Pathogen-induced activation of disease-suppressive functions in the endophytic root microbiome

Microorganisms living inside plants can promote plant growth and health, but their genomic and functional diversity remain largely elusive. Here, metagenomics and network inference show that fungal infection of plant roots enriched for Chitinophagaceae and Flavobacteriaceae in the root endosphere and for chitinase genes and various unknown biosynthetic gene clusters encoding the production of nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKSs). After strain-level genome reconstruction, a consortium of Chitinophaga and Flavobacterium was designed that consistently suppressed fungal root disease. Site-directed mutagenesis then revealed that a previously unidentified NRPS-PKS gene cluster from Flavobacterium was essential for disease suppression by the endophytic consortium. Our results highlight that endophytic root microbiomes harbor a wealth of as yet unknown functional traits that, in concert, can protect the plant inside out.</p

Dissecting disease-suppressive rhizosphere microbiomes using metagenomics

Plants and microbes have coexisted for hundreds of millions of years and have developed deeply intertwined mutually beneficial relations. Among the many benefits of a selected microbial community, pathogen suppression is a particularly desirable trait for both the plant and agronomy industry. Suppressive soils have been described for many years but technology to develop a deep understanding of this phenomenon was only recently introduced. In this thesis, I applied different metagenomic sequencing approaches to study the biological basis of suppressive soils with particular interest toward the biosynthetic potential of the suppressive rhizosphere community. In the introduction, I describe ecology-inspired solutions for biosynthetic gene cluster (BGC) mining and the existing tools and sequencing technologies that can be used to this end.&nbsp;We take a structured approach to the dissection of the suppressive-associated rhizosphere communities. In the first part of the work, we perform the first large-scale soil survey aiming at establishing a soil collection to identify unique characteristics of suppressive soils. Through a combination of phenotyping and marker gene sequencing, we identify four soils with strong Fusarium culmorum-suppressive characteristics. We compare taxonomy composition and volatile profile of both suppressive and non-suppressive soils to identify features that distinguish suppressive soils. The suppressive soils found in this collection do not share physiochemical or categorical characteristics. In addition, diversity and community structure do not separate suppressive and non-suppressive soils. However, network-based analysis shows a group of acidobacteria which co-occur in a suppressive-soil-associated hub.&nbsp;Then, to better understand the secondary metabolite diversity of the suppressive samples we characterize of nonribosomal peptide synthetase diversity in suppressive and conducive soils with the use of functional amplicon sequencing of NRPS adenylation domains. To this end, we developed dom2BGC, a pipeline for the annotation of domains associated with BGCs. We also perform cooccurrence-based clustering of the sequenced domains to restore, through guilt by association, the physical clustering of the different domains annotated to the same (BGC). We identified multiple NRPSes with potentially antifungal activity that occur exclusively in suppressive soils. Furthermore, we sequenced one suppressive sample with 10X metagenomic sequencing, which was used to confirm the presence of dom2BGC reconstructed clusters.After extensive study of suppressive rhizosphere communities, we zoom in and perform a dilution to extinction experiment with a microbial extract from a suppressive soil which progressively loses its phenotype in accordance with the dilution. We evaluate the effect of dilution on the microbial composition and functional profile of the community. Genetic characteristics and taxonomic groups that correlate with the dilution and phenotype loss are investigated for links that can shed light on the key players of suppressive soils. We found multiple metagenome-assembled genomes rich in BGCs and chitin-degrading ability that closely correlate with the loss in pathogen suppression.&nbsp;This work then proceeds to describe the characteristics of a suppressive endosphere. We compare Rhizoctonia solani suppressive and conducive endosphere communities of sugar beet. Shotgun metagenome sequencing showed significant differences in taxonomic abundance and genes associated with Chitinophaga and Flavobacterium bacteria. Additionally, we compose a synthetic community from endosphere isolates that provides disease suppression against Rhizoctoria solani infection. Finally, disease suppression of the synthetic community is lost upon site-directed mutagenesis of a candidate suppressive NRPS in a flavobacterial isolate, providing a model to explain the phenotype.Finally, we detail the development of an assembly tool that aims to improve the assembly of complex BGCs. BGCs often contain repetitive domains that are hard to assemble, but are still very informative as they strongly influence the predicted natural product. Such repetitive domains are sometimes inadvertedly collapsed during the assembly graph formation, which inevitably leads to an erroneous or incomplete cluster. These problems are exacerbated in complex metagenomics assemblies. BiosyntheticSPAdes is designed to identify and isolate BGC-harboring neighborhoods in the assembly graph by finding multiple adjacent BGC-associated domains. Once identified, the BGC subgraph is extracted and collapsed domains are restored based on local coverage. Depending on the subgraph, multiple paths can be traversed to produce a BGC sequence. When multiple putative BGCs are produced, a ranking pipeline shows which candidate BGC is structurally similar to previously assembled BGCs based on sequence similarity and domain order.&nbsp;To conclude, in the discussion I offer some considerations on the effects of sequencing technologies on microbiology and microbial ecology, to then propose experimental and computational strategies that are best fit to identify microbial natural products from complex ecosystems

Mining prokaryotes for antimicrobial compounds : From diversity to function

The bacterial kingdom provides a major source of antimicrobials that can either be directly applied or used as scaffolds to further improve their functionality in the host. The rapidly increasing amount of bacterial genomic, metabolomic and transcriptomic data offers unique opportunities to apply a variety of approaches to mine for existing and novel antimicrobials. Here, we discuss several powerful mining approaches to identify novel molecules with antimicrobial activity across structurally diverse natural products, including ribosomally synthesized and posttranslationally modified peptides, nonribosomal peptides and polyketides. We not only discuss the direct mining of genomes based on identification of biosynthetic gene clusters, but also describe more advanced and integrative approaches in ecology-based mining, functionality-based mining and mode-of-action-based mining. These efforts are likely to accelerate the discovery and development of novel antimicrobial drugs.</p

BiosyntheticSPAdes: reconstructing biosynthetic gene clusters from assembly graphs

Predicting biosynthetic gene clusters (BGCs) is critically important for discovery of antibiotics and other natural products. While BGC prediction from complete genomes is a well-studied problem, predicting BGCs in fragmented genomic assemblies remains challenging. The existing BGC prediction tools often assume that each BGC is encoded within a single contig in the genome assembly, a condition that is violated for most sequenced microbial genomes where BGCs are often scattered through several contigs, making it difficult to reconstruct them. The situation is even more severe in shotgun metagenomics, where the contigs are often short, and the existing tools fail to predict a large fraction of long BGCs. While it is difficult to assemble BGCs in a single contig, the structure of the genome assembly graph often provides clues on how to combine multiple contigs into segments encoding long BGCs. We describe biosyntheticSPAdes, a tool for predicting BGCs in assembly graphs and demonstrate that it greatly improves the reconstruction of BGCs from genomic and metagenomics data sets.</p

Microbial and volatile profiling of soils suppressive to Fusarium culmorum of wheat

In disease-suppressive soils, microbiota protect plants from root infections. Bacterial members of this microbiota have been shown to produce specific molecules that mediate this phenotype. To date, however, studies have focused on individual suppressive soils and the degree of natural variability of soil suppressiveness remains unclear. Here, we screened a large collection of field soils for suppressiveness to Fusarium culmorum using wheat (Triticum aestivum) as a model host plant. A high variation of disease suppressiveness was observed, with 14% showing a clear suppressive phenotype. The microbiological basis of suppressiveness to F. culmorum was confirmed by gamma sterilization and soil transplantation. Amplicon sequencing revealed diverse bacterial taxonomic compositions and no specific taxa were found exclusively enriched in all suppressive soils. Nonetheless, co-occurrence network analysis revealed that two suppressive soils shared an overrepresented bacterial guild dominated by various Acidobacteria. In addition, our study revealed that volatile emission may contribute to suppression, but not for all suppressive soils. Our study raises new questions regarding the possible mechanistic variability of disease-suppressive phenotypes across physico-chemically different soils. Accordingly, we anticipate that larger-scale soil profiling, along with functional studies, will enable a deeper understanding of disease-suppressive microbiomes.</p

Data from: Microbial and volatile profiling of soils suppressive to Fusarium culmorum of wheat

In disease-suppressive soils, microbiota protect plants from root infections. Bacterial members of this microbiota have been shown to produce specific molecules that mediate this phenotype. To date, however, studies have focused on individual suppressive soils and the degree of natural variability of soil suppressiveness remains unclear. Here, we screened a large collection of field soils for suppressiveness to Fusarium culmorum using wheat (Triticum aestivum) as a model host plant. A high variation of disease suppressiveness was observed, with 14% showing a clear suppressive phenotype. The microbiological basis of suppressiveness to F. culmorum was confirmed by gamma sterilization and soil transplantation. Amplicon sequencing revealed diverse bacterial taxonomic compositions and no specific taxa were found exclusively enriched in all suppressive soils. Nonetheless, co-occurrence network analysis revealed that two suppressive soils shared an overrepresented bacterial guild dominated by various Acidobacteria. In addition, our study revealed that volatile emission may contribute to suppression, but not for all suppressive soils. Our study raises new questions regarding the possible mechanistic variability of disease-suppressive phenotypes across physico-chemically different soils. Accordingly, we anticipate that larger-scale soil profiling, along with functional studies, will enable a deeper understanding of disease-suppressive microbiomes.,Ossowicki&amp;Tracanna 16S amplicon data, part 1 Raw FASTQ files of 16S amplicon data. Part 1 of 2-part tarball. Join the two parts using 'cat' before extracting. Ossowicki_Tracanna_16S_amplicon_data.tar.gz.part_aa Ossowicki&amp;Tracanna 16S amplicon data, part 2 Raw FASTQ files of 16S amplicon data. Part 1 of 2-part tarball. Join the two parts using 'cat' before extracting. Ossowicki_Tracanna_16S_amplicon_data.tar.gz.part_ab

Implications of the three-dimensional chromatin organization for genome evolution in a fungal plant pathogen

Abstract The spatial organization of eukaryotic genomes is linked to their biological functions, although it is not clear how this impacts the overall evolution of a genome. Here, we uncover the three-dimensional (3D) genome organization of the phytopathogen Verticillium dahliae, known to possess distinct genomic regions, designated adaptive genomic regions (AGRs), enriched in transposable elements and genes that mediate host infection. Short-range DNA interactions form clear topologically associating domains (TADs) with gene-rich boundaries that show reduced levels of gene expression and reduced genomic variation. Intriguingly, TADs are less clearly insulated in AGRs than in the core genome. At a global scale, the genome contains bipartite long-range interactions, particularly enriched for AGRs and more generally containing segmental duplications. Notably, the patterns observed for V. dahliae are also present in other Verticillium species. Thus, our analysis links 3D genome organization to evolutionary features conserved throughout the Verticillium genus

Exploring the Interspecific Interactions and the Metabolome of the Soil Isolate Hylemonella gracilis

Microbial community analysis of aquatic environments showed that an important component of its microbial diversity consists of bacteria with cell sizes of ~0.1 μm. Such small bacteria can show genomic reductions and metabolic dependencies with other bacteria. However, so far, no study has investigated if such bacteria exist in terrestrial environments like soil. Here, we isolated soil bacteria that passed through a 0.1-μm filter. The complete genome of one of the isolates was sequenced and the bacterium was identified as Hylemonella gracilis. A set of coculture assays with phylogenetically distant soil bacteria with different cell and genome sizes was performed. The coculture assays revealed that H. gracilis grows better when interacting with other soil bacteria like Paenibacillus sp. AD87 and Serratia plymuthica. Transcriptomics and metabolomics showed that H. gracilis was able to change gene expression, behavior, and biochemistry of the interacting bacteria without direct cell-cell contact. Our study indicates that in soil there are bacteria that can pass through a 0.1-μm filter. These bacteria may have been overlooked in previous research on soil microbial communities. Such small bacteria, exemplified here by H. gracilis, can induce transcriptional and metabolomic changes in other bacteria upon their interactions in soil. In vitro, the studied interspecific interactions allowed utilization of growth substrates that could not be utilized by monocultures, suggesting that biochemical interactions between substantially different sized soil bacteria may contribute to the symbiosis of soil bacterial communities

Exploring the Interspecific Interactions and the Metabolome of the Soil Isolate Hylemonella gracilis

Microbial community analysis of aquatic environments showed that an important component of its microbial diversity consists of bacteria with cell sizes of ~0.1 μm. Such small bacteria can show genomic reductions and metabolic dependencies with other bacteria. However, so far, no study has investigated if such bacteria exist in terrestrial environments like soil. Here, we isolated soil bacteria that passed through a 0.1-μm filter. The complete genome of one of the isolates was sequenced and the bacterium was identified as Hylemonella gracilis. A set of coculture assays with phylogenetically distant soil bacteria with different cell and genome sizes was performed. The coculture assays revealed that H. gracilis grows better when interacting with other soil bacteria like Paenibacillus sp. AD87 and Serratia plymuthica. Transcriptomics and metabolomics showed that H. gracilis was able to change gene expression, behavior, and biochemistry of the interacting bacteria without direct cell-cell contact. Our study indicates that in soil there are bacteria that can pass through a 0.1-μm filter. These bacteria may have been overlooked in previous research on soil microbial communities. Such small bacteria, exemplified here by H. gracilis, can induce transcriptional and metabolomic changes in other bacteria upon their interactions in soil. In vitro, the studied interspecific interactions allowed utilization of growth substrates that could not be utilized by monocultures, suggesting that biochemical interactions between substantially different sized soil bacteria may contribute to the symbiosis of soil bacterial communities. IMPORTANCE Analysis of aquatic microbial communities revealed that parts of its diversity consist of bacteria with cell sizes of ~0.1 μm. Such bacteria can show genomic reductions and metabolic dependencies with other bacteria. So far, no study investigated if such bacteria exist in terrestrial environments such as soil. Here, we show that such bacteria also exist in soil. The isolated bacteria were identified as Hylemonella gracilis. Coculture assays with phylogenetically different soil bacteria revealed that H. gracilis grows better when cocultured with other soil bacteria. Transcriptomics and metabolomics showed that H. gracilis was able to change gene expression, behavior, and biochemistry of the interacting bacteria without direct contact. Our study revealed that bacteria are present in soil that can pass through 0.1-μm filters. Such bacteria may have been overlooked in previous research on soil microbial communities and may contribute to the symbiosis of soil bacterial communities