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April 21, 2020  |  

Diversity of phytobeneficial traits revealed by whole-genome analysis of worldwide-isolated phenazine-producing Pseudomonas spp.

Plant-beneficial Pseudomonas spp. competitively colonize the rhizosphere and display plant-growth promotion and/or disease-suppression activities. Some strains within the P. fluorescens species complex produce phenazine derivatives, such as phenazine-1-carboxylic acid. These antimicrobial compounds are broadly inhibitory to numerous soil-dwelling plant pathogens and play a role in the ecological competence of phenazine-producing Pseudomonas spp. We assembled a collection encompassing 63 strains representative of the worldwide diversity of plant-beneficial phenazine-producing Pseudomonas spp. In this study, we report the sequencing of 58 complete genomes using PacBio RS II sequencing technology. Distributed among four subgroups within the P. fluorescens species complex, the diversity of our collection is reflected by the large pangenome which accounts for 25 413 protein-coding genes. We identified genes and clusters encoding for numerous phytobeneficial traits, including antibiotics, siderophores and cyclic lipopeptides biosynthesis, some of which were previously unknown in these microorganisms. Finally, we gained insight into the evolutionary history of the phenazine biosynthetic operon. Given its diverse genomic context, it is likely that this operon was relocated several times during Pseudomonas evolution. Our findings acknowledge the tremendous diversity of plant-beneficial phenazine-producing Pseudomonas spp., paving the way for comparative analyses to identify new genetic determinants involved in biocontrol, plant-growth promotion and rhizosphere competence. © 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.

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April 21, 2020  |  

Genetic map-guided genome assembly reveals a virulence-governing minichromosome in the lentil anthracnose pathogen Colletotrichum lentis.

Colletotrichum lentis causes anthracnose, which is a serious disease on lentil and can account for up to 70% crop loss. Two pathogenic races, 0 and 1, have been described in the C. lentis population from lentil. To unravel the genetic control of virulence, an isolate of the virulent race 0 was sequenced at 1481-fold genomic coverage. The 56.10-Mb genome assembly consists of 50 scaffolds with N50 scaffold length of 4.89 Mb. A total of 11 436 protein-coding gene models was predicted in the genome with 237 coding candidate effectors, 43 secondary metabolite biosynthetic enzymes and 229 carbohydrate-active enzymes (CAZymes), suggesting a contraction of the virulence gene repertoire in C. lentis. Scaffolds were assigned to 10 core and two minichromosomes using a population (race 0 × race 1, n = 94 progeny isolates) sequencing-based, high-density (14 312 single nucleotide polymorphisms) genetic map. Composite interval mapping revealed a single quantitative trait locus (QTL), qClVIR-11, located on minichromosome 11, explaining 85% of the variability in virulence of the C. lentis population. The QTL covers a physical distance of 0.84 Mb with 98 genes, including seven candidate effector and two secondary metabolite genes. Taken together, the study provides genetic and physical evidence for the existence of a minichromosome controlling the C. lentis virulence on lentil. © 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.

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April 21, 2020  |  

Bioinformatic analysis of the complete genome sequence of Pectobacterium carotovorum subsp. brasiliense BZA12 and candidate effector screening

AbstractPectobacterium carotovorum subsp. brasiliense (Pcb) is a gram-negative, plant pathogenic bacterium of the soft rot Enterobacteriaceae (SRE) family. We present the complete genome sequence of Pcb strain BZA12, which reveals that Pcb strain BZA12 carries a single 4,924,809 bp chromosome with 51.97% GC content and comprises 4508 predicted protein-coding genes.Geneannotationofthese genes utilizedGO, KEGG,and COG databases.Incomparison withthree closely related soft-rot pathogens, strain BZA12 has 3797 gene families, among which 3107 gene families are identified as orthologous with those of both P. carotovorum subsp. carotovorum PCC21 and P. carotovorum subsp. odoriferum BCS7, as well as 36 putative Unique Gene Families. We selected five putative effectors from the BZA12 genome and transiently expressed them in Nicotiana benthamiana. Candidate effector A12GL002483 was localized in the cell nucleus and induced cell death. This study provides a foundation for a better understanding of the genomic structure and function of Pcb, particularly in the discovery of potential pathogenic factors and for the development of more effective strategies against this pathogen.

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April 21, 2020  |  

Dynamic virulence-related regions of the plant pathogenic fungus Verticillium dahliae display enhanced sequence conservation.

Plant pathogens continuously evolve to evade host immune responses. During host colonization, many fungal pathogens secrete effectors to perturb such responses, but these in turn may become recognized by host immune receptors. To facilitate the evolution of effector repertoires, such as the elimination of recognized effectors, effector genes often reside in genomic regions that display increased plasticity, a phenomenon that is captured in the two-speed genome hypothesis. The genome of the vascular wilt fungus Verticillium dahliae displays regions with extensive presence/absence polymorphisms, so-called lineage-specific regions, that are enriched in in planta-induced putative effector genes. As expected, comparative genomics reveals differential degrees of sequence divergence between lineage-specific regions and the core genome. Unanticipated, lineage-specific regions display markedly higher sequence conservation in coding as well as noncoding regions than the core genome. We provide evidence that disqualifies horizontal transfer to explain the observed sequence conservation and conclude that sequence divergence occurs at a slower pace in lineage-specific regions of the V. dahliae genome. We hypothesize that differences in chromatin organisation may explain lower nucleotide substitution rates in the plastic, lineage-specific regions of V. dahliae. © 2019 The Authors. Molecular Ecology Published by John Wiley & Sons Ltd.

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April 21, 2020  |  

Snf2 controls pulcherriminic acid biosynthesis and antifungal activity of the biocontrol yeast Metschnikowia pulcherrima.

Metschnikowia pulcherrima synthesises the pigment pulcherrimin, from cyclodileucine (cyclo(Leu-Leu)) as a precursor, and exhibits strong antifungal activity against notorious plant pathogenic fungi. This yeast therefore has great potential for biocontrol applications against fungal diseases; particularly in the phyllosphere where this species is frequently found. To elucidate the molecular basis of the antifungal activity of M. pulcherrima, we compared a wild-type strain with a spontaneously occurring, pigmentless, weakly antagonistic mutant derivative. Whole genome sequencing of the wild-type and mutant strains identified a point mutation that creates a premature stop codon in the transcriptional regulator gene SNF2 in the mutant. Complementation of the mutant strain with the wild-type SNF2 gene restored pigmentation and recovered the strong antifungal activity. Mass spectrometry (UPLC HR HESI-MS) proved the presence of the pulcherrimin precursors cyclo(Leu-Leu) and pulcherriminic acid and identified new precursor and degradation products of pulcherriminic acid and/or pulcherrimin. All of these compounds were identified in the wild-type and complemented strain, but were undetectable in the pigmentless snf2 mutant strain. These results thus identify Snf2 as a regulator of antifungal activity and pulcherriminic acid biosynthesis in M. pulcherrima and provide a starting point for deciphering the molecular functions underlying the antagonistic activity of this yeast. © 2019 The Authors. Molecular Microbiology Published by John Wiley & Sons Ltd.

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April 21, 2020  |  

Function and Distribution of a Lantipeptide in Strawberry Fusarium Wilt Disease-Suppressive Soils.

Streptomyces griseus S4-7 is representative of strains responsible for the specific soil suppressiveness of Fusarium wilt of strawberry caused by Fusarium oxysporum f. sp. fragariae. Members of the genus Streptomyces secrete diverse secondary metabolites including lantipeptides, heat-stable lanthionine-containing compounds that can exhibit antibiotic activity. In this study, a class II lantipeptide provisionally named grisin, of previously unknown biological function, was shown to inhibit F. oxysporum. The inhibitory activity of grisin distinguishes it from other class II lantipeptides from Streptomyces spp. Results of quantitative reverse transcription-polymerase chain reaction with lanM-specific primers showed that the density of grisin-producing Streptomyces spp. in the rhizosphere of strawberry was positively correlated with the number of years of monoculture and a minimum of seven years was required for development of specific soil suppressiveness to Fusarium wilt disease. We suggest that lanM can be used as a diagnostic marker of whether a soil is conducive or suppressive to the disease.

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April 21, 2020  |  

Highly flexible infection programs in a specialized wheat pathogen.

Many filamentous plant pathogens exhibit high levels of genomic variability, yet the impact of this variation on host-pathogen interactions is largely unknown. We have addressed host specialization in the wheat pathogen Zymoseptoria tritici. Our study builds on comparative analyses of infection and gene expression phenotypes of three isolates and reveals the extent to which genomic variation translates into phenotypic variation. The isolates exhibit genetic and genomic variation but are similarly virulent. By combining confocal microscopy, disease monitoring, staining of ROS, and comparative transcriptome analyses, we conducted a detailed comparison of the infection processes of these isolates in a susceptible wheat cultivar. We characterized four core infection stages: establishment, biotrophic growth, lifestyle transition, and necrotrophic growth and asexual reproduction that are shared by the three isolates. However, we demonstrate differentiated temporal and spatial infection development and significant differences in the expression profiles of the three isolates during the infection stages. More than 20% of the genes were differentially expressed and these genes were located significantly closer to transposable elements, suggesting an impact of epigenetic regulation. Further, differentially expressed genes were enriched in effector candidates suggesting that isolate-specific strategies for manipulating host defenses are present in Z. tritici. We demonstrate that individuals of a host-specialized pathogen have highly differentiated infection programs characterized by flexible infection development and functional redundancy. This illustrates how high genetic diversity in pathogen populations results in highly differentiated infection phenotypes, which fact needs to be acknowledged to understand host-pathogen interactions and pathogen evolution.

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April 21, 2020  |  

Toxin and genome evolution in a Drosophila defensive symbiosis.

Defenses conferred by microbial symbionts play a vital role in the health and fitness of their animal hosts. An important outstanding question in the study of defensive symbiosis is what determines long term stability and effectiveness against diverse natural enemies. In this study, we combine genome and transcriptome sequencing, symbiont transfection and parasite protection experiments, and toxin activity assays to examine the evolution of the defensive symbiosis between Drosophila flies and their vertically transmitted Spiroplasma bacterial symbionts, focusing in particular on ribosome-inactivating proteins (RIPs), symbiont-encoded toxins that have been implicated in protection against both parasitic wasps and nematodes. Although many strains of Spiroplasma, including the male-killing symbiont (sMel) of Drosophila melanogaster, protect against parasitic wasps, only the strain (sNeo) that infects the mycophagous fly Drosophila neotestacea appears to protect against parasitic nematodes. We find that RIP repertoire is a major differentiating factor between strains that do and do not offer nematode protection, and that sMel RIPs do not show activity against nematode ribosomes in vivo. We also discovered a strain of Spiroplasma infecting a mycophagous phorid fly, Megaselia nigra. Although both the host and its Spiroplasma are distantly related to D. neotestacea and its symbiont, genome sequencing revealed that the M. nigra symbiont encodes abundant and diverse RIPs, including plasmid-encoded toxins that are closely related to the RIPs in sNeo. Our results suggest that distantly related Spiroplasma RIP toxins may perform specialized functions with regard to parasite specificity and suggest an important role for horizontal gene transfer in the emergence of novel defensive phenotypes.

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April 21, 2020  |  

Population Genome Sequencing of the Scab Fungal Species Venturia inaequalis, Venturia pirina, Venturia aucupariae and Venturia asperata.

The Venturia genus comprises fungal species that are pathogens on Rosaceae host plants, including V. inaequalis and V. asperata on apple, V. aucupariae on sorbus and V. pirina on pear. Although the genetic structure of V. inaequalis populations has been investigated in detail, genomic features underlying these subdivisions remain poorly understood. Here, we report whole genome sequencing of 87 Venturia strains that represent each species and each population within V. inaequalis We present a PacBio genome assembly for the V. inaequalis EU-B04 reference isolate. The size of selected genomes was determined by flow cytometry, and varied from 45 to 93 Mb. Genome assemblies of V. inaequalis and V. aucupariae contain a high content of transposable elements (TEs), most of which belong to the Gypsy or Copia LTR superfamilies and have been inactivated by Repeat-Induced Point mutations. The reference assembly of V. inaequalis presents a mosaic structure of GC-equilibrated regions that mainly contain predicted genes and AT-rich regions, mainly composed of TEs. Six pairs of strains were identified as clones. Single-Nucleotide Polymorphism (SNP) analysis between these clones revealed a high number of SNPs that are mostly located in AT-rich regions due to misalignments and allowed determining a false discovery rate. The availability of these genome sequences is expected to stimulate genetics and population genomics research of Venturia pathogens. Especially, it will help understanding the evolutionary history of Venturia species that are pathogenic on different hosts, a history that has probably been substantially influenced by TEs.Copyright © 2019 Le Cam et al.

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April 21, 2020  |  

Ancestral Admixture Is the Main Determinant of Global Biodiversity in Fission Yeast.

Mutation and recombination are key evolutionary processes governing phenotypic variation and reproductive isolation. We here demonstrate that biodiversity within all globally known strains of Schizosaccharomyces pombe arose through admixture between two divergent ancestral lineages. Initial hybridization was inferred to have occurred ~20-60 sexual outcrossing generations ago consistent with recent, human-induced migration at the onset of intensified transcontinental trade. Species-wide heritable phenotypic variation was explained near-exclusively by strain-specific arrangements of alternating ancestry components with evidence for transgressive segregation. Reproductive compatibility between strains was likewise predicted by the degree of shared ancestry. To assess the genetic determinants of ancestry block distribution across the genome, we characterized the type, frequency, and position of structural genomic variation using nanopore and single-molecule real-time sequencing. Despite being associated with double-strand break initiation points, over 800 segregating structural variants exerted overall little influence on the introgression landscape or on reproductive compatibility between strains. In contrast, we found strong ancestry disequilibrium consistent with negative epistatic selection shaping genomic ancestry combinations during the course of hybridization. This study provides a detailed, experimentally tractable example that genomes of natural populations are mosaics reflecting different evolutionary histories. Exploiting genome-wide heterogeneity in the history of ancestral recombination and lineage-specific mutations sheds new light on the population history of S. pombe and highlights the importance of hybridization as a creative force in generating biodiversity. © The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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April 21, 2020  |  

Evolution of Antibiotic Synthesis Gene Clusters in the Streptomyces globisporus TFH56, Isolated from Tomato Flower.

Streptomyces species are known to produce various bioactive metabolites that can prevent plant diseases. Previously, the Streptomyces strain TFH56 was found to inhibit the gray mold pathogen, Botrytis cinerea, in tomato flower. In this study, the genome sequence of strain TFH56 was acquired using the Pacific Biosciences RS II platform. Three linear sequences (7.67 Mbp in total) were obtained. Based on average nucleotide identity, strain TFH56 was classified as Streptomyces globisporus, which is consistent with the presence of a linear chromosome and linear plasmids. Moreover, as with other examples of S. globisporus, the genome of strain TFH56 included a caryolan-1-ol synthase gene, a conprimycin synthetic gene cluster, and a lidamycin synthetic gene cluster.Copyright © 2019 Cho, Kwak.

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April 21, 2020  |  

Agricultural intensification reduces microbial network complexity and the abundance of keystone taxa in roots.

Root-associated microbes play a key role in plant performance and productivity, making them important players in agroecosystems. So far, very few studies have assessed the impact of different farming systems on the root microbiota and it is still unclear whether agricultural intensification influences the structure and complexity of microbial communities. We investigated the impact of conventional, no-till, and organic farming on wheat root fungal communities using PacBio SMRT sequencing on samples collected from 60 farmlands in Switzerland. Organic farming harbored a much more complex fungal network with significantly higher connectivity than conventional and no-till farming systems. The abundance of keystone taxa was the highest under organic farming where agricultural intensification was the lowest. We also found a strong negative association (R2?=?0.366; P?

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April 21, 2020  |  

Genomic Plasticity Mediated by Transposable Elements in the Plant Pathogenic Fungus Colletotrichum higginsianum.

Phytopathogen genomes are under constant pressure to change, as pathogens are locked in an evolutionary arms race with their hosts, where pathogens evolve effector genes to manipulate their hosts, whereas the hosts evolve immune components to recognize the products of these genes. Colletotrichum higginsianum (Ch), a fungal pathogen with no known sexual morph, infects Brassicaceae plants including Arabidopsis thaliana. Previous studies revealed that Ch differs in its virulence toward various Arabidopsis thaliana ecotypes, indicating the existence of coevolutionary selective pressures. However, between-strain genomic variations in Ch have not been studied. Here, we sequenced and assembled the genome of a Ch strain, resulting in a highly contiguous genome assembly, which was compared with the chromosome-level genome assembly of another strain to identify genomic variations between strains. We found that the two closely related strains vary in terms of large-scale rearrangements, the existence of strain-specific regions, and effector candidate gene sets and that these variations are frequently associated with transposable elements (TEs). Ch has a compartmentalized genome consisting of gene-sparse, TE-dense regions with more effector candidate genes and gene-dense, TE-sparse regions harboring conserved genes. Additionally, analysis of the conservation patterns and syntenic regions of effector candidate genes indicated that the two strains vary in their effector candidate gene sets because of de novo evolution, horizontal gene transfer, or gene loss after divergence. Our results reveal mechanisms for generating genomic diversity in this asexual pathogen, which are important for understanding its adaption to hosts. © The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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April 21, 2020  |  

Smut infection of perennial hosts: the genome and the transcriptome of the Brassicaceae smut fungus Thecaphora thlaspeos reveal functionally conserved and novel effectors.

Biotrophic fungal plant pathogens can balance their virulence and form intricate relationships with their hosts. Sometimes, this leads to systemic host colonization over long time scales without macroscopic symptoms. However, how plant-pathogenic endophytes manage to establish their sustained systemic infection remains largely unknown. Here, we present a genomic and transcriptomic analysis of Thecaphora thlaspeos. This relative of the well studied grass smut Ustilago maydis is the only smut fungus adapted to Brassicaceae hosts. Its ability to overwinter with perennial hosts and its systemic plant infection including roots are unique characteristics among smut fungi. The T. thlaspeos genome was assembled to the chromosome level. It is a typical smut genome in terms of size and genome characteristics. In silico prediction of candidate effector genes revealed common smut effector proteins and unique members. For three candidates, we have functionally demonstrated effector activity. One of these, TtTue1, suggests a potential link to cold acclimation. On the plant side, we found evidence for a typical immune response as it is present in other infection systems, despite the absence of any macroscopic symptoms during infection. Our findings suggest that T. thlaspeos distinctly balances its virulence during biotrophic growth ultimately allowing for long-lived infection of its perennial hosts. © 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.

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April 21, 2020  |  

A Species-Wide Inventory of NLR Genes and Alleles in Arabidopsis thaliana.

Infectious disease is both a major force of selection in nature and a prime cause of yield loss in agriculture. In plants, disease resistance is often conferred by nucleotide-binding leucine-rich repeat (NLR) proteins, intracellular immune receptors that recognize pathogen proteins and their effects on the host. Consistent with extensive balancing and positive selection, NLRs are encoded by one of the most variable gene families in plants, but the true extent of intraspecific NLR diversity has been unclear. Here, we define a nearly complete species-wide pan-NLRome in Arabidopsis thaliana based on sequence enrichment and long-read sequencing. The pan-NLRome largely saturates with approximately 40 well-chosen wild strains, with half of the pan-NLRome being present in most accessions. We chart NLR architectural diversity, identify new architectures, and quantify selective forces that act on specific NLRs and NLR domains. Our study provides a blueprint for defining pan-NLRomes.Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.

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