Erwinia amylovora Auxotrophic Mutant Exometabolomics and Virulence on Apples

Abstract

The Gram-negative bacterium Erwinia amylovora causes fire blight disease of apples and pears. While the virulence systems of E. amylovora have been studied extensively, relatively little is known about its parasitic behavior. The aim of this study was to identify primary metabolites that must be synthesized by this pathogen for full virulence. A series of auxotrophic E. amylovora mutants, representing 21 metabolic pathways, were isolated and characterized for metabolic defects and virulence in apple immature fruits and shoots. On detached apple fruitlets, mutants defective in arginine, guanine, hexosamine, isoleucine/valine, leucine, lysine, proline, purine, pyrimidine, sorbitol, threonine, tryptophan, and glucose metabolism had reduced virulence compared to the wild type, while mutants defective in asparagine, cysteine, glutamic acid, histidine, and serine biosynthesis were as virulent as the wild type. Auxotrophic mutant growth in apple fruitlet medium had a modest positive correlation with virulence in apple fruitlet tissues. Apple tree shoot inoculations with a representative subset of auxotrophs confirmed the apple fruitlet results. Compared to the wild type, auxotrophs defective in virulence caused an attenuated hypersensitive immune response in tobacco, with the exception of an arginine auxotroph. Metabolomic footprint analyses revealed that auxotrophic mutants which grew poorly in fruitlet medium nevertheless depleted environmental resources. Pretreatment of apple flowers with an arginine auxotroph inhibited the growth of the wild-type E. amylovora, while heat-killed auxotroph cells did not exhibit this effect, suggesting nutritional competition with the virulent strain on flowers. The results of our study suggest that certain nonpathogenic E. amylovora auxotrophs could have utility as fire blight biocontrol agents. IMPORTANCE This study has revealed the availability of a range of host metabolites to E. amylovora cells growing in apple tissues and has examined whether these metabolites are available in sufficient quantities to render bacterial de novo synthesis of these metabolites partially or even completely dispensable for disease development. The metabolomics analysis revealed that auxotrophic E. amylovora mutants have substantial impact on their environment in culture, including those that fail to grow appreciably. The reduced growth of virulent E. amylovora on flowers treated with an arginine auxotroph is consistent with the mutant competing for limiting resources in the flower environment. This information could be useful for novel fire blight management tool development, including the application of nonpathogenic E. amylovora auxotrophs to host flowers as an environmentally friendly biocontrol method. Fire blight management options are currently limited mainly to antibiotic sprays onto open blossoms and pruning of infected branches, so novel management options would be attractive to growers.

The guanine–hypoxanthine permease GhxP of Erwinia amylovora facilitates the influx of the toxic guanine derivative 6‐thioguanine

“…2015; Klee et al . 2019a). The ability of the Ea⊿ghxP::Cam r mutant to cause disease was monitored in immature pear and apple fruit assays.…”

“…2014, 2015; Klee et al . 2019a). Some auxotrophs display reduced growth and virulence, while others have minimal effect.…”

“…1997; Wang and Beer, 2006; Klee et al . 2019a). These studies infer that, however limited, purines are available from host tissues and taken up by E. amylovora .…”

Impact of Facultative Bacteria on the Metabolic Function of an Obligate Insect-Bacterial Symbiosis

“…Five days later, the larvae (7-days-old, approximately 25 mg fresh aphid material per sample) were snap-frozen in liquid nitrogen and stored at –80°C. Following extraction of metabolites (protocol in Text S1D ), metabolites were separated using a Waters XSelect HSS T3 column (100 Å, 5 μm, 2.1 mm × 100 mm) fitted with a Restex UltraShield 0.2-μm precolumn filter and analyzed on a Thermo Exactive Plus Orbitrap ( 72 – 74 ). Blanks and a pooled QC sample were included in the analysis.…”

The stringent response regulator (p) ppGpp mediates virulence gene expression and survival in Erwinia amylovora

“…It has been reported that amino acid metabolism might be important for virulence [43][44][45]. In E. amylovora, mutants deficient in arginine, isoleucine/valine, and tryptophan metabolism exhibited reduced virulence [44], and the argD mutant of E. amylovora not only led to arginine auxotrophy, but also exhibited attenuated or no virulence in apples and pears [45]. A methionine metabolism regulator MetR has been identified as a new virulence regulator [46].…”