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item Gray, Stewart

Submitted to: Molecular Plant Microbe Interactions
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/30/2004
Publication Date: 6/1/2004
Citation: Liang, D., Gray, S.M., Palukaitis, P. 2004. Site-directed mutagenesis and generation of chimeric viruses by homologous recombination in yeast to facilitate analysis of plant-virus interactions. Molecular Plant Microbe Interactions. 17:571-576.

Interpretive Summary: Defined changes in plant virus genomes are easily made in the laboratory and are used to study gene function. However, naked genetic material of many plant viruses cannot infect plants by itself. In these cases, the virus genome is inserted into a bacterial genome segment called a plasmid. When the bacteria are introduced into a plant, the plasmid transfers the virus into the plant cell resulting in virus infection. Mutations in the virus genome cannot be made when the virus is contained within the plasmid; thus a complex multi-step procedure is required to introduce mutant viruses into plants. In this study, we developed a novel experimental system to generate mutations in virus genomes while they are in the bacterial plasmid. Adding a yeast gene to the bacterial plasmid allowed the plasmid to reproduce in yeast cells. Small pieces of the virus genome containing a desired mutation are easily synthesized in the laboratory. These, along with the plasmid, are introduced into yeast cells. In yeast, the process of homologous recombination occurs at high frequency. This is essentially an exchange of similar genetic pieces and resulted in an exchange of the normal virus sequence with the mutant sequence. The plasmid containing the mutant virus replicates to high numbers in the yeast and is easily purified. The plasmid is then re-introduced into the bacteria and used transfer the mutant virus into plant cells resulting in virus infection. Subsequently the effects of the introduced mutation on the biology of the virus can be studied.

Technical Abstract: A yeast homologous recombination system was used to generate mutants and chimeras in the genome of Potato leafroll virus (PLRV). A yeast-bacteria shuttle vector was developed that allows mutants and chimeras generated in yeast to be transformed into Escherichia coli for confirmation of the mutations and transformed into Agrobacterium tumefaciens to facilitate agroinfection of plants by the mutant PLRV genomes. The advantages of the system include the high frequency of recovered mutants generated by yeast homologous recombination, the ability to generate over 20 mutants and chimeras using only two restriction endonuclease sites, the ability to introduce multiple additional sequences using three and four DNA fragments, and the mobilization of the same plasmid from yeast to E. coli, A. tumefaciens and plants. The wildtype PLRV genome showed no loss of virulence after sequential propagation in yeast, E. coli and A. tumefaciens. Moreover, many PLRV clones with mutations generated in the capsid protein and readthrough domain of the capsid protein replicated and moved throughout plants. This approach will facilitate the analysis of plant-virus interactions of in vivo-generated mutants for many plant viruses, especially those not transmissible mechanically to plants.