Mutation of theErwinia amylovora argDGene Causes Arginine Auxotrophy, Nonpathogenicity in Apples, and Reduced Virulence in Pears

Abstract

ABSTRACTFire blight is caused byErwinia amylovoraand is the most destructive bacterial disease of apples and pears worldwide. In this study, we found thatE. amylovoraargD(1000)::Tn5, anargDTn5transposon mutant that has the Tn5transposon inserted after nucleotide 999 in theargDgene-coding region, was an arginine auxotroph that did not cause fire blight in apple and had reduced virulence in immature pear fruits. TheE. amylovoraargDgene encodes a predictedN-acetylornithine aminotransferase enzyme, which is involved in the production of the amino acid arginine. A plasmid-borne copy of the wild-typeargDgene complemented both the nonpathogenic and the arginine auxotrophic phenotypes of theargD(1000)::Tn5mutant. However, even when mixed with virulentE. amylovoracells and inoculated onto immature apple fruit, theargD(1000)::Tn5mutant still failed to grow, while the virulent strain grew and caused disease. Furthermore, the pCR2.1-argDcomplementation plasmid was stably maintained in theargD(1000)::Tn5mutant growing in host tissues without any antibiotic selection. Therefore, the pCR2.1-argDcomplementation plasmid could be useful for the expression of genes, markers, and reporters inE. amylovoragrowingin planta, without concern about losing the plasmid over time. The ArgD protein cannot be considered anE. amylovoravirulence factor because theargD(1000)::Tn5mutant was auxotrophic and had a primary metabolism defect. Nevertheless, these results are informative about the parasitic nature of the fire blight disease interaction, since they indicate thatE. amylovoracannot obtain sufficient arginine from apple and pear fruit tissues or from apple vegetative tissues, either at the beginning of the infection process or after the infection has progressed to an advanced state.