High melanization of dark septate endophytes: an advantageous trait for plant colonization and stress tolerance – DARKandSTRONG

The different pathways of melanin synthesis were studied in a panel of Ascomycetes using genomic exploration and phylogenetic studies. In order to identify new inhibitors or inducers of melanization, a screening of chemical compounds was carried out. To obtain genetically modified mutants producing low or high levels of melanin in their hyphae, two genetic approaches were undertaken. The first approach consisted of obtaining KO mutants after transformation with Agrobacterium tumefaciens. The genomic DNA of the mutants was then extracted and sequenced to locate the T-DNA insertion site(s). The second approach consisted of developing the CRISPR/Cas9 strategy. The modification of melanization intensity was also tested using acclimatization tests. The different mutants were tested for their ability to tolerate various abiotic stresses (metals, heat, salt) and biotic stresses (amoeba). In particular, various chemical agents were used to test cell wall integrity and surface hydrophobic properties. Oxidative stress was also analyzed by studying the sensitivity to ROS of melanin-deficient mutants. With regard to biotic interactions, a high-throughput viability test was adapted to monitor the survival of fungi in co-culture with the fungivorous amoeba Protostelium aurantium. In addition, fungus-amoeba interactions were microscopically investigated. Finally, a first study of the outcome of interactions between plants and melanized/non melanized fungi was also conducted in two soils, one contaminated with metals and one not. The morphometric parameters of the plants were measured.

In ascomycete fungi, all three melanin synthesis pathways are present. Indeed, the genes involved in the different stages of melanin synthesis have been identified from the genomic data of the fungal species we selected. However, the DHN-melanin pathway appears to be the most predominant. When a specific inhibitor of this pathway was used, melanization was almost completely suppressed. Only intermediate compounds accumulated in the colonies. Conversely, specific inhibitors of the other pathways had very little effect on the level of melanization in the hyphae. The agrotransformation approach yielded several hundred mutants, and five weakly melanized mutants were identified. A hyperpigmented mutant was also selected using this approach. The number of T-DNA insertions varied from one to three in the mutants we analysed. The insertions were not present in melanogenic genes, but in other genes whose function is not yet known, suggesting an indirect impact of these mutations on the melanization process. The complementary CRISPR/Cas9 approach was initiated by developing specific vectors for the transformation of our model fungal species. The acclimatization approach was successful, as certain abiotic stresses had a positive impact on the level of melanization of the colonies. The screening of chemical compounds was also successful, as it led to the discovery of new melanization-inhibiting molecules. In some rare cases, melanization inducers were also found. Regarding phenotypic tests to study the fungus's tolerance level in response to abiotic stresses, initial results show that melanin plays a key role in surface structural integrity, as melanin-deficient mutants were more sensitive to cell wall disturbances. In biotic interaction tests, the amoeba Protostelium aurantium significantly reduced the viability and radial growth of the fungus, confirming the role of melanin and the cell surface in the fungus's resilience to stress and its importance in ecological interactions. Finally, the DSE Rhexocercosporidium improved the tolerance of its host plant when subjected to metal stress. The growth and fitness of inoculated plants were not affected, whereas numerous symptoms were observed in non-inoculated plants.

In the next phase of the project, the objective will be to continue our research on other albino mutants and other stress factors. The impact of certain chemical compounds on melanin production will be studied in greater detail using time kinetics and dose-response curves. The different mutants will be exposed to various stress factors and omics studies will be conducted to highlight the fine regulation of melanin synthesis pathways, as well as the indirect effect that a variation in melanization could have on the physiology of the fungus. Research on the interaction between melanin levels and environmental stress will be further explored by including additional stress factors and combining radial growth analysis with viability assessments. Surface interaction studies will be carried out using an atomic force microscope. Different plant species will be inoculated with mutants contrasting in their level of melanization, and the outcome of the plant-fungus interaction will be evaluated, particularly in terms of the rate of root colonization by the different strains with varying levels of melanization. All of this research will improve our understanding of melanin-dependent defense mechanisms and propose the development of stress-resistant fungal strains. Ultimately, this will contribute to the application of dark septate endophytes in the fields of sustainable agriculture and biofertilization, in line with the long-term objectives of the project.

Dark septate endophytes (DSEs) are a polyphyletic assemblage of Ascomycetes that colonize plant roots and are originally characterized by the accumulation of high concentrations of melanin in their hyphae. It has been hypothesized that this trait could be advantageous to both partners in plant-DSE associations in response to a variety of biotic and abiotic stresses. However, evidence for the contribution of high melanization of DSEs to stress mitigation is still lacking. Secondly, we hypothesize that melanin plays a role in root penetration by hyphae and subsequent colonization, because melanization is analogously required for virulent fungal pathogens to successfully infect animal and plant tissues. In this French-German collaborative project, we aim to better understand the melanization process in the DSE model Leptodontidium sp., including the study of regulatory mechanisms modulating melanization. Moreover, complementary genetic, pharmacological, physico-chemical, physiological and omics approaches contributed by the Franco-German partners will be used to decipher the role that melanin might play in the competitiveness of Leptodontidium sp. for plant colonization and in the high tolerance of Leptodontidium sp. to a range of abiotic and biotic stresses.
The consortium is composed of researchers from four laboratories having complementary expertise in microbiology, plant-microbe interactions under stress conditions, fungal ecology, multi-omics analyses and bioinformatics. Particular techniques and topics are genetic transformation of DSEs and atomic force microscopy (Université of Lorraine - P1), miRNA analyses and metal stress (Université de Bourgogne Franche-Comté - P2), epigenetics and RNAseq analysis (Friedrich Schiller University Jena - P3), and interactions between fungi and mycoparasites (Wismar University of Applied Sciences - P4). Therefore, answering the questions and testing the hypotheses concerning the role of melanin for DSEs and for DSE-plant interactions can only be achieved by the combined activities of the French-German team.
Understanding the mechanisms that mitigate environmental stress for DSEs and for plants colonized by DSEs could contribute to the exploitation of this relevant fungal resource for the sustainable and economically meaningful production of crops that face increasing constraints, including the presence of mycophagous and plant-pathogenic organisms in the rhizosphere, exposure to contaminants, and climate change impacts such as drought and heat. Consequently, we also aim to ensure wide dissemination of the project results to the scientific community, society and stakeholders in agriculture, horticulture and forestry.