Transcriptome analysis of the Populus trichocarpa-Rhizophagus irregularis Mycorrhizal Symbiosis: Regulation of Plant and Fungal Transportomes under Nitrogen Starvation

Nutrient transfer is a key feature of the arbuscular mycorrhizal (AM) symbiosis. Valuable mineral nutrients are transferred from the AM fungus to the plant, increasing its fitness and productivity, and, in exchange, the AM fungus receives carbohydrates as an energy source from the plant. Here, we analyzed the transcriptome of the Populus trichocarpa-Rhizophagus irregularis symbiosis using RNA-sequencing of non-mycorrhizal or mycorrhizal fine roots, with a focus on the effect of nitrogen (N) starvation. In R. irregularis, we identified 1,015 differentially expressed genes, whereby N starvation led to a general induction of gene expression. Genes of the functional classes of cell growth, membrane biogenesis and cell structural components were highly abundant. Interestingly, N starvation also led to a general induction of fungal transporters, indicating increased nutrient demand upon N starvation. In non-mycorrhizal P. trichocarpa roots, 1,341 genes were differentially expressed under N starvation. Among the 953 down-regulated genes in N starvation, most were involved in metabolic processes including amino acids, carbohydrate and inorganic ion transport, while the 342 up-regulated genes included many defense-related genes. Mycorrhization led to the up-regulation of 549 genes mainly involved in secondary metabolite biosynthesis and transport; only 24 genes were down-regulated. Mycorrhization specifically induced expression of three ammonium transporters and one phosphate transporter, independently of the N conditions, corroborating the hypothesis that these transporters are important for symbiotic nutrient exchange. In conclusion, our data establish a framework of gene expression in the two symbiotic partners under high-N and low-N conditions

The small secreted effector protein MiSSP7.6 of Laccaria bicolor is required for the establishment of ectomycorrhizal symbiosis

To establish and maintain a symbiotic relationship, the ectomycorrhizal fungus Laccaria bicolor releases mycorrhiza-induced small secreted proteins (MiSSPs) into host roots. Here, we have functionally characterized the MYCORRHIZA-iNDUCED SMALL SECRETED PROTEIN OF 7.6 kDa (MiSSP7.6) from L. bicolor by assessing its induced expression in ectomycorrhizae, silencing its expression by RNAi, and tracking in planta subcellular localization of its protein product. We also carried out yeast two-hybrid assays and bimolecular fluorescence complementation analysis to identify possible protein targets of the MiSSP7.6 effector in Populus roots. We showed that MiSSP7.6 expression is upregulated in ectomycorrhizal rootlets and associated extramatrical mycelium during the late stage of symbiosis development. RNAi mutants with a decreased MiSSP7.6 expression have a lower mycorrhization rate, suggesting a key role in the establishment of the symbiosis with plants. MiSSP7.6 is secreted, and it localizes both to the nuclei and cytoplasm in plant cells. MiSSP7.6 protein was shown to interact with two Populus Trihelix transcription factors. Furthermore, when coexpressed with one of the Trihelix transcription factors, MiSSP7.6 is localized to plant nuclei only. Our data suggest that MiSSP7.6 is a novel secreted symbiotic effector and is a potential determinant for ectomycorrhiza formation.Fil: Kang, Heng. Institut National de la Recherche Agronomique; FranciaFil: Chen, Xin. Institut National de la Recherche Agronomique; FranciaFil: Kemppainen, Minna Johanna. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnologia. Instituto de Microbiologia Basica y Aplicada. Laboratorio de Micologia Molecular.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Pardo, Alejandro Guillermo. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnologia. Instituto de Microbiologia Basica y Aplicada. Laboratorio de Micologia Molecular.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Veneault Fourrey, Claire. Institut National de la Recherche Agronomique; FranciaFil: Kohler, Annegret. Institut National de la Recherche Agronomique; FranciaFil: Martin, Francis M.. Institut National de la Recherche Agronomique; Franci

The small secreted effector protein MiSSP7.6 of Laccaria bicolor is required for the establishment of ectomycorrhizal symbiosis

To establish and maintain a symbiotic relationship, the ectomycorrhizal fungus Laccaria bicolor releases mycorrhiza-induced small secreted proteins (MiSSPs) into host roots. Here, we have functionally characterized the MYCORRHIZA-iNDUCED SMALL SECRETED PROTEIN OF 7.6 kDa (MiSSP7.6) from L. bicolor by assessing its induced expression in ectomycorrhizae, silencing its expression by RNAi, and tracking in planta subcellular localization of its protein product. We also carried out yeast two-hybrid assays and bimolecular fluorescence complementation analysis to identify possible protein targets of the MiSSP7.6 effector in Populus roots. We showed that MiSSP7.6 expression is upregulated in ectomycorrhizal rootlets and associated extramatrical mycelium during the late stage of symbiosis development. RNAi mutants with a decreased MiSSP7.6 expression have a lower mycorrhization rate, suggesting a key role in the establishment of the symbiosis with plants. MiSSP7.6 is secreted, and it localizes both to the nuclei and cytoplasm in plant cells. MiSSP7.6 protein was shown to interact with two Populus Trihelix transcription factors. Furthermore, when coexpressed with one of the Trihelix transcription factors, MiSSP7.6 is localized to plant nuclei only. Our data suggest that MiSSP7.6 is a novel secreted symbiotic effector and is a potential determinant for ectomycorrhiza formation.Fil: Kang, Heng. Institut National de la Recherche Agronomique; FranciaFil: Chen, Xin. Institut National de la Recherche Agronomique; FranciaFil: Kemppainen, Minna Johanna. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnologia. Instituto de Microbiologia Basica y Aplicada. Laboratorio de Micologia Molecular.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Pardo, Alejandro Guillermo. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnologia. Instituto de Microbiologia Basica y Aplicada. Laboratorio de Micologia Molecular.; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Veneault Fourrey, Claire. Institut National de la Recherche Agronomique; FranciaFil: Kohler, Annegret. Institut National de la Recherche Agronomique; FranciaFil: Martin, Francis M.. Institut National de la Recherche Agronomique; Franci

An ectomycorrhizal fungus alters sensitivity to jasmonate, salicylate, gibberellin, and ethylene in host roots.

The phytohormones jasmonate, gibberellin, salicylate, and ethylene regulate an interconnected reprogramming network integrating root development with plant responses against microbes. The establishment of mutualistic ectomycorrhizal symbiosis requires the suppression of plant defense responses against fungi as well as the modification of root architecture and cortical cell wall properties. Here, we investigated the contribution of phytohormones and their crosstalk to the ontogenesis of ectomycorrhizae (ECM) between grey poplar (Populus tremula x alba) roots and the fungus Laccaria bicolor. To obtain the hormonal blueprint of developing ECM, we quantified the concentrations of jasmonates, gibberellins, and salicylate via liquid chromatography-tandem mass spectrometry. Subsequently, we assessed root architecture, mycorrhizal morphology, and gene expression levels (RNA sequencing) in phytohormone-treated poplar lateral roots in the presence or absence of L. bicolor. Salicylic acid accumulated in mid-stage ECM. Exogenous phytohormone treatment affected the fungal colonization rate and/or frequency of Hartig net formation. Colonized lateral roots displayed diminished responsiveness to jasmonate but regulated some genes, implicated in defense and cell wall remodelling, that were specifically differentially expressed after jasmonate treatment. Responses to salicylate, gibberellin, and ethylene were enhanced in ECM. The dynamics of phytohormone accumulation and response suggest that jasmonate, gibberellin, salicylate, and ethylene signalling play multifaceted roles in poplar L. bicolor ectomycorrhizal development

The mutualism effector MiSSP7 of Laccaria bicolor alters the interactions between the poplar JAZ6 protein and its associated proteins

Despite the pivotal role of jasmonic acid in the outcome of plant-microorganism interactions, JA-signaling components in roots of perennial trees like western balsam poplar (Populus trichocarpa) are poorly characterized. Here we decipher the poplar-root JA-perception complex centered on PtJAZ6, a co-repressor of JA-signaling targeted by the effector protein MiSSP7 from the ectomycorrhizal basidiomycete Laccaria bicolor during symbiotic development. Through protein–protein interaction studies in yeast we determined the poplar root proteins interacting with PtJAZ6. Moreover, we assessed via yeast triple-hybrid how the mutualistic effector MiSSP7 reshapes the association between PtJAZ6 and its partner proteins. In the absence of the symbiotic effector, PtJAZ6 interacts with the transcription factors PtMYC2s and PtJAM1.1. In addition, PtJAZ6 interacts with it-self and with other Populus JAZ proteins. Finally, MiSSP7 strengthens the binding of PtJAZ6 to PtMYC2.1 and antagonizes PtJAZ6 homo-/heterodimerization. We conclude that a symbiotic effector secreted by a mutualistic fungus may promote the symbiotic interaction through altered dynamics of a JA-signaling-associated protein–protein interaction network, maintaining the repression of PtMYC2.1-regulated genes

Caractérisation de l'étape de pénétration des tissus végétaux via le développement de l'appressorium chez Colletotrichum lindemuthianum, champignon phytopathogène responsable de l'anthracnose du haricot

Colletotrichum lindemuthianum est un champignon phytopathogène, responsable de l'anthracnose du haricot commun, Phaseolus vulgaris, et qui différencie une structure spécialisée pour pénétrer les tissus végétaux : l'appressorium. L'analyse morphologique, biochimique, biophysique et microscopique de trois mutants de pénétration de C. lindemuthianum, a permis de montrer que la mise en place de l'appressorium chez ce champignon se décompose en trois étapes fonctionnelles: la différenciation, la maturation et l'acquisition de la fonctionnalité. De façon surprenante, l'analyse des réponses de défense élicitées chez le haricot par ces trois mutants montre que la maturation de l'appressorium, mais pas sa fonctionnalité, est nécessaire et suffisante pour provoquer la production d'ions superoxides, l'expression de gènes de défense (PAL3, CHS et PvPR2) et la sécrétion dans l'apoplasme de protéines PR. En revanche, la fonctionnalité de l'appressorium (e. g. la pénétration perse du tissu végétal) est nécessaire pour l'obtention d'une réaction hypersensible dans an moins trois relations gène à gène indépendantes. Deux approches de type "transcriptome" (hybridation de filtres moyenne densité a l'aide de sondes différentielles, hybridation soustractive) ont été conduites afin d'identifier des gènes potentiellement impliqués dans la mise en place de l'appressorium, et des gènes potentiellement sous le contrôle de la voie de signalisation à laquelle appartient la Serine/Threonine kinase CLK1, impliquée dans l'acquisition de la fonctionnalité de l'appressorium. Nos résultats suggèrent que la mise en place de l'appressorium chez C. lindemuthianum nécessite la régulation i) du métabolisme des réserves carbonées, ii) de la synthèse des acides amines, iii) de l'endocytose, et iv) du contrôle du cycle cellulaire. Par ailleurs, la voie de signalisation incluant la kinase CLK1 serait impliquée dans i) le choix de la voie de dégradation des protéines (autophagie versus proteasome), ii) la régulation de l'activité mitochondriale, et iii) la réorganisation du cytosquelette via l'actine. Enfin, une approche gène candidat orthologue a été menée sur le gène MgPLS1 de Magnaporthe grisea, champignon phytopathogène qui produit un appressorium, qui code pour une tetraspanine putative impliquée dans la fonctionnalité de I'appressorium. Clpls1, le gène orthologue de C. lindemuthianum est un homologue fonctionnel de MgPLS1 chez M. grisea,; est également nécessaire à la fonctionnalité de l'appressorium chez C. lindemuthianum, et plus précisément an niveau de la formation du pore appressorial.The fungus Colletotrichum lindemuthianum is the causal agent of anthracnose on common bean, Phaseolus vulgaris, that differentiates an appressorium, a specialised structure for penetration of plant tissue. Morphological, biochemical, biophysical and microscopical examination of three C. lindemuthianum penetration mutants allowed to demonstrate that appressorium development by this fungus can be divided in three functional substeps: differentiation; maturation and functionality. Surprisingly, analyses of plant defence responses showed that appressorium maturation but not functionality is necessary for superoxyde ions production, defence genes induction (PAL3, CHS and PvPR2), and accumulation of PR-proteins in the apoplasm. However, appressorium functionality (e.g. genuine penetration within plant tissue) is compulsory for hypersensitive response within at least three independent gene-for-gene relationships. Two transcriptomic-based approaches (hybridisation of macroarrays with differential probes and subtractive hybridisation) have been performed in order to identify genes potentially involved in the appressorium setup and genes putatively under the control of the signal transduction pathway CLK1, a Ser/Thr kinase involved in appressorium functionality, is part of. Results Suggest that appressorium setup in C. lindemuthianum requires regulation of i) metabolism of carbon sources, ii) synthesis of aminoacids, iii) endocytosis, and iv) control of cell cycle. Besides, the signal transduction pathway including CLK1 appears to be involved in i) balance between two protein degradation pathways (autophagy versus proteasome), ii) the regulation of mitochondrial activity, and iii) cytoskeleton reorganisation via actin. A candidate othologue gene approach was done with PLS1, a tetraspanin-encoding gene from the appressorium-producing plant pathogen Magnaporthe grisea where it is involved in appressorium functionality. Clpls1, the orthologue in C. lindemuthianum, is a functional homologue of PLS1 in M. grisea; it is also involved in appressorium functionality in C. lindemuthianum where it is compulsory for the appressorial pore development.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

10 New Insights into Ectomycorrhizal Symbiosis Evolution and Function

International audienc

Who is Controlling whom within the Ectomycorrhizal Symbiosis: Insights from Genomic and Functional Analyses

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Fungal Pls1 tetraspanins as key factors of penetration into host plants: a role in re-establishing polarized growth in the appressorium?

International audienceThe ability of plant pathogenic fungi to infect their host depends on successful penetration into plant tissues. This process often involves the differentiation of a specialized cell, the appressorium. Signalling pathways required for appressorium formation are conserved among fungi. However, the functions involved in appressorium maturation and penetration peg formation are still poorly understood. Recent studies have shown that Pls1 tetraspanins control an appressorial function required for penetration into host plants and are likely conserved among plant pathogenic fungi. Tetraspanins are small membrane proteins widely distributed among ascomycetes and basidiomycetes defining two distinct families; Pls1 tetraspanins are found in both ascomycetes and basidiomycetes and Tsp2 tetraspanins are specific to basidiomycetes. Both fungal tetraspanins families have similar secondary structures shared with animal tetraspanins. Pls1 tetraspanins are present as single genes in genomes of ascomycetes, allowing a unique opportunity to study their function in appressorium mediated penetration. Experimental evidence suggests that Pls1 tetraspanins are required for the formation of the penetration peg at the base of the appressorium, probably through re-establishing cell polarity