Strategies to facilitate the development of uncloned or cloned infectious full-length viral cDNAs: Apple chlorotic leaf spot virus as a case study

Background: Approaches to simplify and streamline the construction of full-length infectious cDNA clones (FLcDNAs) are needed. Among desirable improvements are the ability to use total nucleic acids (TNA) extracts from infected hosts (to bypass viral purification limitations) for the direct one-step amplification of large FL-cDNAs, the possibility to inoculate plants with uncloned FL-cDNAs and the simplified cloning of these large molecules. Results: Using the 7.55 kb genome of Apple chlorotic leaf spot trichovirus (ACLSV) approaches allowing the rapid generation from TNA extracts of FL-cDNAs under the control of the T7 promoter and the successful inoculation of plants using in vitro transcripts obtained from these uncloned amplification products have been developed. We also show that the yeast homologous recombination system permits efficient cloning of FL-cDNAs and the simultaneous one-step tailoring of a ternary Yeast-Escherichia coli-Agrobacterium tumefaciens shuttle vector allowing efficient inoculation of both herbaceous and woody host plants by agroinfiltration. Conclusions: The fast and efficient strategies described here should have broad applications, in particular for the study of difficult plant viruses, such as those infecting woody hosts, and potentially for other, non plant-infecting viral agents.