Construction of Infectious Clones for DNA Viruses: Mastreviruses
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To characterize a virus at the molecular and biological levels, it is necessary to produce an infectious clone. For most of the Geminiviridae , cloning of the genome is relatively easy because of their small genomes and the presence of the virus double-stranded (replicative) DNA form in infected plants. Indeed, the presence of conserved sequences between species in the genera Begomovirus , Curtovirus , and Topocuvirus allows the PCR amplification of most genomes using degenerate “universal” primers. Unlike the other genera, no universal primers are reported that are suitable for all mastreviruses and alternative, more time-consuming methods must be used.
This chapter describes a method that has proven successful for the preparation and testing of infectious clones for a wide range of mastreviruses. It has been designed to ensure its applicability for laboratories throughout the world. Methods are presented for the isolation of total plant DNA and the purification of the replicative (cccDNA) form of the virus using a commercially available plasmid purification kit. Restriction enzyme digestion of the purified DNA using a restriction enzyme with a unique site in the viral genome allows the cloning of a full-length copy of the genome into a high copy number vector, thereby providing a template for sequence analysis and further cloning. The only efficient method for confirming infectivity of mastrevirus clones is using agroinoculation (also termed agroinfection). This requires the production of a multimeric copy of the genome in a T-DNA binary vector, transformation of specific Agrobacterium strains with the binary vector clone, and inoculation of specific regions of seedlings, or seeds, of the appropriate host species. These specific requirements are described and discussed.
