Construction of an infectious clone of a plant RNA virus in a binary vector using one-step Gibson Assembly

J Virol Methods. 2015 Sep 15:222:11-5. doi: 10.1016/j.jviromet.2015.05.003. Epub 2015 May 15.

Abstract

The construction of full-length infectious clones of RNA viruses is often laborious due to the many cloning steps required and the DNA exclusion within the plasmid during Escherichia coli transformation. We demonstrate single-step cloning procedure of an infectious cDNA of the tomato blistering mosaic virus (ToBMV) using Gibson Assembly (GA), which drastically reduces the number of cloning steps. By agro-inoculation with the construct obtained by this procedure, ToBMV was recovered six days post-inoculation in Nicotiana benthamiana plants. The symptoms induced by the recovered virus were indistinguishable from those caused by the wild-type virus. We conclude that the GA is very useful method particularly to construct a full-length cDNA clone of a plant RNA virus in a binary vector.

Keywords: Full-length cDNA clone; Gibson Assembly; Seamless cloning; Tymovirus.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cloning, Molecular / methods*
  • DNA, Complementary / genetics
  • Escherichia coli / genetics
  • Genetic Vectors
  • Nicotiana / virology
  • Plasmids
  • RNA, Viral / genetics
  • Reverse Genetics / methods*
  • Transformation, Genetic
  • Tymovirus / genetics*

Substances

  • DNA, Complementary
  • RNA, Viral