Bacterial pathogenicity relies on a proficient metabolism and there is increasing evidence that metabolic adaptation to exploit host resources is a key property of infectious organisms. In many ...cases, colonization by the pathogen also implies an intensive multiplication and the necessity to produce a large array of virulence factors, which may represent a significant cost for the pathogen. We describe here the existence of a resource allocation trade-off mechanism in the plant pathogen R. solanacearum. We generated a genome-scale reconstruction of the metabolic network of R. solanacearum, together with a macromolecule network module accounting for the production and secretion of hundreds of virulence determinants. By using a combination of constraint-based modeling and metabolic flux analyses, we quantified the metabolic cost for production of exopolysaccharides, which are critical for disease symptom production, and other virulence factors. We demonstrated that this trade-off between virulence factor production and bacterial proliferation is controlled by the quorum-sensing-dependent regulatory protein PhcA. A phcA mutant is avirulent but has a better growth rate than the wild-type strain. Moreover, a phcA mutant has an expanded metabolic versatility, being able to metabolize 17 substrates more than the wild-type. Model predictions indicate that metabolic pathways are optimally oriented towards proliferation in a phcA mutant and we show that this enhanced metabolic versatility in phcA mutants is to a large extent a consequence of not paying the cost for virulence. This analysis allowed identifying candidate metabolic substrates having a substantial impact on bacterial growth during infection. Interestingly, the substrates supporting well both production of virulence factors and growth are those found in higher amount within the plant host. These findings also provide an explanatory basis to the well-known emergence of avirulent variants in R. solanacearum populations in planta or in stressful environments.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Cooperation is associated with major transitions in evolution such as the emergence of multicellularity. It is central to the evolution of many complex traits in nature, including growth and ...virulence in pathogenic bacteria. Whether cells of multicellular parasites function cooperatively during infection remains, however, largely unknown. Here, we show that hyphal cells of the fungal pathogen Sclerotinia sclerotiorum reprogram toward division of labor to facilitate the colonization of host plants. Using global transcriptome sequencing, we reveal that gene expression patterns diverge markedly in cells at the center and apex of hyphae during Arabidopsis thaliana colonization compared with in vitro growth. We reconstructed a genome-scale metabolic model for S. sclerotiorum and used flux balance analysis to demonstrate metabolic heterogeneity supporting division of labor between hyphal cells. Accordingly, continuity between the central and apical compartments of invasive hyphae was required for optimal growth in planta. Using a multicell model of fungal hyphae, we show that this cooperative functioning enhances fungal growth predominantly during host colonization. Our work identifies cooperation in fungal hyphae as a mechanism emerging at the multicellular level to support host colonization and virulence.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
Robustness is a key system-level property of living organisms to maintain their functions while tolerating perturbations. We investigate here how a regulatory network controlling multiple virulence ...factors impacts phenotypic robustness of a bacterial plant pathogen. We reconstruct a cell-scale model of Ralstonia solanacearum connecting a genome-scale metabolic network, a virulence macromolecule network, and a virulence regulatory network, which includes 63 regulatory components. We develop in silico methods to quantify phenotypic robustness under a broad set of conditions in high-throughput simulation analyses. This approach reveals that the virulence regulatory network exerts a control of the primary metabolism to promote robustness upon infection. The virulence regulatory network plugs into the primary metabolism mainly through the control of genes likely acquired via horizontal gene transfer, which results in a functional overlay with ancestral genes. These results support the view that robustness may be a selected trait that promotes pathogenic fitness upon infection.
Summary
In the past 2 decades, progress in molecular analyses of the plant immune system has revealed key elements of a complex response network. Current paradigms depict the interaction of ...pathogen‐secreted molecules with host target molecules leading to the activation of multiple plant response pathways. Further research will be required to fully understand how these responses are integrated in space and time, and exploit this knowledge in agriculture. In this review, we highlight systems biology as a promising approach to reveal properties of molecular plant–pathogen interactions and predict the outcome of such interactions. We first illustrate a few key concepts in plant immunity with a network and systems biology perspective. Next, we present some basic principles of systems biology and show how they allow integrating multiomics data and predict cell phenotypes. We identify challenges for systems biology of plant–pathogen interactions, including the reconstruction of multiscale mechanistic models and the connection of host and pathogen models. Finally, we outline studies on resistance durability through the robustness of immune system networks, the identification of trade‐offs between immunity and growth and in silico plant–pathogen co‐evolution as exciting perspectives in the field. We conclude that the development of sophisticated models of plant diseases incorporating plant, pathogen and climate properties represent a major challenge for agriculture in the future.
Significance Statement
Plant–pathogen interactions involve a complex interplay of molecular events occurring in the plant and the microbe. In this review we argue that systems biology approaches are particularly well suited to tackle the complexity of these interactions and present an overview of current approaches, challenges and applications of systems biology in plant pathology.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Over the last 10 to 15 years, metabolic engineering of microbes has become a versatile tool for high-level de novo synthesis of terpenoids, with the sesquiterpenoids armopha-1,4-diene, farnesene and ...artemisinic acid as prime examples. However, almost all cell factory approaches towards terpenoids to date have been based on sugar as the raw material, which is mainly used as a food resource and subject to high price volatilities. In this study we present de novo synthesis of the sesquiterpenoid α-humulene from the abundantly available non-food carbon source methanol by metabolically engineered Methylobacterium extorquens AM1. Expression of α-humulene synthase from Zingiber zerumbet in combination with farnesyl pyrophosphate (FPP) synthase from Saccharomyces cerevisiae led to concentrations of up to 18mg/L α-humulene. Introduction of a prokaryotic mevalonate pathway from Myxococcus xanthus in combination with ribosome binding site optimization of α-humulene and FPP synthases increased product concentration 3-fold. This value was additionally raised by 30% using a carotenoid synthesis deficient mutant strain. Final product concentrations of up to 1.65g/L were obtained in methanol limited fed-batch cultivations, which is the highest titer of de novo synthesized α-humulene reported to date. This study demonstrates the potential of M. extorquens as a future platform strain for the production of high-value terpenoids from the alternative carbon source methanol.
•First de novo terpenoid production in an engineered methylotrophic bacterium.
•Introduction of the mevalonate pathway from Myxococcus xanthus.•Flux balancing by ribosome binding site optimizations.•Up to 75mg/L α-humulene in shake flask cultivations.•Up to 1.65g/L α-humulene in methanol limited fed-batch fermentation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Quantitative disease resistance (QDR) represents the predominant form of resistance in natural populations and crops. Surprisingly, very limited information exists on the biomolecular network of the ...signaling machineries underlying this form of plant immunity. This lack of information may result from its complex and quantitative nature. Here, we used an integrative approach including genomics, network reconstruction, and mutational analysis to identify and validate molecular networks that control QDR in Arabidopsis thaliana in response to the bacterial pathogen Xanthomonas campestris. To tackle this challenge, we first performed a transcriptomic analysis focused on the early stages of infection and using transgenic lines deregulated for the expression of RKS1, a gene underlying a QTL conferring quantitative and broad-spectrum resistance to X. campestris. RKS1-dependent gene expression was shown to involve multiple cellular activities (signaling, transport, and metabolism processes), mainly distinct from effector-triggered immunity (ETI) and pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) responses already characterized in A. thaliana. Protein–protein interaction network reconstitution then revealed a highly interconnected and distributed RKS1-dependent network, organized in five genemodules. Finally, knockoutmutants for 41 genes belonging to the different functional modules of the network revealed that 76% of the genes and all gene modules participate partially in RKS1-mediated resistance. However, these functional modules exhibit differential robustness to genetic mutations, indicating that, within the decentralized structure of the QDR network, some modules are more resilient than others. In conclusion, our work sheds light on the complexity of QDR and provides comprehensive understanding of a QDR immune network.
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BFBNIB, NMLJ, NUK, PNG, SAZU, UL, UM, UPUK
The global regulator PhcA controls numerous traits associated to virulence and bacterial proliferation in strains of the plant pathogen Ralstonia solanacearum species complex. Here, we conducted a ...genome-wide RNA sequencing study of the GMI1000 wild-type strain and a derived phcA mutant grown in complete medium. The PhcA regulon we identified is the largest regulon described to date in the R. solanacearum species complex with 1581 regulated genes, representing about 30% of the bacterial genome. Among these genes, 166 transcription regulators were identified including known regulators controlling major cellular functions such as the Type 3 secretion system and 27 novel regulators that were not identified in previous transcriptomic studies. This study highlights that PhcA controls other functions beside pathogenicity stricto sensu which participate to the global cell homeostasis (metabolism, energy storage). We then compared the PhcA regulon identified in complete medium to the recently published PhcA regulon obtained in planta. This comparison of the set of GMI1000 genes subjected to PhcA regulation in both conditions revealed 383 common genes. Among them, 326 (85%) had a similar PhcA dependent regulation pattern in complete medium and in planta, and 57 (15%) displayed an opposite regulation pattern. A large majority of the genes repressed by PhcA in complete medium but activated in planta belong to the HrpG-HrpB regulon, which represents a set of key genes required for R. solanacearum pathogenesis. This latter class of genes appears to be specifically induced by PhcA in the plant environment whereas PhcA represses their expression in complete medium. The large set of direct and indirect targets identified in this study will contribute to enrich our knowledge of the intricate regulatory network coordinating the expression of virulence and metabolic functions in the model plant pathogen R. solanacearum.
•The PhcA regulon of R. solanacearum in complete medium (CM) is analysed.•30% of the whole bacterial genes are regulated by PhcA.•PhcA controls functions involved in pathogenicity and global cell homeostasis.•Most of PhcA-regulated genes have similar regulation pattern in CM and in planta.•Genes regulated by HrpG-HrpB are induced by PhcA in planta but repressed in CM.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Methylotrophy is the ability of organisms to grow at the expense of reduced one-carbon compounds, such as methanol or methane. Here, we used transposon sequencing combining hyper-saturated transposon ...mutagenesis with high-throughput sequencing to define the essential methylotrophy genome of Methylobacterium extorquens PA1, a model methylotroph. To distinguish genomic regions required for growth only on methanol from general required genes, we contrasted growth on methanol with growth on succinate, a non-methylotrophic reference substrate. About 500,000 insertions were mapped for each condition, resulting in a median insertion distance of five base pairs. We identified 147 genes and 76 genes as specific for growth on methanol and succinate, respectively, and a set of 590 genes as required under both growth conditions. For the integration of metabolic functions, we reconstructed a genome-scale metabolic model and performed in silico essentiality analysis. In total, the approach uncovered 95 genes not previously described as crucial for methylotrophy, including genes involved in respiration, carbon metabolism, transport, and regulation. Strikingly, regardless of the absence of the Calvin cycle in the methylotroph, the screen led to the identification of the gene for phosphoribulokinase as essential during growth on methanol, but not during growth on succinate. Genetic experiments in addition to metabolomics and proteomics revealed that phosphoribulokinase serves a key regulatory function. Our data support a model according to which ribulose-1,5-bisphosphate is an essential metabolite that induces a transcriptional regulator driving one-carbon assimilation.
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•The essential genome of Methylobacterium extorquens was mapped with high resolution•TnSeq reveals almost 100 novel methylotrophy genes•The Calvin cycle enzyme phosphoribulokinase is essential for growth on methanol•Ribulose-1,5-bisphosphate is crucial for the regulation of one-carbon assimilation
Ochsner et al. use TnSeq to map genomic regions of the model methylotroph Methylobacterium extorquens PA1 that are specifically required during growth on methanol. They identify almost 100 new methylotrophy genes, including the Calvin cycle enzyme phosphoribulokinase, showing that it plays an unprecedented regulatory role for carbon assimilation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Expression of cell phenotypes highly depends on metabolism that supplies matter and energy. To achieve proper utilisation of the different metabolic pathways, metabolism is tightly regulated by a ...complex regulatory network composed of diverse biological entities (genes, transcripts, proteins, signalling molecules…). The integrated analysis of both regulatory and metabolic networks appears very insightful but is not straightforward because of the distinct characteristics of both networks. The classical method used for metabolic flux analysis is Flux Balance Analysis (FBA), which is constraint-based and relies on the assumption of steady-state metabolite concentrations throughout the network. Regarding regulatory networks, a broad spectrum of methods are dedicated to their analysis although logical modelling remains the major method to take charge of large-scale networks.
We present FlexFlux, an application implementing a new way to combine the analysis of both metabolic and regulatory networks, based on simulations that do not require kinetic parameters and can be applied to genome-scale networks. FlexFlux is based on seeking regulatory network steady-states by performing synchronous updates of multi-state qualitative initial values. FlexFlux is then able to use the calculated steady-state values as constraints for metabolic flux analyses using FBA. As input, FlexFlux uses the standards Systems Biology Markup Language (SBML) and SBML Qualitative Models Package ("qual") extension (SBML-qual) file formats and provides a set of FBA based functions.
FlexFlux is an open-source java software with executables and full documentation available online at http://lipm-bioinfo.toulouse.inra.fr/flexflux/. It can be defined as a research tool that enables a better understanding of both regulatory and metabolic networks based on steady-state simulations. FlexFlux integrates well in the flux analysis ecosystem thanks to the support of standard file formats and can thus be used as a complementary tool to existing software featuring other types of analyses.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
Pathogen genetic diversity varies in response to environmental changes. However, it remains unclear whether plant barriers to invasion could be considered a genetic bottleneck for phytopathogen ...populations. Here, we implement a barcoding approach to generate a pool of 90 isogenic and individually barcoded Ralstonia solanacearum strains. We used 90 of these strains to inoculate tomato plants with different degrees of physical permeability to invasion (intact roots, wounded roots and xylem inoculation) and quantify the phytopathogen population dynamics during invasion. Our results reveal that the permeability of plant roots impacts the degree of population bottleneck, genetic diversity, and composition of Ralstonia populations. We also find that selection is the main driver structuring pathogen populations when barriers to infection are less permeable, i.e., intact roots, the removal of root physical and immune barriers results in the predominance of stochasticity in population assembly. Taken together, our study suggests that plant root permeability constitutes a bottleneck for phytopathogen invasion and genetic diversity.
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