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

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.