coupled with the translation of ToMV RNA, while posttranslationally added exogenous 180-kDa protein and replication templates can contribute to replication and can be replicated, respectively. Based on these results, we discuss the mechanisms of ToMV RNA replication complex formation.

The cell-to-cell spread of Tobacco mosaic virus infection depends on virus-encoded movement protein (MP), which is believed to form a ribonucleoprotein complex with viral RNA (vRNA) and to participate in the intercellular spread of infectious particles through plasmodesmata. Previous studies in our laboratory have provided evidence that the vRNA movement process is correlated with the ability of the MP to interact with microtubules, although the exact role of this interaction during infection is not known. Here, we have used a variety of in vivo and in vitro assays to determine that the MP functions as a genuine microtubule-associated protein that binds microtubules directly and modulates microtubule stability. We demonstrate that, unlike MP in whole-cell extract, microtubule-associated MP is not ubiquitinated, which strongly argues against the hypothesis that microtubules target the MP for degradation. In addition, we found that MP interferes with kinesin motor activity in vitro, suggesting that microtubule-associated MP may interfere with kinesin-driven transport processes during infection.

A series of deletion mutants of tobacco mosaic virus movement protein (TMV-MP) was used to identify domains of the protein necessary for membrane association. A membrane fraction was isolated from tobacco BY-2 protoplasts infected with wild-type and mutant TMV that produce MP carrying a 3 aa deletion. Deletions that affected membrane association were clustered around the two major hydrophobic regions of MP that are predicted to be transmembrane. Deletions in other hydrophobic regions also reduced membrane association. In addition, a non-functional mutant of MP, in which one of the known phosphorylation sites was eliminated, was not associated with cellular membranes, while a functional second site revertant restored membrane association. This indicates that MP function requires interaction with membrane; however, membrane association was not sufficient for function. Results are consistent with the hypothesis that TMV-MP is an integral or tightly associated membrane protein that includes two hydrophobic transmembrane domains.

Plant viruses, in particular Tobacco mosaic virus (TMV), are model systems to study RNA and protein trafficking in plants. Although TMV cell-to-cell transport controlled by the 30-kDa movement protein (MP) has been intensively studied, it was only recently demonstrated that the 126/183-kDa replicase proteins are also involved in cell-to-cell movement. Elucidating the role(s) of 126/183-kDa proteins in movement is complicated because these proteins have multiple functions associated with replication and gene expression. To overcome these difficulties we developed a TMV helper virus-defective RNA (dRNA) system to study the role of replicase protein sequences in dRNA cell-to-cell movement. Artificially constructed dRNAs lacking sequences encoding the helicase and polymerase domains of the replicase proteins and portions of the MP were viable in protoplasts and plants in the presence of helper virus. Expression of at least approximately 50% of the methyl transferase (MT) domain was required for efficient dRNA movement in Nicotiana benthamiana. dRNAs that encoded the N-terminal 64 replicase amino acids or lacked a translatable MT domain failed to move or moved poorly. TMV dRNAs expressing 258 amino acids of the replicase protein moved into all specialized non-vascular tissues, whereas dRNAs expressing replicase sequences beyond amino acid 258 were restricted to the epidermis and palisade mesophyll tissues. Furthermore, second-site mutations within the dRNA-encoded truncated replicase protein altered efficiency in dRNA cell-to-cell movement.

The effect of a specific cis-hairpin ribozyme on TMV-based vectors in the infection of tobacco protoplasts was studied. Vectors contained full-length TMV genome cDNA linked to a T7 promoter or a CaMV 35S promoter at the 5'-end and an NOS gene polyadenylation signal at the 3'-end. The coat protein (CP) gene was replaced with the green fluorescent protein (GFPuv) gene allowing quantification of protoplast infection. In plasmids pTMVGFPRIB (T7-driven) and pSTMVGFPRIB (CaMV 35S-driven), the cDNA fragment of the cis-hairpin ribozyme (designed to specifically cleave the transcripts immediately downstream of the 3'-terminus of TMV RNA) was inserted between the 3'-terminus of TMV genome and NOS sequence. The in vitro transcript TMVGFPRIB was three- to fivefold more infectious than the control TMVGFPNOS. Northern blot analysis indicated that the 3'-terminal non-viral sequence had been cleaved from the in vitro transcripts by the cis-hairpin ribozyme soon after in vitro transcription. pSTMVGFPRIB and pSTMVGFPNOS plasmid DNAs were, as expected, less infectious than their in vitro transcript counterparts. However, pSTMVGFPRIB was somewhat more infectious than pSTMVGFPNOS. Northern blot analysis indicated that pSTMVGFPRIB synthesized more genomic and sub-genomic RNAs in the protoplasts. The significant increase in infectivity and viral RNA synthesis is due to the specific activity of the cis-hairpin ribozyme in vivo. Therefore, the cis-hairpin ribozyme described here may improve TMV-based vectors in the expression of foreign protein in plants.

The decapitation depressed the development of the system acquired resistance to reinfection with tobacco mosaic virus in the leaves of hypersensitive tobacco plants Nicotiana tabacum L. (v. Xanthi nc) and in protoplasts, isolated from these leaves.

Microtubules interact strongly with the viral movement protein (MP) of Tobacco mosaic virus (TMV) and are thought to transport the viral genome between plant cells. We describe a functionally enhanced DNA-shuffled movement protein (MP(R3)) that remained bound to the vertices of the cortical endoplasmic reticulum, showing limited affinity for microtubules. A single amino acid change was shown to confer the MP(R3) phenotype. Disruption of the microtubule cytoskeleton in situ with pharmacological agents, or by silencing of the alpha-tubulin gene, had no significant effect on the spread of TMV vectors expressing wild-type MP (MP(WT)) and did not prevent the accumulation of MP(WT) in plasmodesmata. Thus, cell-to-cell trafficking of TMV can occur independently of microtubules. The MP(R3) phenotype was reproduced when infection sites expressing MP(WT) were treated with a specific proteasome inhibitor, indicating that the degradation of MP(R3) is impaired. We suggest that the improved viral transport functions of MP(R3) arise from evasion of a host degradation pathway.

The movement protein (MP) of Tobacco mosaic virus (TMV) facilitates the cell-to-cell transport of the viral RNA genome through plasmodesmata (Pd). A previous report described the functional reversion of a dysfunctional mutation in MP (Pro81Ser) by two additional amino acid substitution mutations (Thr104Ile and Arg167Lys). To further explore the mechanism underlying this intramolecular complementation event, the mutations were introduced into a virus derivative expressing the MP as a fusion to green fluorescent protein (GFP). Microscopic analysis of infected protoplasts and of infection sites in leaves of MP-transgenic Nicotiana benthamiana indicates that MP(P81S)-GFP and MP(P81S;T104I;R167K)-GFP differ in subcellular distribution. MP(P81S)-GFP lacks specific sites of accumulation in protoplasts and, in epidermal cells, exclusively localizes to Pd. MP(P81S;T104I;R167K)-GFP, in contrast, in addition localizes to inclusion bodies and microtubules and thus exhibits a subcellular localization pattern that is similar, if not identical, to the pattern reported for wild-type MP-GFP. Since accumulation of MP to inclusion bodies is not required for function, these observations confirm a role for microtubules in TMV RNA cell-to-cell transport.

Tobacco mosaic virus (TMV) derivatives that encode movement protein (MP) as a fusion to the green fluorescent protein (MP:GFP) were used in combination with antibody staining to identify host cell components to which MP and replicase accumulate in cells of infected Nicotiana benthamiana leaves and in infected BY-2 protoplasts. MP:GFP and replicase colocalized to the endoplasmic reticulum (ER; especially the cortical ER) and were present in large, irregularly shaped, ER-derived structures that may represent "viral factories." The ER-derived structures required an intact cytoskeleton, and microtubules appeared to redistribute MP:GFP from these sites during late stages of infection. In leaves, MP:GFP accumulated in plasmodesmata, whereas in protoplasts, the MP:GFP was targeted to distinct, punctate sites near the plasma membrane. Treating protoplasts with cytochalasin D and brefeldin A at the time of inoculation prevented the accumulation of MP:GFP at these sites. It is proposed that the punctate sites anchor the cortical ER to plasma membrane and are related to sites at which plasmodesmata form in walled cells. Hairlike structures containing MP:GFP appeared on the surface of some of the infected protoplasts and are reminiscent of similar structures induced by other plant viruses. We present a model that postulates the role of the ER and cytoskeleton in targeting the MP and viral ribonucleoprotein from sites of virus synthesis to the plasmodesmata through which infection is spread.

Tobacco mosaic virus strain Cg (TMV-Cg) infects A. thaliana systemically. In order to identify host factors involved in the multiplication of TMV-Cg, we isolated mutant of A. thaliana from an M2 population mutagenized by fast neutron irradiation, in which the accumulation of the coat protein in upper systemic leaves was reduced to low levels. The phenotype of the mutant, YS241, was controlled primarily by a single nuclear recessive mutation named tom2-1, which was distinct from tom1, a separate mutation which also affects TMV-Cg multiplication. The tom2-1 mutation affected the accumulation of TMV-related RNAs in protoplasts in a tobamovirus-specific manner, suggesting that the wild-type TOM2 gene product is necessary for efficient amplification of TMV-related RNAs within a single cell, through specific interaction with virus-coded factors. Furthermore, we found that YS241 contained a single dominant modifier named ttm1, which increased the efficiency of multiplication of TMV-Cg and a tomato strain of TMV in a tom2-1 genetic background, both in plants and in protoplasts. We propose that the ttm1 element might be a translocated form of the TOM2 gene.

Pokeweed antiviral protein (PAP) belongs to a group of ribosome-inactivating proteins (RIPs) that inactivate ribosomes by depurinating rRNA at a specific site. To study the mechanism for the antiviral activity of PAP, the actions of PAP on TMV-infected and uninfected tobacco protoplasts were investigated. The addition of 0.33 microM PAP to TMV-inoculated protoplasts caused a complete inhibition of TMV production. The same concentration of PAP was found to inhibit protein synthesis in the virus-infected protoplasts and to kill the cells, but it had no effect on the uninfected protoplasts. The concentration dependence of protein synthesis-inhibition by PAP was related to that of inhibition of viral multiplication. Furthermore, two other RIPs (ricin A-chain and luffin-a), which showed 240 and 430-fold less activity on tobacco ribosomes than PAP in a cell-free system, did not inhibit viral multiplication even at a concentration of 3.3 microM. The analysis of RNAs from the virus-infected and PAP-treated protoplasts demonstrated that 25S rRNA was depurinated by PAP in the infected cells. These results suggest that PAP, which is normally unable to penetrate the plasma membrane of uninfected protoplasts, gains entrance to the cytosol of infected cells and prevents viral multiplication by inactivating ribosomes.

The movement protein of tobacco mosaic tobamovirus and related viruses is essential for the cell-to-cell spread of infection and, in part, determines the host range of the virus. Movement protein (MP) was fused with the jellyfish green fluorescent protein (GFP), and a modified virus that contained this MP:GFP fusion protein retained infectivity. In protoplasts and leaf tissues, the MP:GFP fusion protein was detected as long filaments shortly after infection. Double-labeling fluorescence microscopy suggests that the MP interacts and coaligns with microtubules. The distribution of the MP is disrupted by treatments that disrupt microtubules, but not by cytochalasin B, which disrupts filamentous F-actin. Microtubules may target the MP to plasmodesmata, the intercellular channels that connect adjacent cells.

p30, the protein required for cell-to-cell movement of tobacco mosaic virus (TMV), has a slightly reduced mobility on SDS-polyacrylamide gels when isolated by immunoprecipitation from TMV-infected protoplasts compared with that of p30 translated from viral RNA in vitro. Further investigation established a probable cause for the difference in mobility between the two: protoplasts incorporate [32P]orthophosphate into p30 at multiple sites, predominantly as phosphoserine. Tryptic peptide mapping reveals at least five internal phosphopeptides in p30, besides the C-terminal tryptic phosphopeptide already reported, involving at least two distinct domains of the protein (at residues 61-114 and residues 212-231), which may be substrates for different protein kinases. These structural results are consistent with a three-domain model for the TMV movement protein with two regulatory domains similar to that recently proposed on genetic grounds for dianthovirus movement proteins.