Erwinia amylovora Auxotrophic Mutant Exometabolomics and Virulence on Apples.

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.

An Erwinia amylovora yjeK mutant exhibits reduced virulence, increased chemical sensitivity and numerous environmentally dependent proteomic alterations.

The Gram-negative bacterium Erwinia amylovora causes fire blight, an economically important disease of apples and pears. Elongation factor P (EF-P) is a highly conserved protein that stimulates the formation of the first peptide bond of certain proteins and facilitates the translation of certain proteins, including those with polyproline motifs. YjeK and YjeA are two enzymes involved in the essential post-translational ß-lysylation of EF-P at a conserved lysine residue, K34. EF-P, YjeA and YjeK have been shown to be essential for the full virulence of Escherichia coli, Salmonella species and Agrobacterium tumefaciens, with efp, yjeA and yjeK mutants having highly similar phenotypes. Here, we identified an E. amylovora yjeK::Tn5 transposon mutant with decreased virulence in apple fruit and trees. The yjeK::Tn5 mutant also showed pleiotropic phenotypes, including reduced growth in rich medium, lower extracellular polysaccharide production, reduced swimming motility and increased chemical sensitivity compared with the wild-type, whilst maintaining wild-type level growth in minimal medium. All yjeK::Tn5 mutant phenotypes were complemented in trans with a plasmid bearing a wild-type copy of yjeK. Comprehensive, quantitative proteomics analyses revealed numerous, environmentally dependent changes in the prevalence of a wide range of proteins, in higher abundance and lower abundance, in yjeK::Tn5 compared with the wild-type, and many of these alterations could be linked to yjeK::Tn5 mutant phenotypes. The environmental dependence of the yjeK::Tn5 mutant proteomic alterations suggests that YjeK could be required for aspects of the environmentally dependent regulation of protein translation. YjeK activity may be critical to overcoming stress, including the challenging host environment faced by invading pathogenic bacteria.

The fire blight pathogen Erwinia amylovora requires the rpoN gene for pathogenicity in apple.

RpoN is a σ(54) factor regulating essential virulence gene expression in several plant pathogenic bacteria, including Pseudomonas syringae and Pectobacterium carotovorum. In this study, we found that mutation of rpoN in the fire blight pathogen Erwinia amylovora caused a nonpathogenic phenotype. The E. amylovora rpoN Tn5 transposon mutant rpoN1250::Tn5 did not cause fire blight disease symptoms on shoots of mature apple trees. In detached immature apple fruits, the rpoN1250::Tn5 mutant failed to cause fire blight disease symptoms and grew to population levels 12 orders of magnitude lower than the wild-type. In addition, the rpoN1250::Tn5 mutant failed to elicit a hypersensitive response when infiltrated into nonhost tobacco plant leaves, and rpoN1250::Tn5 cells failed to express HrpN protein when grown in hrp (hypersensitive response and pathogenicity)-inducing liquid medium. A plasmid-borne copy of the wild-type rpoN gene complemented all the rpoN1250::Tn5 mutant phenotypes tested. The rpoN1250::Tn5 mutant was prototrophic on minimal solid and liquid media, indicating that the rpoN1250::Tn5 nonpathogenic phenotype was not caused by a defect in basic metabolism or growth. This study provides clear genetic evidence that rpoN is an essential virulence gene of E. amylovora, suggesting that rpoN has the same function in E. amylovora as in P. syringae and Pe. carotovorum.

Virulence characteristics accounting for fire blight disease severity in apple trees and seedlings.

The gram-negative bacterium Erwinia amylovora is the causal agent of fire blight, the most destructive bacterial disease of rosaceous plants, including apple and pear. Here, we compared the virulence levels of six E. amylovora strains (Ea273, CFBP1367, Ea581a, E2002a, E4001a, and HKN06P1) on apple trees and seedlings. The strains produced a range of disease severity, with HKN06P1 producing the greatest disease severity in every assay. We then compared virulence characteristic expression among the six strains, including growth rates in immature apple fruit, amylovoran production, levansucrase activity, biofilm formation, carbohydrate utilization, hypersensitive cell death elicitation in tobacco leaves, and protein secretion profiles. Multiple regression analysis indicated that three of the virulence characteristics (amylovoran production, biofilm formation, and growth in immature apple fruit) accounted for >70% of the variation in disease severity on apple seedlings. Furthermore, in greenhouse-grown 'Gala' trees, >75% of the variation in disease severity was accounted for by five of the virulence characteristics: amylovoran production, biofilm formation, growth in immature apple fruit, hypersensitive cell death elicitation, and sorbitol utilization. This study demonstrates that virulence factor expression levels account for differences in disease severity caused by wild isolates of E. amylovora on apple trees.

The C-terminal half of the HrpN virulence protein of the fire blight pathogen Erwinia amylovora is essential for its secretion and for its virulence and avirulence activities.

The HrpN (harpin) protein of the fire blight pathogen Erwinia amylovora is an essential virulence factor secreted via the bacterial type III secretion system. HrpN also has avirulence activity when delivered to tobacco by E. amylovora and has defense elicitor activity when applied to plants as a cell-free protein extract. Here, we characterize a series of random mutations in hrpN that altered the predicted amino acid sequence of the protein. Amino acid substitutions and deletions in the highly conserved, C-terminal portion of HrpN disrupted the virulence and avirulence activities of the protein. Several of these mutations produced a dominant-negative effect on E. amylovora avirulence on tobacco. None of the mutations clearly separated the virulence and avirulence activities of HrpN. Some C-terminal mutations abolished secretion of HrpN by E. amylovora. The results indicate that the C-terminal half of HrpN is essential for its secretion by E. amylovora, for its virulence activity on apple and pear, and for its avirulence activity on tobacco. In contrast, the C-terminal half of HrpN was not required for cell-free elicitor activity. This suggests that the N-terminal and C-terminal halves of HrpN mediate cell-free elicitor activity and avirulence activity, respectively.

Ozone-induced ethylene and foliar injury responses are altered in 1-aminocyclopropane-1-carboxylate synthase antisense potato plants.

• In potato (Solanum tuberosum), two 1-aminocyclopropane-1-carboxylate synthase (ACS) genes are induced by ozone (O3 ). Antisense inhibition of these ethylene (C2 H4 ) biosynthetic genes allowed us to examine the relationship between O3 -induced C2 H4 emission and foliar injury. • Thirty-two lines (antisense for ST-ACS4 or ST-ACS5) were screened in the glasshouse for acute O3 -induced C2 H4 and lesions. Stomatal conductance and ACS transcripts were quantified for selected C2 H4 -altered lines. Six lines were field-tested for chronic O3 effects. • Ten lines produced less, and four lines produced more, acute-O3 -induced C2 H4 than nontransformed (NT) plants. Ethylene levels did not appear to be correlated with stomatal conductance. ST-ACS4 and -5 transcript were reduced in transgenic plants, except in two C2 H4 over-producing lines. In the field, these C2 H4 over-producing lines displayed stunting and leaf rolling in charcoal-filtered (CF) air and chronic O3 , and they sustained the most severe O3 injury. • When C2 H4 production was strongly suppressed or enhanced, corresponding reductions or increases in lesion severity were observed, suggesting a critical role for C2 H4 in the lesion formation process during O3 stress.