Biology subjects

de Jonge, R.

Publications and source records attributed to de Jonge, R..

At least 19 recordsLinked to original sources

bioRxiv2026

Beyond the annotated: protein foundation models enable robust prediction of microbial root competence

BackgroundRoot competence, the ability of soil bacteria to establish and grow on plant roots, is a key ecological trait influencing plant nutrition, growth, and health. However, identifying genomic determinants of root competence across bacteria remains challenging, in part because model generalisability depends strongly on how genomes are represented. Traditional approaches based on curated annotations are incomplete and biased toward well-characterised organisms and functions, limiting generalisation. Sequence-similarity clustering improves coverage but yields high-dimensional features relative to dataset size, hindering training. Foundation models offer an alternative by learning com-pact representations without relying on prior annotation. ResultsHere, we compared pretrained genome representations from protein and DNA foundation models (ESM-2, Bacformer, DNABERT-S) with annotation- and clustering-based features (KEGG orthology, OrthoFinder protein families) for predicting root competence using synthetic microbial community data from Arabidop-sis thaliana and assessed generalisability across bacteria. When training and test sets contained taxonomically related bacteria, most approaches performed similarly. However, when test bacteria belonged to phyla entirely absent from training, reflecting high evolutionary separation across all levels of bacterial classification, only pretrained protein representations retained predictive performance. Bacformer-derived representations, which incorporate genomic context, supported the strongest generalisation, suggesting that conserved genomic organisation contributes to predicting root competence. Feature attribution quantifying protein contributions to model decisions linked root competence to TonB/SusD-dependent receptors, small-molecule transporters, and unannotated proteins with conserved regulatory motifs and homology to carbon starvation-response loci. ConclusionsProtein foundation models support generalisation across evolutionarily distant bacteria and identify genomic determinants of root competence, including unannotated proteins.

genomics↗

bioRxiv2026

The insect- and plant-associated lifestyles of Pseudomonas protegens CHA0 are preserved following serial passage through insect larvae

The plant-beneficial bacterium Pseudomonas protegens CHA0 (CHA0) is widely studied for the biological control of soil-borne plant diseases. Beyond its root-colonising capabilities, CHA0 can also infect and kill insect larvae and thus exhibits a multi-host lifestyle shared with other plant- and insect-colonising bacteria. To better understand the robustness of this multi-host lifestyle, we subjected CHA0 to ten consecutive passages through larvae of the pest insect Plutella xylostella via repeated cycles of insect colonisation and killing forcing it into an insect-only lifestyle. Overall, serial passaging did not result in consistent changes in insect killing speed, larval or root colonisation, plant protection efficiency, microbial antagonism or in vitro growth. This suggests that its multi-host lifestyle was conserved following serial passage. Nonetheless, a few independently passaged lines showed an increase in larval killing speed, which in one case might be linked to choline uptake. To disentangle changes specific to the insect host from those arising due to the experimental system itself, we conducted parallel serial passages through the same system while omitting the insect host. In some of these lines, exposure to the background of the system led to changes in microbial antagonism and in in vitro growth, which likely are associated with mutations in regions encoding for regulatory systems. Our findings indicate that P. protegens CHA0 remains phenotypically stable in complex environments such as an insect host, suggesting that the multi-host lifestyle might also be conserved when applied in the field and supporting CHA0s potential for reliable biocontrol performance against both plant diseases and insect pests. Author summaryControlling insect pests with living organisms, known as biological control, offers an environmentally friendly alternative to chemical pesticides. The plant-beneficial bacterium Pseudomonas protegens CHA0 is a promising biocontrol candidate that not only colonizes plant roots but also infects and kills certain insect larvae. This ability to colonize different hosts appears to be a conserved trait also observed in other bacteria. To better understand the robustness of this multi-host lifestyle, we repeatedly exposed CHA0 to larvae of the insect pest Plutella xylostella and assessed the resulting physiological and genetic changes. Surprisingly, after ten cycles, CHA0 largely retained its insect-killing and plant-protective traits. Although a few populations showed minor changes, including slightly faster insect killing and traits associated with aspects of the experimental system, these changes were limited in scope. Overall, our findings suggest that P. protegens CHA0 does not change rapidly in complex environments such as an insect host, supporting its potential for reliable biocontrol performance in the field.

microbiology↗

bioRxiv2025

ASVNet: Inferring microbes from 16S rRNA amplicon sequencing data

Amplicon sequencing of the variable regions of the 16S rRNA gene is one of the most common techniques in microbiome research. A crucial step in analysing this data is to cluster raw sequences into biologically interpretable groups that approximate the actual organisms in a community, which reduces data complexity, facilitates diversity analyses, and enables clearer ecological and functional interpretations. Most approaches rely on fixed sequence similarity thresholds, typically operational taxonomic units (OTUs) at 97% or amplicon sequence variants (ASVs) at 99-100%. However, finding the correct correspondence between amplicons and the original organisms in a complex community remains challenging, primarily due to the presence of varying copy numbers within genomes and the high similarity of amplicon sequences among species. While the stringent threshold in ASVs prevents merging sequences from different species, it often splits sequences from the same genome into multiple ASVs, inflating species counts. Conversely, the 97% similarity threshold in OTUs tends to frequently collapse multiple distinct organisms into a single unit regardless of their functional or ecological (dis)similarity, and thereby underestimating true diversity. To address these limitations, we introduce ASVNet, an algorithmic framework that leverages amplicon sequence similarity and co-abundance networks to better reflect the underlying organisms in a community. ASVNet builds on the principle that amplicon counts from the same organism co-vary across samples and it thus attempts to cluster these co-occurring, similar sequences as empirical OTUs (eOTUs). Comparative analyses in synthetic and natural root-associated microbial communities with genome-sequenced isolates show that ASVNet-derived eOTUs more accurately represent the original microbes and possess greater biological relevance than OTUs defined at any fixed threshold. By providing a biologically grounded framework to amplicon sequence data interpretation, ASVNet extends our ability to extract meaningful insights from complex microbiome datasets.

bioinformatics↗

bioRxiv2025

Evolution of resistance breaking in spinach downy mildew revealed through pangenome graph analysis

BackgroundFilamentous plant pathogens secrete effectors to successfully establish host infections. In resistant crop varieties, plant immunity can be triggered by immune receptors that recognize these effectors. Resistant crop varieties are grown in large-scale monocultures imposing strong selection pressure on pathogens, driving rapid evolution of effector repertoires resulting in the frequent breakdowns of resistance within just a few growing seasons. The oomycete Peronospora effusa, responsible for downy mildew on spinach, is an example of a rapidly adapting pathogen, but it is yet unknown how P. effusa can successfully overcome resistance of spinach by genomic adaptations. ResultsTo close this knowledge gap, we here generated genome assemblies and constructed a pangenome graph for 19 isolates corresponding to 19 officially denominated resistance-breaking P. effusa races, which can cause disease on a differential set of spinach cultivars. Haplotype-resolved pangenome graph analyses revealed that many isolates emerged from recent sexual recombination, yet others evolved via prolonged asexual reproduction and loss of heterozygosity. By phasing effector candidates to determine their allelic variation, we identified effector candidates associated to resistance breaking of spinach varieties and reconstructed the evolutionary events that led to their diversification. ConclusionsThe here developed and applied computational genomics approaches offer unparalleled insights into the molecular mechanisms of the rapid evolution of P. effusa, and points to potential targets for future resistance breading.

genomics↗

bioRxiv2025

Bacillus: driver of functional states in synthetic plant root bacterial communities

Plant roots release root exudates to attract microbes that form root communities, which in turn promote plant health and growth. Root community assembly arises from millions of interactions between microbes and the plant, leading to robust and stable microbial networks. To manage the complexity of natural root microbiomes for research purposes, scientists have developed reductionist approaches using synthetic microbial inocula, known as SynComs. In recent years, an increasing number of studies employed SynComs to investigate root microbiome assembly and dynamics under various conditions or with specific plant mutants. These studies have identified bacterial traits linked to root competence, but if and how these traits shape root microbiome dynamics across conditions is not well understood. To explore whether bacterial trait selection follows recurrent patterns, we conducted a meta-analysis of nine SynCom studies involving plant roots. Surprisingly, we observed that root communities frequently assemble into two distinct functional states. Further analysis revealed that these states are characterized by differences in the abundance of Bacilli. We propose that these Bacilli-associated functional states are driven by microbial interactions such as quorum sensing and biofilm formation. Additionally, we show that host activities, including root exudation and immune responses, influence the functional state of the root microbiome. Whether natural root communities also organize into distinct functional states remains unclear, but the implications could be significant. Functional diversification within root communities may influence the success and effectiveness of plant-beneficial bioinoculants, particularly Bacilli-based inoculants. To optimize microbiome-driven plant benefits, a deeper understanding of the mechanisms underlying functional state differentiation in root microbiomes is needed.

microbiology↗

bioRxiv2025

SyFi: generating and using sequence fingerprints to distinguish SynCom isolates

The plant root microbiome is a complex community shaped by interactions among bacteria, the plant host, and the environment. Synthetic community (SynCom) experiments help disentangle these interactions by inoculating host plants with a representative set of culturable microbial isolates from the natural root microbiome. Studying these simplified communities provides valuable insights into microbiome assembly and function. However, as SynComs become increasingly complex to better represent natural communities, bioinformatics challenges arise. Specifically, accurately identifying, and quantifying SynCom members based on, for example, 16S rRNA amplicon sequencing becomes more difficult due to the high similarity of the target amplicon, limiting downstream interpretations. Here, we present SynCom Fingerprinting (SyFi), a bioinformatics workflow designed to improve the resolution and accuracy of SynCom member identification. SyFi consists of three modules: the first module constructs a genomic fingerprint for each SynCom member based on its genome sequence, accounting for both copy number and sequence variation in the target gene. The second module then extracts a specific region from this genomic fingerprint to create a secondary fingerprint focused on the target amplicon. The third module uses these fingerprints as a reference to perform pseudoalignment-based quantification of SynCom member abundance from amplicon sequencing reads. We demonstrate that SyFi outperforms standard amplicon analysis by leveraging natural intragenomic variation, enabling more precise differentiation of closely related SynCom members. As a result, SyFi enhances the reliability of microbiome experiments using complex SynComs, which more accurately reflect natural communities. This improved resolution is essential for advancing our understanding of the root microbiome and its impact on plant health and productivity in agricultural and ecological settings. SyFi is available at https://github.com/adriangeerre/SyFi. Impact statementSyFi represents a significant advancement in microbiome research by enhancing the accuracy and resolution of synthetic community (SynCom) member identification. By leveraging natural intragenomic variation, SyFi improves the differentiation of closely related microbial strains, addressing a key challenge in amplicon-based sequencing analysis. This increased precision allows researchers to more reliably track microbial dynamics in complex SynCom experiments, leading to deeper insights into microbiome assembly, function, and host-microbe interactions. As a result, SyFi strengthens the interpretability of microbiome studies, ultimately contributing to a better understanding of plant health and productivity in both agricultural and ecological contexts. Data SummaryThe data reported in this article have been deposited in the National Center for Biotechnology Information Short Read Archive BioProject database. SyFi fingerprint generation was run on a collection of 737 human gut-derived bacterial genomes from Forster et al. (2019) (Genomic read data deposited in the ENA under project numbers ERP105624 and ERP012217) and 447 Arabidopsis-derived bacterial genomes (Selten et al., 2024b) (NCBI Project numbers PRJNA1138681, PRJNA1139421 (Genomes), and PRJNA1131834 (Genomic reads)). A list of the closed genomes used for SyFi validation can be found at https://github.com/adriangeerre/SyFi. Subsequently, SyFi was validated on a complex SynCom dataset by pseudoaligning 16S rRNA V3-V4 and V5-V7 amplicon reads (PRJNA1191388) to SyFi-generated fingerprints and comparing this to shotgun metagenomics-sequenced dataset of the same samples in Selten et al. (2024a) (PRJNA1131994). This complex SynCom dataset included the inoculation of the 447 bacterial isolates on Arabidopsis, Barley, and Lotus roots.

genomics↗

bioRxiv2024

Functional capacities drive recruitment of bacteria into plant root microbiota

Host-associated microbiota follow predictable assembly patterns but show significant variation at the bacterial isolate level depending on the host and environmental context. This variability poses challenges for studying, predicting, and engineering microbiomes. Here we examined how Arabidopsis, Barley, and Lotus plants recruit specific bacteria from highly complex synthetic communities (SynComs) composed of hundreds of bacterial isolates originating from these plants when grown in natural soil. We discovered that, despite their taxonomic diversity, bacteria enriched by these three plant species encode largely overlapping functions. A set of 266 functions common among all host-associated communities was identified at the foundation of the microbiotas functional potential. Analysis of the differences observed between root-associated communities revealed that functions recruited by Arabidopsis and Barley were primarily driven by the SynCom composition, while Lotus selected fewer isolates but with more diverse functionalities, akin to a Swiss army knife strategy. We analysed the variation at the functional level and found this can be explained by the combined functions of bacteria at the family level. Additionally, across major taxa, the isolates covering a broader range of their familys functional diversity achieved higher relative abundance in the root communities. Our work sheds light on key functions and principles guiding the recruitment of bacterial isolates into root microbiota, offering valuable insights for microbiome engineering and inoculant discovery at a previously inaccessible taxonomic level.

plant biology↗

bioRxiv2024

High-resolution quantification of the rhizosphere effect along a soil-to-root gradient shows selection-driven convergence of rhizosphere microbiomes

Plants secrete a complex array of organic compounds, constituting about a third of their photosynthetic products, into the surrounding soil. As a result, concentration gradients are established from the roots into the bulk soil, known as the rhizosphere. Soil microbes benefit from these root exudates for their survival and propagation, and consequently, the composition of the rhizosphere microbial community follows the gradient of available compounds, a phenomenon oftentimes referred to as the rhizosphere effect. However, the fine-grained changes in the microbial community along this soil-root gradient have not been well described. Yet such insights would enable us to underpin the ecological rules underlying root microbial community assembly. Therefore, here we harvested the roots of individual Arabidopsis thaliana plants grown in three different natural soils at high-resolution, such that we could interrogate community assembly and predict microbial growth rate across consecutive, fine-grained, rhizosphere compartments. We found that the strength of the rhizosphere effect depends on root proximity and that microbial communities closer to the roots harbour related microbes. Closer to the roots, microbial community assembly became less random and more driven by selection-based processes. Intriguingly, we observed priority effects, where related microbes that arrive first are more likely to establish, and that microbes might use different ecological growth strategies to colonise the rhizosphere. All effects appeared to be independent from starting conditions as microbial community composition converged on the root despite different soil microbial seed banks. Together, our results provide a high-resolution view of the microbiome changes across the soil-root gradient.

plant biology↗

bioRxiv2024

Pangenome graph analysis reveals extensive effector copy-number variation in spinach downy mildew

Plant pathogens adapt at speeds that challenge contemporary disease management strategies like the deployment of disease resistance genes. The strong evolutionary pressure to adapt, shapes pathogens genomes, and comparative genomics has been instrumental in characterizing this process. With the aim to capture genomic variation at high resolution and study the processes contributing to adaptation, we here leverage and expand on an innovative, multi-genome method to construct, annotate, and analyse the first pangenome graph of an oomycete plant pathogen. We generated telomere-to-telomere genome assemblies of six genetically diverse isolates of the oomycete pathogen Peronospora effusa, the economically most important disease in cultivated spinach worldwide. The pangenome graph demonstrates that P. effusa genomes are highly conserved, both in chromosomal structure and gene content, and revealed the continued activity of transposable elements which are directly responsible for 80% of the observed variation between the isolates. While most genes are generally conserved, pathogenicity related genes are highly variable between the isolates. Most of the variation is found in large gene clusters resulting from extensive copy-number expansion. Pangenome graph-based discovery can thus be effectively used to capture genomic variation at exceptional resolution, thereby providing a framework to study the biology and evolution of plant pathogens. Author SummaryPlant pathogens are known to evolve rapidly and overcome disease resistance of newly introduced crop varieties. This swift adaptation is visible in the genomes of these pathogens, which can be highly variable. Such genomic variation cannot be captured with contemporary comparative genomic methods that rely on a single reference genome or focus solely on protein coding genes. To overcome these limitations and compare multiple genomes in a robust and scalable method, we constructed the first pangenome graph for an oomycete filamentous plant pathogen with six telomere-to-telomere genome assemblies of Peronospora effusa. This high-resolution pangenomic framework enabled detailed comparisons of the genomes at any level, from the nucleotide to the chromosome, and for any subset of protein-coding genes or transposable elements, to discover novel biology and potential mechanisms for the rapid evolution of this devastating pathogen.

genomics↗

bioRxiv2024

Bacterial family-specific enrichment and functions of secretion systems in the rhizosphere

The plant rhizosphere is a highly selective environment where bacteria have developed traits to establish themselves or outcompete other microbes. These traits include bacterial secretion systems (SSs) that range from Type I (T1SS) to Type IX (T9SS) and can play diverse roles. The best known functions are to secrete various proteins or other compounds into the extracellular space or into neighbouring cells, including toxins to attack other microbes or effectors to suppress plant host immune responses. Here, we aimed to determine which bacterial SSs were associated with the plant rhizosphere. We utilised paired metagenomic datasets of rhizosphere and bulk soil samples from five different plant species grown in a wide variety of soil types, amounting to ten different studies. The T3SS and T6SS were generally enriched in the rhizosphere, as observed in studies of individual plant-associated genera. We also identified additional SSs that have received less attention thus far, such as the T2SS, T5SS and Bacteroidetes-specific T6SSiii and T9SS. The predicted secreted proteins of some of these systems (T3SS, T5SS and T6SS) could be linked to functions such as toxin secretion, adhesion to the host and facilitation of plant-host interactions (such as root penetration). The most prominent bacterial taxa with rhizosphere- or soil-enriched SSs included Xanthomonadaceae, Oxalobacteraceae, Comamonadaceae, Caulobacteraceae, and Chitinophagaceae, broadening the scope of known plant-associated taxa that use these systems. We anticipate that the SSs and taxa identified in this study may be utilised for the optimisation of bioinoculants to improve plant productivity.

microbiology↗

bioRxiv2024

Seed tuber microbiome is a predictor of next-season potato vigor

Potato vigor, an important agronomic trait, is heavily influenced by the field of seed tuber production. Soil microbiota vary significantly between fields, impacting plant health and crop yield. Our study demonstrates that seed potato vigor can be predicted based on microbiota associated with seed tuber eyes, the dormant buds that grow out in the next season. By combining time-resolved drone-imaging of potato crop development with microbiome sequencing of seed tuber eyes from 6 varieties produced in 240 fields, we established correlations between microbiome fingerprints and potato vigor parameters. Employing Random Forest algorithms, we developed a predictive "Potato-Microbiome Informed" model, revealing variety-specific relationships between seed tuber microbiome composition and next seasons potato vigor in trial fields. The model accurately predicted vigor of seed tubers to which the model was naive and pinpointed key microbial indicators of potato vigor. By connecting variety-specific microbiome fingerprints to crop performance in the field, we pave the way for microbiome-informed breeding strategies.

microbiology↗

bioRxiv2023

Seed tuber imprinting shapes the next-generation potato microbiome

Potato seed tubers are colonized and inhabited by soil-borne microbes, some of which can positively or negatively impact the performance of the emerging daughter plant in the next season. In this study, we investigated the intergenerational inheritance of microbiota from seed tubers to next-season daughter plants by amplicon sequencing of bacterial and fungal microbiota associated with tubers and roots of two seed potato genotypes produced in six different fields. We observed that field of production and potato genotype significantly affected the seed tuber microbiome composition and that these differences persisted during winter storage of the seed tubers. When seed tubers from different production fields were planted in a single trial field, the microbiomes of daughter tubers and roots of the emerging plants could still be distinguished according to the field of origin of the seed tuber. Remarkably, we found little evidence of direct vertical inheritance of field-unique microbes from the seed tuber to the daughter tubers or roots. Hence, we hypothesize that this intergenerational "memory" is imprinted in the seed tuber, resulting in differential microbiome assembly strategies depending on the field of production of the seed tuber.

microbiology↗

bioRxiv2023

Identification of the conserved iol gene cluster involved in rhizosphere competence in Pseudomonas

The Pseudomonas genus has shown great potential as a sustainable solution to support agriculture through its plant-growth promoting and biocontrol activities. However, their efficacy as bioinoculants is limited by unpredictable colonization in natural conditions. Our study identifies the iol locus, a gene cluster in Pseudomonas involved in inositol catabolism, as a feature enriched among superior root colonizers in natural soil. Further characterization revealed that the iol locus increases competitiveness by inducing swimming motility and fluorescent siderophore production in response to inositol, a plant-derived compound. Public data analyses indicate that the iol locus is broadly conserved in the Pseudomonas genus and linked to diverse host-microbe interactions. Our findings suggest the iol locus as a potential target for developing more effective bioinoculants, given its conservation and association with diverse host-microbe interactions.

microbiology↗

bioRxiv2022

Copiotrophs dominate rhizosphere microbiomes and growth rate potential is a major factor explaining the rhizosphere effect

The structure and function of the root microbial community is shaped by plant root activity, enriching specific microbial taxa and functions from the surrounding soil as the plant root grows. Knowledge of bacterial rhizosphere competence traits are important for predictive microbiome modeling and the development of viable bioinoculants for sustainable agriculture solutions. In this work we compared growth rate potential, a complex trait that recently became predictable from bacterial genome sequences, to functional traits encoded by proteins. We analyzed 84 paired rhizosphere- and soil-derived 16S rRNA metabarcoding datasets from 18 different plants and soil types, performed differential abundance analyses and estimated growth rates for each bacterial genus. This analysis revealed that bacteria with a high growth rate potential consistently dominated the rhizosphere. Next, we analyzed the genome sequences of 3270 bacterial isolates and 6707 MAGs from 1121 plant- and soil-associated metagenomes, confirming this trend in different bacterial phyla. We next investigated which functional traits were enriched in the rhizosphere, expanding the catalog of rhizosphere-associated traits with hundreds of new functions. When we compared the importance of different functional categories to the predicted growth rate potential using a machine learning model, we found that growth rate potential was the main feature for differentiating rhizosphere and soil bacteria, revealing the broad importance of this factor for explaining the rhizosphere effect. Together, we contribute new understanding of the bacterial traits needed for rhizosphere competence. As this trait may be inferred from (meta-) genome data, our work has implications for understanding bacterial community assembly in the rhizosphere, where many uncultivated bacteria reside.

microbiology↗

bioRxiv2022

Cell-type specific transcriptomics reveals roles for root hairs and endodermal barriers in interaction with beneficial rhizobacterium

Growth-promoting bacteria can boost crop productivity in a sustainable way. Pseudomonas simiae WCS417 is a well-studied bacterium that promotes growth of many plant species. Upon colonization, WCS417 affects root system architecture resulting in an expanded root system. Both immunity and root system architecture, are controlled by root-cell-type specific biological mechanisms, but it is unknown how WCS417 affects these mechanisms. Therefore, here, we transcriptionally profiled five Arabidopsis thaliana root cell types following WCS417 colonization. The cortex and endodermis displayed the most differentially expressed genes, even though they were not in direct contact with this epiphytic bacterium. Many of these genes are associated with reduced cell wall biogenesis, possibly facilitating the root architectural changes observed in WCS417-colonized roots. Comparison of the transcriptome profiles in the two epidermal cell types that were in direct contact with WCS417 - trichoblasts that form root hairs and atrichoblasts that dont - imply functional specialization. Whereas basal expression levels of nutrient uptake-related genes and defense-related genes are highest in trichoblasts and atrichoblasts, respectively, upon exposure to WCS417 these roles revert. This suggests that root hairs participate in the activation of root immunity, further supported by attenuation of immunity in a root hairless mutant. Furthermore, we observed elevated expression of suberin biosynthesis genes and increased deposition of suberin in the endodermis in WCS417-colonized roots. Using an endodermal barrier mutant we show the importance of endodermal barrier integrity for optimal plant-beneficial bacterium association. Altogether, we highlight the strength of cell-type-specific transcriptional profiling to uncover "masked" biological mechanisms underlying successful plant-microbe associations.

plant biology↗

bioRxiv2021

Sexual reproduction contributes to the evolution of resistance breaking isolates of the spinach pathogen Peronospora effusa

Peronospora effusa causes downy mildew, the economically most important disease of cultivated spinach worldwide. To date, 19 P. effusa races have been denominated based on their capacity to break spinach resistances, but their genetic diversity and the evolutionary processes that contribute to race emergence are unknown. Here, we performed the first systematic analysis of P. effusa races showing that those emerge by both asexual and sexual reproduction. Specifically, we studied the diversity of 26 P. effusa isolates from 16 denominated races based on mitochondrial and nuclear comparative genomics. Mitochondrial genomes based on long-read sequencing coupled with diversity assessment based on short-read sequencing uncovered two mitochondrial haplogroups, each with distinct genome organization. Nuclear genome-wide comparisons of the 26 isolates revealed that ten isolates from six races could clearly be divided into three asexually evolving groups, in concordance with their mitochondrial phylogeny. The remaining isolates showed signals of reticulated evolution and discordance between nuclear and mitochondrial phylogenies, suggesting that these evolved through sexual reproduction. Increased understanding of this pathogens reproductive modes will provide the framework for future studies into the molecular mechanisms underlying race emergence and into the P. effusa-spinach interaction, thus assisting in sustainable production of spinach through knowledge-driven resistance breeding. Significance statementMany microbial plant pathogens depend on the successful colonization of their hosts to complete their life cycle, thereby damaging food crops worldwide. The most effective way of disease control is to deploy genetic disease resistances. However, the extensive use of resistant crop varieties exerts strong selective pressure on microbial plant pathogens to adapt in order to escape resistance. Through yet unknown mechanisms, the spinach pathogen Peronospora effusa can rapidly break the resistance of newly introduced varieties, often within a single growing season. Thus, there is an urgent need to better understand the mechanisms driving adaptation in P. effusa. This information will lead the way to knowledge-driven resistance breeding. Here, we capture for the first time the genetic variation of 26 P. effusa, 16 of which can break a different combination of host resistances. We demonstrate that P. effusa isolates evolve by both asexual and sexual reproduction, and thereby provide the framework to study the molecular mechanisms of the interactions between P. effusa and spinach.

evolutionary biology↗

bioRxiv2020

Rapid evolution of bacterial mutualism in the plant rhizosphere

Even though beneficial plant-microbe interactions are commonly observed in nature, direct evidence for the evolution of bacterial mutualism in the rhizosphere remains elusive. Here we use experimental evolution to causally show that initially plant-antagonistic Pseudomonas protegens bacterium evolves into mutualists in the rhizosphere of Arabidopsis thaliana within six plant growth cycles (6 months). This evolutionary transition was accompanied with increased mutualist fitness via two mechanisms: i) improved competitiveness for root exudates and ii) enhanced capacity for activating the root-specific transcription factor gene MYB72, which triggers the production of plant-secreted scopoletin antimicrobial for which the mutualists evolved relatively higher tolerance to. Genetically, mutualism was predominantly associated with different mutations in the GacS/GacA two-component regulator system, which conferred high fitness benefits only in the presence of plants. Together, our results show that bacteria can rapidly evolve along the parasitism-mutualism continuum in the plant rhizosphere at an agriculturally relevant evolutionary timescale.

microbiology↗

bioRxiv2020

Experimental evolution-driven identification of Arabidopsis rhizosphere competence genes in Pseudomonas protegens

Beneficial plant root-associated microorganisms carry out a range of functions that are essential for plant performance. Establishment of a bacterium on plant roots, however, requires overcoming several challenges, including competition with neighboring microorganisms and host immunity. Forward and reverse genetics has led to the identification of mechanisms that are used by beneficial microorganisms to overcome these challenges such as the production of iron-chelating compounds, the formation of strong biofilms, or the concealment of characteristic microbial molecular patterns that trigger the host immune system. However, how such mechanisms arose from an evolutionary perspective is much less understood. To study bacterial adaptation in the rhizosphere, we employed experimental evolution to track the physiological and genetic dynamics of root-dwelling Pseudomonas protegens in the Arabidopsis thaliana rhizosphere under axenic conditions. This simplified binary one plant-one bacterium system allows for the amplification of key adaptive mechanisms for bacterial rhizosphere colonization. We identified 35 mutations, including single-nucleotide polymorphisms, insertions, and deletions, distributed over 28 genes. We found that mutations in genes encoding global regulators, and in genes for siderophore production, cell surface decoration, attachment, and motility accumulated in parallel, underlining that bacterial adaptation to the rhizosphere follows multiple strategies. Notably, we observed that motility increased in parallel across multiple independent evolutionary lines. Altogether these results underscore the strength of experimental evolution to identify key genes, pathways, and processes for bacterial rhizosphere colonization, and a methodology for the development of elite beneficial microorganisms with enhanced root-colonizing capacities that can support sustainable agriculture in the future. ImportanceBeneficial root-associated microorganisms carry out many functions that are essential for plant performance. Establishment of a bacterium on plant roots, however, requires overcoming many challenges. Previously, diverse mechanisms that are used by beneficial microorganisms to overcome these challenges were identified. However, how such mechanisms have developed from an evolutionary perspective is much less understood. Here, we employed experimental evolution to track the evolutionary dynamics of a root-dwelling pseudomonad on the root of Arabidopsis. We find that mutations in global regulators, as well as in genes for siderophore production, cell surface decoration, attachment, and motility accumulate in parallel, underlining these strategies for bacterial adaptation to the rhizosphere. We identified 35 mutations distributed over 28 genes. Altogether our results demonstrate the power of experimental evolution to identify key pathways for rhizosphere colonization and a methodology for the development of elite beneficial microorganisms that can support sustainable agriculture.

microbiology↗