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bioRxiv2017

Localized Recombination Drives Diversification of Killing Spectra for Phage Derived Syringacins

To better understand the potential for detrimental interactions between strains of the same bacterial species, we have surveyed bacteriocin killing activity across a diverse suite of strains of the phytopathogen Pseudomonas syringae. Our data demonstrate that killing activity from phage derived bacteriocins of P. syringae (R-type syringacins) is widespread. Despite a high overall diversity of bacteriocin activity, strains can broadly be classified into five main killing types and two main sensitivity types. Furthermore, we show that killing activity switches frequently between strains, and that switches correlate with localized recombination of two genes that together encode the proteins that specify bacteriocin targeting. Lastly, we demonstrate that phage derived bacteriocin killing activity can be swapped between strains simply through expression of these two genes in trans. Overall, our study characterizes extensive diversity of killing activity for phage derived bacteriocins of P. syringae across strains and highlights the power of localized recombination to alter phenotypes that mediate strain interactions during evolution of natural populations and communities.

microbiology↗

bioRxiv2017

Candidatus Nitrosocaldus cavascurensis, an ammonia oxidizing, extremely thermophilic archaeon with a highly mobile genome

Ammonia oxidizing archaea (AOA) of the phylum Thaumarchaeota are widespread in moderate environments but their occurrence and activity has also been demonstrated in hot springs. Here we present the first cultivated thermophilic representative with a sequenced genome, which allows to search for adaptive strategies and for traits that shape the evolution of Thaumarchaeota. Candidatus Nitrosocaldus cavascurensis has been cultivated from a hot spring in Ischia, Italy. It grows optimally at 68{degrees}C under chemolithoautotrophic conditions on ammonia or urea converting ammonia stoichiometrically into nitrite with a generation time of approximately 25h. Phylogenetic analyses based on ribosomal proteins place the organism as a sister group to all known mesophilic AOA. The 1.58 Mb genome of Ca. N. cavascurensis harbors an amoAXCB gene cluster encoding ammonia monooxygenase, genes for a 3-hydroxypropionate/4-hydroxybutyrate pathway for autotrophic carbon fixation, but also genes that indicate potential alternative energy metabolisms. Although a bona fide gene for nitrite reductase is missing, the organism is sensitive to NO-scavenging, underlining the importance of this compound for AOA metabolism. Ca. N. cavascurensis is distinct from all other AOA in its gene repertoire for replication, cell division and repair. Its genome has an impressive array of mobile genetic elements and other recently acquired gene sets, including conjugative systems, a provirus, transposons and cell appendages. Some of these elements indicate recent exchange with the environment, whereas others seem to have been domesticated and might convey crucial metabolic traits.

microbiology↗

bioRxiv2017

Murine polyomavirus microRNAs promote viuria during the acute phase of infection

Polyomaviruses (PyVs) can cause serious disease in immunosuppressed hosts. Several pathogenic PyVs encode microRNAs (miRNAs), small RNAs that regulate gene expression via RNA silencing. Despite recent advances in understanding the activities of PyV miRNAs, the biological functions of PyV miRNAs during in vivo infections are mostly unknown. Studies presented here use murine polyomavirus (MuPyV) as a model to assess the roles of the PyV miRNAs in a natural host. This analysis reveals that a MuPyV mutant that is unable to express miRNAs has enhanced viral DNA loads in select tissues at late times after infection, indicating that during infection of a natural host, PyV miRNAs function to reduce viral replication during the persistent phase of infection. Additionally, MuPyV miRNAs promote viruria during the acute phase of infection as evidenced by a defect in shedding during infection with the miRNA mutant virus. The viruria defect of the miRNA mutant virus could be rescued by infecting Rag2-/-mice. These findings implicate miRNA activity in both the persistent and acute phases of infection and suggest a role for MuPyV miRNA in evading the adaptive immune response.\n\nImportanceMicroRNAs are expressed by diverse viruses, but for only a few is there any understanding of their in vivo function. PyVs can cause serious disease in immunocompromised hosts. Therefore, increased knowledge of how these viruses interact with the immune response is of possible clinical relevance. Here we show a novel activity for a viral miRNA in promoting virus shedding. This work indicates that in addition to any role for the PyV miRNA in long-term persistence, that it also has biological activity during the acute phase. As this mutant phenotype is alleviated by infection of mice lacking an effective adaptive immune response, our work also connects the in vivo activity of a PyV miRNA to the immune response. Given that PyV-associated disease is associated with alterations in the immune response, our findings may help to better understand how the balance between PyV and the immune response becomes altered in pathogenic states.

microbiology↗

bioRxiv2018

Comparative RNA-seq based transcriptomic analysis of Aspergillus nidulans recombinant strains overproducing heterologous glycoside hydrolases

Filamentous fungi are important cellular factories for the production and secretion of homologous and heterologous enzymes such as carbohydrate-active enzymes. However, the regulation of protein secretion in these microorganisms requires more profound studies since the enzyme levels produced are usually below the levels required by industry for profitable processes. Genomic and transcriptomic approaches have been used to understand the overexpression and production of heterologous enzymes and their capacity to induce different cellular biological processes. To investigate this regulation, Aspergillus nidulans recombinant strains were analyzed by transcriptomics. We designed three A. nidulans recombinant strains producing the following heterologous proteins: alpha-arabinofuranosidase (AbfA), beta-glucosidase (BglC) and thermophilic mannanase (Tp-Man5). The heterologous genes abfA and bglC were highly expressed, while tp-man5 mRNA levels were low and similar to those of a reference gene. There was an indirect relationship between mRNA and protein secretion levels, suggesting that transcription is not a bottleneck for target gene expression in this system. Despite the distinct features of the recombinant proteins, 30 differentially expressed genes were common to all the recombinant strains, suggesting that these genes represent a general response to the expression of heterologous genes. We also showed that the early activation of the canonical unfolded protein response (UPR) pathway by hacA alternative splicing was normalized after 8h, except in the strain expressing BglC, suggesting either no accumulation of the BglC misfolded form or the presence of an alternative endoplasmic reticulum (ER) stress and UPR pathway. Finally, to focus our analysis on the secretion pathway, a set of 374 genes was further evaluated. Seventeen genes were common to all the recombinant strains, suggesting again that these genes represent a general response of A. nidulans cells to the overexpression of recombinant genes, even thermophilic genes. Additionally, we reported the possible genetic interactions of these 17 genes based on coexpression network calculations. Interestingly, protein improvements are nongeneric, and improvements in the production of one target protein are not necessarily transferable to another one. Thus, this study may provide genetic and cellular background and targets for genetic manipulation to improve protein secretion by A. nidulans.

microbiology↗

bioRxiv2018

Micro-scale ecology regulates particulate organic matter turnover in model marine microbial communities

The degradation of particulate organic matter in the ocean is a central process in the global carbon cycle, the mode and tempo of which is determined by the bacterial communities that assemble on particle surfaces. Although recent studies have shed light on the dynamics of community assembly on particles -which serve as hotspots of microbial activity in the ocean, the mapping from community composition to function, i.e. particle degradation, remains completely unexplored. Using a collection of marine bacteria cultured from different stages of succession on chitin micro-particles we found that the hydrolytic power of communities is highly dependent on community composition. Different particle degrading taxa -all of which were early successional species during colonization- displayed characteristic particle half-lives that differed by ~170 hours, comparable to the residence time of particles in the oceans mixed layer1. These half-lives were in general longer in multispecies communities, where the growth of obligate cross-feeders limited the ability of degraders to colonize and consume particles. Remarkably, above a certain critical initial ratio of cross-feeder to degrader cells, particle degradation was completely blocked along with the growth of all members of the community. We showed that this interaction occurred between a variety of strains of different taxonomic origins and that it only appears when bacteria interact with particles, suggesting a mechanism by which non-degrading secondary consumers occlude access to the particle resource. Overall, our results show that micro-scale community ecology on particle surfaces can have significant impact on carbon turnover in the ocean.

microbiology↗

bioRxiv2018

Arabidopsis thaliana populations support long-term maintenance and parallel expansions of related Pseudomonas pathogens

Crop disease outbreaks are often associated with clonal expansions of single pathogenic lineages. To determine whether similar boom-and-bust scenarios hold for wild plant pathogens, we carried out a multi-year multi-site survey of Pseudomonas in the natural host Arabidopsis thaliana. The most common Pseudomonas lineage corresponded to a pathogenic clade present in all sites. Sequencing of 1,524 Pseudomonas genomes revealed this lineage to have diversified approximately 300,000 years ago, containing dozens of genetically distinct pathogenic sublineages. These sublineages have expanded in parallel within the same populations and are differentiated both at the level of gene content and disease phenotype. Such coexistence of diverse sublineages indicates that in contrast to crop systems, no single strain has been able to overtake these A. thaliana populations in the recent past. Our results suggest that the selective pressures acting on a plant pathogen in wild hosts may be more complex than those in agricultural systems.

microbiology↗

bioRxiv2018

Listeria monocytogenes InlP interacts with afadin and facilitates basement membrane crossing

During pregnancy, the placenta protects the fetus against the maternal immune response, as well as bacterial and viral pathogens. Bacterial pathogens that have evolved specific mechanisms of breaching this barrier, such as Listeria monocytogenes, present a unique opportunity for learning how the placenta carries out its protective function. We previously identified the L. monocytogenes protein Internalin P (InlP) as a secreted virulence factor critical for placental infection (1). Here, we show that InlP, but not the highly similar L. monocytogenes internalin Lmo2027, binds to human afadin (encoded by AF-6), a protein associated with cell-cell junctions. A crystal structure of InlP reveals several unique features, including an extended leucine-rich repeat (LRR) domain with a distinctive Ca2+-binding site. Despite afadins involvement in the formation of cell-cell junctions, MDCK epithelial cells expressing InlP displayed a decrease in the magnitude of the traction stresses they could exert on deformable substrates, similar to the decrease in traction exhibited by AF-6 knock-out MDCK cells. L. monocytogenes {Delta}inlP mutants were deficient in their ability to form actin-rich protrusions from the basal face of polarized epithelial monolayers, a necessary step in the crossing of such monolayers (transcytosis). A similar phenotype was observed for bacteria expressing an internal in-frame deletion in inlP (inlP DLRR5) that specifically disrupts its interaction with afadin. However, afadin deletion in the host cells did not rescue the transcytosis defect. We conclude that secreted InlP targets cytosolic afadin to specifically promote L. monocytogenes transcytosis across the basal face of epithelial monolayers, which may contribute to the crossing of the basement membrane during placental infection.

microbiology↗

bioRxiv2018

The Tudor protein Veneno assembles the ping-pong amplification complex that produces viral piRNAs in Aedes mosquitoes

TUDOR-domain containing proteins facilitate PIWI interacting (pi)RNA biogenesis in Drosophila melanogaster and other model organisms. In Aedes aegypti mosquitoes, a somatically active piRNA pathway generates piRNAs from viral RNA during acute infection with cytoplasmic RNA viruses. Viral piRNA biogenesis requires ping-pong amplification by the PIWI proteins Ago3 and Piwi5. We hypothesized that Tudor proteins are required for viral piRNA production and performed a knockdown screen targeting all Ae. aegypti Tudor genes. Knockdown of several Tudor genes resulted in reduced viral piRNA levels, with silencing of AAEL012437 having the strongest effect. This protein, which we named Veneno, associates directly with Ago3 in an sDMA-dependent manner and localizes in cytoplasmic foci reminiscent of piRNA processing granules of Drosophila. Veneno-interactome analyses reveal a network of co-factors including the orthologs of the Drosophila piRNA pathway components Vasa and Yb, which in turn interacts directly with Piwi5. We propose that Veneno assembles a multi-protein complex for ping-pong dependent piRNA production from exogenous viral RNA.

microbiology↗

bioRxiv2018

Phylogenetic distribution of roseobacticides in the Roseobacter group and their effect on microalgae

The Roseobacter-group species Phaeobacter inhibens produces the antibacterial tropodithietic acid (TDA) and the algaecidal roseobacticides with both compound classes sharing part of the same biosynthetic pathway. The purpose of this study was to investigate the production of roseobacticides more broadly in TDA-producing roseobacters and to compare the effect of producers and non-producers on microalgae. Of 33 roseobacters analyzed, roseobacticide production was a unique feature of TDA-producing P. inhibens, P. gallaeciensis and P. piscinae strains. One TDA-producing Phaeobacter strain, 27-4, was unable to produce roseobacticides, possibly due to a transposable element. TDA-producing Ruegeria mobilis and Pseudovibrio did not produce roseobacticides. Addition of roseobacticide-containing bacterial extracts affected the growth of the microalgae Rhodomonas salina, Thalassiosira pseudonana and Emiliania huxleyi, while growth of Tetraselmis suecica was unaffected. During co-cultivation, growth of E. huxleyi was initially stimulated by the roseobacticide producer DSM 17395, while the subsequent decline in algal cell numbers during senescence was enhanced. Strain 27-4 that does not produce roseobacticides had no effect on algal growth. Both bacterial strains, DSM 17395 and 27-4, grew during co-cultivation presumably utilizing algal exudates. Furthermore, TDA-producing roseobacters have potential as probiotics in marine larviculture and it is promising that the live feed Tetraselmis was unaffected by roseobacticides-containing extracts.\n\nOriginality-significance statementSome Roseobacter-group bacteria produce the antibacterial compound tropodithetic acid (TDA) and have potential as probiotics in marine aquaculture. However, a few of these strains additionally produce algaecidal compounds, the roseobacticides, which would restrict their use in marine larviculture where algae are used as live feed for fish larvae. We herein found that roseobacticides are limited to TDA-producing Phaeobacter strains and were not biosynthesized by TDA-producers outside this genus. Roseobacticides affected several strains of microalgae, but not the chlorophyte that is used as live feed in the aquaculture industry. Thus, the application of Roseobacter strains as probiotics is not hampered. Furthermore, these results demonstrate how Roseobacter-group strains act as gardeners of microalgae and thereby would be involved in environmental processes on a larger scale.

microbiology↗

bioRxiv2018

Chromatin Functional States Correlate with HIV Latency Reversal in Infected Primary CD4+ T Cells

Human immunodeficiency virus (HIV) infection cannot be cured due to a small reservoir of latently infected CD4+ T cells in treated patients. The \"shock and kill\" approach proposes to eliminate the reservoir by inducing its activation and the direct or indirect killing of infected cells. Current latency reversing agents (LRAs) do not reduce the viral reservoir in treated patients.\n\nWe use a novel dual-fluorescent HIV reporter to identify and purify latent cells, and to determine the fraction of latent cells that undergo viral reactivation after infection of primary CD4+ T cells. Unexpectedly, LRAs reactivate less than 5% of latent proviruses. Analysis of HIV integration sites from induced and non-induced latent populations reveals distinct provirus integration sites between these two populations in terms of chromatin functional states.\n\nThese findings challenge \"shock and kill\", and suggest the need of more potent LRAs in combination with immunomodulatory approaches to eradicate HIV reservoir.

microbiology↗

bioRxiv2018

Cell adhesion and fluid flow jointly initiate genotype spatial distribution in biofilms

Biofilms are microbial collectives that occupy a diverse array of surfaces. The function and evolution of biofilms are strongly influenced by the spatial arrangement of different strains and species within them, but how spatiotemporal distributions of different genotypes in biofilm populations originate is still underexplored. Here, we study the origins of biofilm genetic structure by combining model development, numerical simulations, and microfluidic experiments using the human pathogen Vibrio cholerae. Using spatial correlation functions to quantify the differences between emergent cell lineage segregation patterns, we find that strong adhesion often, but not always, maximizes the size of clonal cell clusters on flat surfaces. Counterintuitively, our model predicts that, under some conditions, investing in adhesion can reduce rather than increase clonal group size. Our results emphasize that a complex interaction of fluid flow and cell adhesiveness can underlie emergent patterns of biofilm genetic structure. This structure, in turn, has an outsize influence on how biofilm-dwelling populations function and evolve.\n\nAuthor summaryBiofilms are bacterial groups, often attached to surfaces, in which a broad variety of cooperative and competitive interactions typically occur. The spatial organization of different strains and species within biofilm communities strongly influences their global functioning, but little is known about how such structure arises. Combining experiments on V. cholerae and simulations of a cellular automaton, we show that the complex interaction between bacterial traits (cell adhesion) and environmental factors (fluid flow intensity) strongly influences the early origins of biofilm spatial structure. In most cases, we found that highly-adhesive strains form larger clusters than the weakly-adhesive ones. Against intuition, however, we also found the opposite outcome: weakly-adhesive tend to form larger clusters than the highly adhesive ones when flows are weak or the population density of colonizing cells is high.

microbiology↗

bioRxiv2018

Microbiota Accessible Carbohydrates Facilitate Clearance of Clostridium difficile

Clostridium difficile (Cd) is an opportunistic diarrheal pathogen and Cd infection (CDI) represents a major healthcare concern, causing an estimated 15,000 deaths per year in the United States alone1. Several enteric pathogens, including Cd, leverage inflammation and the accompanying microbial dysbiosis to thrive in the distal gut2. Although diet is among the most powerful available tools for affecting the health of humans and their relationship with their microbiota, investigation into the effects of diet on CDI has been limited. Here, we show in mice that the consumption of microbiota accessible carbohydrates (MACs) found in dietary plant polysaccharides has a significant impact on CDI. Specifically, using a model of antibiotic-induced CDI that typically resolves within 12 days of infection, we demonstrate that MAC-deficient diets perpetuate CDI. We show that Cd burdens are suppressed through ...

microbiology↗

bioRxiv2018

Synaptic transmission may provide an evolutionary benefit to HIV through modulation of latency

Transmission of HIV is known to occur by two mechanisms in vivo: the free virus pathway, where viral particles bud off an infected cell before attaching to an uninfected cell, and the cell-cell pathway, where infected cells form virological synapses through close contact with an uninfected cell. It has also been shown that HIV replication includes a positive feedback loop controlled by the viral protein Tat, which may act as a stochastic switch in determining whether an infected cell enters latency. In this paper, we introduce a simple mathematical model of HIV replication containing both the free virus and cell-cell pathways. Using this model, we demonstrate that the high multiplicity of infection in cell-cell transmission results in a suppression of latent infection, and that this modulation of latency through balancing the two transmission mechanisms can provide an evolutionary benefit to the virus. This benefit increases with decreasing overall viral fitness, which may provide a within-host evolutionary pressure toward more cell-cell transmission in late-stage HIV infection.

microbiology↗

bioRxiv2018

Cross-feeding modulates antibiotic tolerance in bacterial communities

Microbes frequently rely on metabolites excreted by other bacterial species, but little is known about how this cross-feeding influences the effect of antibiotics. We hypothesized that when species rely on each other for essential metabolites, the minimum inhibitory concentration (MIC) for all species will drop to that of the \"weakest link\" - the species least resistant in monoculture. We tested this hypothesis in an obligate cross-feeding system that was engineered between Escherichia coli, Salmonella enterica, and Methylobacterium extorquens. The effect of tetracycline and ampicillin were tested on both liquid and solid media. In all cases, resistant species were inhibited at significantly lower antibiotic concentrations in the cross-feeding community than in monoculture or a competitive community. However, deviation from the \"weakest link\" hypothesis was also observed in cross-feeding communities apparently as result of changes in the timing of growth and cross-protection. Comparable results were also observed in a clinically relevant system involving facultative cross-feeding between Pseudomonas aeruginosa and an anaerobic consortium found in the lungs of cystic fibrosis patients. P. aeruginosa was inhibited by lower concentrations of ampicillin when cross-feeding than when grown in isolation. These results suggest that cross-feeding significantly alters tolerance to antibiotics in a variety of systems.

microbiology↗

bioRxiv2018

Pyrimidine starvation activates a bistable phenotypic switch leading to ribosome provisioning and rapid exit from stationary phase

Observations of bacteria at the single-cell level have revealed many instances of phenotypic heterogeneity within otherwise clonal populations, but the selective causes, molecular bases and broader ecological relevance remain poorly understood. In an earlier experiment in which the bacterium Pseudomonas fluorescens SBW25 was propagated under a selective regime that mimicked the host immune response, a genotype evolved that stochastically switched between capsulation states. The genetic cause was a mutation in carB that decreased the pyrimidine pool (and growth rate), lowering the activation threshold of a pre-existing but hitherto unrecognised phenotypic switch. Genetic components surrounding bifurcation of UTP flux towards DNA/RNA or UDP-glucose (a precursor of colanic acid forming the capsules) were implicated as key components. Extending these molecular analyses - and based on a combination of genetics, transcriptomics, biochemistry and mathematical modelling - we show that pyrimidine limitation triggers an increase in ribosome biosynthesis and that switching is caused by competition between ribosomes and CsrA/RsmA proteins for the mRNA transcript of a feed-forward regulator of colanic acid biosynthesis. We additionally show that in the ancestral bacterium the switch is part of a programme that determines stochastic entry into the semi-quiescent capsulated state, ensures that such cells are provisioned with excess ribosomes, and enables provisioned cells to exit rapidly from stationary phase under permissive conditions.

microbiology↗

bioRxiv2018

NanoAmpli-Seq: A workflow for amplicon sequencing from mixed microbial communities on the nanopore sequencing platform

BackgroundAmplicon sequencing on Illumina sequencing platforms leverages their deep sequencing and multiplexing capacity, but is limited in genetic resolution due to short read lengths. While Oxford Nanopore or Pacific Biosciences platforms overcome this limitation, their application has been limited due to higher error rates or smaller data output.\n\nResultsIn this study, we introduce an amplicon sequencing workflow, i.e., NanoAmpli-Seq, that builds on Intramolecular-ligated Nanopore Consensus Sequencing (INC-Seq) approach and demonstrate its application for full-length 16S rRNA gene sequencing. NanoAmpli-Seq includes vital improvements to the aforementioned protocol that reduces sample-processing time while significantly improving sequence accuracy. The developed protocol includes chopSeq software for fragmentation and read orientation correction of INC-Seq consensus reads while nanoClust algorithm was designed for read partitioning-based de novo clustering and within cluster consensus calling to obtain full-length 16S rRNA gene sequences.\n\nConclusionsNanoAmpli-Seq accurately estimates the diversity of tested mock communities with average sequence accuracy of 99.5% for 2D and 1D2 sequencing on the nanopore sequencing platform. Nearly all residual errors in NanoAmpli-Seq sequences originate from deletions in homopolymer regions, indicating that homopolymer aware basecalling or error correction may allow for sequencing accuracy comparable to short-read sequencing platforms.

microbiology↗

bioRxiv2018

Agricultural Pollution Risks Influence Microbial Ecology in Honghu Lake

BackgroundAgricultural activities, such as stock-farming, planting industry, and fish aquaculture, can influence the physicochemistry and biology of freshwater lakes. However, the extent to which these agricultural activities, especially those that result in eutrophication and antibiotic pollution, effect water and sediment-associated microbial ecology, remains unclear.\n\nMethodsWe performed a geospatial analysis of water and sediment associated microbial community structure, as well as physicochemical parameters and antibiotic pollution, across 18 sites in Honghu lake, which range from impacted to less-impacted by agricultural pollution. Furthermore, the co-occurrence network of water and sediment were built and compared accorded to the agricultural activities.\n\nResultsPhysicochemical properties including TN, TP, NO3--N, and NO2--N were correlated with microbial compositional differences in water samples. Likewise, in sediment samples, Sed-OM and Sed-TN correlated with microbial diversity. Oxytetracycline and tetracycline concentration described the majority of the variance in taxonomic and predicted functional diversity between impacted and less-impacted sites in water and sediment samples, respectively. Finally, the structure of microbial co-associations was influenced by the eutrophication and antibiotic pollution.\n\nConclusionThese analyses of the composition and structure of water and sediment microbial communities in anthropologically-impacted lakes are imperative for effective environmental pollution monitoring. Likewise, the exploration of the associations between environmental variables (e.g. physicochemical properties, and antibiotics) and community structure is important in the assessment of lake water quality and its ability to sustain agriculture. These results show agricultural practices can negatively influence not only the physicochemical properties, but also the biodiversity of microbial communities associated with the Honghu lake ecosystem. And these results provide compelling evidence that the microbial community can be used as a sentinel of eutrophication and antibiotics pollution risk associated with agricultural activity; and that proper monitoring of this environment is vital to maintain a sustainable environment in Honghu lake.

microbiology↗

bioRxiv2018

Comparative genomics of Pseudomonas syringae reveals convergent gene gain and loss associated with specialisation onto cherry (Prunus avium)

O_LIGenome-wide analyses of the effector- and toxin-encoding genes were used to examine the phylogenetics and evolution of pathogenicity amongst diverse strains of Pseudomonas syringae causing bacterial canker of cherry (Prunus avium) including pathovars P.s pv. morsprunorum (Psm) races 1 and 2, P.s pv. syringae (Pss) and P.s pv. avii.\nC_LIO_LIGenome-based phylogenetic analyses revealed Psm races and P.s pv. avii clades were distinct and were each monophyletic, whereas cherry-pathogenic strains of Pss were interspersed amongst strains from other host species.\nC_LIO_LIA maximum likelihood approach was used to predict effectors associated with host specialisation on cherry. Pss possesses a smaller repertoire of type III effectors but has more toxin biosynthesis clusters compared with Psm and P.s pv. avii. Evolution of cherry pathogenicity was correlated with gain of genes such as hopAR1 and hopBB1 through putative phage transfer and horizontal transfer, respectively. By contrast, loss of the avrPto/hopAB redundant effector group was observed in cherry-pathogenic clades. Ectopic expression of hopAB and hopC1 triggered the hypersensitive reaction in cherry leaves, confirming computational predictions.\nC_LIO_LICherry canker provides a fascinating example of convergent evolution of pathogenicity that is explained by the mix of effector and toxin repertoires acting on a common host.\nC_LI

microbiology↗