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Background: Advances in genomics technologies are making it increasingly feasible to characterize breeding lines that carry traits of agronomic interest. Tobacco germplasm lines that carry loci designated VAM and va have been extensively investigated due to their association with potyvirus resistance (both VAM and va) and defects in leaf surface compounds originating from glandular trichomes (VAM only). Molecular studies and classical genetic analyses are consistent with the model that VAM and va represent deletion mutations in the same chromosomal region. In this study, we used RNA-seq analysis, together with emerging tobacco reference genome sequence information to characterize the genomic regions deleted in tobacco lines containing VAM and va. Results: Tobacco genotypes TI 1406 (VAM), K326-va and K326 (wild type) were analyzed using RNA-seq to generate a list of genes differentially expressed in TI 1406 and K326-va, versus the K326 control. Candidate genes were localized onto tobacco genome scaffolds and validated as being absent in only VAM, or missing in both VAM and va, through PCR analysis. These results enabled the construction of a map that predicted the relative extent of the VAM and va mutations on the distal end of chromosome 21. The RNA-seq analyses lead to the discovery that members of the cembratrienol synthase gene family are deleted in TI 1406. Transformation of TI 1406 with a cembratrienol synthase cDNA, however, did not recover the leaf chemistry phenotype. Common to both TI 1406 and K326-va was the absence of a gene encoding a specific isoform of a eukaryotic translation initiation factor (eiF4E1.S). Transformation experiments showed that ectopic expression of eiF4E1.S is sufficient to restore potyvirus susceptibility in plants possessing either the va or VAM mutant loci. Conclusions: We have demonstrated the feasibility of using RNA-seq and emerging whole genome sequence resources in tobacco to characterize the VAM and va deletion mutants. These results lead to the discovery of genes underlying some of the phenotypic traits associated with these historically important loci. Additionally, initial size estimations were made for the deleted regions, and dominant markers were developed that are very close to one of the deletion junctions that defines va. [ABSTRACT FROM AUTHOR]

Background: Tomato zonate spot virus (TZSV), a dominant species of thrips-transmitted orthotospoviruses in Yunnan and Guangxi provinces in China, causes significant loss of yield in lots of crops and is a major threat to incomes of rural families. However, the detailed molecular mechanism of crop disease caused by TZSV remains obscure., Methods: Next-generation sequencing (NGS)-based transcriptome analysis (RNA-seq) was performed to investigate and compare the gene expression changes in systemic leaves of tobacco upon infection with TZSV and mock-inoculated plants as a control., Results: De novo assembly and analysis of tobacco transcriptome data by RNA-Seq identified 135,395 unigenes. 2102 differentially expressed genes (DEGs) were obtained in tobacco with TZSV infection, among which 1518 DEGs were induced and 584 were repressed. Gene Ontology enrichment analysis revealed that these DEGs were associated with multiple biological functions, including metabolic process, oxidation-reduction process, photosynthesis process, protein kinase activity. The KEGG pathway analysis of these DEGs indicated that pathogenesis caused by TZSV may affect multiple processes including primary and secondary metabolism, photosynthesis and plant-pathogen interactions., Conclusion: Our global survey of transcriptional changes in TZSV infected tobacco provides crucial information into the precise molecular mechanisms underlying pathogenesis and symptom development. This is the first report on the relationships in the TZSV-plant interaction using transcriptome analysis. Findings of present study will significantly help enhance our understanding of the complicated mechanisms of plant responses to orthotospoviral infection.

Background: Roles of microRNAs (miRNAs) and short interfering RNAs (siRNAs) in biotic stress responses, e.g., viral infection, have been demonstrated in plants by many studies. Tomato yellow leaf curl China virus (TYLCCNV) is a monopartite begomovirus that can systemically infect Solanaceae plants, and induces leaf curling, yellowing and enation symptoms when co-inoculated with a betasatellite (TYLCCNB). The released genome sequence of Nicotiana benthamiana provides an opportunity to identify miRNAs and siRNAs responsive to begomovirus-associated betasatellite in N. benthamiana., Results: miRNAs were identified in three small RNA libraries generated using RNA isolated from N. benthamiana plants systemically infected with TYLCCNV (Y10A) alone, co-infected with Y10A and its betasatellite TYLCCNB (Y10β) or a TYLCCNB mutant (Y10mβ) that contains a mutated βC1, the sole betasatellite-encoded protein. A total of 196 conserved miRNAs from 38 families and 197 novel miRNAs from 160 families were identified. Northern blot analysis confirmed that expression of species-specific miRNAs was much lower than that of conserved miRNAs. Several conserved and novel miRNAs were found to be responsive to co-infection of Y10A and Y10β but not to co-infection of Y10A and Y10mβ, suggesting that these miRNAs might play a role unique to interaction between Y10β and N. benthamiana. Additionally, we identified miRNAs that can trigger the production of phased secondary siRNAs (phasiRNAs)., Conclusions: Identification of miRNAs with differential expression profiles in N. benthamiana co-infected with Y10A and Y10β and co-infected with Y10A and Y10mβ indicates that these miRNAs are betasatellite-responsive. Our result also suggested a potential role of miRNA-mediated production of phasiRNAs in interaction between begomovirus and N. benthamiana.

Background: MicroRNAs (miRNAs) and short interfering RNAs (siRNAs) are two major classes of small RNAs. They play important regulatory roles in plants and animals by regulating transcription, stability and/or translation of target genes in a sequence-complementary dependent manner. Over 4,000 miRNAs and several classes of siRNAs have been identified in plants, but in tobacco only computational prediction has been performed and no tobacco-specific miRNA has been experimentally identified. Wounding is believed to induce defensive response in tobacco, but the mechanism responsible for this response is yet to be uncovered., Results: To get insight into the role of small RNAs in damage-induced responses, we sequenced and analysed small RNA populations in roots and leaves from wounding or topping treated tobacco plants. In addition to confirmation of expression of 27 known miRNA families, we identified 59 novel tobacco-specific miRNA members of 38 families and a large number of loci generating phased 21- or 24-nt small RNAs (including ta-siRNAs). A number of miRNAs and phased small RNAs were found to be responsive to wounding or topping treatment. Targets of small RNAs were further surveyed by degradome sequencing., Conclusions: The expression changes of miRNAs and phased small RNAs responsive to wounding or topping and identification of defense related targets for these small RNAs suggest that the inducible defense response in tobacco might be controlled by pathways involving small RNAs.