Production, perception and transport of phytohormones by arbuscular mycorrhizal fungi – Mycormones

Obtention of fungal material: Fungal material (pure spores and fungal exudates) has been produced using cultures of Rhizophagus irregularis on hairy roots.
Task 1: Phytohormones present in germinating spores or their exudates have been analysed. Global and targeted analyses were carried out using the sensitivity and specificity of GC and LC-MS/MS techniques.
Task 2: An image-based method for monitoring the growth of the extra-radical mycelium (ERM) of the fungus has been developed, using a new imaging equipment. This method makes it possible to measure the growth of ERM in response to phytohormones.
Task 3: Recombinant proteins have been produced for some of the candidate strigolactone receptors. Their cleavage and binding properties towards a range of strigolactone analogs have been studied using LC-MS, microscale thermophoresis and differential scanning fluorimetry.
Task 4. To study the transport of phytohormones by mycorrhizal fungi, various experimental systems have been set up.

Task 1. Analysis of phytohormone production by AM fungi. Several phytohormones have been identified in AM germinating spores or their exudates: auxin, cytokinin, ethylene and gibberellin. In the case of ethylene, the fungus carries out biosynthesis, and information has been provided on the synthesis pathway used by the fungus (Pons et al. 2020).
Task 2: Effect of phytohormones on AM fungi. A stimulation of ERM growth in response to two classes of plant hormones has been shown, as well as a stimulating effect of some hormones on spore germination.
Concerning strigolactones, an analysis of structure/function relationships with different analogs has allowed to specify which parts of the molecule are important for its bioactivity on the fungus. This study has also highlighted different biological responses to these analogs (Taulera et al. 2020).
Task 3A. The coding sequences of candidate ethylene and cytokinin receptors have been cloned for functional characterisation.
Task 3B. Characterisation of strigolactone receptor candidates. For one of the candidates, the ability to cleave different strigolactone analogs, and to bind to some of them, has been demonstrated.
Task 4. Study of phytohormone transport. The first tests have given very encouraging results, which need to be confirmed and analysed in further detail.

The remaining project aims will be pursued as planned in the initial proposal.

1. Kabbara et al. (2019) Diversity and evolution of sensor histidine kinases in eukaryotes. Genome Biology and Evolution. 11: 88-108
2. Papon & Binder (2019) An evolutionary perspective on ethylene sensing in microorganisms. Trends in Microbiology. 27: 1-4.
3. Taulera et al. (2020) Initiation of arbuscular mycorrhizal symbiosis involves a novel pathway independent from hyphal branching. Mycorrhiza 30:491-501. doi: 10.1007/s00572-020-00965-9
4. Pons et al. (2020) Phytohormone production by the arbuscular mycorrhizal fungus Rhizophagus irregularis. PLoS One 15(10):e0240886. doi: 10.1371/journal.pone.0240886
5. Kabbara et al. (2020) Cytokinin Sensing in Bacteria. Biomolecules. 10: 186
6. Bidon et al. (2020) Cytokinin and ethylene cell signalling pathways from prokaryotes to eukaryotes. Cells. 9: 2526.

Most land plants live symbiotically with microscopic soil fungi of the order Glomeromycota. This very ancient interaction, called the Arbuscular mycorrhizal (AM) symbiosis, is observed across a wide range of ecosystems and is on a global scale a major player in plant nutrition, soil structure and carbon cycling. AM fungi bear great potential for biofertilization as an alternative to chemical inputs. They develop both within cortical root cells and into the soil, forming a sort of extension of the root system. In host roots the fungi differentiate into specialized structures called arbuscules, where nutrient exchanges take place: the fungus provides its host plant with otherwise inaccessible mineral nutrients, in exchange for organic carbon. In addition, AM fungi and their hosts control each other's development. Furthermore, plants connected by a Common Mycorrhizal Network (CMN) can exchange via the fungus information related to pathogen or herbivore attack.
The symbiosis is controlled through extensive molecular communication between the two partners. The project aims to investigate the role of phytohormones in this communication. Several lines of evidence suggest that the fungal partner could also be a source and target of phytohormone signalling. In particular, we identified in AM fungal genomes homologs of plant cytokinin and ethylene receptors, and detected hormones released by isolated AM fungi. With this project, we will investigate for the first time on a large scale whether AM fungi are themselves able to produce and transport phytohormones, whether they use them as internal regulators and whether they can convey information between plants by transporting hormones in CMNs. To reach these goals, the project brings together two academic groups and an industrial partner specialized in the production of biofertilizers.
We will implement mass spectrometry techniques to detect hormones released in vitro by isolated AM fungi. This will be greatly facilitated by the industrial partner, who will provide large amounts of AM biological material. For the hormones we can detect, we will try to determine whether the fungi performed their biosynthesis or released previously accumulated hormones of plant origin. We will examine the biological effects that exogenously applied phytohormones can have on AM fungi, and try to decipher the underlying cellular and molecular mechanisms. We will also analyse the perception of cytokinins, ethylene and strigolactones, for which we have identified candidate receptors. Finally, we will use labelled phytohormones or analogs to examine their bidirectional transport between host plants and AM fungi, and between plants connected by a CMN.
Since the "normal" situation of plants is to live interaction with AM fungi, our findings will unravel how plant development and physiology are shaped in natural environments. Our project will also yield new knowledge to favour the use of AM fungi as biofertilizers, in line with the current demand for more sustainable agricultural practices.