title: Mutation of the Erwinia amylovora argD gene causes arginine auxotrophy, nonpathogenicity in apples, and reduced virulence in pears.
abstract: Fire blight is caused by Erwinia amylovora and is the most destructive bacterial disease of apples and pears worldwide. In this study, we found that E. amylovora argD(1000)::Tn5, an argD Tn5 transposon mutant that has the Tn5 transposon inserted after nucleotide 999 in the argD gene-coding region, was an arginine auxotroph that did not cause fire blight in apple and had reduced virulence in immature pear fruits. The E. amylovora argD gene encodes a predicted N-acetylornithine aminotransferase enzyme, which is involved in the production of the amino acid arginine. A plasmid-borne copy of the wild-type argD gene complemented both the nonpathogenic and the arginine auxotrophic phenotypes of the argD(1000)::Tn5 mutant. However, even when mixed with virulent E. amylovora cells and inoculated onto immature apple fruit, the argD(1000)::Tn5 mutant still failed to grow, while the virulent strain grew and caused disease. Furthermore, the pCR2.1-argD complementation plasmid was stably maintained in the argD(1000)::Tn5 mutant growing in host tissues without any antibiotic selection. Therefore, the pCR2.1-argD complementation plasmid could be useful for the expression of genes, markers, and reporters in E. amylovora growing in planta, without concern about losing the plasmid over time. The ArgD protein cannot be considered an E. amylovora virulence factor because the argD(1000)::Tn5 mutant 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 that E. amylovora cannot 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.
authors.affiliation: Department of Plant Pathology and Environmental Microbiology, The Pennsylvania State University, University Park, Pennsylvania, USA Graduate Degree Program in Plant Pathology, Department of Plant Pathology and Environmental Microbiology, The Pennsylvania State University, University Park, Pennsylvania, USA.
authors.forename: Laura S
authors.initials: LS
authors.lastname: Ramos
authors.affiliation: The Pennsylvania State University Fruit Research and Extension Center, Biglerville, Pennsylvania, USA.
authors.forename: Brian L
authors.initials: BL
authors.lastname: Lehman
authors.affiliation: Department of Plant Pathology and Environmental Microbiology, The Pennsylvania State University, University Park, Pennsylvania, USA The Pennsylvania State University Fruit Research and Extension Center, Biglerville, Pennsylvania, USA.
authors.forename: Kari A
authors.initials: KA
authors.lastname: Peter
authors.affiliation: Department of Plant Pathology and Environmental Microbiology, The Pennsylvania State University, University Park, Pennsylvania, USA twm4@psu.edu.
authors.forename: Timothy W
authors.initials: TW
authors.lastname: McNellis
mesh_terms: D001120:Arginine / Q000378:metabolism*; D004251:DNA Transposable Elements; D044084:Erwinia amylovora / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D055786:Gene Knockout Techniques; D005816:Genetic Complementation Test; D027845:Malus / Q000382:microbiology*; D016254:Mutagenesis, Insertional; D009154:Mutation; D010935:Plant Diseases / Q000382:microbiology*; D031989:Pyrus / Q000382:microbiology*; D000637:Transaminases / Q000235:genetics / Q000378:metabolism*; D014774:Virulence
chemical_list: D004251:DNA Transposable Elements; D001120:Arginine; D000637:Transaminases; C018731:acetylornithine transaminase