Many plant pathogens secrete toxins that enhance microbial virulence by killing host cells. Usually, these toxins are produced by particular microbial taxa, such as bacteria or fungi. In contrast, ...many bacterial, fungal and oomycete species produce necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) that trigger leaf necrosis and immunity-associated responses in various plants. We have determined the crystal structure of an NLP from the phytopathogenic oomycete Pythium aphanidermatum to 1.35Å resolution. The protein fold exhibits structural similarities to cytolytic toxins produced by marine organisms (actinoporins). Computational modeling of the 3-dimensional structure of NLPs from another oomycete, Phytophthora parasitica, and from the phytopathogenic bacterium, Pectobacterium carotovorum, revealed a high extent of fold conservation. Expression of the 2 oomycete NLPs in an nlp-deficient P. carotovorum strain restored bacterial virulence, suggesting that NLPs of prokaryotic and eukaryotic origins are orthologous proteins. NLP mutant protein analyses revealed that identical structural properties were required to cause plasma membrane permeabilization and cytolysis in plant cells, as well as to restore bacterial virulence. In sum, NLPs are conserved virulence factors whose taxonomic distribution is exceptional for microbial phytotoxins, and that contribute to host infection by plasma membrane destruction and cytolysis. We further show that NLP-mediated phytotoxicity and plant defense gene expression share identical fold requirements, suggesting that toxin-mediated interference with host integrity triggers plant immunity-associated responses. Phytotoxin-induced cellular damage-associated activation of plant defenses is reminiscent of microbial toxin-induced inflammasome activation in vertebrates and may thus constitute another conserved element in animal and plant innate immunity.
When analyzing the secretome of the plant pathogen Pseudomonas syringae pv. tomato DC3000, we identified hemolysin-coregulated protein (Hcp) as one of the secreted proteins. Hcp is assumed to be an ...extracellular component of the type VI secretion system (T6SS). Two copies of hcp genes are present in the P. syringae pv. tomato DC3000 genome, hcp1 (PSPTO_2539) and hcp2 (PSPTO_5435). We studied the expression patterns of the hcp genes and tested the fitness of hcp knockout mutants in host plant colonization and in intermicrobial competition. We found that the hcp2 gene is expressed most actively at the stationary growth phase and that the Hcp2 protein is secreted via the T6SS and appears in the culture medium as covalently linked dimers. Expression of hcp2 is not induced in planta and does not contribute to virulence in or colonization of tomato or Arabidopsis plants. Instead, hcp2 is required for survival in competition with enterobacteria and yeasts, and its function is associated with the suppression of the growth of these competitors. This is the first report on bacterial T6SS-associated genes functioning in competition with yeast. Our results suggest that the T6SS of P. syringae may play an important role in bacterial fitness, allowing this plant pathogen to survive under conditions where it has to compete with other microorganisms for resources.
Bacteriophages vB_YpeM_fEV-1 (fEV-1) and vB_YpeM_fD1 (fD1) were isolated from incoming sewage water samples in Turku, Finland, using
strains EV76 and KIM D27 as enrichment hosts, respectively. ...Genomic analysis and transmission electron microscopy established that fEV-1 is a novel type of dwarf myovirus, while fD1 is a T4-like myovirus. The genome sizes are 38 and 167 kb, respectively. To date, the morphology and genome sequences of some dwarf myoviruses have been described; however, a proteome characterization such as the one presented here, has currently been lacking for this group of viruses. Notably, fEV-1 is the first dwarf myovirus described for
. The host range of fEV-1 was restricted strictly to
strains, while that of fD1 also included other members of Enterobacterales such as
and
. In this study, we present the life cycles, genomes, and proteomes of two
myoviruses, fEV-1 and fD1.
We study the influence of argument sources on argument quality evaluations. Argument source refers to the person who present the argument. We ask whether partial, impartial and reluctant sources ...generate different evaluations of argument quality. We explore the source effect via a survey experiment where participants are asked to evaluate the quality of political arguments. Previous research on source partiality mainly concerns persuasion. The results from these studies suggest that source characteristics such as expertise and trustworthiness affect the persuasiveness of communication. Both impartial and reluctant sources have been observed to promote persuasion, whereas partial sources tend to hinder it. However, the evidence on the difference between impartial and reluctant sources are inconclusive, and research on argument quality evaluations is scarce. In our study, respondents are randomly allocated into four conditions according to who presents a political argument: Control (no argument source); Partial Source, Reluctant Source and Impartial Source. Our results show that overall impartial sources give rise to higher evaluations of argument quality in comparison to reluctant sources, but not in comparison to partial sources. Furthermore, reluctant sources are also perceived low in credibility.
We report here the complete genome sequence and characterization of
Yersinia
bacteriophage vB_YenP_ϕ80-18. ϕ80-18 was isolated in 1991 using a
Y. enterocolitica
serotype O:8 strain 8081 as a host ...from a sewage sample in Turku, Finland, and based on its morphological and genomic features is classified as a podovirus. The genome is 42 kb in size and has 325 bp direct terminal repeats characteristic for podoviruses. The genome contains 57 predicted genes, all encoded in the forward strand, of which 29 showed no similarity to any known genes. Phage particle proteome analysis identified altogether 24 phage particle-associated proteins (PPAPs) including those identified as structural proteins such as major capsid, scaffolding and tail component proteins. In addition, also the DNA helicase, DNA ligase, DNA polymerase, 5′-exonuclease, and the lytic glycosylase proteins were identified as PPAPs, suggesting that they might be injected together with the phage genome into the host cell to facilitate the take-over of the host metabolism. The phage-encoded RNA-polymerase and DNA-primase were not among the PPAPs. Promoter search predicted the presence of four phage and eleven host RNA polymerase –specific promoters in the genome, suggesting that early transcription of the phage is host RNA-polymerase dependent and that the phage RNA polymerase takes over later. The phage tolerates pH values between 2 and 12, and is stable at 50°C but is inactivated at 60°C. It grows slowly with a 50 min latent period and has apparently a low burst size. Electron microscopy revealed that the phage has a head diameter of about 60 nm, and a short tail of 20 nm. Whole-genome phylogenetic analysis confirmed that ϕ80-18 belongs to the
Autographivirinae
subfamily of the
Podoviridae
family, that it is 93.2% identical to
Yersinia
phage fHe-Yen3-01. Host range analysis showed that ϕ80-18 can infect in addition to
Y. enterocolitica
serotype O:8 strains also strains of serotypes O:4, O:4,32, O:20 and O:21, the latter ones representing similar to
Y. enterocolitica
serotype O:8, the American pathogenic biotype 1B strains. In conclusion, the phage ϕ80-18 is a promising candidate for the biocontrol of the American biotype 1B
Y. enterocolitica.
In this study, we characterized a putative Flp/Tad pilus-encoding gene cluster, and we examined its regulation at the transcriptional level and its role in the virulence of potato pathogenic ...enterobacteria of the genus Pectobacterium. The Flp/Tad pilus-encoding gene clusters in Pectobacterium atrosepticum, Pectobacterium wasabiae and Pectobacterium aroidearum were compared to previously characterized flp/tad gene clusters, including that of the well-studied Flp/Tad pilus model organism Aggregatibacter actinomycetemcomitans, in which this pilus is a major virulence determinant. Comparative analyses revealed substantial protein sequence similarity and open reading frame synteny between the previously characterized flp/tad gene clusters and the cluster in Pectobacterium, suggesting that the predicted flp/tad gene cluster in Pectobacterium encodes a Flp/Tad pilus-like structure. We detected genes for a novel two-component system adjacent to the flp/tad gene cluster in Pectobacterium, and mutant analysis demonstrated that this system has a positive effect on the transcription of selected Flp/Tad pilus biogenesis genes, suggesting that this response regulator regulate the flp/tad gene cluster. Mutagenesis of either the predicted regulator gene or selected Flp/Tad pilus biogenesis genes had a significant impact on the maceration ability of the bacterial strains in potato tubers, indicating that the Flp/Tad pilus-encoding gene cluster represents a novel virulence determinant in Pectobacterium. Soft-rot enterobacteria in the genera Pectobacterium and Dickeya are of great agricultural importance, and an investigation of the virulence of these pathogens could facilitate improvements in agricultural practices, thus benefiting farmers, the potato industry and consumers.
Erwinia carotovora subsp. carotovora is a gram-negative bacterium that causes soft rot disease of many cultivated crops. When a collection of E. carotovora subsp. carotovora isolates was analyzed on ...a Southern blot using the harpin-encoding gene hrpN as probe, several harpinless isolates were found. Regulation of virulence determinants in one of these, strain SCC3193, has been characterized extensively. It is fully virulent on potato and in Arabidopsis thaliana. An RpoS (SigmaS) mutant of SCC3193, producing elevated levels of secreted proteins, was found to cause lesions resembling the hypersensitive response when infiltrated into tobacco leaf tissue. This phenotype was evident only when bacterial cells had been cultivated on solid minimal medium at low pH and temperature. The protein causing'the cell death was purified and sequenced, and the corresponding gene was cloned. The deduced sequence of the necrosis-inducing protein (Nip) showed homology to necrosis- and ethylene-inducing elicitors of fungi and oomycetes. A mutant strain of E. carotovora subsp. carotovora lacking the nip gene showed reduced virulence in potato tuber assay but was unaffected in virulence in potato stem or on other tested host plants.
Molecular biological studies on Clavibacter michiganensis subsp. sepedonicus, the causal agent of bacterial ring rot of potato, have gained greater feasibility due to the recent availability of whole ...genomic sequences and genetic tools for related taxa. Here, we describe the first report of construction and characterization of a transposon (Tn) mutant library of C. michiganensis subsp. sepedonicus sp. strain R10. Since virulence of R10 in potato has been shown previously to be associated with elicitation of a nonhost hypersensitive response (HR), the mutant library was screened initially for loss of HR in tobacco. The screen identified two HR-negative mutants containing Tn insertions within the same gene, CMS2989 (chp-7), although at distinct locations. chp-7 is one of 11 pat-1 homologs in C. michiganensis subsp. sepedonicus. HR-negative mutants of R10 multiplied to the same extent as wild type in planta but were less virulent in potato. Complementation with chp-7 restored virulence as well as the HR phenotype. Together, these findings demonstrate a role for chp-7 in C. michiganensis subsp. sepedonicus-plant interactions.
The genomes of hybrid organisms, such as lager yeast (
Saccharomyces cerevisiae
×
Saccharomyces eubayanus
), contain orthologous genes, the functionality and effect of which may differ depending on ...their origin and copy number. How the parental subgenomes in lager yeast contribute to important phenotypic traits such as fermentation performance, aroma production, and stress tolerance remains poorly understood. Here, three de novo lager yeast hybrids with different ploidy levels (allodiploid, allotriploid, and allotetraploid) were generated through hybridization techniques without genetic modification. The hybrids were characterized in fermentations of both high gravity wort (15 °P) and very high gravity wort (25 °P), which were monitored for aroma compound and sugar concentrations. The hybrid strains with higher DNA content performed better during fermentation and produced higher concentrations of flavor-active esters in both worts. The hybrid strains also outperformed both the parent strains. Genome sequencing revealed that several genes related to the formation of flavor-active esters (
ATF1
,
ATF2
¸
EHT1
,
EEB1
, and
BAT1
) were present in higher copy numbers in the higher ploidy hybrid strains. A direct relationship between gene copy number and transcript level was also observed. The measured ester concentrations and transcript levels also suggest that the functionality of the
S. cerevisiae
- and
S. eubayanus
-derived gene products differs. The results contribute to our understanding of the complex molecular mechanisms that determine phenotypes in lager yeast hybrids and are expected to facilitate targeted strain development through interspecific hybridization.
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