Construction of Geminivirus Infectious Clones for Agroinoculation into Plants

The production of geminiviral infectious clones provides a standardized inoculum for use in several host–virus studies. Geminiviruses present either one (monopartite) or two (bipartite) circular single-stranded DNA components, which commonly range from 2.6 to 2.8 kb. Cloning of a monomeric genome is useful for obtaining its precise sequence. For infectious clones, however, it is essential that more than one copy of the genome, more specifically of the origin of replication, is present in order to guarantee the production of full-length progeny DNA. Here, the complete process of preparing infectious geminiviral clones is described starting from the DNA extraction and selection of restriction endonucleases followed by two protocols for constructing dimeric clones: restriction endonuclease digestion and ligation (1) and Gibson Assembly (2).