Tandem metalloenzymes gate plant cell entry by pathogenic fungi

Global food security is endangered by fungal phytopathogens causing devastating crop production losses. Many of these pathogens use specialized appressoria cells to puncture plant cuticles. Here, we unveil a pair of alcohol oxidase–peroxidase enzymes to be essential for pathogenicity. Using Colletotrichum orbiculare, we show that the enzyme pair is cosecreted by the fungus early during plant penetration and that single and double mutants have impaired penetration ability. Molecular modeling, biochemical, and biophysical approaches revealed a fine-tuned interplay between these metalloenzymes, which oxidize plant cuticular long-chain alcohols into aldehydes. We show that the enzyme pair is involved in transcriptional regulation of genes necessary for host penetration. The identification of these infection-specific metalloenzymes opens new avenues on the role of wax-derived compounds and the design of oxidase-specific inhibitors for crop protection. Fungal phytopathogens secrete tandem metalloenzymes that catalyze cuticle oxidation and drive plant cell entryThis study was supported by the “Agence Nationale de la Recherche” and by the Natural Sciences and Engineering Research Council of Canada through the ANR-NSERC project “FUNTASTIC” (ANR-17-CE07-0047, STPGP 493781-16). We are grateful to MANE & Fils and the “Association Nationale Recherche Technologie” (ANRT) for funding the Ph.D. fellowship of D.R. (grant no. 2017/1169). Work in Japan was supported by the Japan Society for the Promotion of Science Grants-in-Aid for Scientific Research—KAKENHI, grant numbers 15H05780 and 20H02989 to Y.K., and 20K15529 to S.K.Peer Reviewed"Article signat per 18 autors/es: Bastien Bissaro and Sayo Kodama and Takumi Nishiuchi and Anna Maria Díaz-Rovira and Hayat Hage and David Ribeaucourt and Mireille Haon and Sacha Grisel and A. Jalila Simaan and Fred Beisson and Stephanie M. Forget and Harry Brumer and Marie-Noëlle Rosso and Victor Guallar and Richard O’Connell and Mickaël Lafond and Yasuyuki Kubo and Jean-Guy Berrin "Postprint (published version

Conserved white-rot enzymatic mechanism for wood decay in the Basidiomycota genus Pycnoporus

White-rot (WR) fungi are pivotal decomposers of dead organic matter in forest ecosystems and typically use a large array of hydrolytic and oxidative enzymes to deconstruct lignocellulose. However, the extent of lignin and cellulose degradation may vary between species and wood type. Here, we combined comparative genomics, transcriptomics and secretome proteomics to identify conserved enzymatic signatures at the onset of wood-decaying activity within the Basidiomycota genus Pycnoporus. We observed a strong conservation in the genome structures and the repertoires of protein-coding genes across the four Pycnoporus species described to date, despite the species having distinct geographic distributions. We further analysed the early response of P. cinnabarinus, P. coccineus and P. sanguineus to diverse (ligno)-cellulosic substrates. We identified a conserved set of enzymes mobilized by the three species for breaking down cellulose, hemicellulose and pectin. The co-occurrence in the exo-proteomes of H2O2-producing enzymes with H2O2-consuming enzymes was a common feature of the three species, although each enzymatic partner displayed independent transcriptional regulation. Finally, cellobiose dehydrogenase-coding genes were systematically co-regulated with at least one AA9 lytic polysaccharide monooxygenase gene, indicative of enzymatic synergy in vivo. This study highlights a conserved core white-rot fungal enzymatic mechanism behind the wood-decaying process.Peer reviewe

Diversité génomique et trajectoires évolutives liées à la dégradation de la lignocellulose chez les champignons dégradeurs du bois

Les champignons dégradeurs du bois jouent un rôle majeur dans la libération du carbone séquestré dans la matière organique des écosystèmes forestiers. Ils ont évolué depuis plus de 290 millions d'années pour s’adapter aux ressources disponibles dans leur niche écologique. En particulier, ils ont acquis la capacité de dégrader les polymères récalcitrants des parois végétales pour en extraire des nutriments carbonés. Malgré de nombreuses découvertes sur les mécanismes enzymatiques impliqués dans la dégradation du bois, notre vision sur l’adaptation évolutive de ces champignons et leur diversité génomique reste incomplète. Ainsi, durant ma thèse nous avons exploré leur diversité fonctionnelle et génomique à différents niveaux taxonomiques, couvrant d'abord un seul genre, Pycnoporus spp., puis un ordre taxonomique, les Polyporales. Ces différentes analyses nous ont permis d’identifier un jeu commun d'enzymes mobilisées en réponse à divers substrats lignocellulosiques ainsi que des expansions de familles de gènes révélant différentes trajectoires d'adaptation à la décomposition du bois. De plus, nous avons développé un outil bioinformatique pour l’identification, dans l’ensemble du règne fongique, des enzymes impliquées dans la cyclisation des sesquiterpènes, métabolites secondaires impliqués dans la communication chimique entre micro-organismes dans leur habitat naturel. Grâce à cet outil, nous avons analysé 1420 génomes fongiques et identifié 11085 gènes candidats codant pour des sesquiterpène synthases (STS). Nos découvertes sur la génomique des champignons dégradeurs du bois contribueront à mieux comprendre leur rôle écologique dans les environnements naturels.Wood-decay fungi play a crucial role in carbon release from dead organic matter in forest ecosystems. They have evolved for over 290 million years to become most efficient in utilizing the available resources of their ecological niches and to survive in harsh environmental conditions. The survival strategy of wood decay fungi largely relies on their ability to degrade recalcitrant plant cell wall polymers, which they use as a carbon source. Despite many discoveries on the enzymatic mechanisms involved in wood decomposition, our vision on the evolutionary adaptation to wood decay and genome diversity remains incomplete. During my thesis, we explored the functional and genomic diversity among wood decayers at different taxonomical levels, covering first a single genus, Pycnoporus, then a taxonomic order, Polyporales. This allowed us to identify a core set of enzymes mobilized by the fungi in response to diverse lignocellulosic substrates as well as gene family expansions that supported different trajectories for adaption to wood decay. In addition, we built a bioinformatic tool to screen the whole fungal kingdom for sesquiterpene synthases (STS), which are involved in the synthesis of secondary metabolite used by the fungi to compete with other microorganisms in their natural habitat. Using this tool, we analyzed 1,420 fungal genomes and identified 11,085 candidate STS, out of which 55% would have been missed by the currently available tools. Overall, our findings on the genomic features of wood decay fungi will contribute to better understanding the ecological roles of these fungi in natural environments

Evolution of Fungal Carbohydrate-Active Enzyme Portfolios and Adaptation to Plant Cell-Wall Polymers

International audienceThe postindustrial era is currently facing two ecological challenges. First, the rise in global temperature, mostly caused by the accumulation of carbon dioxide (CO2) in the atmosphere, and second, the inability of the environment to absorb the waste of human activities. Fungi are valuable levers for both a reduction in CO2 emissions, and the improvement of a circular economy with the optimized valorization of plant waste and biomass. Soil fungi may promote plant growth and thereby increase CO2 assimilation via photosynthesis or, conversely, they may prompt the decomposition of dead organic matter, and thereby contribute to CO2 emissions. The strategies that fungi use to cope with plant-cell-wall polymers and access the saccharides that they use as a carbon source largely rely on the secretion of carbohydrate-active enzymes (CAZymes). In the past few years, comparative genomics and phylogenomics coupled with the functional characterization of CAZymes significantly improved the understanding of their evolution in fungal genomes, providing a framework for the design of nature-inspired enzymatic catalysts. Here, we provide an overview of the diversity of CAZyme enzymatic systems employed by fungi that exhibit different substrate preferences, different ecologies, or belong to different taxonomical groups for lignocellulose degradation

Distribution of methionine sulfoxide reductases in fungi and conservation of the freemethionine-R-sulfoxide reductase in multicellular eukaryotes

Methionine, either as a free amino acid or included in proteins, can be oxidized into methionine sulfoxide (MetO), which exists as R and S diastereomers. Almost all characterized organisms possess thiol-oxidoreductases named methionine sulfoxide reductase (Msr) enzymes to reduce MetO back to Met. MsrA and MsrB reduce the S and R diastereomers of MetO, respectively, with strict stereospecificity and are found in almost all organisms. Another type of thiol-oxidoreductase, the free-methionine- R -sulfoxide reductase (fRMsr), identified so far in prokaryotes and a few unicellular eukaryotes, reduces the R MetO diastereomer of the free amino acid. Moreover, some bacteria possess molybdenum-containing enzymes that reduce MetO, either in the free or protein-bound forms. All these Msrs play important roles in the protection of organisms against oxidative stress. Fungi are heterotrophic eukaryotes that colonize all niches on Earth and play fundamental functions, in organic matter recycling, as symbionts, or as pathogens of numerous organisms. However, our knowledge on fungal Msrs is still limited. Here, we performed a survey of msr genes in almost 700 genomes across the fungal kingdom. We show that most fungi possess one gene coding for each type of methionine sulfoxide reductase: MsrA, MsrB, and fRMsr. However, several fungi living in anaerobic environments or as obligate intracellular parasites were devoid of msr genes. Sequence inspection and phylogenetic analyses allowed us to identify non-canonical sequences with potentially novel enzymatic properties. Finaly, we identified several ocurences of msr horizontal gene transfer from bacteria to fungi

Distribution of methionine sulfoxide reductases in fungi and conservation of the free-methionine-R-sulfoxide reductase in multicellular eukaryotes

International audienceMethionine, either as a free amino acid or included in proteins, can be oxidized into methionine sulfoxide (MetO), which exists as R and S diastereomers. Almost all characterized organisms possess thiol-oxidoreductases named methionine sulfoxide reductase (Msr) enzymes to reduce MetO back to Met. MsrA and MsrB reduce the S and R diastereomers of MetO, respectively, with strict stereospecificity and are found in almost all organisms. Another type of thiol-oxidoreductase, the free-methionine-R-sulfoxide reductase (fRMsr), identified so far in prokaryotes and a few unicellular eukaryotes, reduces the R MetO diastereomer of the free amino acid. Moreover, some bacteria possess molybdenum-containing enzymes that reduce MetO, either in the free or protein-bound forms. All these Msrs play important roles in the protection of organisms against oxidative stress. Fungi are heterotrophic eukaryotes that colonize all niches on Earth and play fundamental functions, in organic matter recycling, as symbionts, or as pathogens of numerous organisms. However, our knowledge on fungal Msrs is still limited. Here, we performed a survey of msr genes in almost 700 genomes across the fungal kingdom. We show that most fungi possess one gene coding for each type of methionine sulfoxide reductase: MsrA, MsrB, and fRMsr. However, several fungi living in anaerobic environments or as obligate intracellular parasites were devoid of msr genes. Sequence inspection and phylogenetic analyses allowed us to identify non-canonical sequences with potentially novel enzymatic properties. Finaly, we identified several ocurences of msr horizontal gene transfer from bacteria to fungi

Ein kleiner Freistrahlprüfstand für aeroakustische Messungen

Beschreibung des Aufbaus des kleinen Strahlprüfstands JExTRA für aeroakustische Experimente beim DLR Institut für Antriebstechnik, Abteilung Triebwerksakustik in Berlin