Melampsora larici-Populina est un champignon biotrophe qui infecte le peuplier et cause la maladie de la rouille foliaire, entraînant d'importants dégâts dans les peupleraies. Un des objectifs de l'UMR Interactions Arbres/Microorganismes est de caractériser les déterminants moléculaires de ce pathosystème. Au cours de cette thèse, des approches post-Génomiques ont permis de mener à bien quatre projets de recherche. Premièrement, l'analyse du transcriptome des temps précoces de l'interaction peuplier/M. larici-Populina a révélé un transporteur de sulfate de peuplier fortement induit par l'infection (chapitre II). Deuxièmement, l'analyse phylogénomique de la famille des thaumatin-Like proteins (TLP) a entre autres mis en évidence certains clades spécifiquement associés aux réponses aux stress chez le peuplier (chapitre III). Troisièmement, le gène codant la petite protéine sécrétée Risp de fonction inconnue est fortement induit lors des réponses de défense du peuplier et n'a pas d'homologue chez les autres plantes. La protéine recombinante est intrinsèquement désordonnée et présente une double activité de protéine antifongique envers M. larici-Populina et d'éliciteur endogène des réponses de défense chez le peuplier (chapitre IV et V). La combinaison de ces deux propriétés n'a jamais été rapportée chez une protéine de plante. Enfin, les gènes MlpP4.1 et MlpH1.1 de M. larici-Populina codent des petites protéines sécrétées riches en cystéines et de fonction inconnue, considérées comme des effecteurs candidats (chapitre VI). L'expression de MlpP4.1 et MlpH1.1 est très fortement induite lors de l'infection des feuilles de peupliers et des activités de virulence ont été observées chez Arabidopsis thaliana. Les analyses biochimique et structurale des protéines recombinantes sont en cours et ont déjà permis de démontrer la forte stabilité de MlpP4.1, probablement liée à la présence de plusieurs ponts disulfures. A l'aide des protéines recombinantes, plusieurs partenaires protéiques ont été identifiés chez les plantes permettant d'établir des hypothèses quant à leur rôle / Melampsora larici-Populina is a biotrophic fungus that infects poplar and causes the foliar rust disease, leading to severe damages in plantations. A major aim of the Tree- Microbe Interactions department is to characterize molecular determinants of the pathosystem. During this thesis, four research projects were achieved through post-Genomic approaches. First, transcriptome analysis of the early interaction between poplar and M. larici-Populina revealed a fungal-Induced host sulfate transporter (chapter II). Secondly, the phylogenomic analysis of the thaumatin-Like protein (TLP) family uncovered some clades specifically associated with stress responses in poplar (chapterIII). Thirdly, the gene encoding the small secreted protein of unknown function Risp is strongly induced during poplar defense reponses and has no homolog in other plants. The recombinant protein is intrinsically disordered and presents a dual activity as an antifungal protein against M. larici-Populina and as an endogenous elicitor of defense responses in poplar (chapter IV and V). The combination of both properties in a single protein has never been reported in plants. Finally, M. larici-Populina MlpP4.1 and MlpH1.1 genes encode cysteine-Rich small-Secreted proteins of unknown fonction, considered as candidate effectors (chapter VI). MlpP4.1 and MlpH1.1 expression is strongly induced during poplar leaf colonization, and virulence activities were observed in Arabidopsis thaliana. Biochemical and structural analyses of recombinant proteins are ongoing and already revealed the strong stability of MlpP4.1, likely due to the presence of several disulfide bridges. Several plant partners of the recombinant proteins were identified and have allowed for setting hypotheses about their role

A perda de eficiência do inóculo fúngico na incorporação ao solo devido ao atrito indica a necessidade de desenvolvimento de um inóculo efetivo, viável, que mantenha a atividade biológica durante transporte e aplicação no solo, e proporcione melhora da remediação do solo contaminado. O projeto visou determinar a melhor formulação de inóculo peletizado de P. castanella afim de melhorar a resistência mecânica do inóculo e garantir as taxas de atividade enzimática e degradação de poluentes em solo. Avaliou-se o uso de agar-agar, fécula de mandioca e carragena na agregação do bagaço de cana-de-açúcar do inóculo por meio de ensaio enzimático, resistência mecânica, biomassa, colonização e degradação de pentaclorofenol (PCF). Fécula de mandioca foi capaz de melhorar a resistência do inóculo, não alterou significativamente o perfil fisiológico do fungo e proporcionou ótima taxa de degradação de PCF em relação aos demais agregantes. Este processo de imobilização mostra-se promissor em relação a outros processos por ser de simples formulação e requerer menos constituintes. / The loss of efficiency of fungal inocula during soil incorporation due to friction indicates the need to develop an effective and viable inoculum, capable to maintain its biological activity during transportation and land application, and improve the remediation of contaminated soil. The project aimed to determine the best formulation of pelleted inoculum of P. castanella in order to improve its mechanical strength and guarantee enzyme activity and degradation of pollutants in soil. We evaluated the use of agar-agar, cassava starch and carrageenan on the aggregation of sugarcane bagasse by carrying enzymatic and mechanical strength assays, biomass quantification, analysis of colonization and pentachlorophenol (PCP) degradation. Cassava starch improved the inoculums mechanical strength, did not significantly alter the physiological profile of the fungus and provided a great rate of PCP degradation when compared to the other compounds. This immobilization process is promising compared to other existing due to its simple formula and for requiring fewer components.

Softwood is the predominant form of land plant biomass in the Northern hemisphere, and is among the most recalcitrant biomass resources to bioprocess technologies. The white rot fungus Phanerochaete carnosa has been isolated almost exclusively from softwoods, while most other known white-rot species, including Phanerochaete chrysosporium, were mainly isolated from hardwoods. Accordingly, it is anticipated that P. carnosa encodes a distinct set of enzymes and proteins that promote softwood decomposition. To elucidate the genetic basis of softwood bioconversion by P. carnosa, its genome was sequenced and transcriptomes were evaluated after growth on wood compared to liquid medium. Results indicate that P. carnosa differs from P. chrysosporium in the number and expression levels of genes that encode lignin peroxidase (LiP) and manganese peroxidase (MnP), two enzymes that modify lignin present in wood. P. carnosa has more genes for MnP with higher expression levels than LiP, while the reverse has been observed for P. chrysosporium. The abundances of transcripts predicted to encode lignocellulose-modifying enzymes were then measured over the course of P. carnosa cultivation on four wood species. Profiles were consistent with decay of lignin before carbohydrates. Transcripts encoding MnP were highly abundant, and those encoding MnP and LiP featured significant substrate-dependent response. Since differences in modes of lignin degradation catalyzed by MnP and LiP could affect the ability of each to degrade lignin from different types of wood, their activity on various hardwoods and softwoods were tested. Results suggest that MnP degrades softwood lignin more effectively than hardwood lignin, consistent with high levels of this enzyme in P. carnosa.

Softwood is the predominant form of land plant biomass in the Northern hemisphere, and is among the most recalcitrant biomass resources to bioprocess technologies. The white rot fungus Phanerochaete carnosa has been isolated almost exclusively from softwoods, while most other known white-rot species, including Phanerochaete chrysosporium, were mainly isolated from hardwoods. Accordingly, it is anticipated that P. carnosa encodes a distinct set of enzymes and proteins that promote softwood decomposition. To elucidate the genetic basis of softwood bioconversion by P. carnosa, its genome was sequenced and transcriptomes were evaluated after growth on wood compared to liquid medium. Results indicate that P. carnosa differs from P. chrysosporium in the number and expression levels of genes that encode lignin peroxidase (LiP) and manganese peroxidase (MnP), two enzymes that modify lignin present in wood. P. carnosa has more genes for MnP with higher expression levels than LiP, while the reverse has been observed for P. chrysosporium. The abundances of transcripts predicted to encode lignocellulose-modifying enzymes were then measured over the course of P. carnosa cultivation on four wood species. Profiles were consistent with decay of lignin before carbohydrates. Transcripts encoding MnP were highly abundant, and those encoding MnP and LiP featured significant substrate-dependent response. Since differences in modes of lignin degradation catalyzed by MnP and LiP could affect the ability of each to degrade lignin from different types of wood, their activity on various hardwoods and softwoods were tested. Results suggest that MnP degrades softwood lignin more effectively than hardwood lignin, consistent with high levels of this enzyme in P. carnosa.

A perda de eficiência do inóculo fúngico na incorporação ao solo devido ao atrito indica a necessidade de desenvolvimento de um inóculo efetivo, viável, que mantenha a atividade biológica durante transporte e aplicação no solo, e proporcione melhora da remediação do solo contaminado. O projeto visou determinar a melhor formulação de inóculo peletizado de P. castanella afim de melhorar a resistência mecânica do inóculo e garantir as taxas de atividade enzimática e degradação de poluentes em solo. Avaliou-se o uso de agar-agar, fécula de mandioca e carragena na agregação do bagaço de cana-de-açúcar do inóculo por meio de ensaio enzimático, resistência mecânica, biomassa, colonização e degradação de pentaclorofenol (PCF). Fécula de mandioca foi capaz de melhorar a resistência do inóculo, não alterou significativamente o perfil fisiológico do fungo e proporcionou ótima taxa de degradação de PCF em relação aos demais agregantes. Este processo de imobilização mostra-se promissor em relação a outros processos por ser de simples formulação e requerer menos constituintes. / The loss of efficiency of fungal inocula during soil incorporation due to friction indicates the need to develop an effective and viable inoculum, capable to maintain its biological activity during transportation and land application, and improve the remediation of contaminated soil. The project aimed to determine the best formulation of pelleted inoculum of P. castanella in order to improve its mechanical strength and guarantee enzyme activity and degradation of pollutants in soil. We evaluated the use of agar-agar, cassava starch and carrageenan on the aggregation of sugarcane bagasse by carrying enzymatic and mechanical strength assays, biomass quantification, analysis of colonization and pentachlorophenol (PCP) degradation. Cassava starch improved the inoculums mechanical strength, did not significantly alter the physiological profile of the fungus and provided a great rate of PCP degradation when compared to the other compounds. This immobilization process is promising compared to other existing due to its simple formula and for requiring fewer components.

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