Putative transcription antiterminator RfaH contributes to Erwinia amylovora virulence

Abstract

The gram-negative bacterium Erwinia amylovora causes fire blight disease of apple and pear trees. The exopolysaccharide amylovoran and lipopolysaccharides are essential E. amylovora virulence factors. Production of amylovoran and lipopolysaccharide is specified in part by genes that are members of long operons. Here, we show that full virulence of E. amylovora in apple fruitlets and tree shoots depends on the predicted transcription antiterminator RfaH. RfaH reduces pausing in the production of long transcripts having an operon polarity suppressor regulatory element within their promoter region. In E. amylovora, only the amylovoran operon and a lipopolysaccharide operon have such regulatory elements within their promoter regions and in the correct orientation. These operons showed dramatically increased polarity in the ΔrfaH mutant compared to the wild type as determined by RNA sequencing. Amylovoran and lipopolysaccharide production in vitro was reduced in rfaH mutants compared to the wild type, which probably contributes to the rfaH mutant virulence phenotype. Furthermore, type VI secretion cluster 1, which contributes to E. amylovora virulence, showed reduced expression in ΔrfaH compared to the wild type, although without an increase in polarity. The data suggest that E. amylovora RfaH directly, specifically, and exclusively suppresses operon polarity in the amylovoran operon and a lipopolysaccharide operon.

Keywords: apple; fire blight; operon; pear; type VI secretion.

FIGURE 1

The rfaH gene is essential for full virulence of Erwinia amylovora. (a) Genomic position and context of rfaH in the CFBP 1430 reference genome, with Tn5 transposon insertion locations, ΔrfaH deletion region, and complementation region indicated. bp, base pairs. (b) Disease symptoms on Gala apple fruitlets 1 week after inoculation with 2 × 10⁶ colony‐forming units (cfu) of the indicated E. amylovora strains. (c) Disease symptoms on gala apple fruitlets 1 week after inoculation with 2 × 10⁶ cfu of the indicated strains. (d) Disease severity on greenhouse‐grown Gala apple tree shoots at 1, 2 and 3 weeks postinoculation. At each time point, bars not sharing a letter have a statistically significant difference as determined by analysis of variance with post hoc Tukey tests; p ≤ 0.01, n = 20 shoots per strain. For all plots, error bars indicate standard error. Each assay was performed at least twice with the same result.

FIGURE 2

Effect of deletion of rfaH on expression of the Erwinia amylovora amylovoran ams and lipopolysaccharide rfb operons and type VI secretion system cluster 1 (T6SS‐1), as determined by RNA sequencing. (a) Relative transcript abundance for ams operon genes in ΔrfaH compared to the wild type. The ops element position is indicated. (b) Relative transcript abundance for rfb operon genes in ΔrfaH compared to the wild type. The ops element position is indicated. (c) Relative transcript abundance for T6SS‐1 genes in ΔrfaH compared to the wild type. For clarity, the commonly used E. amylovora locus names as given in GenBank annotation FN434113.1 are used wherever available; locus names or numbers from NCBI reference annotation NC_013961.1 are used for all other loci.

FIGURE 3

Deletion of rfaH exacerbates 5′ operon polarity in the Erwinia amylovora ams and rfb gene clusters. (a) RNA‐Seq coverage (reads per base) maps of the indicated gene clusters in wild‐type and ΔrfaH E. amylovora. (b) 5′ polarity of the indicated genes and gene clusters in wild‐type and ΔrfaH E. amylovora. Operon polarity was calculated as the ratio of mRNA reads mapping to the 5′‐most 15% (“start”) of the gene or gene cluster to reads mapping to the 3′‐most 15% (“end”) gene regions indicated in panel (a). Asterisks indicate statistically significant differences by t test (p ≤ 0.01, n = 3) comparison. n.s., not significant; kb, kilobase pairs.

FIGURE 4

Virulence‐related phenotypes of Erwinia amylovora rfaH mutants. (a) Amylovoran production of indicated strains after 24 h growth in amylovoran minimal medium. Asterisks indicate a statistically significant difference from the wild type by pairwise t tests (p ≤ 0.01, n = 3). (b) Lipopolysaccharide (LPS) production by indicated strains as visualized by polyacrylamide gel electrophoresis and silver staining. (c) Hypersensitive response elicitation by indicated strains infiltrated at 1 × 10⁸ cfu/ml into Nicotiana tabacum ‘Glurk’, photographed 48 h after infiltration. Four leaves were inoculated for this experiment, with all four leaves showing identical results. (d) Swimming motility in 0.3% Luria Bertani agar at 36 h postinoculation with indicated strains; the Mot⁻ negative control strain is a nonmotile Tn5 mutant of HNK06P1. Average diameter of swimming area indicated with standard deviations; the asterisk indicates the statistically significant difference from the wild type by pairwise t test (p ≤ 0.01, n = 5) comparison. Each assay was performed at least twice with the same result.