New approach for the construction of infectious clones of a circular DNA plant virus using Gibson Assembly.

Viruses belonging to the genus Begomovirus (family Geminiviridae) have circular single-strand DNA genomes encapsidated into quasi-icosahedral particles, and are transmitted by whiteflies of the Bemisia tabaci complex. Biological and molecular properties of begomoviruses have been studied efficiently with infectious clones containing dimeric genomic components. However, current approaches employing enzymatic digestion and ligation to binary vectors are laborious, mostly due to many cloning steps or partial digestion by restriction enzyme. Here, an infectious clone of the bipartite begomovirus Bean golden mosaic virus (BGMV) was obtained using PCR and Gibson Assembly (GA). Common bean (Phaseolus vulgaris) seedlings displayed severe yellow mosaic and stunt symptoms 15 days after agroinoculation with DNA-A and DNA-B of BGMV. The approach based on PCR-GA protocol is a fast and useful tool to obtain infectious clones of a circular DNA plant virus.