Pathogenicity Mutants of Erwinia Amylovora Caused by Tn5 Insertion

Abstract

To study the genetics of pathogenicity of Erwinia amylovora, strains altered in pathogenicity through single-site mutations are needed. The transposon Tn5 (Kmr) was introduced into Ea322 with the suicidal plasmid vector pJB4JI. Among 11,000 prototrophic Kmr strains tested, 23 were altered in their pathogenic capabilities on apple and pear tissue. Three of these mutants also failed to induce a hypersensitive reaction when infiltrated into tobacco leaves. The location of the transposon in the genome was determined by Southern blot hybridization with Tn5. Ten mutants have single Tn5 insertions in chromosomal, rather than plasmid DNA. Thirteen mutants contain more than one DNA sequence that hybridized with Tn5. In three cases, two independent mutants were found with Tn5 insertions in the same EcoRI fragment. Among the 23 mutants, the transposon had inserted into at least eight different sites in chromosomal DNA, indicating that the mechanism for pathogenicity is complex. To confirm that the Tn5 was responsible for the mutations, parental wild-type genes were replaced with genes rendered nonfunctional through Tn5 insertion. Cotransfer of Kmr with the altered pathogenic phenotype was demonstrated. Complementation experiments are in progress in which cloned wild-type DNA fragments are transferred to Tn5-induced mutants of E. amylovora.KeywordsHypersensitive ReactionPathogenicity MutantTobacco LeaveSouthern Blot HybridizationEcoRI FragmentThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.