Articles
CLONING OF ERWINIA AMYLOVORA DNA INVOLVED IN PATHOGENICITY AND INDUCTION OF THE HYPERSENSITIVE REACTION
The HR is considered a plant defense mechanism active against potential pathogens.
It is characterized by the rapid collapse (< 18 h) and death of the plant tissues into which bacteria are introduced.
The reaction results in localization of the pathogenic bacteria and thus spread to adjacent healthy tissue does not occur.
Three mutants of E. amylovora that are unable to induce the HR in tobacco were produced by transposon mutagenesis using Tn5. These mutants also were nonpathogenic to immature pear fruits.
Probing of Southern blots of genomic DNA from the mutants with 32P-labeled Tn5 revealed that the transposon had inserted in a different site of the DNA of each mutant.
The Tn5-containing EcoRI fragment from one mutant, strain Ea322-T101, was cloned in pBR322, resulting in pCPP101. Two libraries of DNA from the wild-type parent, strain Ea322, were constructed in pBR322 and pBR325. The Tn5-containing fragment was used to probe the libraries to identify members containing wild-type DNA homologous to the probe.
The plasmids containing homologous DNA were introduced into Ea322-T101 by transformation.
The recombinant strains were tested for their ability to induce the HR in tobacco and for pathogenicity to immature pear fruits.
Three plasmids weakly restored to the mutant both the ability to induce the HR in tobacco leaves and pathogenicity to immature pear fruit.
The smallest plasmid, pCPP120, containing a 16.3 kb EcoR1 fragment of Ea322 DNA was chosen for further study.
The DNA of strain Ea322 contained in pCPP120 was found to include one or more genes that inhibit the growth of E. amylovora. Strains containing pCPP120 grow at about one-half the rate of the same strain without the plasmid.
When grown in the absence of antibiotic selection pressure (as in plant tissue), pCPP120 is rapidly lost from the bacterial population.
This may explain why mutants complemented