Marc-henri Lebrun | National Institute of Agricultural Research (INRA) (original) (raw)

Papers by Marc-henri Lebrun

Frontiers in Cellular and Infection Microbiology, 2014

The concept of pathogenesis has evolved considerably over recent years, and the scenario "a micro... more The concept of pathogenesis has evolved considerably over recent years, and the scenario "a microbe + virulence factors = disease" is probably far from reality in a number of cases. Actual pathogens have extremely broad biological diversity and are found in all major groups of microorganisms (viruses, bacteria, fungi, protozoa. . .). Their pathogenicity results from strong and often highly specific interactions they have with either their microbial environment, hosts and/or arthropod vectors. In this review, we explore the contribution of metagenomic approaches toward understanding pathogens within the context of microbial communities. With this broader view, we discussed the concept of "pathobiome" and the research questions that this raises.

New Phytologist, 2007

During the breeding process of cultivated crops, resistance genes to pests and diseases are commo... more During the breeding process of cultivated crops, resistance genes to pests and diseases are commonly introgressed from wild species. The size of these introgressions is predicted by theoretical models but has rarely been measured in cultivated varieties. By combining resistance tests with isogenic strains, genotyping and sequencing of different rice accessions, it was shown that, in the elite rice variety IR64, the resistance conferring allele of the rice blast resistance gene Pi33 was introgressed from the wild rice Oryza rufipogon (accession IRGC101508). Further characterization of this introgression revealed a large introgression at this locus in IR64 and the related variety IR36. The introgressed fragment represents approximately half of the short arm of rice chromosome 8. This is the first report of a large introgression in a cultivated variety of rice. Such a large introgression is likely to have been maintained during backcrossing only if a selection pressure was exerted on this genomic region. The possible traits that were selected are discussed.

Genome Biology and Evolution, 2015

Deciphering the genetic bases of pathogen adaptation to its host is a key question in ecology and... more Deciphering the genetic bases of pathogen adaptation to its host is a key question in ecology and evolution. To understand how the fungus Magnaporthe oryzae adapts to different plants, we sequenced eight M. oryzae isolates differing in host specificity (rice, foxtail millet, wheat, and goosegrass), and one Magnaporthe grisea isolate specific of crabgrass. Analysis of Magnaporthe genomes revealed small variation in genome sizes (39-43 Mb) and gene content (12,283-14,781 genes) between isolates. The whole set of Magnaporthe genes comprised 14,966 shared families, 63% of which included genes present in all the nine M. oryzae genomes. The evolutionary relationships among Magnaporthe isolates were inferred using 6,878 single-copy orthologs. The resulting genealogy was mostly bifurcating among the different host-specific lineages, but was reticulate inside the rice lineage. We detected traces of introgression from a nonrice genome in the rice reference 70-15 genome. Among M. oryzae isolates and host-specific lineages, the genome composition in terms of frequencies of genes putatively involved in pathogenicity (effectors, secondary metabolism, cazome) was conserved. However, 529 shared families were found only in nonrice lineages, whereas the rice lineage possessed 86 specific families absent from the nonrice genomes. Our results confirmed that the host specificity of M. oryzae isolates was associated with a divergence between lineages without major gene flow and that, despite the strong conservation of gene families between lineages, adaptation to different hosts, especially to rice, was associated with the presence of a small number of specific gene families. All information was gathered in a public database (http://genome.jouy.inra.fr/gemo).

Recent Developments in Management of Plant Diseases, 2009

... Valérie Toquin, François Barja, Catherine Sirven, Stéphanie Gamet, Lucile Mauprivez, Philippe... more ... Valérie Toquin, François Barja, Catherine Sirven, Stéphanie Gamet, Lucile Mauprivez, Philippe Peret, Marie-Pascale Latorse, Jean-Luc Zundel, Frédéric Schmitt, Marc ... spectrin plays a crucial role in membrane integrity and dynamics (An et al., 2004; De Matteis and Morrow, 2000 ...

IMA fungus, 2014

To ensure a stable platform for fungal taxonomy, it is of paramount importance that the genetic a... more To ensure a stable platform for fungal taxonomy, it is of paramount importance that the genetic application of generic names be based on their DNA sequence data, and wherever possible, not morphology or ecology alone. To facilitate this process, a new database, accessible at www.GeneraofFungi.org (GoF) was established, which will allow deposition of metadata linked to holo-, lecto-, neo- or epitype specimens, cultures and DNA sequence data of the type species of genera. Although there are presently more than 18 000 fungal genera described, we aim to initially focus on the subset of names that have been placed on the "Without-prejudice List of Protected Generic Names of Fungi" (see IMA Fungus 4(2): 381-443, 2013). To enable the global mycological community to keep track of typification events and avoid duplication, special MycoBank Typification identfiers (MBT) will be issued upon deposit of metadata in MycoBank. MycoBank is linked to GoF, thus deposited metadata of generic...

Molecular Plant-Microbe Interactions, 2010

Comparative analyses of genome sequences from several plant-infecting fungi have shown conservati... more Comparative analyses of genome sequences from several plant-infecting fungi have shown conservation and expansion of protein families with plant disease-related functions. Here, we show that this hypothesis can be extended to mutualistic symbiotic fungi. We have identified a gene encoding an Era (Escherichia coli Ras)-like GTPase in the rice blast fungus Magnaporthe oryzae and found that it is orthologous to the mature amino terminal part of the Gin1 protein from the arbuscular mycorrhizal (AM) fungus Glomus intraradices. M. oryzae Erl1 is required for full root virulence. Appressoria formation was not severely affected in Δerl1strains, but invasive hyphae grew slower than in the wild type. Root browning defect of Δerl1 strains could be complemented by the AM gene under the control of the ERL1 promoter. Erl1 and Gin-N localized to the nucleus when carboxy-terminally labeled with green fluorescent protein (GFP). However, amino-terminal GFP-tagged versions of the proteins expressed in Aspergillus nidulans were shown to localize in the cytoplasm and to cause polarity defects. These data suggest that Erl1 and Gin-N are orthologs and might be involved in the control of hyphal growth in planta. This is the first characterization of an Era-like GTPase in filamentous fungi.

Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2008

The secreted proteins (secretome) of fungi play a key role in interactions of pathogenic and symb... more The secreted proteins (secretome) of fungi play a key role in interactions of pathogenic and symbiotic fungi with plants. Using the plant pathogenic fungus Leptosphaeria maculans and symbiont Laccaria bicolor grown in culture, we have established a proteomic protocol for extraction, concentration and resolution of the fungal secretome. As no proteomic data were available on mycelium tissues from both L. maculans and L. bicolor, mycelial proteins were studied; they also helped verifying the purity of secretome samples. The quality of protein extracts was initially assessed by both 1-DE and 2-DE using first a broad pH range for IEF, and then narrower acidic and basic pH ranges, prior to 2-DE. Compared with the previously published protocols for which only dozens of 2-D spots were recovered from fungal secretome samples, up to approximately 2000 2-D spots were resolved by our method. MS identification of proteins along several pH gradients confirmed this high resolution, as well as the presence of major secretome markers such as endopolygalacturonases, b-glucanosyltransferases, pectate lyases and endoglucanases. Shotgun proteomic experiments evidenced the enrichment of secreted protein within the liquid medium. This is the first description of the proteome of L. maculans and L. bicolor, and the first application of liquid-phase IEF to any fungal extracts.

by Kim Plummer, Darren Soanes, Muriel Viaud, Linda Kohn, Marc-henri Lebrun, Jeffrey Rollins, Nicholas Talbot, Oded Yarden, Luis Aguirrezabal Christine Granier, Sabine Fillinger, Emmanuel Quévillon, Baptiste Brault, Ulrich Güldener, and Pascale Cotton

PLoS Genetics, 2011

Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic f... more Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of one strain of S. sclerotiorum and two strains of B. cinerea. The comparative analysis of these genomes relative to one another and to other sequenced fungal genomes is provided here. Their 38-39 Mb genomes include 11,860-14,270 predicted genes, which share 83% amino acid identity on average between the two species. We have mapped the S. sclerotiorum assembly to 16 chromosomes and found large-scale co-linearity with the B. cinerea genomes. Seven percent of the S. sclerotiorum genome comprises transposable elements compared to ,1% of B. cinerea. The arsenal of genes associated with necrotrophic processes is similar between the species, including genes involved in plant cell wall degradation and oxalic acid production. Analysis of secondary metabolism gene clusters revealed an expansion in number and diversity of B. cinerea-specific secondary metabolites relative to S. sclerotiorum. The potential diversity in secondary metabolism might be involved in adaptation to specific ecological niches. Comparative genome analysis revealed the basis of differing sexual mating compatibility systems between S. sclerotiorum and B. cinerea. The organization of the mating-type loci differs, and their structures provide evidence for the evolution of heterothallism from homothallism. These data shed light on the evolutionary and mechanistic bases of the genetically complex traits of necrotrophic pathogenicity and sexual mating. This resource should facilitate the functional studies designed to better understand what makes these fungi such successful and persistent pathogens of agronomic crops.

Fungal Genetics and Biology, 2015

Studying essential genes in haploid fungi requires specific tools. Conditional promoter replaceme... more Studying essential genes in haploid fungi requires specific tools. Conditional promoter replacement (CPR) is an efficient method for testing gene essentiality. However, this tool requires promoters that can be strongly down-regulated. To this end, we tested the nitrate reductase promoters of Magnaporthe oryzae (pMoNIA1) and Zymoseptoria tritici (pZtNIA1) for their conditional expression in Z. tritici. Expression of EGFP driven by pMoNIA1 or pZtNIA1 was induced on nitrate and down-regulated on glutamate (10-fold less than nitrate). Levels of differential expression were similar for both promoters, demonstrating that the Z. tritici nitrogen regulatory network functions with a heterologous promoter similarly to a native promoter. To establish CPR, the promoter of Z. tritici BGS1, encoding a b-1,3-glucan synthase, was replaced by pZtNIA1 using targeted sequence replacement. Growth of pZtNIA1::BGS1 CPR transformants was strongly reduced in conditions repressing pZtNIA1, while their growth was similar to wild type in conditions inducing pZtNIA1. This differential phenotype demonstrates that BGS1 is important for growth in Z. tritici. In addition, in inducing conditions, pZtNIA1::BGS1 CPR transformants were hyper-sensitive to Calcofluor white, a cell wall disorganizing agent. Nitrate reductase promoters are therefore suitable for conditional promoter replacement in Z. tritici. This tool is a major step toward identifying novel fungicide targets.

Genome Biology, 2008

Horizontal transfer between fungi

Phylogenetic and comparative genomic analysis of orthologs o... more Horizontal transfer between fungi

Phylogenetic and comparative genomic analysis of orthologs of the Magnaporthe grisea ACE1 cluster reveals evidence for horizontal transfer of part of this cluster from an M. grisea-like ancestor into an ancestor of Aspergillus clavatus.

Proceedings of the National Academy of Sciences, 2001

Molecular Plant-microbe Interactions, 2000

Three genetically independent avirulence genes, AVR1-Irat7, AVRI-MedNoi; and AVR1-Ku86, were iden... more Three genetically independent avirulence genes, AVR1-Irat7, AVRI-MedNoi; and AVR1-Ku86, were identified in a cross involving isolates Guy11 and 2/0/3 of the rice blast fungus, Magnaporthe grisea. Using 76 random progeny, we constructed a partial genetic map with restriction fragment length polymorphism (RFLP) markers revealed by probes such as the repeated sequences MGL/MGR583 and Pot3/MGR586, cosmids from the M. grisea genetic map, and a telomere sequence oligonucleotide. Avirulence genes AVR1-MedNoi and AVR1-Ku86 were closely linked to telomere RFLPs such as marker TelG (6 cM from AVR1-MedNoi) and TelF (4.5 cM from AVR1-Ku86). Avirulence gene AVR1-Irat7 was linked to a cosmid RFLP located on chromosome 1 and mapped at 20 cM from the avirulence gene AVR1-CO39. Using bulked segregant analysis, we identified 11 random amplified polymorphic DNA (RAPD) markers closely linked (0 to 10 cM) to the avirulence genes segregating in this cross. Most of these RAPD markers corresponded to junction fragments between known or new transposons and a single-copy sequence. Such junctions or the whole sequences of single-copy RAPD markers were frequently absent in one parental isolate. Single-copy sequences from RAPD markers tightly linked to avirulence genes will be used for positional cloning.

Communicative & Integrative Biology, 2008

The clonal structure of Magnaporthe oryzae populations was observed in various countries. This in... more The clonal structure of Magnaporthe oryzae populations was observed in various countries. This information was used to propose new resistance deployment strategies. However, our understanding of how new virulent races appear and spread remains limited. Population genetic analyses, with neutral and selected markers, provide tools to evaluate such events. We used microsatellite markers to study populations at the worldwide scale.

Frontiers in Cellular and Infection Microbiology, 2014

The concept of pathogenesis has evolved considerably over recent years, and the scenario "a micro... more The concept of pathogenesis has evolved considerably over recent years, and the scenario "a microbe + virulence factors = disease" is probably far from reality in a number of cases. Actual pathogens have extremely broad biological diversity and are found in all major groups of microorganisms (viruses, bacteria, fungi, protozoa. . .). Their pathogenicity results from strong and often highly specific interactions they have with either their microbial environment, hosts and/or arthropod vectors. In this review, we explore the contribution of metagenomic approaches toward understanding pathogens within the context of microbial communities. With this broader view, we discussed the concept of "pathobiome" and the research questions that this raises.

Fungal Genetics and Biology, 2014

Fungi have the capacity to cause devastating diseases of both plants and animals, causing signifi... more Fungi have the capacity to cause devastating diseases of both plants and animals, causing significant harvest losses that threaten food security and human mycoses with high mortality rates. As a consequence, there is a critical need to promote development of new antifungal drugs, which requires a comprehensive molecular knowledge of fungal pathogenesis. In this review, we critically evaluate current knowledge of seven fungal organisms used as major research models for fungal pathogenesis. These include pathogens of both animals and plants; Ashbya gossypii, Aspergillus fumigatus, Candida albicans, Fusarium oxysporum, Magnaporthe oryzae, Ustilago maydis and Zymoseptoria tritici. We present key insights into the virulence mechanisms deployed by each species and a comparative overview of key insights obtained from genomic analysis. We then consider current trends and future challenges associated with the study of fungal pathogenicity.

PLOS Genetics, 2011

Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic f... more Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of

THE PLANT CELL ONLINE, 2010

Magnaporthe oryzae is the most damaging fungal pathogen of rice (Oryza sativa). In this study, we... more Magnaporthe oryzae is the most damaging fungal pathogen of rice (Oryza sativa). In this study, we characterized the TIG1 transducin b-like gene required for infectious growth and its interacting genes that are required for plant infection in this model phytopathogenic fungus. Tig1 homologs in yeast and mammalian cells are part of a conserved histone deacetylase (HDAC) transcriptional corepressor complex. The tig1 deletion mutant was nonpathogenic and defective in conidiogenesis. It had an increased sensitivity to oxidative stress and failed to develop invasive hyphae in plant cells. Using affinity purification and coimmunoprecipitation assays, we identified several Tig1-associated proteins, including two HDACs that are homologous to components of the yeast Set3 complex. Functional analyses revealed that TIG1, SET3, SNT1, and HOS2 were core components of the Tig1 complex in M. oryzae. The set3, snt1, and hos2 deletion mutants displayed similar defects as those observed in the tig1 mutant, but deletion of HST1 or HOS4 had no detectable phenotypes. Deletion of any of these core components of the Tig1 complex resulted in a significant reduction in HDAC activities. Our results showed that TIG1, like its putative yeast and mammalian orthologs, is one component of a conserved HDAC complex that is required for infectious growth and conidiogenesis in M. oryzae and highlighted that chromatin modification is an essential regulatory mechanism during plant infection.

Science, 2010

Powdery mildews are phytopathogens whose growth and reproduction are entirely dependent on living... more Powdery mildews are phytopathogens whose growth and reproduction are entirely dependent on living plant cells. The molecular basis of this life-style, obligate biotrophy, remains unknown. We present the genome analysis of barley powdery mildew, Blumeria graminis f.sp. hordei (Blumeria), as well as a comparison with the analysis of two powdery mildews pathogenic on dicotyledonous plants. These genomes display massive retrotransposon proliferation, genome-size expansion, and gene losses. The missing genes encode enzymes of primary and secondary metabolism, carbohydrate-active enzymes, and transporters, probably reflecting their redundancy in an exclusively biotrophic life-style. Among the 248 candidate effectors of pathogenesis identified in the Blumeria genome, very few (less than 10) define a core set conserved in all three mildews, suggesting that most effectors represent species-specific adaptations.

PROTEOMICS, 2010

Plant secretomics is a newly emerging area of the plant proteomics field. It basically describes ... more Plant secretomics is a newly emerging area of the plant proteomics field. It basically describes the global study of secreted proteins into the extracellular space by plant cell or tissue at any given time and under certain conditions through various secretory mechanisms. A combination of biochemical, proteomics and bioinformatics approaches has been developed to isolate, identify and profile secreted proteins using complementary in vitro suspensioncultured cells and in planta systems. Developed inventories of secreted proteins under normal, biotic and abiotic conditions revealed several different types of novel secreted proteins, including the leaderless secretory proteins (LSPs). On average, LSPs can account for more than 50% of the total identified secretome, supporting, as in other eukaryotes, the existence of novel secretory mechanisms independent of the classical endoplasmic reticulum-Golgi secretory pathway, and suggesting that this non-classical mechanism of protein expression is, for as yet unknown reasons, more massively used than in other eukaryotic systems. Plants LSPs, which seem to be potentially involved in the defense/stress responses, might have dual (extracellular and/or intracellular) roles as most of them have established intracellular functions, yet presently unknown extracellular functions. Evidence is emerging on the role of glycosylation in the apical sorting and trafficking of secretory proteins. These initial secretome studies in plants have considerably advanced our understanding on secretion of different types of proteins and their underlying mechanisms, and opened a door for comparative analysis of plant secretomes with those of other organisms. In this first review on plant secretomics, we summarize and discuss the secretome definition, the applied approaches for unlocking secrets of the secreted proteins in the extracellular fluid, the possible functional significance and secretory mechanisms of LSPs, as well as glycosylation of secreted proteins and challenges involved ahead. Further improvements in existing and developing strategies and techniques will continue to drive forward plant secretomics research to building comprehensive and confident data sets of secreted proteins. This will lead to an increased understanding on how cells couple the concerted action of secreted protein network to their internal and external environments.

Frontiers in Cellular and Infection Microbiology, 2014

The concept of pathogenesis has evolved considerably over recent years, and the scenario "a micro... more The concept of pathogenesis has evolved considerably over recent years, and the scenario "a microbe + virulence factors = disease" is probably far from reality in a number of cases. Actual pathogens have extremely broad biological diversity and are found in all major groups of microorganisms (viruses, bacteria, fungi, protozoa. . .). Their pathogenicity results from strong and often highly specific interactions they have with either their microbial environment, hosts and/or arthropod vectors. In this review, we explore the contribution of metagenomic approaches toward understanding pathogens within the context of microbial communities. With this broader view, we discussed the concept of "pathobiome" and the research questions that this raises.

New Phytologist, 2007

During the breeding process of cultivated crops, resistance genes to pests and diseases are commo... more During the breeding process of cultivated crops, resistance genes to pests and diseases are commonly introgressed from wild species. The size of these introgressions is predicted by theoretical models but has rarely been measured in cultivated varieties. By combining resistance tests with isogenic strains, genotyping and sequencing of different rice accessions, it was shown that, in the elite rice variety IR64, the resistance conferring allele of the rice blast resistance gene Pi33 was introgressed from the wild rice Oryza rufipogon (accession IRGC101508). Further characterization of this introgression revealed a large introgression at this locus in IR64 and the related variety IR36. The introgressed fragment represents approximately half of the short arm of rice chromosome 8. This is the first report of a large introgression in a cultivated variety of rice. Such a large introgression is likely to have been maintained during backcrossing only if a selection pressure was exerted on this genomic region. The possible traits that were selected are discussed.

Genome Biology and Evolution, 2015

Deciphering the genetic bases of pathogen adaptation to its host is a key question in ecology and... more Deciphering the genetic bases of pathogen adaptation to its host is a key question in ecology and evolution. To understand how the fungus Magnaporthe oryzae adapts to different plants, we sequenced eight M. oryzae isolates differing in host specificity (rice, foxtail millet, wheat, and goosegrass), and one Magnaporthe grisea isolate specific of crabgrass. Analysis of Magnaporthe genomes revealed small variation in genome sizes (39-43 Mb) and gene content (12,283-14,781 genes) between isolates. The whole set of Magnaporthe genes comprised 14,966 shared families, 63% of which included genes present in all the nine M. oryzae genomes. The evolutionary relationships among Magnaporthe isolates were inferred using 6,878 single-copy orthologs. The resulting genealogy was mostly bifurcating among the different host-specific lineages, but was reticulate inside the rice lineage. We detected traces of introgression from a nonrice genome in the rice reference 70-15 genome. Among M. oryzae isolates and host-specific lineages, the genome composition in terms of frequencies of genes putatively involved in pathogenicity (effectors, secondary metabolism, cazome) was conserved. However, 529 shared families were found only in nonrice lineages, whereas the rice lineage possessed 86 specific families absent from the nonrice genomes. Our results confirmed that the host specificity of M. oryzae isolates was associated with a divergence between lineages without major gene flow and that, despite the strong conservation of gene families between lineages, adaptation to different hosts, especially to rice, was associated with the presence of a small number of specific gene families. All information was gathered in a public database (http://genome.jouy.inra.fr/gemo).

Recent Developments in Management of Plant Diseases, 2009

... Valérie Toquin, François Barja, Catherine Sirven, Stéphanie Gamet, Lucile Mauprivez, Philippe... more ... Valérie Toquin, François Barja, Catherine Sirven, Stéphanie Gamet, Lucile Mauprivez, Philippe Peret, Marie-Pascale Latorse, Jean-Luc Zundel, Frédéric Schmitt, Marc ... spectrin plays a crucial role in membrane integrity and dynamics (An et al., 2004; De Matteis and Morrow, 2000 ...

IMA fungus, 2014

To ensure a stable platform for fungal taxonomy, it is of paramount importance that the genetic a... more To ensure a stable platform for fungal taxonomy, it is of paramount importance that the genetic application of generic names be based on their DNA sequence data, and wherever possible, not morphology or ecology alone. To facilitate this process, a new database, accessible at www.GeneraofFungi.org (GoF) was established, which will allow deposition of metadata linked to holo-, lecto-, neo- or epitype specimens, cultures and DNA sequence data of the type species of genera. Although there are presently more than 18 000 fungal genera described, we aim to initially focus on the subset of names that have been placed on the "Without-prejudice List of Protected Generic Names of Fungi" (see IMA Fungus 4(2): 381-443, 2013). To enable the global mycological community to keep track of typification events and avoid duplication, special MycoBank Typification identfiers (MBT) will be issued upon deposit of metadata in MycoBank. MycoBank is linked to GoF, thus deposited metadata of generic...

Molecular Plant-Microbe Interactions, 2010

Comparative analyses of genome sequences from several plant-infecting fungi have shown conservati... more Comparative analyses of genome sequences from several plant-infecting fungi have shown conservation and expansion of protein families with plant disease-related functions. Here, we show that this hypothesis can be extended to mutualistic symbiotic fungi. We have identified a gene encoding an Era (Escherichia coli Ras)-like GTPase in the rice blast fungus Magnaporthe oryzae and found that it is orthologous to the mature amino terminal part of the Gin1 protein from the arbuscular mycorrhizal (AM) fungus Glomus intraradices. M. oryzae Erl1 is required for full root virulence. Appressoria formation was not severely affected in Δerl1strains, but invasive hyphae grew slower than in the wild type. Root browning defect of Δerl1 strains could be complemented by the AM gene under the control of the ERL1 promoter. Erl1 and Gin-N localized to the nucleus when carboxy-terminally labeled with green fluorescent protein (GFP). However, amino-terminal GFP-tagged versions of the proteins expressed in Aspergillus nidulans were shown to localize in the cytoplasm and to cause polarity defects. These data suggest that Erl1 and Gin-N are orthologs and might be involved in the control of hyphal growth in planta. This is the first characterization of an Era-like GTPase in filamentous fungi.

Comparative Biochemistry and Physiology Part A: Molecular & Integrative Physiology, 2008

The secreted proteins (secretome) of fungi play a key role in interactions of pathogenic and symb... more The secreted proteins (secretome) of fungi play a key role in interactions of pathogenic and symbiotic fungi with plants. Using the plant pathogenic fungus Leptosphaeria maculans and symbiont Laccaria bicolor grown in culture, we have established a proteomic protocol for extraction, concentration and resolution of the fungal secretome. As no proteomic data were available on mycelium tissues from both L. maculans and L. bicolor, mycelial proteins were studied; they also helped verifying the purity of secretome samples. The quality of protein extracts was initially assessed by both 1-DE and 2-DE using first a broad pH range for IEF, and then narrower acidic and basic pH ranges, prior to 2-DE. Compared with the previously published protocols for which only dozens of 2-D spots were recovered from fungal secretome samples, up to approximately 2000 2-D spots were resolved by our method. MS identification of proteins along several pH gradients confirmed this high resolution, as well as the presence of major secretome markers such as endopolygalacturonases, b-glucanosyltransferases, pectate lyases and endoglucanases. Shotgun proteomic experiments evidenced the enrichment of secreted protein within the liquid medium. This is the first description of the proteome of L. maculans and L. bicolor, and the first application of liquid-phase IEF to any fungal extracts.

by Kim Plummer, Darren Soanes, Muriel Viaud, Linda Kohn, Marc-henri Lebrun, Jeffrey Rollins, Nicholas Talbot, Oded Yarden, Luis Aguirrezabal Christine Granier, Sabine Fillinger, Emmanuel Quévillon, Baptiste Brault, Ulrich Güldener, and Pascale Cotton

PLoS Genetics, 2011

Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic f... more Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of one strain of S. sclerotiorum and two strains of B. cinerea. The comparative analysis of these genomes relative to one another and to other sequenced fungal genomes is provided here. Their 38-39 Mb genomes include 11,860-14,270 predicted genes, which share 83% amino acid identity on average between the two species. We have mapped the S. sclerotiorum assembly to 16 chromosomes and found large-scale co-linearity with the B. cinerea genomes. Seven percent of the S. sclerotiorum genome comprises transposable elements compared to ,1% of B. cinerea. The arsenal of genes associated with necrotrophic processes is similar between the species, including genes involved in plant cell wall degradation and oxalic acid production. Analysis of secondary metabolism gene clusters revealed an expansion in number and diversity of B. cinerea-specific secondary metabolites relative to S. sclerotiorum. The potential diversity in secondary metabolism might be involved in adaptation to specific ecological niches. Comparative genome analysis revealed the basis of differing sexual mating compatibility systems between S. sclerotiorum and B. cinerea. The organization of the mating-type loci differs, and their structures provide evidence for the evolution of heterothallism from homothallism. These data shed light on the evolutionary and mechanistic bases of the genetically complex traits of necrotrophic pathogenicity and sexual mating. This resource should facilitate the functional studies designed to better understand what makes these fungi such successful and persistent pathogens of agronomic crops.

Fungal Genetics and Biology, 2015

Studying essential genes in haploid fungi requires specific tools. Conditional promoter replaceme... more Studying essential genes in haploid fungi requires specific tools. Conditional promoter replacement (CPR) is an efficient method for testing gene essentiality. However, this tool requires promoters that can be strongly down-regulated. To this end, we tested the nitrate reductase promoters of Magnaporthe oryzae (pMoNIA1) and Zymoseptoria tritici (pZtNIA1) for their conditional expression in Z. tritici. Expression of EGFP driven by pMoNIA1 or pZtNIA1 was induced on nitrate and down-regulated on glutamate (10-fold less than nitrate). Levels of differential expression were similar for both promoters, demonstrating that the Z. tritici nitrogen regulatory network functions with a heterologous promoter similarly to a native promoter. To establish CPR, the promoter of Z. tritici BGS1, encoding a b-1,3-glucan synthase, was replaced by pZtNIA1 using targeted sequence replacement. Growth of pZtNIA1::BGS1 CPR transformants was strongly reduced in conditions repressing pZtNIA1, while their growth was similar to wild type in conditions inducing pZtNIA1. This differential phenotype demonstrates that BGS1 is important for growth in Z. tritici. In addition, in inducing conditions, pZtNIA1::BGS1 CPR transformants were hyper-sensitive to Calcofluor white, a cell wall disorganizing agent. Nitrate reductase promoters are therefore suitable for conditional promoter replacement in Z. tritici. This tool is a major step toward identifying novel fungicide targets.

Genome Biology, 2008

Horizontal transfer between fungi

Phylogenetic and comparative genomic analysis of orthologs o... more Horizontal transfer between fungi

Phylogenetic and comparative genomic analysis of orthologs of the Magnaporthe grisea ACE1 cluster reveals evidence for horizontal transfer of part of this cluster from an M. grisea-like ancestor into an ancestor of Aspergillus clavatus.

Proceedings of the National Academy of Sciences, 2001

Molecular Plant-microbe Interactions, 2000

Three genetically independent avirulence genes, AVR1-Irat7, AVRI-MedNoi; and AVR1-Ku86, were iden... more Three genetically independent avirulence genes, AVR1-Irat7, AVRI-MedNoi; and AVR1-Ku86, were identified in a cross involving isolates Guy11 and 2/0/3 of the rice blast fungus, Magnaporthe grisea. Using 76 random progeny, we constructed a partial genetic map with restriction fragment length polymorphism (RFLP) markers revealed by probes such as the repeated sequences MGL/MGR583 and Pot3/MGR586, cosmids from the M. grisea genetic map, and a telomere sequence oligonucleotide. Avirulence genes AVR1-MedNoi and AVR1-Ku86 were closely linked to telomere RFLPs such as marker TelG (6 cM from AVR1-MedNoi) and TelF (4.5 cM from AVR1-Ku86). Avirulence gene AVR1-Irat7 was linked to a cosmid RFLP located on chromosome 1 and mapped at 20 cM from the avirulence gene AVR1-CO39. Using bulked segregant analysis, we identified 11 random amplified polymorphic DNA (RAPD) markers closely linked (0 to 10 cM) to the avirulence genes segregating in this cross. Most of these RAPD markers corresponded to junction fragments between known or new transposons and a single-copy sequence. Such junctions or the whole sequences of single-copy RAPD markers were frequently absent in one parental isolate. Single-copy sequences from RAPD markers tightly linked to avirulence genes will be used for positional cloning.

Communicative & Integrative Biology, 2008

The clonal structure of Magnaporthe oryzae populations was observed in various countries. This in... more The clonal structure of Magnaporthe oryzae populations was observed in various countries. This information was used to propose new resistance deployment strategies. However, our understanding of how new virulent races appear and spread remains limited. Population genetic analyses, with neutral and selected markers, provide tools to evaluate such events. We used microsatellite markers to study populations at the worldwide scale.

Frontiers in Cellular and Infection Microbiology, 2014

The concept of pathogenesis has evolved considerably over recent years, and the scenario "a micro... more The concept of pathogenesis has evolved considerably over recent years, and the scenario "a microbe + virulence factors = disease" is probably far from reality in a number of cases. Actual pathogens have extremely broad biological diversity and are found in all major groups of microorganisms (viruses, bacteria, fungi, protozoa. . .). Their pathogenicity results from strong and often highly specific interactions they have with either their microbial environment, hosts and/or arthropod vectors. In this review, we explore the contribution of metagenomic approaches toward understanding pathogens within the context of microbial communities. With this broader view, we discussed the concept of "pathobiome" and the research questions that this raises.

Fungal Genetics and Biology, 2014

Fungi have the capacity to cause devastating diseases of both plants and animals, causing signifi... more Fungi have the capacity to cause devastating diseases of both plants and animals, causing significant harvest losses that threaten food security and human mycoses with high mortality rates. As a consequence, there is a critical need to promote development of new antifungal drugs, which requires a comprehensive molecular knowledge of fungal pathogenesis. In this review, we critically evaluate current knowledge of seven fungal organisms used as major research models for fungal pathogenesis. These include pathogens of both animals and plants; Ashbya gossypii, Aspergillus fumigatus, Candida albicans, Fusarium oxysporum, Magnaporthe oryzae, Ustilago maydis and Zymoseptoria tritici. We present key insights into the virulence mechanisms deployed by each species and a comparative overview of key insights obtained from genomic analysis. We then consider current trends and future challenges associated with the study of fungal pathogenicity.

PLOS Genetics, 2011

Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic f... more Sclerotinia sclerotiorum and Botrytis cinerea are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of

THE PLANT CELL ONLINE, 2010

Magnaporthe oryzae is the most damaging fungal pathogen of rice (Oryza sativa). In this study, we... more Magnaporthe oryzae is the most damaging fungal pathogen of rice (Oryza sativa). In this study, we characterized the TIG1 transducin b-like gene required for infectious growth and its interacting genes that are required for plant infection in this model phytopathogenic fungus. Tig1 homologs in yeast and mammalian cells are part of a conserved histone deacetylase (HDAC) transcriptional corepressor complex. The tig1 deletion mutant was nonpathogenic and defective in conidiogenesis. It had an increased sensitivity to oxidative stress and failed to develop invasive hyphae in plant cells. Using affinity purification and coimmunoprecipitation assays, we identified several Tig1-associated proteins, including two HDACs that are homologous to components of the yeast Set3 complex. Functional analyses revealed that TIG1, SET3, SNT1, and HOS2 were core components of the Tig1 complex in M. oryzae. The set3, snt1, and hos2 deletion mutants displayed similar defects as those observed in the tig1 mutant, but deletion of HST1 or HOS4 had no detectable phenotypes. Deletion of any of these core components of the Tig1 complex resulted in a significant reduction in HDAC activities. Our results showed that TIG1, like its putative yeast and mammalian orthologs, is one component of a conserved HDAC complex that is required for infectious growth and conidiogenesis in M. oryzae and highlighted that chromatin modification is an essential regulatory mechanism during plant infection.

Science, 2010

Powdery mildews are phytopathogens whose growth and reproduction are entirely dependent on living... more Powdery mildews are phytopathogens whose growth and reproduction are entirely dependent on living plant cells. The molecular basis of this life-style, obligate biotrophy, remains unknown. We present the genome analysis of barley powdery mildew, Blumeria graminis f.sp. hordei (Blumeria), as well as a comparison with the analysis of two powdery mildews pathogenic on dicotyledonous plants. These genomes display massive retrotransposon proliferation, genome-size expansion, and gene losses. The missing genes encode enzymes of primary and secondary metabolism, carbohydrate-active enzymes, and transporters, probably reflecting their redundancy in an exclusively biotrophic life-style. Among the 248 candidate effectors of pathogenesis identified in the Blumeria genome, very few (less than 10) define a core set conserved in all three mildews, suggesting that most effectors represent species-specific adaptations.

PROTEOMICS, 2010

Plant secretomics is a newly emerging area of the plant proteomics field. It basically describes ... more Plant secretomics is a newly emerging area of the plant proteomics field. It basically describes the global study of secreted proteins into the extracellular space by plant cell or tissue at any given time and under certain conditions through various secretory mechanisms. A combination of biochemical, proteomics and bioinformatics approaches has been developed to isolate, identify and profile secreted proteins using complementary in vitro suspensioncultured cells and in planta systems. Developed inventories of secreted proteins under normal, biotic and abiotic conditions revealed several different types of novel secreted proteins, including the leaderless secretory proteins (LSPs). On average, LSPs can account for more than 50% of the total identified secretome, supporting, as in other eukaryotes, the existence of novel secretory mechanisms independent of the classical endoplasmic reticulum-Golgi secretory pathway, and suggesting that this non-classical mechanism of protein expression is, for as yet unknown reasons, more massively used than in other eukaryotic systems. Plants LSPs, which seem to be potentially involved in the defense/stress responses, might have dual (extracellular and/or intracellular) roles as most of them have established intracellular functions, yet presently unknown extracellular functions. Evidence is emerging on the role of glycosylation in the apical sorting and trafficking of secretory proteins. These initial secretome studies in plants have considerably advanced our understanding on secretion of different types of proteins and their underlying mechanisms, and opened a door for comparative analysis of plant secretomes with those of other organisms. In this first review on plant secretomics, we summarize and discuss the secretome definition, the applied approaches for unlocking secrets of the secreted proteins in the extracellular fluid, the possible functional significance and secretory mechanisms of LSPs, as well as glycosylation of secreted proteins and challenges involved ahead. Further improvements in existing and developing strategies and techniques will continue to drive forward plant secretomics research to building comprehensive and confident data sets of secreted proteins. This will lead to an increased understanding on how cells couple the concerted action of secreted protein network to their internal and external environments.