Project description:TMV-resistant (N), -susceptible (n), and enhanced susceptible mutant sun1-1 (N) tomato were grown in asceptic conditions in growth chambers (25º, 16hr/8hr light dark). 4-5 week old tomato were treated with TMV leaf sap or mock leaf sap, and leaf tissue was collected after 3, 9, and 27 hours. RNA was isolated from each sample (leaf tissue from one plant/one treatment/one timepoint), using standard TIGR protocols, and 25ug of total RNA was labeled using TIGR indirect labeling protocols. The experiment was repeated three times. Keywords: Direct comparison
Project description:BackgroundSynthetic biology is a discipline that includes making life forms artificially from chemicals. Here, a DNA molecule was enzymatically synthesized in vitro from DNA templates made from oligonucleotides representing the text of the first Tobacco mosaic virus (TMV) sequence elucidated in 1982. No infectious DNA molecule of that seminal reference sequence exists, so the goal was to synthesize it and then build viral chimeras.ResultsRNA was transcribed from synthetic DNA and encapsidated with capsid protein in vitro to make synthetic virions. Plants inoculated with the virions did not develop symptoms. When two nucleotide mutations present in the original sequence, but not present in most other TMV sequences in GenBank, were altered to reflect the consensus, the derivative synthetic virions produced classic TMV symptoms. Chimeras were then made by exchanging TMV capsid protein DNA with Tomato mosaic virus (ToMV) and Barley stripe mosaic virus (BSMV) capsid protein DNA. Virus expressing ToMV capsid protein exhibited altered, ToMV-like symptoms in Nicotiana sylvestris. A hybrid ORF6 protein unknown to nature, created by substituting the capsid protein genes in the virus, was found to be a major symptom determinant in Nicotiana benthamiana. Virus expressing BSMV capsid protein did not have an extended host range to barley, but did produce novel symptoms in N. benthamiana.ConclusionsThis first report of the chemical synthesis and artificial assembly of a plant virus corrects a long-standing error in the TMV reference genome sequence and reveals that unnatural hybrid virus proteins can alter symptoms unexpectedly.
Project description:Strigolactones (SLs) are plant hormones that regulate diverse developmental processes and environmental responses in plants. It has been discovered that SLs play an important role in regulating plant immune resistance to pathogens, but there are currently no reports on their role in the interaction between Nicotiana benthamiana and Tobacco mosaic virus (TMV). In this study, the exogenous application of SLs weakened the resistance of N. benthamiana to TMV, promoting TMV infection, whereas the exogenous application of Tis108, an SL inhibitor, resulted in the opposite effect. Virus-induced gene silencing (VIGS) inhibition of two key SL synthesis enzyme genes, NtCCD7 and NtCCD8, enhanced the resistance of N. benthamiana to TMV. Additionally, we conducted a screening of N. benthamiana related to TMV infection. TMV-infected plants treated with SLs were compared to the control by using RNA-seq. KEGG enrichment analysis and weighted gene co-expression network analysis (WGCNA) of differentially expressed genes (DEGs) suggested that plant hormone signaling transduction may play a significant role in the SL-TMV-N. benthamiana interactions. This study reveals new functions of SLs in regulating plant immunity and provides a reference for controlling TMV diseases in production.
Project description:Tobacco mild green mosaic virus (TMGMV) isolates from different plant families show no evidence of differential adaptation to their host of origin