Mutation of the Erwinia amylovora argD Gene Causes Arginine Auxotrophy, Nonpathogenicity in Apples, and Reduced Virulence in Pears

Abstract

c 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.

Expression of the dspA/E gene of Erwinia amylovora in non-host plant Arabidopsis thaliana

“…This is in agreement with previous reports that dspA/E mutants trigger HR in tobacco [6,27,28]. Moreover, in a study by Laura et al [28], Figure 1. Arabidopsis leaves stained for GUS activity.…”

“…At each sampling point, cells were recovered from necrotic tissues cuts by suspension in NGA broth and plating on NGA agar medium at appropriate dilution. The bacterial growth following two days of incubation at [26][27][28] C was scored as colony-forming units per millilitre (CFU/mL). This was carried out in a completely randomized plot design, and the infiltration assay was performed twice.…”

Erwinia amylovora pyrC mutant causes fire blight despite pyrimidine auxotrophy

“…The Tn5 mutants were created by the mutagenesis of Erw. amylovora strain HKN06P1 (Lee et al 2010; Table 1) with an engineered Tn5 transposon, according to the manufacturer's instructions (EZ-Tn5<R6KcoriKan-2> Transposome Kit; Epicentre, Madison, WI), and mutant screening was performed as described elsewhere (Ramos et al 2014). The location of the Tn5 insertion in pyrC244::Tn5 was determined by Tn5 plasmid rescue according to the kit manufacturer's instructions, sequencing of the DNA flanking the Tn5 transposon in plasmid EZ-Tn5-pyrC using primers FP and RP (Table 1; Fig.…”

“…Growth in M9N media and uracil auxotrophy tests were performed as described elsewhere (Ramos et al 2014).…”

“…amylovora arginine auxotroph was not pathogenic on apple and pear, indicating that an intact arginine biosynthesis pathway is required for Erw. amylovora to grow in host tissues and cause disease (Ramos et al 2014). An Erw.…”