Differential responses to virus challenge of laboratory and wild accessions of australian species of nicotiana, and comparative analysis of RDR1 gene sequences.
ABSTRACT: Nicotiana benthamiana is a model plant utilised internationally in plant virology because of its apparent hyper-susceptibility to virus infection. Previously, others showed that all laboratory accessions of N. benthamiana have a very narrow genetic basis, probably originating from a single source. It is unknown if responses to virus infection exhibited by the laboratory accession are typical of the species as a whole. To test this, 23 accessions of N. benthamiana were collected from wild populations and challenged with one to four viruses. Additionally, accessions of 21 other Nicotiana species and subspecies from Australia, one from Peru and one from Namibia were tested for susceptibility to the viruses, and for the presence of a mutated RNA-dependent RNA polymerase I allele (Nb-RDR1m) described previously from a laboratory accession of N. benthamiana. All Australian Nicotiana accessions tested were susceptible to virus infections, although there was symptom variability within and between species. The most striking difference was that plants of a laboratory accession of N. benthamiana (RA-4) exhibited hypersensitivity to Yellow tailflower mild mottle tobamovirus infection and died, whereas plants of wild N. benthamiana accessions responded with non-necrotic symptoms. Plants of certain N. occidentalis accessions also exhibited initial hypersensitivity to Yellow tailflower mild mottle virus resembling that of N. benthamiana RA-4 plants, but later recovered. The mutant Nb-RDR1m allele was identified from N. benthamiana RA-4 but not from any of 51 other Nicotiana accessions, including wild accessions of N. benthamiana, demonstrating that the accession of N. benthamiana used widely in laboratories is unusual.
Project description:Salicylic acid (SA), an essential secondary messenger for plant defence responses, plays a role in maintaining a balance (trade-off) between plant growth and resistance induction, but the detailed mechanism has not been explored. Because the SA mimic benzothiadiazole (BTH) is a more stable inducer of plant defence than SA after exogenous application, we analysed expression profiles of defence genes after BTH treatment to better understand SA-mediated immune induction. Transcript levels of the salicylic acid glucosyltransferase (SAGT) gene were significantly lower in BTH-treated Nicotiana tabacum (Nt) plants than in SA-treated Nt control plants, suggesting that SAGT may play an important role in SA-related host defence responses. Treatment with BTH followed by SA suppressed SAGT transcription, indicating that the inhibitory effect of BTH is not reversible. In addition, in BTH-treated Nt and Nicotiana benthamiana (Nb) plants, an early high accumulation of SA and SA 2-O-?-d-glucoside was only transient compared to the control. This observation agreed well with the finding that SAGT-overexpressing (OE) Nb lines contained less SA and jasmonic acid (JA) than in the Nb plants. When inoculated with a virus, the OE Nb plants showed more severe symptoms and accumulated higher levels of virus, while resistance increased in SAGT-silenced (IR) Nb plants. In addition, the IR plants restricted bacterial spread to the inoculated leaves. After the BTH treatment, OE Nb plants were slightly larger than the Nb plants. These results together indicate that SAGT has a pivotal role in the balance between plant growth and SA/JA-mediated defence for optimum plant fitness.
Project description:Citrus leaf blotch virus (CLBV), a member of the family Flexiviridae, has a ~9-kb single-stranded, positive-sense genomic RNA encapsidated by a 41-kDa coat protein. CLBV isolates are associated with symptom production in citrus including leaf blotching of Dweet tangor and stem pitting in Etrog citron (Dweet mottle disease), and some isolates are associated with bud union crease on trifoliate rootstocks, but Koch's postulates for this virus were not fulfilled. A full-genome cDNA of CLBV isolate SRA-153, which induces bud union crease, was placed under the T7 promoter (clone T7-CLBV), or between the 35S promoter and the Nos-t terminator, with or without a ribozyme sequence downstream of the CLBV sequence (clones 35SRbz-CLBV and 35S-CLBV). RNA transcripts from T7-CLBV failed to infect Etrog citron and Nicotiana occidentalis and N. benthamiana plants, whereas agro-inoculation with binary vectors carrying 35SRbz-CLBV or 35S-CLBV, and the p19 silencing suppressor, caused systemic infection and production of normal CLBV virions. Virus accumulation was similar in citron plants directly agro-infiltrated, or mechanically inoculated with wild-type or 35SRbz-CLBV-derived virions from Nicotiana, and the three sources incited the symptoms characteristic of Dweet mottle disease, but not bud union crease. Our results show that (1) virions derived from an infectious clone show the same replication, movement and pathogenicity characteristics as the wild-type CLBV; (2) CLBV is the causal agent of Dweet mottle disease but not of the bud union crease syndrome; and (3) for the first time an RNA virus could be successfully agro-inoculated on citrus plants. This infectious clone may become a useful viral vector for citrus genomic studies.
Project description:Actin filament, also known as microfilament, is one of two major cytoskeletal elements in plants and plays important roles in various biological processes. Like in animal cells, actin filaments have been thought to participate in autophagy in plants. However, surprisingly, in this study we found that actin filaments are dispensable for the occurrence of autophagy in plants. Disruption of actin filaments by short term treatment with actin polymerization inhibitors, cytochalasin D and latrunculin B, or transient overexpression of Profilin 3 in Nicotiana benthamiana had no effect on basal autophagy as well as the upregulation of nocturnal autophagy and salt stress-induced autophagy. Furthermore, anti-microfilament drug treatment affected neither basal nor salt stress-induced autophagy in Arabidopsis. In addition, prolonged perturbation of actin filaments by silencing Actin7 or 24-h treatment with microfilament-disrupting agents in N. benthamiana caused endoplasmic reticulum (ER) disorganization and subsequent degradation via autophagy involving ATG2, 3, 5, 6 and 7. Our findings reveal that, unlike mammalian cells, actin filaments are unnecessary for bulk autophagy in plants.Abbreviations: ATG: autophagy-related; CD: cytochalasin D; Cvt pathway: cytoplasm to vacuole targeting pathway; DMSO: dimethyl sulfoxide; ER: endoplasmic reticulum; LatB: latrunculin B; Nb: Nicotiana benthamiana; PAS: phagophore assembly site; PRF3: Profilin 3; RER: rough ER; SER: smooth ER; TEM: transmission electron microscopy; TRV: Tobacco rattle virus; VIGS: virus-induced gene silencing; wpi: weeks post-agroinfiltration.
Project description:Rice stripe virus (RSV) is naturally transmitted by the small brown planthopper and infects plants of the family Poaceae. Under laboratory conditions, RSV can infect Nicotiana benthamiana by mechanical inoculation, providing a useful system to study RSV-plant interactions. Measurements of CO2 assimilation ability and PSII photochemical efficiency showed that these were both reduced in N. benthamiana plants infected by RSV. These plants also had decreased expression of the N. benthamiana Phosphoribulokinases (NbPRKs), the key gene in the Calvin cycle. When the NbPRKs were silenced using the TRV-Virus Induced Gene Silencing system, the plants had decreased CO2 assimilation ability, indicating that the downregulated expression of NbPRKs contributes to the reduced CO2 assimilation ability of RSV-infected plants. Additionally, NbPRKs-silenced plants were more resistant to RSV. Similarly, resistance was enhanced by silencing of either N. benthamiana Rubisco small subunit (NbRbCS) or Phosphoglycerate kinase (NbPGK), two other key genes in the Calvin cycle. Conversely, transgenic plants overexpressing NbPRK1 were more susceptible to RSV infection. The results suggest that a normally functional Calvin cycle may be necessary for RSV infection of N. benthamiana.
Project description:<i>Tobamovirus</i> is a group of viruses that have become serious pathogens of crop plants. As part of a study informing risk of wild plant virus spill over to crops, we investigated the capacity of a solanaceous-infecting tobamovirus from an isolated indigenous flora to adapt to new exotic hosts. Yellow tailflower mild mottle virus (YTMMV) (genus <i>Tobamovirus</i>, family <i>Virgaviridae</i>) was isolated from a wild plant of yellow tailflower (<i>Anthocercis littoria</i>, family Solanaceae) and initially passaged through a plant of <i>Nicotiana benthamiana</i>, then one of <i>Nicotiana glutinosa</i> where a single local lesion was used to inoculate a <i>N. benthamiana</i> plant. Sap from this plant was used as starting material for nine serial passages through three plant species. The virus titre was recorded periodically, and 85% of the virus genome was sequenced at each passage for each host. Six polymorphic sites were found in the YTMMV genome across all hosts and passages. At five of these, the alternate alleles became fixed in the viral genome until the end of the experiment. Of these five alleles, one was a non-synonymous mutation (U1499C) that occurred only when the virus replicated in tomato. The mutant isolate harbouring U1499C, designated YTMMV-?, increased its titre over passages in tomato and outcompeted the wild-type isolate when both were co-inoculated to tomato. That YTMMV-? had greater reproductive fitness in an exotic host than did the wild type isolate suggests YTMMV evolution is influenced by host changes.
Project description:The potential transmission of plant pathogenic viruses through processed foods could be a source of concern for global crop production; however, there is a lack of supporting evidence. The present study was conducted to investigate the presence of plant pathogenic viruses in five samples of gochujang (fermented red pepper paste) manufactured in Korea. Several viruses infecting pepper were detected by reverse transcriptionpolymerase chain reaction, among which the pepper mild mottle virus (PMMoV) was detected in all five samples, at concentrations ranging from 2.8 to 7.0 (log10 copies/ml). In addition, PMMoV was observed by transmission electron microscopy in all five samples. The samples exhibited viral pathogenicity to Nicotiana benthamiana plants, indicating that global trade of processed products could be a possible source of the transmission of plant viruses.
Project description:In plants, endogenes are less prone to RNA silencing than transgenes. While both can be efficiently targeted by small RNAs for post-transcriptional gene silencing (PTGS), generally only transgene PTGS is accompanied by transitivity, RNA-directed DNA methylation (RdDM) and systemic silencing. In order to investigate whether a transgene could mimick an endogene and thus be less susceptible to RNA silencing, we generated an intron-containing, endogene-resembling GREEN FLUORESCENT PROTEIN (GFP) transgene (GFP(endo)). Upon agroinfiltration of a hairpin GFP (hpF) construct, transgenic Nicotiana benthamiana plants harboring GFP(endo) (Nb-GFP(endo)) were susceptible to local PTGS. Yet, in the local area, PTGS was not accompanied by RdDM of the GFP(endo) coding region. Importantly, hpF-agroinfiltrated Nb-GFP(endo) plants were resistant to systemic silencing. For reasons of comparison, transgenic N. benthamiana plants (Nb-GFP(cDNA)) carrying a GFP cDNA transgene (GFP(cDNA)) were included in the analysis. HpF-agroinfiltrated Nb-GFP(cDNA) plants exhibited local PTGS and RdDM. In addition, systemic silencing was established in Nb-GFP(cDNA) plants. In agreement with previous reports using grafted scions, in systemically silenced tissue, siRNAs mapping to the 3' of GFP were predominantly detectable by Northern blot analysis. Yet, in contrast to other reports, in systemically silenced leaves, PTGS was also accompanied by dense RdDM comprising the entire GFP(cDNA) coding region. Overall, our analysis indicated that cDNA transgenes are prone to systemic PTGS and RdDM, while endogene-resembling ones are resistant to RNA silencing.
Project description:Efforts to control viral diseases in crop production include several types of physical or chemical treatments; antiviral extracts of a number of plants have also been examined to inhibit plant viral infection. However, treatments utilizing naturally selected microorganisms with activity against plant viruses are poorly documented. Here we report isolation of a soil inhabiting bacterium, Pseudomonas oleovorans strain KBPF-004 (developmental code KNF2016) which showed antiviral activity against mechanical transmission of tobamoviruses. Antiviral activity was also evaluated in seed transmission of two tobamoviruses, Pepper mild mottle virus (PMMoV) and Cucumber green mottle mosaic virus (CGMMV), by treatment of seed collected from infected pepper and watermelon, respectively. Pepper and watermelon seeds were treated with culture supernatant of P. oleovorans strain KBPF-004 or control strain ATCC 8062 before planting. Seeds germinated after treatment with water or ATCC 8062 yielded about 60% CGMMV or PMMoV positive plants, whereas < 20% of KBPF-004-treated seeds were virus-infected, a significantly reduced seed transmission rate. Furthermore, supernatant of P. oleovorans strain KBPF-004 remodeled aggregation of PMMoV 126 kDa protein and subcellular localization of movement protein in Nicotiana benthamiana, diminishing aggregation of the 126 kDa protein and essentially abolishing association of the movement protein with the microtubule network. In leaves agroinfiltrated with constructs expressing the coat protein (CP) of either PMMoV or CGMMV, less full-size CP was detected in the presence of supernatant of P. oleovorans strain KBPF-004. These changes may contribute to the antiviral effects of P. oleovorans strain KBPF-004.
Project description:BACKGROUND:Pepper mild mottle virus (PMMoV) is a member in the genus Tobamovirus and infects mainly solanaceous plants. However, the mechanism of virus-host interactions remains unclear. To explore the responses of pepper plants to PMMoV infection, we analyzed the transcriptomic changes in pepper plants after PMMoV infection using a high-throughput RNA sequencing approach and explored the roles of host autophagy in regulating PMMoV infection. RESULTS:A total of 197 differentially expressed genes (DEGs) were obtained after PMMoV infection, including 172 significantly up-regulated genes and 25 down-regulated genes. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed that most up-regulated DEGs were involved in plant abiotic and biotic stresses. Further analyses showed the expressions of multiple autophagy-related genes (ATGs) were increased after PMMoV infection in pepper and Nicotiana benthamiana plants. Through confocal microscopy and transmission electron microscopy, we have found that PMMoV infection in plant can induce autophagy, evidenced by the increased number of GFP-ATG8a fluorescent punctate and the appearance of double membrane autophagic structures in cells of N. benthamiana. Additionally, inhibition of autophagy significantly increased PMMoV RNA accumulation and aggravated systemic PMMoV symptoms through autophagy inhibitor (3-MA and E64d) treatment and silencing of NbATG expressions by a Tobacco rattle virus-induced gene silencing assays. These results indicated that autophagy played a positive role in plant resistance to PMMoV infection. CONCLUSIONS:Taken together, our results provide a transcriptomic insight into pepper responding to PMMoV infection and reveal that autophagy induced by PMMoV infection has an antiviral role in regulating PMMoV infection. These results also help us to better understand the mechanism controlling PMMoV infection in plants and to develop better strategies for breeding projects for virus-resistant crops.
Project description:Plant viral movement proteins facilitate virion movement mainly through interaction with a number of factors from the host. We report the association of a cell wall localized ascorbate oxidase (CsAO4) from Cucumis sativus with the movement protein (MP) of Cucumber mosaic virus (CMV). This was identified first in a yeast two-hybrid screen and validated by in vivo pull down and bimolecular fluorescence complementation (BiFC) assays. The BiFC assay showed localization of the bimolecular complexes of these proteins around the cell wall periphery as punctate spots. The expression of CsAO4 was induced during the initial infection period (up to 72 h) in CMV infected Nicotiana benthamiana plants. To functionally validate its role in viral spread, we analyzed the virus accumulation in CsAO4 overexpressing Arabidopsis thaliana and transiently silenced N. benthamiana plants (through a Tobacco rattle virus vector). Overexpression had no evident effect on virus accumulation in upper non-inoculated leaves of transgenic lines in comparison to WT plants at 7 days post inoculation (dpi). However, knockdown resulted in reduced CMV accumulation in systemic (non-inoculated) leaves of Nb?AO-pTRV2 silenced plants as compared to TRV inoculated control plants at 5 dpi (up to 1.3 fold difference). In addition, functional validation supported the importance of AO in plant development. These findings suggest that AO and viral MP interaction helps in early viral movement; however, it had no major effect on viral accumulation after 7 dpi. This study suggests that initial induction of expression of AO on virus infection and its association with viral MP helps both towards targeting of the MP to the apoplast and disrupting formation of functional AO dimers for spread of virus to nearby cells, reducing the redox defense of the plant during initial stages of infection.