Publications by authors named "Marie-Noelle Rosso"

42 Publications

Gene family expansions and transcriptome signatures uncover fungal adaptations to wood decay.

Environ Microbiol 2021 Feb 4. Epub 2021 Feb 4.

INRAE, Aix Marseille Univ, UMR1163, Biodiversité et Biotechnologie Fongiques, Marseille, 13009, France.

Because they comprise some of the most efficient wood-decayers, Polyporales fungi impact carbon cycling in forest environment. Despite continuous discoveries on the enzymatic machinery involved in wood decomposition, the vision on their evolutionary adaptation to wood decay and genome diversity remains incomplete. We combined the genome sequence information from 50 Polyporales species, including 26 newly sequenced genomes and sought for genomic and functional adaptations to wood decay through the analysis of genome composition and transcriptome responses to different carbon sources. The genomes of Polyporales from different phylogenetic clades showed poor conservation in macrosynteny, indicative of genome rearrangements. We observed different gene family expansion/contraction histories for plant cell wall degrading enzymes in core polyporoids and phlebioids and captured expansions for genes involved in signalling and regulation in the lineages of white rotters. Furthermore, we identified conserved cupredoxins, thaumatin-like proteins and lytic polysaccharide monooxygenases with a yet uncharacterized appended module as new candidate players in wood decomposition. Given the current need for enzymatic toolkits dedicated to the transformation of renewable carbon sources, the observed genomic diversity among Polyporales strengthens the relevance of mining Polyporales biodiversity to understand the molecular mechanisms of wood decay.
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http://dx.doi.org/10.1111/1462-2920.15423DOI Listing
February 2021

Genomic Analysis Enlightens Agaricales Lifestyle Evolution and Increasing Peroxidase Diversity.

Mol Biol Evol 2020 Nov 19. Epub 2020 Nov 19.

Centro de Investigaciones Biológicas Margarita Salas (CIB), CSIC, Madrid, Spain.

As actors of global carbon cycle, Agaricomycetes (Basidiomycota) have developed complex enzymatic machineries that allow them to decompose all plant polymers, including lignin. Among them, saprotrophic Agaricales are characterized by an unparalleled diversity of habitats and lifestyles. Comparative analysis of 52 Agaricomycetes genomes (14 of them sequenced de novo) reveals that Agaricales possess a large diversity of hydrolytic and oxidative enzymes for lignocellulose decay. Based on the gene families with the predicted highest evolutionary rates -namely cellulose-binding CBM1, glycoside hydrolase GH43, lytic polysaccharide monooxygenase AA9, class-II peroxidases, glucose-methanol-choline oxidase/dehydrogenases, laccases, and unspecific peroxygenases- we reconstructed the lifestyles of the ancestors that led to the extant lignocellulose-decomposing Agaricomycetes. The changes in the enzymatic toolkit of ancestral Agaricales are correlated with the evolution of their ability to grow not only on wood but also on leaf-litter and decayed wood, with grass-litter decomposers as the most recent eco-physiological group. In this context, the above families were analyzed in detail in connection with lifestyle diversity. Peroxidases appear as a central component of the enzymatic toolkit of saprotrophic Agaricomycetes, consistent with their essential role in lignin degradation and high evolutionary rates. This includes not only expansions/losses in peroxidase genes common to other basidiomycetes, but also the widespread presence in Agaricales (and Russulales) of new peroxidases types not found in wood-rotting Polyporales, and other Agaricomycetes orders. Therefore, we analyzed the peroxidase evolution in Agaricomycetes by ancestral-sequence reconstruction revealing several major evolutionary pathways, and mapped the appearance of the different enzyme types in a time-calibrated species tree.
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http://dx.doi.org/10.1093/molbev/msaa301DOI Listing
November 2020

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

DNA Res 2020 Apr;27(2)

INRAE, UMR1163, Biodiversity and Biotechnology of Fungi, Aix Marseille University, 13009 Marseille, France.

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.
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http://dx.doi.org/10.1093/dnares/dsaa011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7406137PMC
April 2020

Rational Design of Mechanism-Based Inhibitors and Activity-Based Probes for the Identification of Retaining α-l-Arabinofuranosidases.

J Am Chem Soc 2020 03 26;142(10):4648-4662. Epub 2020 Feb 26.

York Structural Biology Laboratory, Department of Chemistry, The University of York, Heslington, York YO10 5DD, U.K.

Identifying and characterizing the enzymes responsible for an observed activity within a complex eukaryotic catabolic system remains one of the most significant challenges in the study of biomass-degrading systems. The debranching of both complex hemicellulosic and pectinaceous polysaccharides requires the production of α-l-arabinofuranosidases among a wide variety of coexpressed carbohydrate-active enzymes. To selectively detect and identify α-l-arabinofuranosidases produced by fungi grown on complex biomass, potential covalent inhibitors and probes which mimic α-l-arabinofuranosides were sought. The conformational free energy landscapes of free α-l-arabinofuranose and several rationally designed covalent α-l-arabinofuranosidase inhibitors were analyzed. A synthetic route to these inhibitors was subsequently developed based on a key Wittig-Still rearrangement. Through a combination of kinetic measurements, intact mass spectrometry, and structural experiments, the designed inhibitors were shown to efficiently label the catalytic nucleophiles of retaining GH51 and GH54 α-l-arabinofuranosidases. Activity-based probes elaborated from an inhibitor with an aziridine warhead were applied to the identification and characterization of α-l-arabinofuranosidases within the secretome of grown on arabinan. This method was extended to the detection and identification of α-l-arabinofuranosidases produced by eight biomass-degrading basidiomycete fungi grown on complex biomass. The broad applicability of the cyclophellitol-derived activity-based probes and inhibitors presented here make them a valuable new tool in the characterization of complex eukaryotic carbohydrate-degrading systems and in the high-throughput discovery of α-l-arabinofuranosidases.
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http://dx.doi.org/10.1021/jacs.9b11351DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7068720PMC
March 2020

A fungal family of lytic polysaccharide monooxygenase-like copper proteins.

Nat Chem Biol 2020 03 13;16(3):345-350. Epub 2020 Jan 13.

INRA, Biodiversité et Biotechnologie Fongiques (BBF), UMR1163, Aix Marseille Université, Marseille, France.

Lytic polysaccharide monooxygenases (LPMOs) are copper-containing enzymes that play a key role in the oxidative degradation of various biopolymers such as cellulose and chitin. While hunting for new LPMOs, we identified a new family of proteins, defined here as X325, in various fungal lineages. The three-dimensional structure of X325 revealed an overall LPMO fold and a His brace with an additional Asp ligand to Cu(II). Although LPMO-type activity of X325 members was initially expected, we demonstrated that X325 members do not perform oxidative cleavage of polysaccharides, establishing that X325s are not LPMOs. Investigations of the biological role of X325 in the ectomycorrhizal fungus Laccaria bicolor revealed exposure of the X325 protein at the interface between fungal hyphae and tree rootlet cells. Our results provide insights into a family of copper-containing proteins, which is widespread in the fungal kingdom and is evolutionarily related to LPMOs, but has diverged to biological functions other than polysaccharide degradation.
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http://dx.doi.org/10.1038/s41589-019-0438-8DOI Listing
March 2020

Insights into an unusual Auxiliary Activity 9 family member lacking the histidine brace motif of lytic polysaccharide monooxygenases.

J Biol Chem 2019 11 30;294(45):17117-17130. Epub 2019 Aug 30.

Department of Chemistry, University of Copenhagen, 2100 Copenhagen, Denmark

Lytic polysaccharide monooxygenases (LPMOs) are redox-enzymes involved in biomass degradation. All characterized LPMOs possess an active site of two highly conserved histidine residues coordinating a copper ion (the histidine brace), which are essential for LPMO activity. However, some protein sequences that belong to the AA9 LPMO family display a natural N-terminal His to Arg substitution (Arg-AA9). These are found almost entirely in the phylogenetic fungal class , associated with wood decay, but no function has been demonstrated for any Arg-AA9. Through bioinformatics, transcriptomic, and proteomic analyses we present data, which suggest that Arg-AA9 proteins could have a hitherto unidentified role in fungal degradation of lignocellulosic biomass in conjunction with other secreted fungal enzymes. We present the first structure of an Arg-AA9, AA9B, a naturally occurring protein from The AA9B structure reveals gross changes in the region equivalent to the canonical LPMO copper-binding site, whereas features implicated in carbohydrate binding in AA9 LPMOs have been maintained. We obtained a structure of AA9B with xylotetraose bound on the surface of the protein although with a considerably different binding mode compared with other AA9 complex structures. In addition, we have found indications of protein phosphorylation near the N-terminal Arg and the carbohydrate-binding site, for which the potential function is currently unknown. Our results are strong evidence that Arg-AA9s function markedly different from canonical AA9 LPMO, but nonetheless, may play a role in fungal conversion of lignocellulosic biomass.
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http://dx.doi.org/10.1074/jbc.RA119.009223DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6851306PMC
November 2019

Tracking of enzymatic biomass deconstruction by fungal secretomes highlights markers of lignocellulose recalcitrance.

Biotechnol Biofuels 2019 1;12:76. Epub 2019 Apr 1.

2INRA, Aix Marseille Univ., UMR1163, BBF, Biodiversité et Biotechnologie Fongiques, Marseille, France.

Background: Lignocellulose biomass is known as a recalcitrant material towards enzymatic hydrolysis, increasing the process cost in biorefinery. In nature, filamentous fungi naturally degrade lignocellulose, using an arsenal of hydrolytic and oxidative enzymes. Assessment of enzyme hydrolysis efficiency generally relies on the yield of glucose for a given biomass. To better understand the markers governing recalcitrance to enzymatic degradation, there is a need to enlarge the set of parameters followed during deconstruction.

Results: Industrially-pretreated biomass feedstocks from wheat straw, miscanthus and poplar were sequentially hydrolysed following two steps. First, standard secretome from was used to maximize cellulose hydrolysis, producing three recalcitrant lignin-enriched solid substrates. Then fungal secretomes from three basidiomycete saprotrophs ( and ) displaying various hydrolytic and oxidative enzymatic profiles were applied to these recalcitrant substrates, and compared to the secretome. As a result, most of the glucose was released after the first hydrolysis step. After the second hydrolysis step, half of the remaining glucose amount was released. Overall, glucose yield after the two sequential hydrolyses was more dependent on the biomass source than on the fungal secretomes enzymatic profile. Solid residues obtained after the two hydrolysis steps were characterized using complementary methodologies. Correlation analysis of several physico-chemical parameters showed that released glucose yield was negatively correlated with lignin content and cellulose crystallinity while positively correlated with xylose content and water sorption. Water sorption appears as a pivotal marker of the recalcitrance as it reflects chemical and structural properties of lignocellulosic biomass.

Conclusions: Fungal secretomes applied to highly recalcitrant biomass samples can further extend the release of the remaining glucose. The glucose yield can be correlated to chemical and physical markers, which appear to be independent from the biomass type and secretome. Overall, correlations between these markers reveal how nano-scale properties (polymer content and organization) influence macro-scale properties (particle size and water sorption). Further systematic assessment of these markers during enzymatic degradation will foster the development of novel cocktails to unlock the degradation of lignocellulose biomass.
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http://dx.doi.org/10.1186/s13068-019-1417-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6442405PMC
April 2019

Broad-specificity GH131 β-glucanases are a hallmark of fungi and oomycetes that colonize plants.

Environ Microbiol 2019 08 21;21(8):2724-2739. Epub 2019 Apr 21.

INRA, Aix-Marseille Univ, UMR1163, Biodiversité et Biotechnologie Fongiques, BBF, Marseille, France.

Plant-tissue-colonizing fungi fine-tune the deconstruction of plant-cell walls (PCW) using different sets of enzymes according to their lifestyle. However, some of these enzymes are conserved among fungi with dissimilar lifestyles. We identified genes from Glycoside Hydrolase family GH131 as commonly expressed during plant-tissue colonization by saprobic, pathogenic and symbiotic fungi. By searching all the publicly available genomes, we found that GH131-coding genes were widely distributed in the Dikarya subkingdom, except in Taphrinomycotina and Saccharomycotina, and in phytopathogenic Oomycetes, but neither other eukaryotes nor prokaryotes. The presence of GH131 in a species was correlated with its association with plants as symbiont, pathogen or saprobe. We propose that GH131-family expansions and horizontal-gene transfers contributed to this adaptation. We analysed the biochemical activities of GH131 enzymes whose genes were upregulated during plant-tissue colonization in a saprobe (Pycnoporus sanguineus), a plant symbiont (Laccaria bicolor) and three hemibiotrophic-plant pathogens (Colletotrichum higginsianum, C. graminicola, Zymoseptoria tritici). These enzymes were all active on substrates with β-1,4, β-1,3 and mixed β-1,4/1,3 glucosidic linkages. Combined with a cellobiohydrolase, GH131 enzymes enhanced cellulose degradation. We propose that secreted GH131 enzymes unlock the PCW barrier and allow further deconstruction by other enzymes during plant tissue colonization by symbionts, pathogens and saprobes.
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http://dx.doi.org/10.1111/1462-2920.14596DOI Listing
August 2019

Integrative visual omics of the white-rot fungus exposes the biotechnological potential of its oxidative enzymes for delignifying raw plant biomass.

Biotechnol Biofuels 2018 23;11:201. Epub 2018 Jul 23.

1Aix Marseille Univ, INRA, UMR 1163, Biodiversité et Biotechnologie Fongiques, BBF, Marseille, France.

Background: Plant biomass conversion for green chemistry and bio-energy is a current challenge for a modern sustainable bioeconomy. The complex polyaromatic lignin polymers in raw biomass feedstocks (i.e., agriculture and forestry by-products) are major obstacles for biomass conversions. White-rot fungi are wood decayers able to degrade all polymers from lignocellulosic biomass including cellulose, hemicelluloses, and lignin. The white-rot fungus efficiently breaks down lignin and is regarded as having a high potential for the initial treatment of plant biomass in its conversion to bio-energy. Here, we describe the extraordinary ability of for lignin degradation using its enzymatic arsenal to break down wheat straw, a lignocellulosic substrate that is considered as a biomass feedstock worldwide.

Results: We performed integrative multi-omics analyses by combining data from the fungal genome, transcriptomes, and secretomes. We found that the fungus possessed an unexpectedly large set of genes coding for Class II peroxidases involved in lignin degradation (19 genes) and GMC oxidoreductases/dehydrogenases involved in generating the hydrogen peroxide required for lignin peroxidase activity and promoting redox cycling of the fungal enzymes involved in oxidative cleavage of lignocellulose polymers (36 genes). The examination of interrelated multi-omics patterns revealed that eleven Class II Peroxidases were secreted by the fungus during fermentation and eight of them where tightly co-regulated with redox cycling enzymatic partners.

Conclusion: As a peculiar feature of , we observed gene family extension, up-regulation and secretion of an abundant set of versatile peroxidases and manganese peroxidases, compared with other Polyporales species. The orchestrated secretion of an abundant set of these delignifying enzymes and redox cycling enzymatic partners could contribute to the delignification capabilities of the fungus. Our findings highlight the diversity of wood decay mechanisms present in Polyporales and the potentiality of further exploring this taxonomic order for enzymatic functions of biotechnological interest.
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http://dx.doi.org/10.1186/s13068-018-1198-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6055342PMC
July 2018

The ectomycorrhizal basidiomycete Laccaria bicolor releases a secreted β-1,4 endoglucanase that plays a key role in symbiosis development.

New Phytol 2018 12 6;220(4):1309-1321. Epub 2018 Apr 6.

UMR 1136 INRA-Université de Lorraine 'Interactions Arbres/Microorganismes', Laboratoire d'Excellence ARBRE, Centre INRA-Lorraine, 54280, Champenoux, France.

In ectomycorrhiza, root ingress and colonization of the apoplast by colonizing hyphae is thought to rely mainly on the mechanical force that results from hyphal tip growth, but this could be enhanced by secretion of cell-wall-degrading enzymes, which have not yet been identified. The sole cellulose-binding module (CBM1) encoded in the genome of the ectomycorrhizal Laccaria bicolor is linked to a glycoside hydrolase family 5 (GH5) endoglucanase, LbGH5-CBM1. Here, we characterize LbGH5-CBM1 gene expression and the biochemical properties of its protein product. We also immunolocalized LbGH5-CBM1 by immunofluorescence confocal microscopy in poplar ectomycorrhiza. We show that LbGH5-CBM1 expression is substantially induced in ectomycorrhiza, and RNAi mutants with a decreased LbGH5-CBM1 expression have a lower ability to form ectomycorrhiza, suggesting a key role in symbiosis. Recombinant LbGH5-CBM1 displays its highest activity towards cellulose and galactomannans, but no activity toward L. bicolor cell walls. In situ localization of LbGH5-CBM1 in ectomycorrhiza reveals that the endoglucanase accumulates at the periphery of hyphae forming the Hartig net and the mantle. Our data suggest that the symbiosis-induced endoglucanase LbGH5-CBM1 is an enzymatic effector involved in cell wall remodeling during formation of the Hartig net and is an important determinant for successful symbiotic colonization.
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http://dx.doi.org/10.1111/nph.15113DOI Listing
December 2018

Lytic xylan oxidases from wood-decay fungi unlock biomass degradation.

Nat Chem Biol 2018 03 29;14(3):306-310. Epub 2018 Jan 29.

INRA, Aix Marseille University, Biodiversité et Biotechnologie Fongiques (BBF), Marseille, France.

Wood biomass is the most abundant feedstock envisioned for the development of modern biorefineries. However, the cost-effective conversion of this form of biomass into commodity products is limited by its resistance to enzymatic degradation. Here we describe a new family of fungal lytic polysaccharide monooxygenases (LPMOs) prevalent among white-rot and brown-rot basidiomycetes that is active on xylans-a recalcitrant polysaccharide abundant in wood biomass. Two AA14 LPMO members from the white-rot fungus Pycnoporus coccineus substantially increase the efficiency of wood saccharification through oxidative cleavage of highly refractory xylan-coated cellulose fibers. The discovery of this unique enzyme activity advances our knowledge on the degradation of woody biomass in nature and offers an innovative solution for improving enzyme cocktails for biorefinery applications.
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http://dx.doi.org/10.1038/nchembio.2558DOI Listing
March 2018

Fungal secretomics to probe the biological functions of lytic polysaccharide monooxygenases.

Carbohydr Res 2017 Aug 17;448:155-160. Epub 2017 May 17.

Protein Glycoscience and Biotechnology, Department of Biotechnology and Biomedicine, Technical University of Denmark, Elektrovej 375, 2800 Kgs Lyngby, Denmark. Electronic address:

Enzymatic degradation of plant biomass is of growing interest for the development of a sustainable bio-based industry. Filamentous fungi, which degrade complex and recalcitrant plant polymers, are proficient secretors of enzymes acting on the lignocellulose composite of plant cell walls in addition to starch, the main carbon storage reservoir. In this review, we focus on the identification of lytic polysaccharide monooxygenases (LPMOs) and their redox partners in fungal secretomes to highlight the biological functions of these remarkable enzyme systems and we discuss future trends related to LPMO-potentiated bioconversion.
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http://dx.doi.org/10.1016/j.carres.2017.05.010DOI Listing
August 2017

The integrative omics of white-rot fungus Pycnoporus coccineus reveals co-regulated CAZymes for orchestrated lignocellulose breakdown.

PLoS One 2017 10;12(4):e0175528. Epub 2017 Apr 10.

Aix-Marseille Université, INRA, UMR 1163, Biodiversité et Biotechnologie Fongiques, BBF, Marseille, France.

Innovative green technologies are of importance for converting plant wastes into renewable sources for materials, chemicals and energy. However, recycling agricultural and forestry wastes is a challenge. A solution may be found in the forest. Saprotrophic white-rot fungi are able to convert dead plants into consumable carbon sources. Specialized fungal enzymes can be utilized for breaking down hard plant biopolymers. Thus, understanding the enzymatic machineries of such fungi gives us hints for the efficient decomposition of plant materials. Using the saprotrophic white-rot fungus Pycnoporus coccineus as a fungal model, we examined the dynamics of transcriptomic and secretomic responses to different types of lignocellulosic substrates at two time points. Our integrative omics pipeline (SHIN+GO) enabled us to compress layers of biological information into simple heatmaps, allowing for visual inspection of the data. We identified co-regulated genes with corresponding co-secreted enzymes, and the biological roles were extrapolated with the enriched Carbohydrate-Active Enzyme (CAZymes) and functional annotations. We observed the fungal early responses for the degradation of lignocellulosic substrates including; 1) simultaneous expression of CAZy genes and secretion of the enzymes acting on diverse glycosidic bonds in cellulose, hemicelluloses and their side chains or lignin (i.e. hydrolases, esterases and oxido-reductases); 2) the key role of lytic polysaccharide monooxygenases (LPMO); 3) the early transcriptional regulation of lignin active peroxidases; 4) the induction of detoxification processes dealing with biomass-derived compounds; and 5) the frequent attachments of the carbohydrate binding module 1 (CBM1) to enzymes from the lignocellulose-responsive genes. Our omics combining methods and related biological findings may contribute to the knowledge of fungal systems biology and facilitate the optimization of fungal enzyme cocktails for various industrial applications.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0175528PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5386290PMC
September 2017

Visual Comparative Omics of Fungi for Plant Biomass Deconstruction.

Front Microbiol 2016 24;7:1335. Epub 2016 Aug 24.

Aix-Marseille Université, INRA, UMR1163, Biodiversité et Biotechnologie Fongiques Marseille, France.

Wood-decay fungi contain the cellular mechanisms to decompose such plant cell wall components as cellulose, hemicellulose, and lignin. A multi-omics approach to the comparative analysis of wood-decay fungi gives not only new insights into their strategies for decomposing recalcitrant plant biomass, but also an understanding of how to exploit these mechanisms for biotechnological applications. We have developed an analytical workflow, Applied Biomass Conversion Design for Efficient Fungal Green Technology (ABCDEFGT), to simplify the analysis and interpretation of transcriptomic and secretomic data. ABCDEFGT utilizes self-organizing maps for grouping genes with similar transcription patterns, and an overlay with secreted proteins. The key feature of ABCDEFGT is simple graphic outputs of genome-wide transcriptomic and secretomic topographies, which enables visual inspection without a priori of the omics data and facilitates discoveries of co-regulated genes and proteins. Genome-wide omics landscapes were built with the newly sequenced fungal species Pycnoporus coccineus, Pycnoporus sanguineus, and Pycnoporus cinnabarinus grown on various carbon sources. Integration of the post-genomic data revealed a global overlap, confirming the pertinence of the genome-wide approach. ABCDEFGT was evaluated by comparison with the latest clustering method for ease of output interpretation, and ABCDEFGT gave a better biological representation of fungal behaviors. The genome-wide multi-omics strategy allowed us to determine the potential synergy of particular enzymes decomposing cellulose, hemicellulose, and lignin such as Lytic Polysaccharide Monooxygenases, modular enzymes associated with a cellulose binding module1, and Class II Peroxidase isoforms co-regulated with oxido-reductases. Overall, ABCDEFGT was capable of visualizing genome-wide transcriptional and secretomic profiles for intuitive interpretations and is suitable for exploration of newly-sequenced organisms.
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http://dx.doi.org/10.3389/fmicb.2016.01335DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4996036PMC
September 2016

Enhanced degradation of softwood versus hardwood by the white-rot fungus Pycnoporus coccineus.

Biotechnol Biofuels 2015 18;8:216. Epub 2015 Dec 18.

Aix Marseille Université, UMR1163 Biodiversité et Biotechnologie Fongiques, 163 avenue de Luminy, 13288 Marseille, France ; INRA, UMR1163 Biodiversité et Biotechnologie Fongiques, 163 avenue de Luminy, 13288 Marseille, France ; Polytech'Marseille, UMR1163 Biodiversité et Biotechnologie Fongiques, 163 avenue de Luminy, 13288 Marseille, France.

Background: White-rot basidiomycete fungi are potent degraders of plant biomass, with the ability to mineralize all lignocellulose components. Recent comparative genomics studies showed that these fungi use a wide diversity of enzymes for wood degradation. Deeper functional analyses are however necessary to understand the enzymatic mechanisms leading to lignocellulose breakdown. The Polyporale fungus Pycnoporus coccineus BRFM310 grows well on both coniferous and deciduous wood. In the present study, we analyzed the early response of the fungus to softwood (pine) and hardwood (aspen) feedstocks and tested the effect of the secreted enzymes on lignocellulose deconstruction.

Results: Transcriptomic and proteomic analyses revealed that P. coccineus grown separately on pine and aspen displayed similar sets of transcripts and enzymes implicated in lignin and polysaccharide degradation. In particular, the expression of lignin-targeting oxidoreductases, such as manganese peroxidases, increased upon cultivation on both woods. The sets of enzymes secreted during growth on both pine and aspen were more efficient in saccharide release from pine than from aspen, and characterization of the residual solids revealed polysaccharide conversion on both pine and aspen fiber surfaces.

Conclusion: The combined analysis of soluble sugars and solid residues showed the suitability of P. coccineus secreted enzymes for softwood degradation. Analyses of solubilized products and residual surface chemistries of enzyme-treated wood samples pointed to differences in fiber penetration by different P. coccineus secretomes. Accordingly, beyond the variety of CAZymes identified in P. coccineus genome, transcriptome and secretome, we discuss several parameters such as the abundance of manganese peroxidases and the potential role of cytochrome P450s and pectin degradation on the efficacy of fungi for softwood conversion.
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http://dx.doi.org/10.1186/s13068-015-0407-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4683735PMC
December 2015

A PCR-based method to quantify fungal growth during pretreatment of lignocellulosic biomass.

J Microbiol Methods 2015 Aug 30;115:67-70. Epub 2015 May 30.

INRA, UMR1163 Biodiversity and Biotechnology of Fungi, F-13009 Marseille, France; Aix-Marseille Université, Polytech Marseille, UMR1163 Biodiversity and Biotechnology of Fungi, F-13009 Marseille, France. Electronic address:

Filamentous fungi have shown great potential in the pretreatment of lignocellulosic biomass and their use in bio-processes based on Solid State Fermentation (SSF) opens promising perspectives for plant biomass valorization. Obviously, quantification of the fungal biomass throughout the fermentation is a crucial parameter for SSF evaluation and control, both at the laboratory and industrial scale. Here we provide a qPCR-based method as a reliable alternative to conventional methods to estimate mycelial growth during plant biomass treatment. For the three strains analyzed, the lowest detection limit ranged from 58 to 272 μg mycelium dry weight per gram biomass (dry weight). We show that the qPCR-based method allows fungal growth monitoring during fermentation and provides relevant information for selection of the most appropriate fungal strains in specific SSF/reactor conditions.
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http://dx.doi.org/10.1016/j.mimet.2015.05.024DOI Listing
August 2015

L-lactic acid production by Aspergillus brasiliensis overexpressing the heterologous ldha gene from Rhizopus oryzae.

Microb Cell Fact 2015 May 3;14:66. Epub 2015 May 3.

Aix-Marseille Université, Unité de Recherche sur les Maladies Infectieuses et Tropicales Emergentes (URMITE), UM63, CNRS 7278, IRD 198, INSERM U1095, IHU Méditerranée Infection, Pôle des Maladies Infectieuses, Assistance Publique-Hôpitaux de Marseille, Faculté de Médecine, 27 Bd Jean Moulin, 13005, Marseille, France.

Background: Lactic acid is the building block of poly-lactic acid (PLA), a biopolymer that could be set to replace petroleum-based plastics. To make lactic acid production cost-effective, the production process should be carried out at low pH, in low-nutrient media, and with a low-cost carbon source. Yeasts have been engineered to produce high levels of lactic acid at low pH from glucose but not from carbohydrate polymers (e.g. cellulose, hemicellulose, starch). Aspergilli are versatile microbial cell factories able to naturally produce large amounts of organic acids at low pH and to metabolize cheap abundant carbon sources such as plant biomass. However, they have never been used for lactic acid production.

Results: To investigate the feasibility of lactic acid production with Aspergillus, the NAD-dependent lactate dehydrogenase (LDH) responsible for lactic acid production by Rhizopus oryzae was produced in Aspergillus brasiliensis BRFM103. Among transformants, the best lactic acid producer, A. brasiliensis BRFM1877, integrated 6 ldhA gene copies, and intracellular LDH activity was 9.2 × 10(-2) U/mg. At a final pH of 1.6, lactic acid titer reached 13.1 g/L (conversion yield: 26%, w/w) at 138 h in glucose-ammonium medium. This extreme pH drop was subsequently prevented by switching nitrogen source from ammonium sulfate to Na-nitrate, leading to a final pH of 3 and a lactic acid titer of 17.7 g/L (conversion yield: 47%, w/w) at 90 h of culture. Final titer was further improved to 32.2 g/L of lactic acid (conversion yield: 44%, w/w) by adding 20 g/L glucose to the culture medium at 96 h. This strain was ultimately able to produce lactic acid from xylose, arabinose, starch and xylan.

Conclusion: We obtained the first Aspergillus strains able to produce large amounts of lactic acid by inserting recombinant ldhA genes from R. oryzae into a wild-type A. brasiliensis strain. pH regulation failed to significantly increase lactic acid production, but switching nitrogen source and changing culture feed enabled a 1.8-fold increase in conversion yields. The strain produced lactic acid from plant biomass. Our findings make A. brasiliensis a strong contender microorganism for low-pH acid production from various complex substrates, especially hemicellulose.
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http://dx.doi.org/10.1186/s12934-015-0249-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4425913PMC
May 2015

Fast solubilization of recalcitrant cellulosic biomass by the basidiomycete fungus Laetisaria arvalis involves successive secretion of oxidative and hydrolytic enzymes.

Biotechnol Biofuels 2014 8;7(1):143. Epub 2014 Oct 8.

INRA, UMR1163 Biotechnologie des Champignons Filamenteux, 13288 Marseille, France ; Aix-Marseille Université, Polytech Marseille, UMR1163 Biotechnologie des Champignons Filamenteux, 13288 Marseille, France.

Background: Enzymatic breakdown of lignocellulosic biomass is a known bottleneck for the production of high-value molecules and biofuels from renewable sources. Filamentous fungi are the predominant natural source of enzymes acting on lignocellulose. We describe the extraordinary cellulose-deconstructing capacity of the basidiomycete Laetisaria arvalis, a soil-inhabiting fungus.

Results: The L. arvalis strain displayed the capacity to grow on wheat straw as the sole carbon source and to fully digest cellulose filter paper. The cellulolytic activity exhibited in the secretomes of L. arvalis was up to 7.5 times higher than that of a reference Trichoderma reesei industrial strain, resulting in a significant improvement of the glucose release from steam-exploded wheat straw. Global transcriptome and secretome analyses revealed that L. arvalis produces a unique repertoire of carbohydrate-active enzymes in the fungal taxa, including a complete set of enzymes acting on cellulose. Temporal analyses of secretomes indicated that the unusual degradation efficiency of L. arvalis relies on its early response to the carbon source, and on the finely tuned sequential secretion of several lytic polysaccharide monooxygenases and hydrolytic enzymes targeting cellulose.

Conclusions: The present study illustrates the adaptation of a litter-rot fungus to the rapid breakdown of recalcitrant plant biomass. The cellulolytic capabilities of this basidiomycete fungus result from the rapid, selective and successive secretion of oxidative and hydrolytic enzymes. These enzymes expressed at critical times during biomass degradation may inspire the design of improved enzyme cocktails for the conversion of plant cell wall resources into fermentable sugars.
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http://dx.doi.org/10.1186/s13068-014-0143-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4197297PMC
October 2014

A role for LATERAL ORGAN BOUNDARIES-DOMAIN 16 during the interaction Arabidopsis-Meloidogyne spp. provides a molecular link between lateral root and root-knot nematode feeding site development.

New Phytol 2014 Jul 6;203(2):632-45. Epub 2014 May 6.

Facultad de Ciencias Ambientales y Bioquímica, Universidad de Castilla-La Mancha, Av. Carlos III s/n, E-45071, Toledo, Spain.

Plant endoparasitic nematodes induce the formation of their feeding cells by injecting effectors from the esophageal glands into root cells. Although vascular cylinder cells seem to be involved in the formation of root-knot nematode (RKN) feeding structures, molecular evidence is scarce. We address the role during gall development of LATERAL ORGAN BOUNDARIES-DOMAIN 16 (LBD16), a key component of the auxin pathway leading to the divisions in the xylem pole pericycle (XPP) for lateral root (LR) formation. Arabidopsis T-DNA tagged J0192 and J0121 XPP marker lines, LBD16 and DR5::GUS promoter lines, and isolated J0192 protoplasts were assayed for nematode-dependent gene expression. Infection tests in LBD16 knock-out lines were used for functional analysis. J0192 and J0121 lines were activated in early developing galls and giant cells (GCs), resembling the pattern of the G2/M-transition specific ProC yc B 1;1 :CycB1;1(NT)-GUS line. LBD16 was regulated by auxins in galls as in LRs, and induced by RKN secretions. LBD16 loss of function mutants and a transgenic line with defective XPP cells showed a significantly reduced infection rate. The results show that genes expressed in the dividing XPP, particularly LBD16, are important for gall formation, as they are for LR development.
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http://dx.doi.org/10.1111/nph.12826DOI Listing
July 2014

Identification of novel target genes for safer and more specific control of root-knot nematodes from a pan-genome mining.

PLoS Pathog 2013 Oct 31;9(10):e1003745. Epub 2013 Oct 31.

INRA, UMR 1355 ISA, Institut Sophia Agrobiotech, Sophia-Antipolis, France ; CNRS, UMR 7254 ISA, Institut Sophia Agrobiotech, Sophia-Antipolis, France ; Université de Nice Sophia-Antipolis, UMR ISA, Institut Sophia Agrobiotech, Sophia-Antipolis, France.

Root-knot nematodes are globally the most aggressive and damaging plant-parasitic nematodes. Chemical nematicides have so far constituted the most efficient control measures against these agricultural pests. Because of their toxicity for the environment and danger for human health, these nematicides have now been banned from use. Consequently, new and more specific control means, safe for the environment and human health, are urgently needed to avoid worldwide proliferation of these devastating plant-parasites. Mining the genomes of root-knot nematodes through an evolutionary and comparative genomics approach, we identified and analyzed 15,952 nematode genes conserved in genomes of plant-damaging species but absent from non target genomes of chordates, plants, annelids, insect pollinators and mollusks. Functional annotation of the corresponding proteins revealed a relative abundance of putative transcription factors in this parasite-specific set compared to whole proteomes of root-knot nematodes. This may point to important and specific regulators of genes involved in parasitism. Because these nematodes are known to secrete effector proteins in planta, essential for parasitism, we searched and identified 993 such effector-like proteins absent from non-target species. Aiming at identifying novel targets for the development of future control methods, we biologically tested the effect of inactivation of the corresponding genes through RNA interference. A total of 15 novel effector-like proteins and one putative transcription factor compatible with the design of siRNAs were present as non-redundant genes and had transcriptional support in the model root-knot nematode Meloidogyne incognita. Infestation assays with siRNA-treated M. incognita on tomato plants showed significant and reproducible reduction of the infestation for 12 of the 16 tested genes compared to control nematodes. These 12 novel genes, showing efficient reduction of parasitism when silenced, constitute promising targets for the development of more specific and safer control means.
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http://dx.doi.org/10.1371/journal.ppat.1003745DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3814813PMC
October 2013

Effectors of root sedentary nematodes target diverse plant cell compartments to manipulate plant functions and promote infection.

Plant Signal Behav 2013 Sep 1;8(9). Epub 2013 Jul 1.

Cell and Molecular Sciences; The James Hutton Institute; Dundee, UK.

Sedentary plant-parasitic nematodes maintain a biotrophic relationship with their hosts over a period of several weeks and induce the differentiation of root cells into specialized feeding cells. Nematode effectors, which are synthesized in the esophageal glands and injected into the plant tissue through the syringe-like stylet, play a central role in these processes. Previous work on nematode effectors has shown that the apoplasm is targeted during invasion of the host while the cytoplasm is targeted during the induction and the maintenance of the feeding site. A large number of candidate effectors potentially secreted by the nematode into the plant tissues to promote infection have now been identified. This work has shown that the targeting and the role of effectors are more complex than previously thought. This review will not cover the prolific recent findings in nematode effector function but will instead focus on recent selected examples that illustrate the variety of plant cell compartments that effectors are addressed to in order reach their plant targets.
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http://dx.doi.org/10.4161/psb.25507DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4002590PMC
September 2013

Lateral gene transfers have polished animal genomes: lessons from nematodes.

Front Cell Infect Microbiol 2012 6;2:27. Epub 2012 Mar 6.

Institut National de la Recherche Agronomique, UMR 1355 ISA, 400 route des Chappes Sophia-Antipolis, France. etienne.danchin@ sophia.inra.fr

It is now accepted that lateral gene transfers (LGT), have significantly contributed to the composition of bacterial genomes. The amplitude of the phenomenon is considered so high in prokaryotes that it challenges the traditional view of a binary hierarchical tree of life to correctly represent the evolutionary history of species. Given the plethora of transfers between prokaryotes, it is currently impossible to infer the last common ancestral gene set for any extant species. For this ensemble of reasons, it has been proposed that the Darwinian binary tree of life may be inappropriate to correctly reflect the actual relations between species, at least in prokaryotes. In contrast, the contribution of LGT to the composition of animal genomes is less documented. In the light of recent analyses that reported series of LGT events in nematodes, we discuss the importance of this phenomenon in the evolutionary history and in the current composition of an animal genome. Far from being neutral, it appears that besides having contributed to nematode genome contents, LGT have favored the emergence of important traits such as plant-parasitism.
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http://dx.doi.org/10.3389/fcimb.2012.00027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3417587PMC
December 2013

An immunocytochemical procedure for protein localization in various nematode life stages combined with plant tissues using methylacrylate-embedded specimens.

Phytopathology 2012 Oct;102(10):990-6

Universite de Nice-Sophia Antipolis, Sophia-Antipolis, France.

Plant-parasitic nematodes possess a large number of proteins that are secreted in planta, allowing them to be successful parasites of plants. The majority of these proteins are synthesized mainly in the nematode subventral and dorsal glands as well as in other organs. To improve the immunovisualization of these proteins, we adapted a methacrylate embedding method for the localization of proteins inside nematode tissues, and extracellularly when secreted in planta or within plant cells. An important advantage is that the method is applicable for all nematode stages: preparasitic as well as parasitic stages, including large mature females. Herein, the method has been successfully applied for the localization of four nematode secreted proteins, such as Mi-MAP-1, Mi-CBM2-bearing proteins, Mi-PEL3, and Mi-6D4. In addition, we could also localize 14-3-3 proteins, as well as two cytoskeletal proteins, by double-immunolabeling on preparasitic juveniles. Superior preservation of nematode and plant morphology, allowed more accurate protein localization as compared with other methods. Besides excellent epitope preservation, dissolution of methacrylate from tissue sections unmasks target proteins and thereby drastically increases antibody access.
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http://dx.doi.org/10.1094/PHYTO-02-12-0031-RDOI Listing
October 2012

A root-knot nematode-secreted protein is injected into giant cells and targeted to the nuclei.

New Phytol 2012 Jun 27;194(4):924-31. Epub 2012 Apr 27.

INRA UMR 1301, CNRS UMR 6243, Université de Nice Sophia Antipolis, 400 route des Chappes, F-06903 Sophia-Antipolis, France INRA, Université Aix-Marseille, UMR1163 Biotechnologie des Champignons Filamenteux, F-13288 Marseille, France.

Root-knot nematodes (RKNs) are obligate endoparasites that maintain a biotrophic relationship with their hosts over a period of several weeks and induce the differentiation of root cells into specialized feeding cells. Nematode effectors synthesized in the oesophageal glands and injected into the plant tissue through the syringe-like stylet certainly play a central role in these processes. In a search for nematode effectors, we used comparative genomics on expressed sequence tag (EST) datasets to identify Meloidogyne incognita genes encoding proteins potentially secreted upon the early steps of infection. We identified three genes specifically expressed in the oesophageal glands of parasitic juveniles that encode predicted secreted proteins. One of these genes, Mi-EFF1 is a pioneer gene that has no similarity in databases and a predicted nuclear localization signal. We demonstrate that RKNs secrete Mi-EFF1 within the feeding site and show Mi-EFF1 targeting to the nuclei of the feeding cells. RKNs were previously shown to secrete proteins in the apoplasm of infected tissues. Our results show that nematodes sedentarily established at the feeding site also deliver proteins within plant cells through their stylet. The protein Mi-EFF1 injected within the feeding cells is targeted at the nuclei where it may manipulate nuclear functions of the host cell.
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http://dx.doi.org/10.1111/j.1469-8137.2012.04164.xDOI Listing
June 2012

siRNAs Trigger Efficient Silencing of a Parasitism Gene in Plant Parasitic Root-Knot Nematodes.

Genes (Basel) 2012 Jul 10;3(3):391-408. Epub 2012 Jul 10.

INRA, UMR 1355 Institut Sophia Agrobiotech, Interactions Plantes-Nematodes, Sophia Antipolis F-06903, France.

Expanding genomic data on plant pathogens open new perspectives for the development of specific and environment friendly pest management strategies based on the inhibition of parasitism genes that are essential for the success of infection. Identifying such genes relies on accurate reverse genetics tools and the screening of pathogen knock-down phenotypes. Root-knot nematodes are major cosmopolitan crop pests that feed on a wide range of host plants. Small interfering RNAs (siRNAs) would provide a powerful tool for reverse genetics of nematode parasitism genes provided that they could (1) target genes expressed in inner tissues of infective nematodes and (2) target genes expressed during parasitism. In this study, we show that siRNAs can access inner tissues of the infective juveniles during soaking and accumulate in the esophagus, amphidial pouches and related neurons of the nematode. We provide evidence that siRNAs can trigger knock-down of the parasitism gene Mi-CRT, a calreticulin gene expressed in the esophageal glands of Meloidogyne incognita. Mi-CRT knock-down in infective juveniles affected nematode virulence. However, Mi-CRT knock-down was not persistent after plant infection, indicating that siRNA-mediated RNAi is best suited for functional analysis of genes involved in pre-parasitic stages or in the early steps of infection.
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http://dx.doi.org/10.3390/genes3030391DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3899990PMC
July 2012

Proteins secreted by root-knot nematodes accumulate in the extracellular compartment during root infection.

Plant Signal Behav 2011 Aug 1;6(8):1232-4. Epub 2011 Aug 1.

INRA UMR 1301, CNRS UMR 6243, UNSA, Sophia-Antipolis, France.

Root-knot nematodes are biotrophic parasites that invade the root apex of host plants and migrate towards the vascular cylinder where they induce the differentiation of root cells into hypertrophied multinucleated giant cells. Giant cells are part of the permanent feeding site required for nematode development into the adult stage. To date, a repertoire of candidate effectors potentially secreted by the nematode into the plant tissues to promote infection has been identified. However, the precise role of these candidate effectors during root invasion or during giant cell induction and maintenance remains largely unknown. Primarily, the identification of the destination of nematode effectors within plant cell compartment(s) is crucial to decipher their actual functions. We analysed the fine localization in root tissues of five nematode effectors throughout the migratory and sedentary phases of parasitism using an adapted immunocytochemical method that preserves host and pathogen tissues.  We showed that secretion of effectors from the amphids or the oesophageal glands is tightly regulated during the course of infection. The analysed effectors accumulated in the root tissues along the nematode migratory path and along the cell wall of giant cells, showing the apoplasm as an important destination compartment for these effectors during migration and feeding cell formation.
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http://dx.doi.org/10.4161/psb.6.8.16290DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3260731PMC
August 2011

Targeting protein-protein interactions for parasite control.

PLoS One 2011 Apr 27;6(4):e18381. Epub 2011 Apr 27.

Department of Genetics, The Genome Center, Washington University School of Medicine, St. Louis, Missouri, United States of America.

Finding new drug targets for pathogenic infections would be of great utility for humanity, as there is a large need to develop new drugs to fight infections due to the developing resistance and side effects of current treatments. Current drug targets for pathogen infections involve only a single protein. However, proteins rarely act in isolation, and the majority of biological processes occur via interactions with other proteins, so protein-protein interactions (PPIs) offer a realm of unexplored potential drug targets and are thought to be the next-generation of drug targets. Parasitic worms were chosen for this study because they have deleterious effects on human health, livestock, and plants, costing society billions of dollars annually and many sequenced genomes are available. In this study, we present a computational approach that utilizes whole genomes of 6 parasitic and 1 free-living worm species and 2 hosts. The species were placed in orthologous groups, then binned in species-specific orthologous groups. Proteins that are essential and conserved among species that span a phyla are of greatest value, as they provide foundations for developing broad-control strategies. Two PPI databases were used to find PPIs within the species specific bins. PPIs with unique helminth proteins and helminth proteins with unique features relative to the host, such as indels, were prioritized as drug targets. The PPIs were scored based on RNAi phenotype and homology to the PDB (Protein DataBank). EST data for the various life stages, GO annotation, and druggability were also taken into consideration. Several PPIs emerged from this study as potential drug targets. A few interactions were supported by co-localization of expression in M. incognita (plant parasite) and B. malayi (H. sapiens parasite), which have extremely different modes of parasitism. As more genomes of pathogens are sequenced and PPI databases expanded, this methodology will become increasingly applicable.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0018381PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3083401PMC
April 2011

The Ma gene for complete-spectrum resistance to Meloidogyne species in Prunus is a TNL with a huge repeated C-terminal post-LRR region.

Plant Physiol 2011 Jun 11;156(2):779-92. Epub 2011 Apr 11.

INRA, UMR Interactions Biotiques et Santé Végétale INRA 1301, CNRS, UMR CNRS 6243, and Université de Nice Sophia-Antipolis, F-06903 Sophia-Antipolis, France.

Root-knot nematode (RKN) Meloidogyne species are major polyphagous pests of most crops worldwide, and cultivars with durable resistance are urgently needed because of nematicide bans. The Ma gene from the Myrobalan plum (Prunus cerasifera) confers complete-spectrum, heat-stable, and high-level resistance to RKN, which is remarkable in comparison with the Mi-1 gene from tomato (Solanum lycopersicum), the sole RKN resistance gene cloned. We report here the positional cloning and the functional validation of the Ma locus present at the heterozygous state in the P.2175 accession. High-resolution mapping totaling over 3,000 segregants reduced the Ma locus interval to a 32-kb cluster of three Toll/Interleukin1 Receptor-Nucleotide Binding Site-Leucine-Rich Repeat (LRR) genes (TNL1-TNL3), including a pseudogene (TNL2) and a truncated gene (TNL3). The sole complete gene in this interval (TNL1) was validated as Ma, as it conferred the same complete-spectrum and high-level resistance (as in P.2175) using its genomic sequence and native promoter region in Agrobacterium rhizogenes-transformed hairy roots and composite plants. The full-length cDNA (2,048 amino acids) of Ma is the longest of all Resistance genes cloned to date. Its TNL structure is completed by a huge post-LRR (PL) sequence (1,088 amino acids) comprising five repeated carboxyl-terminal PL exons with two conserved motifs. The amino-terminal region (213 amino acids) of the LRR exon is conserved between alleles and contrasts with the high interallelic polymorphisms of its distal region (111 amino acids) and of PL domains. The Ma gene highlights the importance of these uncharacterized PL domains, which may be involved in pathogen recognition through the decoy hypothesis or in nuclear signaling.
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http://dx.doi.org/10.1104/pp.111.176230DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3177275PMC
June 2011

Agrobacterium rhizogenes-mediated transformation of Prunus as an alternative for gene functional analysis in hairy-roots and composite plants.

Plant Cell Rep 2011 Jul 17;30(7):1313-26. Epub 2011 Mar 17.

Institut National de la Recherche Agronomique, UMR 1301, Sophia-Antipolis, France.

Resistant rootstocks offer an alternative to pesticides for the control of soil pests. In Prunus spp., resistance loci to root-knot nematodes (RKN) have been mapped and a transformation method is needed to validate candidate genes. Our efforts have focused on the generation of transformed hairy-roots and composite plants appropriate for nematode infection assays. An efficient and reliable method using the A4R strain of Agrobacterium rhizogenes for the transformation of Prunus roots with an Egfp reporter gene is given. The rooting efficiency, depending on the genotypes, was maximal for the interspecific hybrid 253 (Myrobalan plum × almond-peach), susceptible to RKN, that was retained for subsequent studies. From the agro-inoculated cuttings, 72% produced roots, mainly at the basal section of the stem. Transformed roots were screened by microscope detection of Egfp fluorescence and molecular analyses of the integration of the transgene. The absence of residual agrobacteria in the plants was checked by the non-amplification of the chromosomal gene chvH. Egfp was expressed visually in 76% of the rooted plants. Isolated hairy roots in Petri dishes and composite plants (transformed roots and non-transformed aerial part) in soil containers were inoculated with the RKN Meloidogyne incognita. In both cases, root transformation did not affect the ability of the nematodes to develop in the root tissues. Our results showed that isolated hairy-roots can be used to validate candidate genes and the conditions in which composite plants offer a complementary system for studying the function of root genes in physiological conditions of whole plants are discussed.
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http://dx.doi.org/10.1007/s00299-011-1043-9DOI Listing
July 2011

Identifying discriminative classification-based motifs in biological sequences.

Bioinformatics 2011 May 3;27(9):1231-8. Epub 2011 Mar 3.

Katholieke Universiteit Leuven, Department of Computer Science, Celestijnenlaan 200A, Leuven, Belgium.

Motivation: Identification of conserved motifs in biological sequences is crucial to unveil common shared functions. Many tools exist for motif identification, including some that allow degenerate positions with multiple possible nucleotides or amino acids. Most efficient methods available today search conserved motifs in a set of sequences, but do not check for their specificity regarding to a set of negative sequences.

Results: We present a tool to identify degenerate motifs, based on a given classification of amino acids according to their physico-chemical properties. It returns the top K motifs that are most frequent in a positive set of sequences involved in a biological process of interest, and absent from a negative set. Thus, our method discovers discriminative motifs in biological sequences that may be used to identify new sequences involved in the same process. We used this tool to identify candidate effector proteins secreted into plant tissues by the root knot nematode Meloidogyne incognita. Our tool identified a series of motifs specifically present in a positive set of known effectors while totally absent from a negative set of evolutionarily conserved housekeeping proteins. Scanning the proteome of M. incognita, we detected 2579 proteins that contain these specific motifs and can be considered as new putative effectors.

Availability And Implementation: The motif discovery tool and the proteins used in the experiments are available at http://dtai.cs.kuleuven.be/ml/systems/merci.
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http://dx.doi.org/10.1093/bioinformatics/btr110DOI Listing
May 2011