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Accueil du site → Doctorat → Allemagne → Molecular and cytological investigations of the fungal endophyte Piriformospora indica and its interactions with the crop plant barley

Justus-Liebig-Universität Gießen (2007)

Molecular and cytological investigations of the fungal endophyte Piriformospora indica and its interactions with the crop plant barley

Deshmukh, Sachin D.

Titre : Molecular and cytological investigations of the fungal endophyte Piriformospora indica and its interactions with the crop plant barley

Auteur : Deshmukh, Sachin D.

Université de soutenance : Justus-Liebig-Universität Gießen

Grade : Doktorgrade Dr. Rer. Nat. 2007

Résumé
Plant roots are potential hosts of a plethora of beneficial microorganisms including mycorrhizal fungi, rhizobial bacteria, and endophytic fungi of the newly defined order Sebacinales (Basidiomycota). The recently discovered root endophyte Piriformospora indica represents a model organism of this fungal order. The axenically cultivable P. indica colonizes roots of a large number of monocotyledonous and dicotyledonous plant species including cereals and Brassicaceae. The symbiosis is characterized by increased biomass and grain yield of interacting plants. In barley, the endophyte induces local and systemic resistance to fungal diseases and to abiotic stress. To further elucidate the lifestyle of P. indica, fungal development and host reactions were analyzed during the mutualistic symbiosis with barley roots. It was revealed that like other mutualistic endophytes, P. indica colonizes roots in an asymptomatic manner. The fungal colonization increases with root tissue maturation. The root tip meristem showed no colonization and the elongation zone showed mainly intercellular colonization. The differentiation zone was heavily infested by inter- and intracellular hyphae and intracellular chlamydospores. The majority of hyphae were present in intercellular spaces, dead rhizodermal and cortical cells that became completely filled with chlamydospores at later interaction stages. In some cases, hyphae penetrated cells and built a meshwork around plasmolyzed protoplasts, suggesting that the fungus either actively kills cells or senses cells undergoing endogenous programmed cell death. However, the expression of the barley BAX inhibitor-1 (HvBI-1) gene, an inhibitor of plant cell death, was suppressed during colonization. Consistently, fungal proliferation was strongly inhibited in transgenic barley lines overexpressing GFP-tagged HvBI-1. The cytological observations and responses of the host genetic factor to fungal infestation showed that P. indica requires host cell death for proliferation in differentiated barley roots and the endophyte interferes with the host cell death program to form a mutualistic interaction with plants. Infection of plant roots with pathogenic necrotrophic fungi of the genus Fusarium leads to necrotized roots and severe reduction of root and shoot biomass. Recently, it has been shown that P. indica infested plants are more resistant to Fusarium culmorum. In the current study, the interaction of P. indica with Fusarium graminearum in barley roots was analyzed. Upon infestation with P. indica, roots were protected from Fusarium infections as evidenced by reduced root rot symptoms. Consistently, Fusarium quantification using quantitative polymerase chain reaction (Q-PCR) revealed a correlation between reduced root rot symptoms and the relative amount of fungal DNA in the roots. Expression of pathogenesis related (PR) genes, which strongly increased in response to F. graminearum infections, was diminished in the presence of P. indica. This finding indicates that PR proteins do not play a crucial role in the P. indica-mediated resistance response to Fusarium. While P. indica is emerging as a model fungus to study mutualistic plant-fungus interactions, genetic transformation has not been accomplished yet. Transformation of the fungus by biolistic gene transfer and analysis of transgene expression in its saprophytic phase was performed. The transcriptionally active nature of the constitutive Aspergillus nidulans glyceraldehyde 3-phosphate (gpd) promoter and suitability of red fluorescent protein DsRed as a reporter gene in P. indica was established. The genes for hygromycin (Hyg-B) resistance and DsRed were employed as markers. Patches of fluorescent hyphae and chlamydospores were observed upon fluorescence microscopic examination of hygromycin-resistant mycelia. The procedure described here lays the ground for the use of yet unavailable molecular genetic tools in analyzing a plant root endophytic fungus.

Mots clés : biodiversity, mycorrhiza, rhizosphere, Sebacinales, systemic resistance, Fungal transformation, Symbiosis, mutualistic

Présumé

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Page publiée le 17 mars 2008, mise à jour le 8 janvier 2019