Arbuscular mycorrhizal symbiosis is a mutualistic interaction between most land plants and fungi of the glomeromycotina subphylum. The initiation, development and regulation of this symbiosis involve numerous signalling events between and within the symbiotic partners. Among other signals, phytohormones are known to play important roles at various stages of the interaction. During presymbiotic steps, plant roots exude strigolactones which stimulate fungal spore germination and hyphal branching, and promote the initiation of symbiosis. At later stages, different plant hormone classes can act as positive or negative regulators of the interaction. Although the fungus is known to reciprocally emit regulatory signals, its potential contribution to the phytohormonal pool has received little attention, and has so far only been addressed by indirect assays. In this study, using mass spectrometry, we analyzed phytohormones released into the medium by germinated spores of the arbuscular mycorrhizal fungus Rhizophagus irregularis. We detected the presence of a cytokinin (isopentenyl adenosine) and an auxin (indole-acetic acid). In addition, we identified a gibberellin (gibberellin A 4) in spore extracts. We also used gas chromatography to show that R. irregularis produces ethylene from methionine and the α-keto γ-methylthio butyric acid pathway. These results highlight the possibility for AM fungi to use phytohormones to interact with their host plants, or to regulate their own development.

20 * Correspondence to: puech@lrsv.ups-tlse.fr, +33 (0)5 34 32 38 31 (VPP) and frei-dit-21 frey@lrsv.ups-tlse.fr (NFdF), +33 (0)5 34 32 38 60 22 23 Text word count: 5465 24 Number of figure: 3 25 -Fig. 1 Detection by LC-MS of iPR and IAA exuded by R. irregularis spores (colour print) 26 -Fig. 2 GA 4 detection in multiple reactions monitoring (MRM) mode (colour print) HIGLIGHT 31 In this work, we provide for the first time direct experimental evidence, by mass 32 spectrometry and gas chromatography analyses, that the model AM fungus Rhizophagus 33 irregularis produces four different phytohormones. 34 ABSTRACT 35 Arbuscular Mycorrhizal (AM) symbiosis is a mutualistic interaction between most land 36 plants and fungi of the glomeromycotina subphylum. The initiation, development and regulation 37 of this symbiosis involve numerous signalling events between and within the symbiotic partners. 38 Among other signals, phytohormones are known to play important roles at various stages of the 39 interaction. During presymbiotic steps, plant roots exude strigolactones which stimulate the 40 fungus, and favour the initiation of symbiosis. At later stages, different plant hormone classes 41 can act as positive or negative regulators of the interaction. Although the fungus is known to 42 reciprocally emit regulatory signals, its potential contribution to the phytohormonal pool has 43 received little attention, and has so far only been addressed by indirect assays. In this study, 44 using mass spectrometry, we detected the presence of a cytokinin (isopentenyl-adenosine), an 45 auxin (indole-acetic acid), and a gibberellin (gibberellic acid 4), in germinating spores or 46 exudates of Rhizophagus irregularis. We also used gas chromatography to show that R. 47 irregularis produces ethylene from methionine and the α -keto γ -methylthiobutyric acid (KMBA) 48 pathway. These results highlight the possibility for AM fungi to use phytohormones to interact 49 with their host plants, or to regulate their own development. 50 51 KEYWORDS: 52 Arbuscular mycorrhizal fungi Abscisic acid (ABA) 63 Aminocyclopropane-carboxylate (ACC) 64Aminocyclopropane-carboxylate oxidase (ACO) 65 93 Arbuscular Mycorrhizal (AM) symbiosis is a 460 million-year-old interaction (Redecker, 94 2000) between glomeromycotina fungi and over 70% of land plants (Brundrett and Tedersoo, 95 2018). In angiosperms, AM fungi colonize the inner root cortex of their host to develop 96 intracellular ramified structures called arbuscules. These arbuscules are the main site for nutrient 97 exchange between the plant and the fungus. AM fungi provide their host plant with water and 98 minerals, in return for photosynthetic carbon (mainly sugars and lipids) (Smith and Read, 2008; 99