Molecular regulation of virulence in Legionella pneumophila.

Legionella pneumophila is a gram-negative bacteria found in natural and anthropogenic aquatic environments such as evaporative cooling towers, where it reproduces as an intracellular parasite of cohabiting protozoa. If L. pneumophila is aerosolized and inhaled by a susceptible person, bacteria may colonize their alveolar macrophages causing the opportunistic pneumonia Legionnaires' disease. L. pneumophila utilizes an elaborate regulatory network to control virulence processes such as the Dot/Icm Type IV secretion system and effector repertoire, responding to changing nutritional cues as their host becomes depleted. The bacteria subsequently differentiate to a transmissive state that can survive in the environment until a replacement host is encountered and colonized. In this review, we discuss the lifecycle of L. pneumophila and the molecular regulatory network that senses nutritional depletion via the stringent response, a link to stationary phase-like metabolic changes via alternative sigma factors, and two-component systems that are homologous to stress sensors in other pathogens, to regulate differentiation between the intracellular replicative phase and more transmissible states. Together, we highlight how this prototypic intracellular pathogen offers enormous potential in understanding how molecular mechanisms enable intracellular parasitism and pathogenicity.

Tissue-specific mRNA profiling of the Brassica napus-Sclerotinia sclerotiorum interaction uncovers novel regulators of plant immunity.

White mold is caused by the fungal pathogen Sclerotinia sclerotiorum and leads to rapid and significant loss in plant yield. Among its many brassicaceous hosts, including Brassica napus (canola) and Arabidopsis, the response of individual tissue layers directly at the site of infection has yet to be explored. Using laser microdissection coupled with RNA sequencing, we profiled the epidermis, mesophyll, and vascular leaf tissue layers of B. napus in response to S. sclerotiorum. High-throughput tissue-specific mRNA sequencing increased the total number of detected transcripts compared with whole-leaf assessments and provided novel insight into the conserved and specific roles of ontogenetically distinct leaf tissue layers in response to infection. When subjected to pathogen infection, the epidermis, mesophyll, and vasculature activate both specific and shared gene sets. Putative defense genes identified through transcription factor network analysis were then screened for susceptibility against necrotrophic, hemi-biotrophic, and biotrophic pathogens. Arabidopsis deficient in PR5-like RECEPTOR KINASE (PR5K) mRNA levels were universally susceptible to all pathogens tested and were further characterized to identify putative interacting partners involved in the PR5K signaling pathway. Together, these data provide insight into the complexity of the plant defense response directly at the site of infection.

The effect of polyhydroxyalkanoates in Pseudomonas chlororaphis PA23 biofilm formation, stress endurance, and interaction with the protozoan predator Acanthamoeba castellanii.

Pseudomonas chlororaphis PA23 is a biocontrol agent capable of protecting canola against the fungal pathogen Sclerotinia sclerotiorum. In addition to producing antifungal compounds, this bacterium synthesizes and accumulates polyhydroxyalkanoate (PHA) polymers as carbon and energy storage compounds. Because the role of PHA in PA23 physiology is currently unknown, we investigated the impact of this polymer on stress resistance, adherence to surfaces, and interaction with the protozoan predator Acanthamoeba castellanii. Three PHA biosynthesis mutants were created, PA23phaC1, PA23phaC1ZC2, and PA23phaC1ZC2D, which accumulated reduced PHA. Our phenotypic assays revealed that PA23phaC1ZC2D produced less phenazine (PHZ) compared with the wild type (WT) and the phaC1 and phaC1ZC2 mutants. All three mutants exhibited enhanced sensitivity to UV irradiation, starvation, heat stress, cold stress, and hydrogen peroxide. Moreover, motility, exopolysaccharide production, biofilm formation, and root attachment were increased in strains with reduced PHA levels. Interaction studies with the amoeba A. castellanii revealed that the WT and the phaC1 and phaC1ZC2 mutants were consumed less than the phaC1ZC2D mutant, likely due to decreased PHZ production by the latter. Collectively these findings indicate that PHA accumulation enhances PA23 resistance to a number of stresses in vitro, which could improve the environmental fitness of this bacterium in hostile environments.

Microbial Community Dynamics of Soybean (Glycine max) Is Affected by Cropping Sequence.

The microbial composition of the rhizosphere soil could be an important determinant of crop yield, pathogen resistance, and other beneficial attributes in plants. However, little is known about the impact of cropping sequences on microbial community dynamics, especially in economically important species like soybean. Using 2-year crop sequences of corn-soybean, canola-soybean, and soybean-soybean, we investigated how crops from the previous growing season influenced the structure of the microbiome in both the bulk soil and soybean rhizosphere. A combination of marker-based Illumina sequencing and bioinformatics analyses was used to show that bacterial species richness and evenness in the soybean rhizosphere soil were similar following canola and soybean compared to a previous corn sequence. However, fungal species richness and evenness remained unaffected by crop sequence. In addition, bacterial and fungal species diversity in both the bulk and soybean rhizosphere soil were not influenced by crop sequence. Lastly, the corn-soybean sequence significantly differed in the relative abundance of certain bacterial and fungal classes in both the soybean rhizosphere and bulk soil. While canola-soybean and a continuous soybean sequence did not, suggesting that a preceding corn sequence may reduce the occurrence of overall bacterial and fungal community members. For the present study, crop sequence impacts bacterial diversity and richness in both the bulk soil and soybean rhizosphere soil whereas fungal diversity and richness are resilient to crop sequence practices. Together, these findings could help drive decision making for annual crop and soil management practices.

Degradation of BTEX mixture by a new Pseudomonas putida strain: role of the quorum sensing in the modulation of the upper BTEX oxidative pathway.

A new Pseudomonas putida strain (AQ8) was isolated from a decommissioned oil refinery's soil in Italy and characterized for its ability to degrade BTEX. The draft genome of the new strain was sequenced and annotated for genes that encode enzymes putatively involved in BTEX degradation and quorum sensing. The strain was transformed with a plasmid expressing lactonase, which cleaves the autoinducer quorum sensing signal molecule, the acyl-homoserine lactone, to obtain a quorum sensing minus strain. P. putida AQ8 depleted the 40% on average of all the components of the initial BTEX concentration in 36 h. The quorum sensing minus strain, in the same time interval, depleted only the 10% of the initial BTEX concentration. The role of quorum sensing in regulating the expression of the annotated benzene/toluene dioxygenase gene (benzA) and biphenyl/toluene/benzene dioxygenase (bphA) genes, which are involved in BTEX degradation, was studied by quantitative RT-real-time quantitative (q)PCR analysis. The qPCR data showed decreased levels of expression of the benzA and bphA genes in the quorum sensing minus strain. Our results showed, for the first time, quorum sensing modulation of the level of transcription of dioxygenase genes in the upper BTEX oxidation pathway.

Investigation of the quorum-sensing regulon of the biocontrol bacterium Pseudomonas chlororaphis strain PA23.

Pseudomonas chlororaphis strain PA23 is a biocontrol agent capable of protecting canola from stem rot disease caused by the fungal pathogen Sclerotinia sclerotiorum. PA23 produces several inhibitory compounds that are under control of a complex regulatory network. Included in this cascade is the PhzRI quorum sensing (QS) system, which plays an essential role in PA23 biocontrol, as well as CsaRI and AurRI, which have not yet been characterized in PA23. The focus of the current study was to employ RNA sequencing to explore the spectrum of PA23 genes under QS control. In this work, we investigated genes under the control of the main QS transcriptional regulator, PhzR, as well as those differentially expressed in an AHL-deficient strain, PA23-6863, which constitutively expresses an AiiA lactonase, rendering the strain QS defective. Transcriptomic profiling revealed 545 differentially expressed genes (365 downregulated; 180 upregulated) in the phzR mutant and 534 genes (382 downregulated; 152 upregulated) in the AHL-deficient PA23-6863. In both strains, decreased expression of phenazine, pyrrolnitrin, and exoprotease biosynthetic genes was observed. We have previously reported that QS activates expression of these genes and their encoded products. In addition, elevated siderophore and decreased chitinase gene expression was observed in the QS-deficient stains, which was confirmed by phenotypic analysis. Inspection of the promoter regions revealed the presence of "phz-box" sequences in only 58 of the 807 differentially expressed genes, suggesting that much of the QS regulon is indirectly regulated. Consistent with this notion, 41 transcriptional regulators displayed altered expression in one or both of the QS-deficient strains. Collectively, our findings indicate that QS governs expression of approximately 13% of the PA23 genome affecting diverse functions ranging from secondary metabolite production to general metabolism.

Quorum sensing and the anaerobic regulator (ANR) control polyhydroxyalkanoate (PHA) production in Pseudomonas chlororaphis PA23.

Pseudomonas chlororaphis PA23 is a biocontrol agent that, in addition to producing antifungal compounds, synthesizes polyhydroxyalkanoate (PHA) polymers as a carbon and energy sink. Quorum sensing (QS) and the anaerobic regulator (ANR) are required for PA23-mediated fungal suppression; however, the role of these regulators in PHA production is unknown. Strains lacking either QS or ANR accumulated less PHA polymers when propagated on Ramsay's minimal medium (RMM) with glucose or octanoate as the carbon source. In the acyl-homoserine lactone (AHL)-deficient background, all six of the genes in the pha locus (phaC1, phaC2, phaZ, phaD, phaF, phaI) showed reduced expression in RMM glucose, and all except phaC2 were repressed in RMM octanoate. Although changes in gene activity were observed in the anr mutant, they were less pronounced. Analysis of the promoter regions for QS- and ANR-binding consensus sequences revealed putative phzboxes upstream of phaZ and phaI, but no anr boxes were identified. Our findings indicate that altered pha gene expression likely contributes to the lower PHA accumulation in the QS- and ANR-deficient strains, which may be in part indirectly mediated. This study is the first to show that mcl-PHA production is under QS and ANR control.

A comparative study of the gut microbiota in immune-mediated inflammatory diseases-does a common dysbiosis exist?

BACKGROUND: Immune-mediated inflammatory disease (IMID) represents a substantial health concern. It is widely recognized that IMID patients are at a higher risk for developing secondary inflammation-related conditions. While an ambiguous etiology is common to all IMIDs, in recent years, considerable knowledge has emerged regarding the plausible role of the gut microbiome in IMIDs. This study used 16S rRNA gene amplicon sequencing to compare the gut microbiota of patients with Crohn's disease (CD; N = 20), ulcerative colitis (UC; N = 19), multiple sclerosis (MS; N = 19), and rheumatoid arthritis (RA; N = 21) versus healthy controls (HC; N = 23). Biological replicates were collected from participants within a 2-month interval. This study aimed to identify common (or unique) taxonomic biomarkers of IMIDs using both differential abundance testing and a machine learning approach. RESULTS: Significant microbial community differences between cohorts were observed (pseudo F = 4.56; p = 0.01). Richness and diversity were significantly different between cohorts (pFDR < 0.001) and were lowest in CD while highest in HC. Abundances of Actinomyces, Eggerthella, Clostridium III, Faecalicoccus, and Streptococcus (pFDR < 0.001) were significantly higher in all disease cohorts relative to HC, whereas significantly lower abundances were observed for Gemmiger, Lachnospira, and Sporobacter (pFDR < 0.001). Several taxa were found to be differentially abundant in IMIDs versus HC including significantly higher abundances of Intestinibacter in CD, Bifidobacterium in UC, and unclassified Erysipelotrichaceae in MS and significantly lower abundances of Coprococcus in CD, Dialister in MS, and Roseburia in RA. A machine learning approach to classify disease versus HC was highest for CD (AUC = 0.93 and AUC = 0.95 for OTU and genus features, respectively) followed by MS, RA, and UC. Gemmiger and Faecalicoccus were identified as important features for classification of subjects to CD and HC. In general, features identified by differential abundance testing were consistent with machine learning feature importance. CONCLUSIONS: This study identified several gut microbial taxa with differential abundance patterns common to IMIDs. We also found differentially abundant taxa between IMIDs. These taxa may serve as biomarkers for the detection and diagnosis of IMIDs and suggest there may be a common component to IMID etiology.

Identification and application of exogenous dsRNA confers plant protection against Sclerotinia sclerotiorum and Botrytis cinerea.

Sclerotinia sclerotiorum, the causal agent of white stem rot, is responsible for significant losses in crop yields around the globe. While our understanding of S. sclerotiorum infection is becoming clearer, genetic control of the pathogen has been elusive and effective control of pathogen colonization using traditional broad-spectrum agro-chemical protocols are less effective than desired. In the current study, we developed species-specific RNA interference-based control treatments capable of reducing fungal infection. Development of a target identification pipeline using global RNA sequencing data for selection and application of double stranded RNA (dsRNA) molecules identified single gene targets of the fungus. Using this approach, we demonstrate the utility of this technology through foliar applications of dsRNAs to the leaf surface that significantly decreased fungal infection and S. sclerotiorum disease symptoms. Select target gene homologs were also tested in the closely related species, Botrytis cinerea, reducing lesion size and providing compelling evidence of the adaptability and flexibility of this technology in protecting plants against devastating fungal pathogens.

Polyhydroxyalkanoate (PHA) Polymer Accumulation and pha Gene Expression in Phenazine (phz⁻) and Pyrrolnitrin (prn⁻) Defective Mutants of Pseudomonas chlororaphis PA23.

Pseudomonas chlororaphis PA23 was isolated from the rhizosphere of soybeans and identified as a biocontrol bacterium against Sclerotinia sclerotiorum, a fungal plant pathogen. This bacterium produces a number of secondary metabolites, including phenazine-1-carboxylic acid, 2-hydroxyphenazine, pyrrolnitrin (PRN), hydrogen cyanide, proteases, lipases and siderophores. It also synthesizes and accumulates polyhydroxyalkanoate (PHA) polymers as carbon and energy storage compounds under nutrient-limited conditions. Pseudomonads like P. chlororaphis metabolize glucose via the Entner-Doudoroff and Pentose Phosphate pathways, which provide precursors for phenazine production. Mutants defective in phenazine (PHZ; PA23-63), PRN (PA23-8), or both (PA23-63-1) accumulated higher concentrations of PHAs than the wild-type strain (PA23) when cultured in Ramsay's Minimal Medium with glucose or octanoic acid as the carbon source. Expression levels of six pha genes, phaC1, phaZ, phaC2, phaD, phaF, and phaI, were compared with wild type PA23 by quantitative real time polymerase chain reaction (qPCR). The qPCR studies indicated that there was no change in levels of transcription of the PHA synthase genes phaC1 and phaC2 in the phz⁻ (PA23-63) and phz⁻ prn⁻ (PA23-63-1) mutants in glucose medium. There was a significant increase in expression of phaC2 in octanoate medium. Transcription of phaD, phaF and phaI increased significantly in the phz⁻ prn⁻ (PA23-63-1) mutant. Mutations in regulatory genes like gacS, rpoS, and relA/spoT, which affect PHZ and PRN production, also resulted in altered gene expression. The expression of phaC1, phaC2, phaF, and phaI genes was down-regulated significantly in gacS and rpoS mutants. Thus, it appears that PHZ, PRN, and PHA production is regulated by common mechanisms. Higher PHA production in the phz⁻ (PA23-63), prn- (PA23-8), and phz⁻ prn⁻ (PA23-63-1) mutants in octanoic medium could be correlated with higher expression of phaC2. Further, the greater PHA production observed in the phz⁻ and prn⁻ mutants was not due to increased transcription of PHA synthase genes in glucose medium, but due to more accessibility of carbon substrates and reducing power, which were otherwise used for the synthesis of PHZ and PRN.

RNA sequencing of Brassica napus reveals cellular redox control of Sclerotinia infection.

Brassica napus is one of the world's most valuable oilseeds and is under constant pressure by the necrotrophic fungal pathogen, Sclerotinia sclerotiorum, the causal agent of white stem rot. Despite our growing understanding of host pathogen interactions at the molecular level, we have yet to fully understand the biological processes and underlying gene regulatory networks responsible for determining disease outcomes. Using global RNA sequencing, we profiled gene activity at the first point of infection on the leaf surface 24 hours after pathogen exposure in susceptible (B. napus cv. Westar) and tolerant (B. napus cv. Zhongyou 821) plants. We identified a family of ethylene response factors that may contribute to host tolerance to S. sclerotiorum by activating genes associated with fungal recognition, subcellular organization, and redox homeostasis. Physiological investigation of redox homeostasis was further studied by quantifying cellular levels of the glutathione and ascorbate redox pathway and the cycling enzymes associated with host tolerance to S. sclerotiorum. Functional characterization of an Arabidopsis redox mutant challenged with the fungus provides compelling evidence into the role of the ascorbate-glutathione redox hub in the maintenance and enhancement of plant tolerance against fungal pathogens.

Synthesis of polyhydroxyalkanoates (PHAs) from vegetable oils and free fatty acids by wild-type and mutant strains of Pseudomonas chlororaphis.

Pseudomonas chlororaphis PA23 was isolated from soybean roots as a plant-growth-promoting rhizobacterium. This strain secretes a wide range of compounds, including the antibiotics phenazine-1-carboxylic acid (PCA), pyrrolnitrin, and 2-hydroxyphenazine. We have determined that P. chlororaphis PA23 can synthesize medium-chain-length polyhydroxyalkanoate (PHA) polymers utilizing free fatty acids, such as octanoic acid and nonanoic acid, as well as vegetable oils as sole carbon sources. Genome analysis identified a pha operon containing 7 genes in P. chlororaphis PA23 that were highly conserved. A nonpigmented strain that does not synthesize PCA, P. chlororaphis PA23-63, was also studied for PHA production. Pseudomonas chlororaphis PA23-63 produced 2.42-5.14 g/L cell biomass and accumulated PHAs from 11.7% to 32.5% cdm when cultured with octanoic acid, nonanoic acid, fresh canola oil, waste canola fryer oil, or biodiesel-derived waste free fatty acids under batch culture conditions. The subunit composition of the PHAs produced from fresh canola oil, waste canola fryer oil, or biodiesel-derived free fatty acids did not differ significantly. Addition of octanoic acid and nonanoic acid to canola oil cultures increased PHA production, but addition of glucose did not. PHA production in the phz mutant, P. chlororaphis PA23-63, was greater than that in the parent strain.

The biocontrol agent Pseudomonas chlororaphis PA23 primes Brassica napus defenses through distinct gene networks.

BACKGROUND: The biological control agent Pseudomonas chlororaphis PA23 is capable of protecting Brassica napus (canola) from the necrotrophic fungus Sclerotinia sclerotiorum via direct antagonism. While we have elucidated bacterial genes and gene products responsible biocontrol, little is known about how the host plant responds to bacterial priming on the leaf surface, including global changes in gene activity in the presence and absence of S. sclerotiorum. RESULTS: Application of PA23 to the aerial surfaces of canola plants reduced the number of S. sclerotiorum lesion-forming petals by 91.1%. RNA sequencing of the host pathogen interface showed that pretreatment with PA23 reduced the number of genes upregulated in response to S. sclerotiorum by 16-fold. By itself, PA23 activated unique defense networks indicative of defense priming. Genes encoding MAMP-triggered immunity receptors detecting flagellin and peptidoglycan were downregulated in PA23 only-treated plants, consistent with post-stimulus desensitization. Downstream, we observed reactive oxygen species (ROS) production involving low levels of H2O2 and overexpression of genes associated with glycerol-3-phosphate (G3P)-mediated systemic acquired resistance (SAR). Leaf chloroplasts exhibited increased thylakoid membrane structures and chlorophyll content, while lipid metabolic processes were upregulated. CONCLUSION: In addition to directly antagonizing S. sclerotiorum, PA23 primes the plant defense response through induction of unique local and systemic defense networks. This study provides novel insight into the effects of biocontrol agents applied to the plant phyllosphere. Understanding these interactions will aid in the development of biocontrol systems as an alternative to chemical pesticides for protection of important crop systems.

The global regulator ANR is essential for Pseudomonas chlororaphis strain PA23 biocontrol.

Pseudomonas chlororaphis PA23 is a biocontrol agent capable of protecting canola from stem rot disease caused by the fungus Sclerotinia sclerotiorum. The focus of the current study was to elucidate the role of the transcriptional regulator ANR in the biocontrol capabilities of this bacterium. An anr mutant was created, PA23anr, that was devoid antifungal activity. In other pseudomonads, ANR is essential for regulating HCN production. Characterization of PA23anr revealed that, in addition to HCN, ANR controls phenazine (PHZ), pyrrolnitrin (PRN), protease and autoinducer (AHL) signal molecule production. In gene expression studies, hcnA, phzA, prnA and phzI were found to be downregulated, consistent with our endproduct analysis. Because the phenotype of PA23anr closely resembles that of quorum sensing (QS)-deficient strains, we explored whether there is a connection between ANR and the PhzRI QS system. Both phzI and phzR are positively regulated by ANR, whereas PhzR represses anr transcription. Complementation of PA23anr with pUCP-phzR, C6-HSL or both yielded no change in phenotype. Conversely, PA23phzR harbouring pUCP23-anr exhibited partial-to-full restoration of antifungal activity, HCN, PRN and AHL production together with hcnA, prnA, phzI and rpoS expression. PHZ and protease production remained unchanged indicating that ANR can complement the QS-deficient phenotype with respect to some but not all traits. Our experiments were conducted at atmospheric O2 levels underscoring the fact that ANR has a profound effect on PA23 physiology under aerobic conditions.

PtrA Is Functionally Intertwined with GacS in Regulating the Biocontrol Activity of Pseudomonas chlororaphis PA23.

In vitro inhibition of the fungal pathogen Sclerotinia sclerotiorum by Pseudomonas chlororaphis PA23 is reliant upon a LysR-type transcriptional regulator (LTTR) called PtrA. In the current study, we show that Sclerotinia stem rot and leaf infection are significantly increased in canola plants inoculated with the ptrA-mutant compared to the wild type, establishing PtrA as an essential regulator of PA23 biocontrol. LTTRs typically regulate targets that are upstream of and divergently transcribed from the LTTR locus. We identified a short chain dehydrogenase (scd) gene immediately upstream of ptrA. Characterization of a scd mutant revealed that it is phenotypically identical to the wild type. Moreover, scd transcript abundance was unchanged in the ptrA mutant. These findings indicate that PtrA regulation does not involve scd, rather this LTTR controls genes located elsewhere on the chromosome. Employing a combination of complementation and transcriptional analysis we investigated whether connections exist between PtrA and other regulators of biocontrol. Besides ptrA, gacS was the only gene able to partially rescue the wild-type phenotype, establishing a connection between PtrA and the sensor kinase GacS. Transcriptomic analysis revealed decreased expression of biosynthetic (phzA, prnA) and regulatory genes (phzI, phzR, rpoS, gacA, rsmX, rsmZ, retS) in the ptrA mutant; conversely, rsmE, and rsmY were markedly upregulated. The transcript abundance of ptrA was nine-fold higher in the mutant background indicating that this LTTR negatively autoregulates itself. In summary, PtrA is an essential regulator of genes required for PA23 biocontrol that is functionally intertwined with GacS.

Pseudomonas brassicacearum strain DF41 kills Caenorhabditis elegans through biofilm-dependent and biofilm-independent mechanisms.

Pseudomonas brassicacearum DF41 is a biocontrol agent that suppresses disease caused by the fungal pathogen Sclerotinia sclerotiorum A number of exometabolites are produced by DF41 including the lipopeptide sclerosin, hydrogen cyanide (HCN) and degradative enzymes. Production of these compounds is controlled at both the transcriptional and posttranscriptional level by quorum sensing (QS) and the Gac-two component regulatory system. In order to be successful, a biocontrol agent must persist in the environment at levels sufficient for pathogen control. Bacterivorous predators, including nematodes, represent a challenge to the establishment of introduced microorganisms. In the current study, DF41 was investigated for its ability to resist predation by Caenorhabditis elegans. We discovered that this bacterium is capable of killing C. elegans through two different mechanisms: the first involves exposure to toxic metabolites; and the second entails biofilm formation on the nematode head blocking the buccal cavity. Biofilm formation on nematodes, which has only been reported for Yersinia spp. and Xenorhabdus nematophila, is dependent upon the Gac system. Biofilms were not observed when bacteria were grown on NaCl-containing media, and on C. elegans biofilm-resistant mutants. Co-culturing with nematodes lead to increased expression of the pdfRI-rfiA QS genes and hcnA which is under QS control. HCN was the most nematicidal of the exometabolites, suggesting that this bacterium can respond to predator cues and upregulate expression of toxins accordingly. In summary, DF41 is able to respond to the presence of C. elegans and through two distinct mechanisms it can escape predation. IMPORTANCE: Pseudomonas brassicacearum DF41 can suppress fungal pathogens through a process known as biocontrol. To be successful, a biocontrol agent must be able to persist in the environment at levels sufficient for pathogen control. Predators including the nematode Caenorhabditis elegans represent a threat to persistence. The aim of the current study was to investigate the DF41-C. elegans interaction. We discovered that DF41 is able to escape predation through two distinct mechanisms. The first involves exposure to toxic bacterial metabolites and the second entails formation of a sticky coating on the nematode head, called a biofilm, which blocks feeding and causes starvation. This is the first report of a pseudomonad forming biofilms on the C. elegans surface. When grown with C. elegans, DF41 exhibits altered gene expression and metabolite production indicating that this bacterium can sense the presence of these predators and adjust its physiology accordingly.

Integrating Large-Scale Data and RNA Technology to Protect Crops from Fungal Pathogens.

With a rapidly growing human population it is expected that plant science researchers and the agricultural community will need to increase food productivity using less arable land. This challenge is complicated by fungal pathogens and diseases, many of which can severely impact crop yield. Current measures to control fungal pathogens are either ineffective or have adverse effects on the agricultural enterprise. Thus, developing new strategies through research innovation to protect plants from pathogenic fungi is necessary to overcome these hurdles. RNA sequencing technologies are increasing our understanding of the underlying genes and gene regulatory networks mediating disease outcomes. The application of invigorating next generation sequencing strategies to study plant-pathogen interactions has and will provide unprecedented insight into the complex patterns of gene activity responsible for crop protection. However, questions remain about how biological processes in both the pathogen and the host are specified in space directly at the site of infection and over the infection period. The integration of cutting edge molecular and computational tools will provide plant scientists with the arsenal required to identify genes and molecules that play a role in plant protection. Large scale RNA sequence data can then be used to protect plants by targeting genes essential for pathogen viability in the production of stably transformed lines expressing RNA interference molecules, or through foliar applications of double stranded RNA.

Elucidating the Role of Effectors in Plant-Fungal Interactions: Progress and Challenges.

Pathogenic fungi have diverse growth lifestyles that support fungal colonization on plants. Successful colonization and infection for all lifestyles depends upon the ability to modify living host plants to sequester the necessary nutrients required for growth and reproduction. Secretion of virulence determinants referred to as "effectors" is assumed to be the key governing factor that determines host infection and colonization. Effector proteins are capable of suppressing plant defense responses and alter plant physiology to accommodate fungal invaders. This review focuses on effector molecules of biotrophic and hemibiotrophic plant pathogenic fungi, and the mechanism required for the release and uptake of effector molecules by the fungi and plant cells, respectively. We also place emphasis on the discovery of effectors, difficulties associated with predicting the effector repertoire, and fungal genomic features that have helped promote effector diversity leading to fungal evolution. We discuss the role of specific effectors found in biotrophic and hemibiotrophic fungi and examine how CRISPR/Cas9 technology may provide a new avenue for accelerating our ability in the discovery of fungal effector function.

Pyrrolnitrin and Hydrogen Cyanide Production by Pseudomonas chlororaphis Strain PA23 Exhibits Nematicidal and Repellent Activity against Caenorhabditis elegans.

Pseudomonas chlororaphis strain PA23 is a biocontrol agent able to suppress growth of the fungal pathogen Sclerotinia sclerotiorum. This bacterium produces an arsenal of exometabolites including pyrrolnitrin (PRN), phenazine (PHZ), hydrogen cyanide (HCN), and degradative enzymes. Production of these compounds is controlled at both the transcriptional and posttranscriptional levels by the Gac-Rsm system, RpoS, PsrA, and the Phz quorum-sensing system. Beyond pathogen-suppression, the success of a biocontrol agent is dependent upon its ability to establish itself in the environment where predation by bacterivorous organisms, including nematodes, may threaten persistence. The focus of this study was to investigate whether PA23 is able to resist grazing by Caenorhabditis elegans and to define the role played by exoproducts in the bacterial-nematode interaction. We discovered that both PRN and HCN contribute to fast- and slow-killing of C. elegans. HCN is well-established as having lethal effects on C. elegans; however, PRN has not been reported to be nematicidal. Exposure of L4 stage nematodes to purified PRN reduced nematode viability in a dose-dependent fashion and led to reduced hatching of eggs laid by gravid adults. Because bacterial metabolites can act as chemoattractants or repellents, we analyzed whether PA23 exhibited attractant or repulsive properties towards C. elegans. Both PRN and HCN were found to be potent repellents. Next we investigated whether the presence of C. elegans would elicit changes in PA23 gene activity. Co-culturing the two organisms increased expression of a number of genes associated with biocontrol, including phzA, hcnA, phzR, phzI, rpoS and gacS. Exoproduct analysis showed that PHZ and autoinducer signals were upregulated, consistent with the gene expression profiles. Collectively, these findings indicate that PA23 is able to sense the presence of C. elegans and it is able to both repel and kill the nematodes, which should facilitate environmental persistence and ultimately biocontrol.