Genetic characterization of the HrpL regulon of the fire blight pathogenErwinia amylovorareveals novel virulence factors

Abstract

The bacterial pathogen Erwinia amylovora is the causal agent of fire blight, an economically significant disease of apple and pear. Disease initiation by E. amylovora requires the translocation of effector proteins into host cells via the hypersensitive response and pathogenicity (hrp) type III secretion system (T3SS). The alternative sigma factor HrpL positively regulates the transcription of structural and translocated components of the T3SS via hrp promoter elements. To characterize genome-wide HrpL-dependent gene expression in E. amylovora Ea1189, wild-type and Ea1189ΔhrpL strains were cultured in hrp-inducing minimal medium, and total RNA was compared using a custom microarray designed to represent the annotated genes of E. amylovora ATCC 49946. The results revealed 24 genes differentially regulated in Ea1189ΔhrpL relative to Ea1189 with fold-change expression ratios greater than 1.5; of these, 19 genes exhibited decreased transcript abundance and five genes showed increased transcript abundance relative to Ea1189. To expand our understanding of the HrpL regulon and to elucidate direct versus indirect HrpL-mediated effects on gene expression, the genome of E. amylovora ATCC 49946 was examined in silico using a hidden Markov model assembled from known Erwinia spp. hrp promoters. This technique identified 15 putative type III novel hrp promoters, seven of which were validated with quantitative polymerase chain reaction based on expression analyses. It was found that HrpL-regulated genes encode all known components of the hrp T3SS, as well as five putative type III effectors. Eight genes displayed apparent indirect HrpL regulation, suggesting that the HrpL regulon is connected to downstream signalling networks. The construction of deletion mutants of three novel HrpL-regulated genes resulted in the identification of additional virulence factors as well as mutants displaying abnormal motility and biofilm phenotypes.

HopX1 in Erwinia amylovora Functions as an Avirulence Protein in Apple and Is Regulated by HrpL

“…Forty-four percent of the set of genes predicted in our work probably differ because the profiles used differ in the set of hrp boxes and the alignment of the training set. As in previous work (16,17,26,37,40), our profiles are conserved in the Ϫ35 region of the promoter sequences; however, they differ in the spacer region length and the Ϫ10 region (GGAAC-19-20-ACNNA). These differences are enough to identify different promoter sequences on the same genome or produce different scores.…”

“…Interestingly, hopX1 Ea and hopAK1 Ea were not present in the D. dadantii genome, as orthologs of the latter sequences were sought in the ASAP database, indicating that the two pathogens have diverged in their pathogenic strategies. Recent independent analyses of the HrpL regulon of Ea1189 using the published genome of ATCC 49946 for the bioinformatics analysis identified 16 genes directly regulated by HrpL (26). Our work predicted nine common genes with the latter, including four encoding the T3SS-associated proteins: avrRpt2 Ea , hopPtoC Ea , hopX1 Ea (eop3), and hopAK1 Ea (eop2) (see Table S1 in the supplemental material).…”

Global Small RNA Chaperone Hfq and Regulatory Small RNAs Are Important Virulence Regulators in Erwinia amylovora

“…The T3SS of E. amylovora is mainly required for the translocation of the effector protein DspE into plant cells; DspE is required for the pathogenicity of E. amylovora, multiplication in planta, and disease promotion by the alteration of host cell defenses (6)(7)(8). Genes associated with production of the T3SS and type III effector genes, including the alternate sigma factor hrpL, type III pilus hrpA, translocator hrpN, and effector dspE, are expressed at between 6 and 48 h of inoculation to flower stigmas (9), which correlates well with regulatory studies of the HrpL regulon performed in vitro (10). In addition to the T3SS and DspE, a third pathogenicity factor produced by E. amylovora is the exopolysaccharide amylovoran, which is an important component of biofilms (11,12).…”

“…Compared to previously characterized regulators such as HrpL, the master regulator for T3SS (10), and RcsBCD, the key regulator of amylovoran production (42), Hfq appears to more broadly regulate many virulence determinants, including amylovoran production, biofilm formation, T3SS translocation and secretion, and bacterial motility. Thus, Hfq and the sRNAs that it regulates likely play a central role in the fine-tuning of virulence gene expression in E. amylovora.…”

“…In order to successfully establish infections, E. amylovora utilizes a complicated regulatory network involving two-component signal transduction systems, alternate sigma factors, quorum sensing, and the second messenger cyclic di-GMP to collectively control the expression of virulence genes at different stages of infection (1,4,10,(15)(16)(17)(18). In addition, the requirement at different times of infection for the T3SS and biofilm formation and the importance of cellular motility suggest that E. amylovora cells must be able to rapidly alter the production of distinct virulence factors in response to specific host cues.…”