Article: FEMS Microbiology Ecology

Tracking the invasion: dispersal of Hymenoscyphus fraxineus airborne inoculum at different scales. M Grosdidier, R Ioos, C Husson, O Cael, T Scordia… FEMS Microbiology Ecology.


Ash dieback is caused by an invasive pathogen, Hymenoscyphus fraxineus, which emerged in Europe in the 1990s and jeopardizes the management of ash stands. Although the biological cycle of the pathogen is well understood, its dispersal patterns via airborne spores remain poorly described. We investigated the seasonal and spatial patterns of dispersal in France using both a passive spore-trapping method coupled with a real-time PCR assay and reports of ash dieback based on symptom observations. Spores detection varies from year to year, with a detection ability of 30 to 47%, depending on meteorological conditions, which affect both production of inoculum and efficiency of the trapping. Nevertheless, our results are consistent and we showed that sporulation peak occurred from June to August and that spores were detected up to 50-100 km ahead of the disease front, proving the presence of the pathogen before any observation of symptoms. The spore dispersal gradient was steep, most of inoculum remaining within 50 m of infected ashes. Two dispersal kernels were fitted using Bayesian methods to estimate the mean dispersal distance of H. fraxineus from inoculum sources. The estimated mean distances of dispersal, either local or regional scale, were 1.4 km and 2.6 km, respectively, the best fitting kernel being the inverse power-law. This information may help to design disease management strategies.

Article: Current opinion in microbiology

Show me the way: rust effector targets in heterologous plant systems. C Lorrain, B Petre, S Duplessis. Current opinion in microbiology 46, 19-25


For years, the study of rust fungal effectors has been impeded by the lack of molecular genetic tools in rust pathosystems. The recent use of heterologous plants to perform effector screens (effectoromics)-including effector localisation (cellular targets) and protein interactors (molecular targets) in plant cells-has changed the game. These screens revealed that many candidate effectors from various rust fungi target specific plant cell compartments, including chloroplasts, and associate with specific plant protein complexes. Such information represents unparalleled opportunities to understand how effectors sustain extreme parasitic interactions and obligate biotrophy. Despite their limitations, we here portray how the use of heterologous expression systems has been essential for gaining new insight into rust effectors.

Article: FEMS Microbiology Reviews

Bacterial-Fungal Interactions: ecology, mechanisms and challenges A Deveau, G Bonito, J Uehling, M Paoletti, M Becker, S Bindschedler, … FEMS microbiology reviews


Fungi and bacteria are found living together in a wide variety of environments. Their interactions are significant drivers of many ecosystem functions and are important for the health of plants and animals. A large number of fungal and bacterial families are engaged in complex interactions that lead to critical behavioural shifts of the microorganisms ranging from mutualism to pathogenicity. The importance of bacterial-fungal interactions (BFI) in environmental science, medicine and biotechnology has led to the emergence of a dynamic and multidisciplinary research field that combines highly diverse approaches including molecular biology, genomics, geochemistry, chemical and microbial ecology, biophysics and ecological modelling. In this review, we discuss most recent advances that underscore the roles of BFI across relevant habitats and ecosystems. A particular focus is placed on the understanding of BFI within complex microbial communities and in regards of the metaorganism concept. We also discuss recent discoveries that clarify the (molecular) mechanisms involved in bacterial-fungal relationships, and the contribution of new technologies to decipher generic principles of BFI in terms of physical associations and molecular dialogues. Finally, we discuss future directions for researches in order to catalyse a synergy within the BFI research area and to resolve outstanding questions.

Article: Applied and Environmental Microbiology

Enzyme activities of two recombinant heme-including peroxidases TvDyP1 and TvVP2 identified from the secretome of Trametes versicolor S Amara, T Perrot, D Navarro, A Deroy, A Benkhelfallah, A Chalak, … Applied and Environmental Microbiology, AEM. 02826-17


Trametes versicolor is a wood inhabiting Agaricomycete known for its ability to cause strong white rot decay on hardwood and for its high tolerance toward phenolic compounds. The goal of the present work was to give insights on the molecular biology and biochemistry of heme-including class-II and dye-decolorizing peroxidases secreted from this fungus. Proteomic analysis of the secretome of T. versicolor BRFM1218 grown on oak wood revealed a set of 200 secreted proteins among which were a dye-decolorizing peroxidase TvDyP1 and a versatile peroxidase TvVP2. Both peroxidases were heterologously produced in E. coli, and were biochemically characterized and tested for their capacity to oxidize complex substrates. Both peroxidases were found to be active against several substrates in acidic conditions, and TvDyP1 was very stable in a relatively large range of pH (pH 2.0 to 6.0) while TvVP2 was more stable at pH 5.0-6.0 only. Thermostability of both enzymes was also tested and TvDyP1 was globally found to be more stable than TvVP2. After 180 min of incubation at T°C ranging from 30°C to 50°C, activities of TvVP2 drastically decreased retaining 10% to 30% of the its initial activity. In the same conditions, TvDyP1 retained 20% to 80% of enzyme activity. The two proteins were catalytically characterized and TvVP2 was shown to accept a wider range of reducing substrates than TvDyP1. Furthermore, both enzymes were found to be active against two flavonoids, quercetin and catechin, found in oak wood, TvVP2 displaying a more rapid oxidation of the two compounds. They were tested for their potential interest in dye decolourization of five industrial dyes and TvVP2 presented a wider oxidation and decolourization capacity towards the dye substrates than TvDyP1.

IMPORTANCE Trametes versicolor is a wood inhabiting Agaricomycete known for its ability to cause strong white rot decay on hardwood and for its high tolerance toward phenolic compounds. Among white-rot fungi, the basidiomycete T. versicolor has been extensively studied for its efficiency to degrade wood, and specifically lignin, thanks to an extracellular oxidative enzymatic system. The corresponding oxidative system was previously studied in several works for classical lignin and manganese peroxidases, and in this study, two new components of the oxidative system of T. versicolor, one dye-decoloririzing peroxidase and one versatile peroxidase were biochemically characterized in depth and compare to other fungal peroxidases.

Article: Frontiers in Microbiology

Genome-Wide Analysis of Corynespora cassiicola Leaf Fall Disease Putative Effectors. D Lopez, S Ribeiro, P Label, B Fumanal, JS Venisse, A Kohler, … Frontiers in Microbiology 9, 276


Corynespora cassiicola is an Ascomycetes fungus with a broad host range and diverse life styles. Mostly known as a necrotrophic plant pathogen, it has also been associated with rare cases of human infection. In the rubber tree, this fungus causes the Corynespora leaf fall (CLF) disease, which increasingly affects natural rubber production in Asia and Africa. It has also been found as an endophyte in South American rubber plantations where no CLF outbreak has yet occurred. The C. cassiicola species is genetically highly diverse, but no clear relationship has been evidenced between phylogenetic lineage and pathogenicity. Cassiicolin, a small glycosylated secreted protein effector, is thought to be involved in the necrotrophic interaction with the rubber tree but some virulent C. cassiicola isolates do not have a cassiicolin gene. This study set out to identify other putative effectors involved in CLF. The genome of a highly virulent C. cassiicola isolate from the rubber tree (CCP) was sequenced and assembled. In silico prediction revealed 2870 putative effectors, comprising CAZymes, lipases, peptidases, secreted proteins and enzymes associated with secondary metabolism. Comparison with the genomes of 44 other fungal species, focusing on effector content, revealed a striking proximity with phylogenetically unrelated species (Colletotrichum acutatum, Colletotrichum gloesporioides, Fusarium oxysporum, nectria hematococca and Botrosphaeria dothidea) sharing life style plasticity and broad host range. Candidate effectors involved in the compatible interaction with the rubber tree were identified by transcriptomic analysis. Differentially expressed genes included 92 putative effectors, among which cassiicolin and two other secreted singleton proteins. Finally, the genomes of 35 C. cassiicola isolates representing the genetic diversity of the species were sequenced and assembled, and putative effectors identified. At the intraspecific level, effector-based classification was found to be highly consistent with the phylogenomic trees. Identification of lineage-specific effectors is a key step toward understanding C. cassiicola virulence and host specialization mechanisms.

Article: Journal of Biological Chemistry

Function and maturation of the Fe–S center in dihydroxyacid dehydratase from Arabidopsis H Gao, T Azam, S Randeniya, J Couturier, N Rouhier, MK Johnson. Journal of Biological Chemistry, jbc. RA117. 001592


Dihydroxyacid dehydratase (DHAD) is the third enzyme required for branched-chain amino acid biosynthesis in bacteria, fungi, and plants. DHAD enzymes contain two distinct types of active-site Fe–S clusters. The best characterized examples are Escherichia coli DHAD, which contains an oxygen-labile [Fe4S4] cluster, and spinach DHAD, which contains an oxygen-resistant [Fe2S2] cluster. Although the Fe–S cluster is crucial for DHAD function, little is known about the cluster-coordination environment or the mechanism of catalysis and cluster biogenesis. Here, using the combination of UV-visible absorption and circular dichroism, resonance Raman and electron paramagnetic resonance, we spectroscopically characterized the Fe–S center in DHAD from Arabidopsis thaliana (At). Our results indicated that AtDHAD can accommodate [Fe2S2] and [Fe4S4] clusters. However, only the [Fe2S2] cluster–bound form is catalytically active. We found that the [Fe2S2] cluster is coordinated by at least one non-cysteinyl ligand, which can be replaced by the thiol group(s) of dithiothreitol.In vitro cluster transfer and reconstitution reactions revealed that [Fe2S2] cluster–containing NFU2 protein is likely the physiological cluster donor for in vivo maturation of AtDHAD. In summary, AtDHAD binds either one [Fe4S4] or one [Fe2S2] cluster, with only the latter being catalytically competent and capable of substrate and product binding, and NFU2 appears to be the physiological [Fe2S2] cluster donor for DHAD maturation. This work represents the first in vitro characterization of recombinant AtDHAD, providing new insights into the properties, biogenesis, and catalytic role of the active-site Fe–S center in a plant DHAD.

Article: Molecular Plant-Microbe Interactions

The rust fungus Melampsora larici-populina expresses a conserved genetic program and distinct sets of secreted protein genes during infection of its two host plants,…C Lorrain, C Marchal, S Hacquard, C Delaruelle, J Pétrowski, B Petre, …Molecular Plant-Microbe Interactions


Mechanisms required for broad spectrum or specific host colonization of plant parasites are poorly understood. As a perfect illustration, heteroecious rust fungi require two alternate host plants to complete their life cycles. Melampsora larici populina infects two taxonomically unrelated plants, larch on which sexual reproduction is achieved and poplar on which clonal multiplication occurs leading to severe epidemics in plantations. We applied deep RNA sequencing to three key developmental stages of M. larici-populina infection on larch: basidia, pycnia and aecia; and we performed comparative transcriptomics of infection on poplar and larch hosts using available expression data. Secreted protein was the only significantly over-represented category among differentially expressed M. larici-populina genes between the basidial, the pycnial and the aecial stages, highlighting their probable involvement in the infection process. Comparison of fungal transcriptomes in larch and poplar revealed a majority of rust genes commonly expressed on the two hosts and a fraction exhibiting host-specific expression. More particularly, gene families encoding small secreted proteins presented striking expression profiles that highlight probable candidate effectors specialized on each host. Our results bring valuable new information about the biological cycle of rust fungi and identify genes that may contribute to host specificity.

Article: New Phytologist

High intraspecific genome diversity in the model arbuscular mycorrhizal symbiont Rhizophagus irregularis ECH Chen, E Morin, D Beaudet, J Noel, G Yildirir, S Ndikumana, … New Phytologist


Arbuscular mycorrhizal fungi (AMF) are known to improve plant fitness through the estab- lishment of mycorrhizal symbioses. Genetic and phenotypic variations among closely related AMF isolates can significantly affect plant growth, but the genomic changes underlying this variability are unclear.

Toaddressthisissue,weimprovedthegenomeassemblyandgeneannotationofthemodel strain Rhizophagus irregularis DAOM197198, and compared its gene content with five iso- lates of R. irregularis sampled in the same field.
All isolates harbor striking genome variations, with large numbers of isolate-specific genes, gene family expansions, and evidence of interisolate genetic exchange. The observed variabil- ity affects all gene ontology terms and PFAM protein domains, as well as putative mycor- rhiza-induced small secreted effector-like proteins and other symbiosis differentially expressed genes. High variability is also found in active transposable elements.

Overall, these findings indicate a substantial divergence in the functioning capacity of iso- lates harvested from the same field, and thus their genetic potential for adaptation to biotic and abiotic changes. Our data also provide a first glimpse into the genome diversity that resides within natural populations of these symbionts, and open avenues for future analyses of plant–AMF interactions that link AMF genome variation with plant phenotype and fitness.

Article: Journal of Biological Inorganic Chemistry

Roles and maturation of iron–sulfur proteins in plastids J Przybyla-Toscano, M Roland, F Gaymard, J Couturier, N Rouhier JBIC Journal of Biological Inorganic Chemistry, 1-2

One reason why iron is an essential element for most organisms is its presence in prosthetic groups such as hemes or iron–sulfur (Fe–S) clusters, which are notably required for electron transfer reactions. As an organelle with an intense metabolism in plants, chloroplast relies on many Fe–S proteins. This includes those present in the electron transfer chain which will be, in fact, essential for most other metabolic processes occurring in chloroplasts, e.g., carbon fixation, nitrogen and sulfur assimilation, pigment, amino acid, and vitamin biosynthetic pathways to cite only a few examples. The maturation of these Fe–S proteins requires a complex and specific machinery named SUF (sulfur mobilisation). The assembly process can be split in two major steps, (1) the de novo assembly on scaffold proteins which requires ATP, iron and sulfur atoms, electrons, and thus the concerted action of several proteins forming early acting assembly complexes, and (2) the transfer of the preformed Fe–S cluster to client proteins using a set of late-acting maturation factors. Similar machineries, having in common these basic principles, are present in the cytosol and in mitochondria. This review focuses on the currently known molecular details concerning the assembly and roles of Fe–S proteins in plastids.

Article: Frontiers in Microbiology

Secretome analysis from the ectomycorrhizal ascomycete Cenococcum geophilum MDF Pereira, C Veneault-Fourrey, P Vion, F Guinet, E Morin, K Barry, … Frontiers in Microbiology 9, 141


Cenococcum geophilum is an ectomycorrhizal fungus with global distribution in numerous habitats and associates with a large range of host species including gymnosperm and angiosperm trees. Moreover, C. geophilum is the unique ectomycorrhizal species within the clade Dothideomycetes, the largest class of Ascomycetes containing predominantly saprotrophic and many devastating phytopathogenic fungi. Recent studies highlight that mycorrhizal fungi, as pathogenic ones, use effectors in form of Small Secreted Proteins (SSPs) as molecular keys to promote symbiosis. In order to better understand the biotic interaction of C. geophilum with its host plants, the goal of this work was to characterize mycorrhiza-induced small-secreted proteins (MiSSPs) that potentially play a role in the ectomycorrhiza formation and functioning of this ecologically very important species. We combined different approaches such as gene expression profiling, genome localization and conservation of MiSSP genes in different C. geophilum strains and closely related species as well as protein subcellular localization studies of potential targets of MiSSPs in interacting plants using in tobacco leaf cells. Gene expression analyses of C. geophilum interacting with Pinus sylvestris and Populus sp. showed that similar sets of genes coding for secreted proteins were up-regulated and only few were specific to each host. Whereas pine induced more carbohydrate active enzymes (CAZymes), the interaction with poplar induced the expression of specific SSPs.
We identified a set of 22 MiSSPs, which are located in both, gene-rich, repeat-poor or gene-sparse, repeat-rich regions of the C. geophilum genome, a genome showing a bipartite architecture as seen for some pathogens but not yet for an ectomycorrhizal fungus. Genome re-sequencing data of 15 C. geophilum strains and two close relatives Glonium stellatum and Lepidopterella palustris were used to study sequence conservation of MiSSP-encoding genes. The 22 MiSSPs showed a high presence-absence polymorphism among the studied C. geophilum strains suggesting an evolution through gene gain/gene loss. Finally, we showed that six CgMiSSPs target four distinct sub-cellular compartments such as endoplasmic reticulum, plasma membrane, cytosol and tonoplast. Overall, this work presents a comprehensive analysis of secreted proteins and MiSSPs in different genetic level of C. geophilum opening a valuable resource to future functional analysis.