Erwinia amylovora causes the devastating fire blight disease of apple and pear trees. During systemic infection of host trees, pathogen cells must rapidly respond to changes in their environment as they move through different host tissues that present distinct challenges and sources of nutrition. Growing evidence indicates that small RNAs (sRNAs) play an important role in disease progression as posttranscriptional regulators. The sRNA ArcZ positively regulates the motility phenotype and transcription of flagellar genes in E. amylovora Ea1189 yet is a direct repressor of translation of the flagellar master regulator, FlhD. We utilized transposon mutagenesis to conduct a forward genetic screen and identified suppressor mutations that increase motility in the Ea1189ΔarcZ mutant background. This enabled us to determine that the mechanism of transcriptional activation of the flhDC mRNA by ArcZ is mediated by the leucine-responsive regulatory protein, Lrp. We show that Lrp contributes to expression of virulence and several virulence-associated traits, including production of the exopolysaccharide amylovoran, levansucrase activity, and biofilm formation. We further show that Lrp is regulated posttranscriptionally by ArcZ through destabilization of lrp mRNA. Thus, ArcZ regulation of FlhDC directly and indirectly through Lrp forms an incoherent feed-forward loop that regulates levansucrase activity and motility as outputs. This work identifies Lrp as a novel participant in virulence regulation in E. amylovora and places it in the context of a virulence-associated regulatory network. IMPORTANCE Fire blight disease continues to plague the commercial production of apples and pears despite more than a century of research into disease epidemiology and disease control. The causative agent of fire blight, Erwinia amylovora coordinates turning on or off specific virulence-associated traits at the appropriate time during disease development. The development of novel control strategies requires an in-depth understanding of E. amylovora regulatory mechanisms, including regulatory control of virulence-associated traits. This study investigates how the small RNA ArcZ regulates motility at the transcriptional level and identifies the transcription factor Lrp as a novel participant in the regulation of several virulence-associated traits. We report that ArcZ and Lrp together affect key virulence-associated traits through integration of transcriptional and posttranscriptional mechanisms. Further understanding of the topology of virulence regulatory networks can uncover weak points that can subsequently be exploited to control E. amylovora.

Fire blight, caused by the bacterial phytopathogen Erwinia amylovora, is an economically important and mechanistically complex disease that affects apple and pear production in most geographic production hubs worldwide. We compile, assess, and present a genetic outlook on the progression of an E. amylovora infection in the host. We discuss the key aspects of type III secretion–mediated infection and systemic movement, biofilm formation in xylem, and pathogen dispersal via ooze droplets, a concentrated suspension of bacteria and exopolysaccharide components. We present an overall outlook on the genetic elements contributing to E. amylovora pathogenesis, including an exploration of the impact of floral microbiomes on E. amylovora colonization, and summarize the current knowledge of host responses to an incursion and how this response stimulates further infection and systemic spread. We hope to facilitate the identification of new, unexplored areas of research in this pathosystem that can help identify evolutionarily susceptible genetic targets to ultimately aid in the design of sustainable strategies for fire blight disease mitigation. Expected final online publication date for the Annual Review of Phytopathology, Volume 59 is August 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

To successfully infect plant hosts, the collective regulation of virulence factors in a bacterial pathogen is crucial. Hfq is an RNA chaperone protein that facilitates the small RNA (sRNA) regulation of global gene expression at the post-transcriptional level. In this study, the functional role of Hfq in a broad host range phytopathogen Pantoea ananatis was determined. Inactivation of the hfq gene in P. ananatis LMG 2665T resulted in the loss of pathogenicity and motility. In addition, there was a significant reduction of quorum sensing signal molecule acyl-homoserine lactone (AHL) production and biofilm formation. Differential sRNA expression analysis between the hfq mutant and wild-type strains of P. ananatis revealed 276 sRNAs affected in their abundance by the loss of hfq at low (OD600 = 0.2) and high cell (OD600 = 0.6) densities. Further analysis identified 25 Hfq-dependent sRNAs, all showing a predicted Rho-independent terminator of transcription and mapping within intergenic regions of the P. ananatis genome. These included known sRNAs such as ArcZ, FnrS, GlmZ, RprA, RyeB, RyhB, RyhB2, Spot42, and SsrA, and 16 novel P. ananatis sRNAs. The current study demonstrated that Hfq is an important component of the collective regulation of virulence factors and sets a foundation for understanding Hfq-sRNA mediated regulation in the phytopathogen P. ananatis.

Erwinia amylovora, the causative agent of fire blight disease of apple and pear trees, causes disease on flowers by invading natural openings at the base of the floral cup. To reach these openings, the bacteria use flagellar motility to swim from stigma tips to the hypanthium and through nectar. We have previously shown that the Hfq-dependent sRNAs ArcZ, OmrAB and RmaA regulate swimming motility in E. amylovora. Here, we tested these three sRNAs to determine at what regulatory level they exert their effects and to what extent they can complement each other. We found that ArcZ and OmrAB repress the flagellar master regulator flhD post-transcriptionally. We also found that ArcZ and RmaA positively regulate flhD at the transcriptional level. The role of ArcZ as an activator of flagellar motility appears to be unique to E. amylovora and may have recently evolved. Our results suggest that the Hfq-dependent sRNAs ArcZ, OmrAB and RmaA play an integral role in regulation of flagellar motility by acting primarily on the master regulator, FlhD, but also through additional factors.