Molecular cloning of Indian tomato leaf curl vims genome following a simple method of concentrating the supercoiled replicative form of viral DNA

Abstract

DNA-A and DNA-B components of the genome of a whitefly transmitted virus causing yellowing and leaf curl in tomato (ITLCV) were cloned following a simple procedure for isolation of the double stranded replicative form of viral DNA from infected tomato plants. The method is based on extraction of total DNA from infected plants followed by concentration of the double stranded replicative form of viral DNA by an alkaline denaturation procedure identical to that used for isolati.on of plasmid DNA from Escherichia coli. The attempted cloning of DNA showed that 95% of the transformants contained plasmids with an insert of either DNA-A (2.75 kb) or DNA-B (2.55 kb). Cloned DNA-A and DNA-B when used as probes could detect DNA-A/DNA-B in total nucleic acid obtained from fresh diseased tissue. Both DNA-A and DNA-B are needed for infection and they have a common region of 166 bases with about 94% nucleotide sequence homology, a characteristic of all bipartite geminiviruses. Comparison of the amino acid sequence of the putative coat protein product of ITLCV with some other mono- and bipartite geminiviruses revealed a maximum of 86% homology with Indian cassava mosaic virus.