Submitted to: Molecular Plant-Microbe Interactions
Publication Type: Peer reviewed journal
Publication Acceptance Date: 4/30/2014
Publication Date: 5/12/2014
Publication URL: http://handle.nal.usda.gov/10113/59041
Citation: Cooper, B. 2014. Tobacco mosaic virus: Proof by synthesis. Molecular Plant-Microbe Interactions. 15:R67. Interpretive Summary: The ribonucleic acid sequence of Tobacco mosaic virus (TMV) was discovered in 1982. Oddly, no known DNA clone of the sequenced strain exists. Therefore, it is unknown if the sequence reported in 1982 is for an infectious virus. In this study, DNA of the TMV sequence was constructed using oligonucleotides. The DNA was converted to RNA and used to inoculate tobacco plants, but the plants did not develop symptoms. Mutations were discovered in the original sequence. When the mutations were corrected the virus became infectious. The corrected TMV DNA was then combined with DNA from Tomato mosaic virus and the new virus elicited a resistant reaction on Nicotiana sylvestris whereas the corrected TMV did not. These experiments show that a gene from N. sylvestris causes resistance to ToMV but not TMV. The corrected TMV DNA was also combined with DNA from Barley stripe mosaic virus, but the new virus did not infect barley. This report corrects a long-standing error in the DNA sequence of TMV, shows which virus genes elicit disease resistance in N. sylvestris, and shows that the host range of TMV cannot be easily expanded to other plants just by adding DNA from another virus. These data are most likely to influence scientists at universities, government agencies and companies who are interested in making new molecules and genes.
Technical Abstract: A linear, non-self-replicating DNA molecule encoding Tobacco mosaic virus (TMV) was enzymatically synthesized in vitro from DNA templates made from overlapping oligonucleotides. The molecule was a replica of the alphabetic text rendering of the first TMV genome sequence elucidated by Goelet et al. (1982). RNA was transcribed from it, encapsidated with purified capsid protein (CP) in vitro, and inoculated to tobacco. The plants did not develop symptoms. When two nucleotide mutations present in the Goelet 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. Next, encapsidation complementation was evaluated by substituting the TMV CP gene with oligonucleotides for the homologous Tomato mosaic virus (ToMV) CP and the heterologous Barley stripe mosaic virus (BSMV) CP genes. The former chimera exhibited altered, ToMV-like symptoms on Nicotiana sylvestris, showing the ToMV CP was the elicitor of the N. sylvestris N’ gene. A hybrid TMV-ToMV ORF6 protein unknown to nature, which was created by substituting the ToMV CP gene, was found to be a major symptom determinant in N. benthamiana. On the other hand, BSMV CP did not extend the host range of TMV to barley. These results correct a long-standing error in the TMV reference genome sequence and address historical observations of symptomology and transencapsidation of TMV.