Details

Autor(en) / Beteiligte

• Blawid, Rosana
• Nagata, Tatsuya

Titel

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

Ist Teil von

• Journal of virological methods, 2015-09, Vol.222, p.11-15

Ort / Verlag

Netherlands: Elsevier B.V

Erscheinungsjahr

2015

Link zum Volltext

Quelle

MEDLINE

Beschreibungen/Notizen

• •The Gibson Assembly can be used to construct infectious full length cDNA clones of plant RNA viruses with high efficiency.•The use of the proper Escherichia coli strain to avoid DNA deletion is important.•The constructed agro-infectious clones caused the same symptoms compared with the wild-type virus. 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.