Identification of novel virulence factors in Erwinia amylovora through temporal transcriptomic analysis of infected apple flowers under field conditions.

The enterobacterial pathogen Erwinia amylovora uses multiple virulence-associated traits to cause fire blight, a devastating disease of apple and pear trees. Many virulence-associated phenotypes have been studied that are critical for virulence and pathogenicity. Despite the in vitro testing that has revealed how these systems are transcriptionally regulated, information on when and where in infected tissues these genes are being expressed is lacking. Here, we used a high-throughput sequencing approach to characterize the transcriptome of E. amylovora during disease progression on apple flowers under field infection conditions. We report that type III secretion system genes and flagellar genes are strongly co-expressed. Likewise, genes involved in biosynthesis of the exopolysaccharide amylovoran and sorbitol utilization had similar expression patterns. We further identified a group of 16 genes whose expression is increased and maintained at high levels throughout disease progression across time and tissues. We chose five of these genes for mutational analysis and observed that deletion mutants lacking these genes all display reduced symptom development on apple shoots. Furthermore, these induced genes were over-represented for genes involved in sulphur metabolism and cycling, suggesting the possibility of an important role for maintenance of oxidative homeostasis during apple flower infection.

Complete Genomes of Two Xanthomonas translucens pv. translucens Strains Isolated from Barley in North Dakota.

Xanthomonas translucens causes the disease bacterial leaf streak in several cereal crops and grasses. Here, we report the complete genome sequences of two isolates of X. translucens pv. translucens that were isolated from barley in an important cereal crop production region.

Implication of the Type III Effector RipS1 in the Cool-Virulence of Ralstonia solanacearum Strain UW551.

Members of the Ralstonia solanacearum species complex cause a variety of wilting diseases across a wide range of hosts by colonizing and blocking xylem vessels. Of great concern are race 3 biovar 2 strains of R. solanacearum capable of causing brown rot of potato at cool temperatures, which are select agents in the United States. To gain a better understanding of cool-virulence mechanisms, we generated libraries of transposon mutants in the cool-virulent R. solanacearum strain UW551 and screened 10,000 mutants using our seedling assay for significantly reduced virulence at 20°C. We found several mutants that exhibited reduced virulence at 28 and 20°C and also mutants that were only affected at the cooler temperature. One mutant of the latter chosen for further study had the transposon inserted in an intergenic region between a type III secretion system effector gene ripS1 and a major facilitator superfamily (MFS) protein gene. Gene expression analysis showed that expression of ripS1 was altered by the transposon insertion, but not the MFS protein gene. An independent mutant with this insertion upstream of ripS1 was generated and used to confirm virulence and gene expression phenotypes. The effector, RipS1, has unknown function and is part of a family of effectors belonging to the largest known type III effectors. The functional connection between RipS1 and cool-virulence of R. solanacearum UW551 suggests that RipS1 (and/or its upstream promoter element) may serve as a potential target for development of cool-virulence-specific diagnostic tools to differentiate the highly regulated cool-virulent strains from non-cool-virulent strains of R. solanacearum. Our results provide important information for continued work toward a better understanding of cool-virulence of R. solanacearum and development of proper control strategies to combat this important plant pathogen.

A high-throughput virulence screening method for the Ralstonia solanacearum species complex.

Ralstonia solanacearum species complex strains are the causative agents for wilting diseases of many plants, including the economically important brown rot of potato. We developed a high-throughput virulence screen that is implemented in 96-well microtiter plates using seedlings grown in soft water agar to save space, effort, and resources. Nicotiana glutinosa was determined to be the most effective host for this assay, and we confirmed bacterial growth and systemic spread in inoculated seedlings. In our assay, N. glutinosa seeds were sown quickly and easily on top of individual water agar wells of a 96-well plate by pipetting out desired number of seeds in an aqueous suspension. They were inoculated on the same day by first touching a bacterial colony with an autoclaved toothpick and then stabbing the toothpick into the center of the water agar well. Such inoculation method resulted in inocula above a threshold of 2 × 104 CFU per well achieving consistent virulence results and enabling reduction of inoculum preparation efforts to facilitate high-throughput screening. Our assay is suitable for forward genetic screening of a large number of strains, isolates or mutants for disease symptoms under both cool (20 °C) and warm (28 °C) temperature conditions before detailed studies can be narrowed down to a manageable number of desired candidates. Our virulence screen method provides a valuable tool for future work in understanding genetics of virulence of Rssc, especially cool virulence of the highly regulated race 3 biovar 2 group of R. solanacearum, leading toward development of effective control strategies.

Genetic Dissection of the Erwinia amylovora Disease Cycle.

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.

Orchestration of virulence factor expression and modulation of biofilm dispersal in Erwinia amylovora through activation of the Hfq-dependent small RNA RprA.

Erwinia amylovora is the causative agent of the devastating disease fire blight of pome fruit trees. After infection of host plant leaves at apple shoot tips, E. amylovora cells form biofilms in xylem vessels, restrict water flow, and cause wilting symptoms. Although E. amylovora is well known to be able to cause systemic infection, how biofilm cells of E. amylovora transit from the sessile mode of growth in xylem to the planktonic mode of growth in cortical parenchyma remains unknown. Increasing evidence has suggested the important modulatory roles of Hfq-dependent small RNAs (sRNAs) in the pathogenesis of E. amylovora. Here, we demonstrate that the sRNA RprA acts as a positive regulator of amylovoran exopolysaccharide production, the type III secretion system (T3SS), and flagellar-dependent motility, and as a negative regulator of levansucrase activity and cellulose production. We also show that RprA affects the promoter activity of multiple virulence factor genes and regulates hrpS, a critical T3SS regulator, at the posttranscriptional level. We determined that rprA expression can be activated by the Rcs phosphorelay, and that expression is active during T3SS-mediated host infection in an immature pear fruit infection model. We further showed that overexpression of rprA activated the in vitro dispersal of E. amylovora cells from biofilms. Thus, our investigation of the varied role of RprA in affecting E. amylovora virulence provides important insights into the functions of this sRNA in biofilm control and systemic infection.

Small RNA ArcZ Regulates Oxidative Stress Response Genes and Regulons in Erwinia amylovora.

Erwinia amylovora, causative agent of fire blight disease of apple and pear trees, has evolved to use small RNAs for post-transcriptional regulation of virulence traits important for disease development. The sRNA ArcZ regulates several virulence traits, and to better understand its roles, we conducted a transcriptomic comparison of wild-type and ΔarcZ mutant E. amylovora. We found that ArcZ regulates multiple cellular processes including genes encoding enzymes involved in mitigating the threat of reactive oxygen species (katA, tpx, osmC), and that the ΔarcZ mutant has reduced catalase activity and is more susceptible to exogenous hydrogen peroxide. We quantified hydrogen peroxide production by apple leaves inoculated with E. amylovora and found that the while wild-type E. amylovora cells produce enough catalase to cope with defense peroxide, the ΔarcZ mutant is likely limited in virulence because of inability to cope with peroxide levels in host leaves. We further found that the ArcZ regulon overlaps significantly with the regulons of transcription factors involved in oxidative sensing including Fnr and ArcA. In addition, we show that ArcZ regulates arcA at the post-transcriptional level suggesting a role for this system in mediating adaptations to oxidative state, especially during disease development.

Functional Characterization of a Global Virulence Regulator Hfq and Identification of Hfq-Dependent sRNAs in the Plant Pathogen Pantoea ananatis.

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.

Chromosomally Encoded hok-sok Toxin-Antitoxin System in the Fire Blight Pathogen Erwinia amylovora: Identification and Functional Characterization.

Toxin-antitoxin (TA) systems are genetic elements composed of a protein toxin and a counteracting antitoxin that is either a noncoding RNA or protein. In type I TA systems, the antitoxin is a noncoding small RNA (sRNA) that base pairs with the cognate toxin mRNA interfering with its translation. Although type I TA systems have been extensively studied in Escherichia coli and a few human or animal bacterial pathogens, they have not been characterized in plant-pathogenic bacteria. In this study, we characterized a chromosomal locus in the plant pathogen Erwinia amylovora Ea1189 that is homologous to the hok-sok type I TA system previously identified in the Enterobacteriaceae-restricted plasmid R1. Phylogenetic analysis indicated that the chromosomal location of the hok-sok locus is, thus far, unique to E. amylovora We demonstrated that ectopic overexpression of hok is highly toxic to E. amylovora and that the sRNA sok reversed the toxicity of hok through mok, a reading frame presumably translationally coupled with hok We also identified the region that is essential for maintenance of the main toxicity of Hok. Through a hok-sok deletion mutant (Ea1189Δhok-sok), we determined the contribution of the hok-sok locus to cellular growth, micromorphology, and catalase activity. Combined, our findings indicate that the hok-sok TA system, besides being potentially self-toxic, provides fitness advantages to E. amylovoraIMPORTANCE Bacterial toxin-antitoxin systems have received great attention because of their potential as targets for antimicrobial development and as tools for biotechnology. Erwinia amylovora, the causal agent of fire blight disease on pome fruit trees, is a major plant-pathogenic bacterium. In this study, we identified and functionally characterized a unique chromosomally encoded hok-sok toxin-antitoxin system in E. amylovora that resembles the plasmid-encoded copies of this system in other Enterobacteriaceae This study of a type I toxin-antitoxin system in a plant-pathogenic bacterium provides the basis to further understand the involvement of toxin-antitoxin systems during infection by a plant-pathogenic bacterium. The new linkage between the hok-sok toxin-antitoxin system and the catalase-mediated oxidative stress response leads to additional considerations of targeting this system for antimicrobial development.

The Leucine-Responsive Regulatory Protein Lrp Participates in Virulence Regulation Downstream of Small RNA ArcZ in Erwinia amylovora.

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.

Three Hfq-dependent small RNAs regulate flagellar motility in the fire blight pathogen Erwinia amylovora.

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.

Yersinia pseudotuberculosis BarA-UvrY Two-Component Regulatory System Represses Biofilms via CsrB.

The formation of biofilms by Yersinia pseudotuberculosis (Yptb) and Y. pestis requires the hmsHFRS genes, which direct production of a polysaccharide extracellular matrix (Hms-ECM). Despite possessing identical hmsHFRS sequences, Yptb produces much less Hms-ECM than Y. pestis. The regulatory influences that control Yptb Hms-ECM production and biofilm formation are not fully understood. In this study, negative regulators of biofilm production in Yptb were identified. Inactivation of the BarA/UvrY two-component system or the CsrB regulatory RNA increased binding of Congo Red dye, which correlates with extracellular polysaccharide production. These mutants also produced biofilms that were substantially more cohesive than the wild type strain. Disruption of uvrY was not sufficient for Yptb to cause proventricular blockage during infection of Xenopsylla cheopis fleas. However, this strain was less acutely toxic toward fleas than wild type Yptb. Flow cytometry measurements of lectin binding indicated that Yptb BarA/UvrY/CsrB mutants may produce higher levels of other carbohydrates in addition to poly-GlcNAc Hms-ECM. In an effort to characterize the relevant downstream targets of the BarA/UvrY system, we conducted a proteomic analysis to identify proteins with lower abundance in the csrB::Tn5 mutant strain. Urease subunit proteins were less abundant and urease enzymatic activity was lower, which likely reduced toxicity toward fleas. Loss of CsrB impacted expression of several potential regulatory proteins that may influence biofilms, including the RcsB regulator. Overexpression of CsrB did not alter the Congo-red binding phenotype of an rcsB::Tn5 mutant, suggesting that the effect of CsrB on biofilms may require RcsB. These results underscore the regulatory and compositional differences between Yptb and Y. pestis biofilms. By activating CsrB expression, the Yptb BarA/UvrY two-component system has pleiotropic effects that impact biofilm production and stability.