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PLoS Pathogens Aug 2019Necrotrophic plant pathogens acquire nutrients from dead plant cells, which requires the disintegration of the plant cell wall and tissue structures by the pathogen....
Necrotrophic plant pathogens acquire nutrients from dead plant cells, which requires the disintegration of the plant cell wall and tissue structures by the pathogen. Infected plants lose tissue integrity and functional immunity as a result, exposing the nutrient rich, decayed tissues to the environment. One challenge for the necrotrophs to successfully cause secondary infection (infection spread from an initially infected plant to the nearby uninfected plants) is to effectively utilize nutrients released from hosts towards building up a large population before other saprophytes come. In this study, we observed that the necrotrophic pathogen Dickeya dadantii exhibited heterogeneity in bacterial cell length in an isogenic population during infection of potato tuber. While some cells were regular rod-shape (<10μm), the rest elongated into filamentous cells (>10μm). Short cells tended to occur at the interface of healthy and diseased tissues, during the early stage of infection when active attacking and killing is occurring, while filamentous cells tended to form at a later stage of infection. Short cells expressed all necessary virulence factors and motility, whereas filamentous cells did not engage in virulence, were non-mobile and more sensitive to environmental stress. However, compared to the short cells, the filamentous cells displayed upregulated metabolic genes and increased growth, which may benefit the pathogens to build up a large population necessary for the secondary infection. The segregation of the two subpopulations was dependent on differential production of the alarmone guanosine tetraphosphate (ppGpp). When exposed to fresh tuber tissues or freestanding water, filamentous cells quickly transformed to short virulent cells. The pathogen adaptation of cell length heterogeneity identified in this study presents a model for how some necrotrophs balance virulence and vegetative growth to maximize fitness during infection.
Topics: Bacterial Proteins; Cell Wall; Enterobacteriaceae; Gene Expression Regulation, Bacterial; Guanosine Tetraphosphate; Host-Pathogen Interactions; Plant Diseases; Solanum tuberosum; Virulence; Virulence Factors
PubMed: 31381590
DOI: 10.1371/journal.ppat.1007703 -
Molecules (Basel, Switzerland) Jun 2019Bacterial stem and root rot disease of sweet potato caused by recently broke out in major sweet potato planting areas in China and calls for effective approaches to...
Bacterial stem and root rot disease of sweet potato caused by recently broke out in major sweet potato planting areas in China and calls for effective approaches to control the pathogen and disease. Here, we developed a simple method for green synthesis of silver nanoparticles (AgNPs) using bacterial culture supernatants. AgNPs synthesized with the cell-free culture supernatant of a bacterium displayed the characteristic surface plasmon resonance peak at 420-430 nm and as nanocrystallites in diameters of 20-100 nm determined by transmission electron microscopy, scanning electron microscopy, and X-ray diffraction spectroscopy. Functional groups associated with proteins in the culture supernatant may reduce silver ions and stabilize AgNPs. The AgNPs showed antibacterial activities against growth, swimming motility, biofilm formation, and maceration of sweet potato tubers whereas the culture supernatant of did not. AgNPs (12 µg∙ml) and AgNO (50 µg∙ml) showed close antibacterial activities. The antibacterial activities increased with the increase of AgNP concentrations. The green-synthesized AgNPs can be used to control the soft rot disease by control of pathogen contamination of sweet potato seed tubers.
Topics: Anti-Bacterial Agents; China; Culture Media; Enterobacteriaceae; Green Chemistry Technology; Metal Nanoparticles; Microbial Sensitivity Tests; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Pseudomonas; Silver; Spectroscopy, Fourier Transform Infrared; Surface Plasmon Resonance
PubMed: 31234369
DOI: 10.3390/molecules24122303 -
Nucleic Acids Research Sep 2019Small non-coding RNAs (sRNAs) regulate numerous cellular processes in all domains of life. Several approaches have been developed to identify them from RNA-seq data,...
Small non-coding RNAs (sRNAs) regulate numerous cellular processes in all domains of life. Several approaches have been developed to identify them from RNA-seq data, which are efficient for eukaryotic sRNAs but remain inaccurate for the longer and highly structured bacterial sRNAs. We present APERO, a new algorithm to detect small transcripts from paired-end bacterial RNA-seq data. In contrast to previous approaches that start from the read coverage distribution, APERO analyzes boundaries of individual sequenced fragments to infer the 5' and 3' ends of all transcripts. Since sRNAs are about the same size as individual fragments (50-350 nucleotides), this algorithm provides a significantly higher accuracy and robustness, e.g., with respect to spontaneous internal breaking sites. To demonstrate this improvement, we develop a comparative assessment on datasets from Escherichia coli and Salmonella enterica, based on experimentally validated sRNAs. We also identify the small transcript repertoire of Dickeya dadantii including putative intergenic RNAs, 5' UTR or 3' UTR-derived RNA products and antisense RNAs. Comparisons to annotations as well as RACE-PCR experimental data confirm the precision of the detected transcripts. Altogether, APERO outperforms all existing methods in terms of sRNA detection and boundary precision, which is crucial for comprehensive genome annotations. It is freely available as an open source R package on https://github.com/Simon-Leonard/APERO.
Topics: Algorithms; Datasets as Topic; Enterobacteriaceae; Escherichia coli; Genome, Bacterial; High-Throughput Nucleotide Sequencing; Internet; RNA, Antisense; RNA, Bacterial; RNA, Messenger; RNA, Small Untranslated; Salmonella enterica; Sequence Analysis, RNA; Software
PubMed: 31147705
DOI: 10.1093/nar/gkz485 -
Environmental Microbiology Aug 2019Dickeya dadantii is a plant pathogen that causes soft rot disease on vegetable and potato crops. To successfully cause infection, this pathogen needs to coordinately...
Dickeya dadantii is a plant pathogen that causes soft rot disease on vegetable and potato crops. To successfully cause infection, this pathogen needs to coordinately modulate the expression of genes encoding several virulence determinants, including plant cell wall degrading enzymes (PCWDEs), type III secretion system (T3SS) and flagellar motility. Here, we uncover a novel feed-forward signalling circuit for controlling virulence. Global RNA chaperone Hfq interacts with an Hfq-dependent sRNA ArcZ and represses the translation of pecT, encoding a LysR-type transcriptional regulator. We demonstrate that the ability of ArcZ to be processed to a 50 nt 3'- end fragment is essential for its regulation of pecT. PecT down-regulates PCWDE and the T3SS by repressing the expression of a global post-transcriptional regulator- (RsmA-) associated sRNA encoding gene rsmB. In addition, we show that the protein levels of two cyclic di-GMP (c-di-GMP) diguanylate cyclases (DGCs), GcpA and GcpL, are repressed by Hfq. Further studies show that both DGCs are essential for the Hfq-mediated post-transcriptional regulation on RsmB. Overall, our report provides new insights into the interplays between ubiquitous signalling transduction systems that were most studied independently and sheds light on multitiered regulatory mechanisms for a precise disease regulation in bacteria.
Topics: Bacterial Proteins; Cell Wall; Cyclic GMP; Enterobacteriaceae; Gene Expression Regulation, Bacterial; Plant Diseases; RNA, Bacterial; RNA, Small Untranslated; RNA-Binding Proteins; Signal Transduction; Type III Secretion Systems; Virulence; Virulence Factors
PubMed: 30895662
DOI: 10.1111/1462-2920.14603 -
The Journal of Biological Chemistry Feb 2019In the quest for a sustainable economy of the Earth's resources and for renewable sources of energy, a promising avenue is to exploit the vast quantity of polysaccharide...
In the quest for a sustainable economy of the Earth's resources and for renewable sources of energy, a promising avenue is to exploit the vast quantity of polysaccharide molecules contained in green wastes. To that end, the decomposition of pectin appears to be an interesting target because this polymeric carbohydrate is abundant in many fruit pulps and soft vegetables. To quantitatively study this degradation process, here we designed a bioreactor that is continuously fed with de-esterified pectin (PGA). Thanks to the pectate lyases produced by bacteria cultivated in the vessel, the PGA is depolymerized into oligogalacturonates (UGA), which are continuously extracted from the tank. A mathematical model of our system predicted that the conversion efficiency of PGA into UGA increases in a range of coefficients of dilution until reaching an upper limit where the fraction of UGA that is extracted from the bioreactor is maximized. Results from experiments with a continuous reactor hosting a strain of the plant pathogenic bacterium and in which the dilution coefficients were varied quantitatively validated the predictions of our model. A further theoretical analysis of the system enabled an comparison of the efficiency of eight other pectate lyase-producing microorganisms with that of Our findings suggest that is the most efficient microorganism and therefore the best candidate for a practical implementation of our scheme for the bioproduction of UGA from PGA.
Topics: Bacterial Proteins; Bioreactors; Enterobacteriaceae; Models, Biological; Oligosaccharides; Pectins; Polysaccharide-Lyases; Polysaccharides; Virulence Factors
PubMed: 30510137
DOI: 10.1074/jbc.RA118.004615 -
Frontiers in Microbiology 2018Osmoregulated periplasmic glucans (OPGs) are general constituents of alpha-, beta-, and gamma-Proteobacteria. This polymer of glucose is required for full virulence of...
Osmoregulated periplasmic glucans (OPGs) are general constituents of alpha-, beta-, and gamma-Proteobacteria. This polymer of glucose is required for full virulence of many pathogens including . The phytopathogenic enterobacterium causes soft-rot disease in a wide range of plants. An OPG-defective mutant is impaired in environment sensing. We previously demonstrated that (i) fluctuation of OPG concentration controlled the activation level of the RcsCDB system, and (ii) RcsCDB along with EnvZ/OmpR controlled the mechanism of OPG succinylation. These previous data lead us to explore whether OPGs are required for other two-component systems. In this study, we demonstrate that inactivation of the EnvZ/OmpR system in an OPG-defective mutant restores full synthesis of pectinase but only partial virulence. Unlike for the RcsCDB system, the EnvZ-OmpR system is not controlled by OPG concentration but requires OPGs for proper activation.
PubMed: 30425688
DOI: 10.3389/fmicb.2018.02459 -
BMC Genomics Oct 2018Dickeya sp. strain PA1 is the causal agent of bacterial soft rot in Phalaenopsis, an important indoor orchid in China. PA1 and a few other strains were grouped into a...
BACKGROUND
Dickeya sp. strain PA1 is the causal agent of bacterial soft rot in Phalaenopsis, an important indoor orchid in China. PA1 and a few other strains were grouped into a novel species, Dickeya fangzhongdai, and only the orchid-associated strains have been shown to cause soft rot symptoms.
METHODS
We constructed the complete PA1 genome sequence and used comparative genomics to explore the differences in genomic features between D. fangzhongdai and other Dickeya species.
RESULTS
PA1 has a 4,979,223-bp circular genome with 4269 predicted protein-coding genes. D. fangzhongdai was phylogenetically similar to Dickeya solani and Dickeya dadantii. The type I to type VI secretion systems (T1SS-T6SS), except for the stt-type T2SS, were identified in D. fangzhongdai. The three phylogenetically similar species varied significantly in terms of their T5SSs and T6SSs, as did the different D. fangzhongdai strains. Genomic island (GI) prediction and synteny analysis (compared to D. fangzhongdai strains) of PA1 also indicated the presence of T5SSs and T6SSs in strain-specific regions. Two typical CRISPR arrays were identified in D. fangzhongdai and in most other Dickeya species, except for D. solani. CRISPR-1 was present in all of these Dickeya species, while the presence of CRISPR-2 varied due to species differentiation. A large polyketide/nonribosomal peptide (PK/NRP) cluster, similar to the zeamine biosynthetic gene cluster in Dickeya zeae rice strains, was discovered in D. fangzhongdai and D. solani. The D. fangzhongdai and D. solani strains might recently have acquired this gene cluster by horizontal gene transfer (HGT).
CONCLUSIONS
Orchid-associated strains are the typical members of D. fangzhongdai. Genomic analysis of PA1 suggested that this strain presents the genomic characteristics of this novel species. Considering the absence of the stt-type T2SS, the presence of CRISPR loci and the zeamine biosynthetic gene cluster, D. fangzhongdai is likely a transitional form between D. dadantii and D. solani. This is supported by the later acquisition of the zeamine cluster and the loss of CRISPR arrays by D. solani. Comparisons of phylogenetic positions and virulence determinants could be helpful for the effective quarantine and control of this emerging species.
Topics: Bacterial Secretion Systems; Base Composition; Clustered Regularly Interspaced Short Palindromic Repeats; Computational Biology; Conserved Sequence; Enterobacteriaceae; Evolution, Molecular; Gene Order; Genes, Bacterial; Genome, Bacterial; Genomics; Molecular Sequence Annotation; Open Reading Frames; Orchidaceae; Phylogeny; Plant Diseases; Whole Genome Sequencing
PubMed: 30373513
DOI: 10.1186/s12864-018-5154-3 -
Molecular Plant Pathology Feb 2019The identification of the virulence factors of plant-pathogenic bacteria has relied on the testing of individual mutants on plants, a time-consuming process. Transposon...
The identification of the virulence factors of plant-pathogenic bacteria has relied on the testing of individual mutants on plants, a time-consuming process. Transposon sequencing (Tn-seq) is a very powerful method for the identification of the genes required for bacterial growth in their host. We used this method in a soft-rot pathogenic bacterium to identify the genes required for the multiplication of Dickeya dadantii in chicory. About 100 genes were identified showing decreased or increased fitness in the plant. Most had no previously attributed role in plant-bacterium interactions. Following our screening, in planta competition assays confirmed that the uridine monophosphate biosynthesis pathway and the purine biosynthesis pathway were essential to the survival of D. dadantii in the plant, as the mutants ∆carA, ∆purF, ∆purL, ∆guaB and ∆pyrE were unable to survive in the plant in contrast with the wild-type (WT) bacterium. This study also demonstrated that the biosynthetic pathways of leucine, cysteine and lysine were essential for bacterial survival in the plant and that RsmC and GcpA were important in the regulation of the infection process, as the mutants ∆rsmC and ∆gcpA were hypervirulent. Finally, our study showed that D. dadantii flagellin was glycosylated and that this modification conferred fitness to the bacterium during plant infection. Assay by this method of the large collections of environmental pathogenic strains now available will allow an easy and rapid identification of new virulence factors.
Topics: Bacterial Proteins; Cichorium intybus; DNA Transposable Elements; Enterobacteriaceae; Gene Expression Regulation, Bacterial; Glycosylation; Virulence
PubMed: 30267562
DOI: 10.1111/mpp.12754 -
Frontiers in Plant Science 2018Cyclotides are cyclic plant polypeptides of 27-37 amino acid residues. They have been extensively studied in bioengineering and drug development contexts. However, less...
Cyclotides are cyclic plant polypeptides of 27-37 amino acid residues. They have been extensively studied in bioengineering and drug development contexts. However, less is known about the relevance of cyclotides for the plants producing them. The anti-insect larvae effects of kB1 and antibacterial activity of cyO2 suggest that cyclotides are a part of plant host defense. The sweet violet ( L.) produces a wide array of cyclotides, including kB1 (kalata B1) and cyO2 (cycloviolacin O2), with distinct presumed biological roles. Here, we evaluate V. odorata cyclotides' potency against plant pathogens and their mode of action using bioassays, liposome experiments and immunogold labeling for transmission electron microscopy (TEM). We explore the link between the biological activity and distribution in plant generative, vegetative tissues and seeds, depicted by immunohistochemistry and matrix assisted laser desorption ionization mass spectrometry imaging (MALDI-MSI). Cyclotides cyO2, cyO3, cyO13, and cyO19 are shown to have potent activity against model fungal plant pathogens () and fungi isolated from violets (), with minimal inhibitory concentrations (MICs) ranging from 0.8 μM to 25 μM. Inhibition of phytopathogenic bacteria - pv. and - is also observed with MIC = 25-100 μM. A membrane-disrupting antifungal mode of action is shown. Finding cyO2 inside the fungal spore cells in TEM images may indicate that other, intracellular targets may be involved in the mechanism of toxicity. Fungi can not break down cyclotides in the course of days. varv A (kalata S) and kB1 show little potency against pathogenic fungi when compared with the tested cycloviolacins. cyO2, cyO3, cyO19 and kB1 are differentially distributed and found in tissues vulnerable to pathogen (epidermis, rizodermis, vascular bundles, protodermis, procambium, ovary walls, outer integuments) and pest (ground tissues of leaf and petiole) attacks, respectively, indicating a link between the cyclotides' sites of accumulation and biological role. Cyclotides emerge as a comprehensive defense system in , in which different types of peptides have specific targets that determine their distribution in plant tissues.
PubMed: 30254654
DOI: 10.3389/fpls.2018.01296 -
Frontiers in Microbiology 2018is a bacterial plant pathogen that causes soft rot disease on a wide range of host plants. The type III secretion system (T3SS) is an important virulence factor in ....
Development of a Method to Monitor Gene Expression in Single Bacterial Cells During the Interaction With Plants and Use to Study the Expression of the Type III Secretion System in Single Cells of in Potato.
is a bacterial plant pathogen that causes soft rot disease on a wide range of host plants. The type III secretion system (T3SS) is an important virulence factor in . Expression of the T3SS is induced in the plant apoplast or in -inducing minimal medium (hrp-MM), and is repressed in nutrient-rich media. Despite the understanding of induction conditions, how individual cells in a clonal bacterial population respond to these conditions and modulate T3SS expression is not well understood. In our previous study, we reported that in a clonal population, only a small proportion of bacteria highly expressed T3SS genes while the majority of the population did not express T3SS genes under hrp-MM condition. In this study, we developed a method that enabled observation and quantification of gene expression in single bacterial cells . Using this technique, we observed that the expression of the T3SS genes and is restricted to a small proportion of cells during the infection of potato. We also report that the expression of T3SS genes is higher at early stages of infection compared to later stages. This expression modulation is achieved through adjusting the ratio of T3SS and T3SS cells and the expression intensity of T3SS cells. Our findings not only shed light into how bacteria use a bi-stable gene expression manner to modulate an important virulence factor, but also provide a useful tool to study gene expression in individual bacterial cells .
PubMed: 30002651
DOI: 10.3389/fmicb.2018.01429