Random mutagenesis in a plant viral genome using a DNA repair-deficient mutator Escherichia coli strain

Random mutagenesis in a plant viral genome is valuable for generating attenuated strains or for analyzing viral gene function at the molecular level. A DNA repair-deficient mutator Escherichia coli strain was used for random mutagenesis of a plant viral genome. A full-length infectious cDNA clone of Citrus tatter leaf virus (genus Capillovirus) L strain (CTLV-L) genomic RNA under the T7 promoter sequence (pITCL) was introduced into the mutator E. coli strain XL1-Red and mutagenized overnight. To fix mutations, the mixture of plasmid DNA isolated from colonies of the mutator bacteria was introduced into another E. coli strain, JM109, which has normal DNA repair function. Infectious viral genomic RNA was transcribed in vitro from each mutagenized pITCL clone and inoculated on host plants. Phenotypic mutants were selected for altered pathogenicity in the inoculated plants. Nucleotide sequence analysis of each mutant revealed that mutations were introduced randomly into the CTLV-L genome regardless of the function of the viral gene. The nucleotide substitutions were biased towards single point mutations, which consisted of more transitions than transversions or single-base frameshifts. These mutations were preserved stably in plants subject to sequential mechanical inoculation. The strategy presented below is a simple and very efficient way to generate virus mutants for analyzing the functions of viral genes.