Abstract
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.
Highlights
Synthetic biology is a discipline that includes making life forms artificially from chemicals
The sequences confirmed that the virus encodes at least four proteins (Figure 1): the 126 kDa replicase component; the 183 kDa replicase component that arises from translational read-though of the amber termination codon of the 126 kDa protein gene; a 30 kDa movement protein (MP) required for Tobacco mosaic virus (TMV) RNA translocation
Goelet sequence DNA synthesis from modular clones Thirty sets of long-oligonucleotides with complementary, overlapping ends were chemically synthesized for construction of a linear DNA molecule encoding the Goelet TMV sequence (Figure 1)
Summary
Synthetic biology is a discipline that includes making life forms artificially from chemicals. The sequence of TMV variant V01408.1, dubbed the Goelet sequence, became a reference by which all other plant viruses were compared, and it facilitated the creation of TMV cDNA from which infectious RNA transcripts could be expressed [11], opening new possibilities for characterizing the encoded genes and for transiently expressing foreign genes in plants [12]. Amidst all this success, is the almost forgotten fact that the Goelet sequence does not represent a single molecule of a TMV genome. It is a composite consensus of more than 400 independent clones of cDNA fragments reverse transcribed from a population of TMV RNA molecules exhibiting nucleotide polymorphisms consistent with the inherent error rate of viral RNA-dependent replication
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