Thesis (MScBosb)--University of Stellenbosch, 2007. / Bibliography. Also available via the Internet.

Thesis (Ph.D.)--University of Western Sydney, 2007. / A thesis submitted to the University of Western Sydney, College of Health and Science, Centre for Plant and Food Science, in fulfilment of the requirements for the degree of Doctor of Philosophy. Includes bibliographical references.

Dans les sols, les champignons ectomycorhiziens (ECM) forment une symbiose très répandue avec les racines des arbres et contribuent ainsi à leur croissance et à leur santé. Des études antérieures ont montré que certaines bactéries pouvaient influencer positivement la symbiose entre les ECM et les arbres, appelées BAM pour Bactéries Auxiliaires de la Mycorhization. Les mécanismes de l’effet auxiliaire des BAM sont encore peu connus. En amont de cette thèse, il avait été montré in vitro que la BAM Pseudomonas fluorescens BBc6 formait des structures similaires à des biofilms sur les hyphes de l'ECM Laccaria bicolor. Dans ce contexte, afin d'enrichir les connaissances concernant les interactions entre les ECM et les BAM, cette thèse a porté sur l'interaction physique entre ces deux organismes. L'étude a en partie été réalisée via une méthode d'analyse par microscopie confocale, développée durant cette thèse. Les résultats obtenus ont montré que cette bactérie formait des biofilms localisés préférentiellement sur la région apicale des colonies de l'ECM ce qui pourrait indiquer une interaction trophique. L'existence d'une telle interaction a d'ailleurs par la suite été confirmée. Les résultats ont également montré que l'interaction physique entre Laccaria bicolor et BBc6 n'est pas spécifique puisque l'ensemble des treize autres souches bactériennes testées a formé des biofilms sur les hyphes de Laccaria bicolor. En revanche, BBc6 s'est montrée incapable de former des biofilms sur certains champignons appartenant aux Ascomycètes, suggérant des mécanismes d'inhibition. De plus, l'étude de la matrice des biofilms formés par BBc6 a révélé la présence de réseaux de filaments constitués d'ADN qui semblent structurer ces biofilms et qui ont aussi été observés chez l'ensemble des souches bactériennes testées. Ces résultats révèlent un rôle structural de la molécule d'ADN qui, bien qu'il semble répandu, n'a que peu été reporté jusqu'à présent. Enfin, il a été montré que des mutants de BBc6 qui ont perdu leur effet auxiliaire forment des biofilms différents de la souche sauvage sur une surface abiotique suggérant un lien potentiel entre l'effet auxiliaire et la formation de biofilms / In soil ecosystems, ectomycorrhizal fungi (ECM) form a widespread symbiosis with roots of trees, contributing to tree growth and health. It has been shown that some bacteria, called mycorrhization helper bacteria (MHB), stimulate mycorrhizal symbiosis. The mechanisms of this helper effect are poorly understood. Previous studies have shown that the MHB Pseudomonas fluorescens BBc6 formed biofilm-like structures around the hyphae of the ECM Laccaria bicolor during their in vitro interaction. In this context, in order to increase knowledge concerning MHB/ECM interactions, the work presented here focuses on the physical interaction between these two organisms. To this purpose, a method of analysis based on confocal microscopy was developed. The results showed that the bacteria formed biofilms preferentially localized on the apical region of the ECM colonies, which could indicate a trophic interaction. Such an interaction has been subsequently confirmed. The results also showed that the physical interaction between L. bicolor and BBc6 is not specific since all thirteen other bacterial strains tested formed biofilms on the hyphae of L. bicolor. On the other hand, BBc6 was unable to form biofilms on some fungi belonging to Ascomycetes, suggesting the existence of inhibition mechanisms. Moreover, the study of the BBc6 biofilm matrix revealed networks of DNA-containing filaments which seem to structure these biofilms and which have also been observed in all the bacterial strains tested. These results reveal a structural role of the DNA molecule, a role that has been rarely reported so far despite its probable high occurrence. Finally, it has been shown that BBc6 mutants having lost their helper effect presented a modified phenotype concerning their biofilm formation on abiotic surface, suggesting a potential link between the helper effect and the biofilms formation

La Truffe noire (Tuber melanosporum Vittad.) est un champignon ectomycorhizien spontanément présent dans les groupements végétaux ouverts en cours de reforestation. Alors que ces milieux ont fortement régressé au XXème siècle dans la zone méditerranéenne, 80 % de la production actuelle provient de boisements artificiels où des arbres inoculés par la Truffe sont plantés. Malgré un grand corpus de connaissances empiriques, la production reste souvent aléatoire et les connaissances fondamentales d'écologie et de biologie de la Truffe restent fragmentaires. Dans ce travail, nous avons d'abord étudié la distribution de la diversité fongique ectomycorhizienne sur les différents hôtes présents dans les garrigues pré-forestières à Truffe. Nous avons ensuite testé les interactions entre la Truffe et les plantes endo- ou non-mycorhiziennes, qui se matérialisent par la création d'un brûlé, où la flore est localement affectée. Dans un troisième volet, nous avons cherché à mieux comprendre la diversité génétique des populations de Truffes, et plus spécifiquement l'appariement sexuel et la dispersion de cette espèce à vie végétative haploïde et à fructification hypogée. Par une approche comparative entre truffières plantées et spontanées, nous avons finalement évalué les modifications liées au processus de proto-domestication en cours. Ainsi, en combinant écologie des communautés, expériences en conditions contrôlées et génétique des populations, nous avons montré qu'en région méditerranéenne :1) La Truffe est présente de façon fugace dans des communautés ectomycorhiziennes riches, avec de nombreuses espèces multi-hôtes, mais où la Truffe montre une préférence d'hôte marquée pour le chêne vert (Q. ilex). 2) Certaines plantes endo- ou non-mycorhiziennes, dont l'effet positif sur la Truffe a empiriquement été observé par les trufficulteurs, favorisent le développement du mycélium de Truffe dans le sol, agissant indirectement sur les interactions plante-plante (chêne – plantes endomycorhiziennes). L'effet inhibiteur de la Truffe observé sur la germination des graines peut-être une des causes précoces du brûlé. Par ailleurs, la Truffe semble effectivement coloniser les racines de plantes herbacées non-ectomycorhiziennes.3) Les flux de gènes sont limités à l'échelle de la truffière, l'appariement sexuel réunit des individus proches génétiquement et physiquement, et bien que la Truffe soit probablement hermaphrodite, les parents paternels sont peu détectables, probablement de taille plus réduite que les parents maternels (formant la gléba). Les pratiques culturales pourraient entraîner un brassage génétique plus important en plantation, mais à l'échelle régionale, aucune différence de diversité génétique n'a été détectée entre populations spontanées et cultivées. Ce travail montre la richesse des interactions biotiques impliquant la Truffe et les diversités végétale et fongique des truffières artificielles et spontanées de la région méditerranéenne. Les résultats acquis contribuent à lever le voile sur sa biologie reproductive, et jalonnent le chemin pour des pratiques intégratrices de la diversité biologique des truffières, ainsi que pour le développement de futures expérimentations in situ. Mots clés : appariement sexuel, Arbutus unedo, Cistus albidus, domestication, écologie des communautés, forêts méditerranéennes, génétique des populations, isolement par la distance, ITS, microsatellites, mésocosmes, mycorhizes, pratiques empiriques, qPCR, Quercus coccifera, Quercus ilex, successions secondaires / The Black Truffle (Tuber melanosporum Vittad.) is an ectomycorrhizal fungus spontaneously growing in open woodlands before canopy closure. Such open landscapes drastically regressed during the last century in the Mediterranean regions, and nowadays 80% of the production comes from man-made plantations where the Truffle is inoculated. Despite a large corpus of local knowledge and empirical practices, the production remains largely sporadic and unpredictable, and our knowledge of the biology and ecology of the Truffle is still fragmentary. In this work, we first analyzed the distribution of the ectomycorrhizal fungal diversity among host plants co-existing in the shrub-dominated landscapes where Truffle naturally occurs. We then analyzed the interactions between the Truffle and endo- or non-mycorrhizal plants, as they typically occur in the so-called brûlés, zones with scarce vegetation. A third part aimed at better understanding the genetic diversity of Truffle populations, with special focus on fertilization and dispersal process of this fungus with haploid lifecycle and hypogeous fruiting. In a multi-scale approach combining community ecology, experimentation and population genetics, we found that in the Mediterranean region:1) The Truffle is transiently present in rich ectomycorrhizal communities, showing a significant host preference for Q. ilex, in assemblies made of numerous multi-host fungal species. 2) Some endo- or non-mycorrhizal plants species, that were supposed to provide beneficial effect on the Truffle, can be experimentally shown to stimulate the development of T. melanosporum mycelium in soil, and indirectly trigger plant-plant interactions (between oak and endomycorrhizal plants). The early brûlé symptoms could at least partially result from a Truffle's inhibitor effect on seed germination.3) At truffle ground scale, gene flow is limited, and mating occurs between genetically and physically close parents. Despite probable hermaphroditism of the Truffle, paternal parents are poorly detectable, certainly of smaller size than maternal ones (these forming nourishing tissue of the ascocarp). Cultural practices could favor genetic mixing/diversity at brûlé scale, but at the regional scale, no difference in genetic diversity was found between spontaneous and planted compartments.This work revealed the richness of biotic interactions involving the black Truffle and the plant as well as the fungal diversity in both artificial and spontaneous truffle-ground of the Mediterranean region. These results enlighten the reproductive biology of the species, and pave the way for practices integrating the biological diversity of truffle-grounds and the development of further in situ experimentations. Key words: Arbutus unedo, Cistus albidus, community ecology, domestication, empirical practices, local knowledge, experimental approach, inbreeding, isolation by distance, ITS, Mediterranean forests, mesocosms, microsatellites, mycorrhiza, population genetics, qPCR, Quercus ilex, Q. coccifera, secondary successions.

Ectomycorrhizal (ECM) associations are important elements of forest biomes, connecting and transferring nutrients through an intricate and complex system of hyphal networks, ensuring plants of the nutrients they require, in nutrient poor soil. ECM research and particularly investigations into the diversity of the fungal partners has not received much attention in South Africa, hindering the advance of research in this field. This has been attributed to the difficulty of identifying the mycobionts involved in the symbiosis. The objectives of this study were to examine the ECM fungal diversity associating with Pinus patula, in selected forest plantations in Mpumalanga, South Africa. Both morphological and molecular techniques were used to identify specimens of both sporocarp collections and ECM root tip morphotypes. Morphological analysis of the ECM root tips involved characterisation of root morphology such as colour, branching and texture, and anatomical analysis examined hyphal arrangement in the root mantle and rhizomorphs. Molecular analysis involved sequencing of the Internal Transcribed Spacer (ITS) region and comparative BLAST analysis. Twenty-four sporocarp species were identified from 13 genera, namely: Amanita, Boletus, Clavulina, Inocybe, Lactarius, Rhizopogon, Russula, Scleroderma, Suillus, Tricholoma, Thelephora, Tomentella and Xerocomus. ECM root tip analysis led to the characterisation of 7 wild-type morphotypes identified as an Albatrellus sp., 2 Amanita species, a Rhizopogon sp., Thelephora terrestris, a Tomentella sp. and Scleroderma citrinum. A secondary objective was to determine whether fertilisation treatments within the study sites were responsible for differences in fungal species community structure. No evidence of a change in species diversity or shift in species composition was encountered. It is envisaged that these comprehensive ECM descriptions will be used as reference material to stimulate continued research in this field in South Africa.

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The contamination of soil for copper had refusal contribute to plants establishment in mining and vineyards areas of South Brazil. Mycorrhyzal forestry species could be one alternative to viable the development of plants in contaminated areas for heavy metals. The work was developed at Soil Department and Botanical Garden, in Federal University of Santa Maria; FEPAGRO- FLORESATA, Santa Maria-RS and Camaquã Mines, RS. Firstly, it was evaluated the mycorrhizal occurrence in forestry species like Luehea divricata Mart. et Zucc., Parapiptadenia rigida (Benth) Brenan, Peltophorum dubium (Sprengel) Tauber., Cedrela fissilis Vell., Eugenia involucrata Dc., Fícus puschnathiana (Miq.) Miq., Apuleia Ieiocarpa (J. Vogel) J.F. Macbr., Inga marginata Willd., Tabebuia Alba (Cham.) Sandwith, Tabebuia heptaphylla (Vell.) Toledo, Enterolobium contortisiliquum (Vell.) Morong., Tipuana tipu (Benth.) O. Kuntze and Phytolacca dioica L. Roots and soil sampling were performed at Botanical Garden of Federal University of Santa Maria and in native forest on Boca do Monte, district of Santa Maria, RS. No one of studied species had found ectomycorrhizal association in natural environment conditions. Roots of Luehea divricata, Parapiptadenia rigida, Peltophorum dubium, Cedrela fissilis, Fícus puschnathiana, Ingá marginata, Tabebuia Alba, Tabebuia heptaphylla, Enterolobium contortisiliquum, Tipuana tipu and Phytolacca dioica had arbuscular mycorrhizal. An experiment in liquid and solid culture medium was conducted to select copper tolerant ectomycorrhizal fungi. The fungi tested in solid medium were: Pisolitus microcarpus UFSC-Pt116, Suillus sp. UFSM RA2.8, Suillus sp. UFSM RA118, Scleroderma sp. UFSM SC3.6 and Scleroderma sp. UFSC-Sc124. In liquid culture medium were tested the species: Pisolitus microcarpus UFSC-Pt116, Pisolitus tinctorius UFSC-Pt24, Scleroderma sp. UFSC-Sc124, UFSM MC and UFSM RA2.8. The fungi UFSC-Pt116 showed copper tolerance and produced high amount of fungi mycelium. Latter, it was characterized ectomycorrizal on Parapiptadenia rigida, Peltophorum dubium, Apuleia Ieiocarpa and Enterolobium contortisiliquum seedlings in vitro conditions, cultivated and previously inoculated with the ectomycorrhizal fungi UFSC-Pt116. Seedlings of Parapiptadenia rigida, Peltophorum dubium and Enterolobium contortisiliquum was formed association with UFSC Pt 116. The region of transcribed internal space was studied and evaluated the differences in sequencing polymorphism of this region of isolated UFSC-Pt116, it was compared to other ectomycorrhizal fungi like Pisolithus, Sclerodrema, Rhizopogon and Suillus available in the GenBank data. It was confirmed the morphological identification of the isolates as belonging to the genus Pisolithus. However, with molecular results did not confirm the species of this fungus. The second part, it was evaluated the tolerance to copper for forestry species like Parapiptadenia rigida, Peltophorum dubium, Cedrela fissilis and Enterolobium contortisiliquum. Seedlings of Cedrela fissilis was evidenced copper accumulation on leaves, stem and roots. Seedlings of Enterolobium contortisiliquum was accumulated copper on roots, but the metal was not translocated to stem and leaves. It is possible to consider Parapiptadenia rigida as a cooper excluding plant. Peltophorum dubium, Parapiptadenia rigida and Enterolobium contortisiliquum was showed high tolerance to contaminated soil with copper. The thirdly part, it was evaluated the behavior Peltophorum dubium seedlings inoculated with the UFSC Pt 116 ectomycorrhizal fungi in soil with high copper concentrations. The increasing of copper doses was reduced root length, specific root surface area, height and stem diameter of Peltophorum plants. Seedlings of inoculated Peltophorum were showed less copper concentration on tissues and reduced copper absorption on more elevated doses. It was concluded that isolates UFSC-Pt116, UFSC-Pt24 and UFSC-SC124 were efficient on mycelium production in medium with high copper amounts. The isolated UFSC-Pt116 was not efficient to promote Peltophorum growth in soil with high copper concentration, but this isolated was reduced the copper absorption for plant. / A contaminação do solo por cobre tem contribuído negativamente para o estabelecimento de plantas em áreas de mineração e áreas vinícolas do sul do Brasil. Espécies florestais micorrizadas podem ser uma alternativa para viabilizar o desenvolvimento de plantas em áreas contaminadas por metais. O trabalho foi desenvolvido no Departamento de Solos, no Jardim Botânico da Universidade Federal de Santa Maria; na FEPAGRO FLORESTA, Santa Maria e nas Minas do Camaquã, Caçapava do Sul, RS. A primeira etapa objetivou avaliar a ocorrência de associação micorrízica nas espécies florestais Luehea divricata Mart. et Zucc. (açoita-cavalo), Parapiptadenia rigida (Benth) Brenan (angico-vermelho), Peltophorum dubium (Sprengel) Tauber. (canafístula), Cedrela fissilis Vell. (cedro), Eugenia involucrata Dc. (cerejeira), Fícus puschnathiana (Miq.) Miq. (figueira-do-mato), Apuleia Ieiocarpa (J. Vogel) J.F. Macbr. (grápia), Inga marginata Willd. (ingá-feijão), Tabebuia Alba (Cham.) Sandwith (ipê-ouro), Tabebuia heptaphylla (Vell.) Toledo (ipê-roxo), Enterolobium contortisiliquum (Vell.) Morong. (timbaúva), Tipuana tipu (Benth.) O. Kuntze (tipuana) e Phytolacca dioica L. (umbu). As coletas de raízes e de solo foram realizadas no Jardim Botânico da Universidade Federal de Santa Maria e em mata nativa no distrito municipal de Boca do Monte, Santa Maria, RS. Nenhuma das espécies estudadas apresentaram associação com fungos ectomicorrízicos em ambiente natural. Raízes de açoita-cavalo, angico-vermelho, canafístula, cedro, figueira, ingá-feijão, ipê-ouro, ipê roxo, timbaúva, tipuana e umbu apresentam micorrizas arbusculares. Um segundo experimento foi desenvolvido para selecionar fungos ectomicorrízicos tolerante a cobre em meio de cultura líquido e sólido. Os fungos testados no meio de cultura sólido foram: Pisolithus microcarpus UFSC-Pt116, Suillus sp. UFSM RA2.8, Suillus sp. UFSM RA118, Scleroderma sp. UFSM SC3.6 e Scleroderma sp. UFSC-Sc124. No meio de cultura líquido foram testados os fungos: Pisolithus microcarpus UFSC-Pt116, Pisolithus tinctorius UFSC-Pt24, Scleroderma sp. UFSC Sc124, o fungo UFSM MC e o UFSM RA2.8. O fungo UFSC-Pt116 apresentou tolerância ao cobre e produziu maior quantidade de massa seca do micélio fúngico. Posteriormente, caracterizou-se a formação de ectomicorrizas em plântulas de angicovermelho, canafístula, grápia e timbaúva cultivadas in vitro, inoculadas com o fungo UFSCPt116. Plântulas de angico-vermelho, canafístula e timbaúva formam associação com fungo ectomicorrízico UFSC-Pt116. Estudou-se a região do espaço interno transcrito e analisaramse as diferenças no polimorfismo da seqüência desta região no isolado UFSC-Pt116 com as seqüências dos isolados de Pisolithus, Scleroderma, Rhizopogon e Suillus do GenBank. Confirmou-se a identificação morfológica do isolado como pertencente ao gênero Pisolithus. Contudo, com os resultados moleculares não foi possível confirmar a espécie do fungo. A segunda etapa objetivou avaliar o comportamento de angico-vermelho, canafístula, cedro e timbaúva tolerantes ao cobre. Mudas de cedro acumulam cobre na parte aérea e radicular. Mudas de timbaúva acumulam cobre na raiz da planta, não o translocam para a parte aérea. O angico-vermelho pode ser considerado uma planta exclusora de cobre. Canafístula, angicovermelho e timbaúva apresentaram maior tolerância a solo contaminado por cobre. A terceira etapa objetivou avaliar o comportamento de mudas de canafístula inoculadas com fungo ectomicorrízico em solo com excesso de cobre. O fungo ectomicorrízico utilizado como inoculante foi o isolado UFSC-Pt116. O incremento das doses de cobre reduziu o comprimento radicular, área superficial específica radicular, altura e diâmetro das plantas de canafístula. Mudas de canafístula inoculadas com fungo ectomicorrízico apresentaram menor teor de cobre em seu tecido e reduziram a absorção de cobre em dose mais elevada. Concluiuse que os isolados UFSC-Pt116, UFSC-Pt24 e UFSC-Sc124 foram eficientes in vitro na produção de micélio com elevada quantidade de cobre. O isolado UFSC-Pt116 não respondeu suficientemente para favorecer o desenvolvimento de canafístula em solo com excesso de cobre. Entretanto, este isolado reduziu a absorção de cobre pela planta.

La symbiose ectomycorhizienne associe les racines d’un arbre aux hyphes d’un champignon, conduisant à un échange réciproque de nutriments entre les deux partenaires. La colonisation fongique massive du cortex racinaire est caractérisée par la formation d’une interface symbiotique, le réseau de Hartig. L’acquisition du génome du symbionte ectomycorhizien Laccaria bicolor a permis d’identifier des petites protéines prédites sécrétées, les MiSSPs (Mycorrhiza-induced Small Secreted Proteins). Mon projet de thèse avait pour objectifs la comparaison des sécrétomes, notamment les petites protéines sécrétées (SSPs), de champignons ectomycorhiziens et saprotrophes, la localisation subcellulaire in planta et l’analyse fonctionnelle de MiSSPs de L. bicolor. L’analyse bioinformatique a notamment permis de révéler des clusters de SSPs conservées entre champignons ectomycorhiziens et saprotrophes ou spécifiques de champignons ectomycorhiziens, mettant en lumière que les champignons ectomycorhiziens partagent des SSPs avec leurs ancêtres saprotrophes mais possèdent aussi d’autres SSPs spécifiques à leur mode de vie. Un jeu de MiSSPs de L. bicolor ont été localisées in planta. Trois d’entre eux ciblent spécifiquement des compartiments subcellulaires de la cellule végétale. Le motif répété DWRR présent dans la séquence de MiSSP8 est partagé par une famille de protéine fongique de champignons majoritairement saprotrophes. Ces résultats suggèrent que l’utilisation de SSPs comme moyen de communication est générique chez les champignons et démontrent aussi qu’au moins une petite protéine sécrétée requise pour la symbiose de L. bicolor a évoluée à partir de protéines de champignons saprotrophes / Roots of most trees form ectomycorrhizal (ECM) symbiosis with mutualistic soil-borne fungi, relying on a bi-directional exchange of nutrients between the two partners. Fungal colonization of cortical root cells form the Hartig net, a symbiotic interface. Functioning of this symbiotic interface is not well known. However, Laccaria bicolor genome sequencing sheds the light on upregulated small-secreted proteins, so-called MiSSPs (Mycorrhiza-Small Secreted Proteins). Several L. bicolor MiSSPs were demonstrated as symbiosis effectors. My PhD project aims to compare secretomes, in particular SSPs, of fungal with different lifestyles and pursue functional analysis of MiSSPs of L. bicolor. Based on the clustering analysis, we identified clusters of SSPs shared between saprotrophic and ECM fungi and clusters of SSPs specific to ECM-fungi. This study highlights that ECM fungi share SSPs with their saprotrophic ancestors but also possess lifestyle specific SSPs. In planta subcellular localization of a set of MiSSPs belonging to a core-regulon showed that three of them are able to target different plant subcellular compartments. Functional analysis of the symbiosis effector MiSSP8 does not lead to the identification of a putative interactor but the repetitive motif DWRR of MiSSP8 protein sequence is unique to fungi and is shared with SSPs from saprotrophic ancestors. Our results suggest the use of SSPs as mean of communication is common and generic and show at least one SSPs required for ectomycorhizal symbiosis of L. bicolor has evolved from SSPs found in saprotrophic fungi

La symbiose ectomycorhizienne se définie comme une association mutualiste entre les racines des plantes ligneuses et le mycélium de champignons du sol. Elle est majoritaire dans les écosystèmes forestiers de l'hémisphère nord et permet l'amélioration de la nutrition hydrominérale des plantes ligneuses, notamment lorsque la disponibilité en ressources se fait rare. Des données transcriptomiques et génomiques du champignon ectomycorhizien Hebeloma cylindrosporum ont permis d'identifier différents gènes codant pour des protéines capables de transporter des nutriments. L'implication éventuelle de ces systèmes de transport dans la nutrition potassique et phosphatée ectomycorhize-dépendante de la plante hôte Pinus pinaster reste à évaluer. Dans cette étude, deux gènes candidats codant pour des systèmes de transport de potassium (K+), HcTrk1 et HcSKC, et deux autres pour des transporteurs de phosphate inorganique (Pi), HcPT1.1 et HcPT2, ont été analysés. Des approches permettant la localisation dans l'ectomycorhize des transcrits (hybridation in situ) et des protéines (fusion traductionnelle) de ces candidats ont été générés. Ces différents outils ont permis de montrer que le transporteur HcTrk1 et le canal HcSKC étaient respectivement localisés dans l'ectomycorhize au niveau des sites de prélèvement et de relargage du K+. D'autre part, des lignées transgéniques d'H. cylindrosporum sur- et/ou sous-exprimant ces gènes ont été produites afin de voir si la nutrition végétale en K+ et en Pi était impactée. Ainsi, l'utilisation en mycorhization de lignées surexprimant HcTrk1 et sous-exprimant HcSKC ont montré une diminution de la nutrition potassique et de l'homéostasie du phosphore de la plante hôte. Les mêmes types d'approches ont été utilisés avec les transporteurs de Pi HcPT1.1 et HcPT2. En utilisant des lignées transgéniques, il a ainsi pu être montré que la surexpression de HcPT1.1 en condition carencée décrite précédemment était liée à l'activité de son promoteur. Quant au transporteur HcPT2, des analyses préliminaires de localisation suggèrent qu'il pourrait être impliqué dans le prélèvement de Pi du sol et dans son relargage au niveau du réseau de Hartig. Des études complémentaires restent cependant à mener. Enfin, cinq autres systèmes de transport de K+ et trois de phosphate ont été récemment identifiés à partir du génome d'H. cylindrosporum, ouvrant la voie à la dissection fine des mécanismes moléculaires régissant la nutrition potassique et phosphatée ectomycorhize dépendante de P. pinaster. / Ectomycorrhizal symbiosis is defined as a mutual association between the roots of woody plants and the mycelium of soil fungi. This symbiosis is widespread in northern forests and plays a major role in nutrient and water uptake of woody plants, especially when resources become scarce. Transcriptomic and genomic data of the ectomycorrhizal fungus Hebeloma cylindrosporum allowed the identification of several genes coding for nutrient transport proteins. Their putative involvement in ectomycorrhiza-dependent potassium and phosphate nutrition of the host plant Pinus pinaster needs to be assessed. In this study, two candidate genes coding for potassium (K+) transport systems, HcTrk1 and HcSKC, and two other genes coding for inorganic phosphate (Pi) transporters, HcPT1.1 and HcPT2, were analyzed. Molecular approaches allowing the localization of transcripts (in situ hybridization) and proteins (translational fusion) of these candidates in ectomycorrhiza were obtained. These tools allowed us to show that the HcTrk1 transporter and the HcSKC channel were localized in K+ uptake and release sites of the ectomycorrhiza, respectively. In order to know whether these proteins play a role in plant K+ and Pi nutrition, H. cylindrosporum transgenic lines with up- and/or down-regulated expression of candidate genes were produced. In mycorrhizal assays, the use of fungal strains with up- or down- regulated expression of HcTrk1 and HcSKC, respectively, affects the K+ nutrition and phosphorus homeostasis of the host plant. The same approaches were used for HcPT1.1 and HcPT2 Pi transporters. Therefore, using transgenic strategies, we demonstrated that the previously shown up-regulation of HcPT1.1 expression under Pi shortage is related to its promoter activity. Concerning HcPT2, preliminary localization analysis suggested that this transporter might be involved in Pi uptake from soil and in release in the Hartig net. However, complementary studies are needed. Five and three novel K+ and Pi transport systems, respectively, were identified from the recent genome accession of H. cylindrosporum, opening the way to a fine dissection of molecular mechanisms controlling the ectomycorrhiza-dependent K+ and phosphate nutrition of Pinus pinaster.