Truffles Regulate Plant Root Morphogenesis via the Production of Auxin and Ethylene

Truffles are symbiotic fungi that form ectomycorrhizas with plant roots. Here we present evidence that at an early stage of the interaction, i.e. prior to physical contact, mycelia of the white truffle Tuber borchii and the black truffle Tuber melanopsorum induce alterations in root morphology of the host Cistus incanus and the nonhost Arabidopsis (Arabidopsis thaliana; i.e. primary root shortening, lateral root formation, root hair stimulation). This was most likely due to the production of indole-3-acetic acid (IAA) and ethylene by the mycelium. Application of a mixture of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid and IAA fully mimicked the root morphology induced by the mycelium for both host and nonhost plants. Application of the single hormones only partially mimicked it. Furthermore, primary root growth was not inhibited in the Arabidopsis auxin transport mutant aux1-7 by truffle metabolites while root branching was less effected in the ethylene-insensitive mutant ein2-LH. The double mutant aux1-7;ein2-LH displayed reduced sensitivity to fungus-induced primary root shortening and branching. In agreement with the signaling nature of truffle metabolites, increased expression of the auxin response reporter DR5∷GFP in Arabidopsis root meristems subjected to the mycelium could be observed, confirming that truffles modify the endogenous hormonal balance of plants. Last, we demonstrate that truffles synthesize ethylene from l-methionine probably through the α-keto-γ-(methylthio)butyric acid pathway. Taken together, these results establish the central role of IAA and ethylene as signal molecules in truffle/plant interactions.Ectomycorrhizal symbioses are mutualistic interactions between filamentous fungi and plant roots. Truffles, which are ascomycete fungi renown for their aromatic fruiting bodies, form ectomycorrhizas (ECM) in temperate climates predominantly with trees (i.e. hazel Corylus avellana], oaks Quercus spp.]).In soil, microorganisms communicate with plants by exchanging chemical signals throughout the rhizosphere. Depending on the nature of the interaction, these molecules can be either volatiles or solutes (dissolved solids). For example, rhizobacteria induce growth promotion in Arabidopsis (Arabidopsis thaliana) through the action of the volatile compound 2,3-butanediol (Ryu et al., 2003). The sesquiterpene volatile (E)-β-caryophyllene is produced by maize (Zea mays) roots fed upon by arthropods, and serves as attractant to natural enemies of the insects (Rasmann et al., 2005). Nonvolatile signal molecules can also dissolve in water and diffuse in the soil. Indeed the nitrogen-fixing bacteria Rhizobium secrete a nodulation factor that induces changes in root morphology of legumes (Dénarié and Cullimore, 1993; Heidstra and Bisseling, 1996). Mycelium branching is induced in the mycorrhizal fungus Gigaspora margarita by 5-deoxy-strigol, a strigolactone exuded from the roots of the legume host Lotus japonicus (Akiyama et al., 2005).Also ectomycorrhizal fungi engage in a molecular dialogue with plants and produce chemical signals that modulate plant root/ECM morphogenesis. The indole alkaloid hypaphorine produced by ectomycorrhizal fungus Pisolithus tinctorius inhibits root hair elongation in the host Eucalyptus globules and the nonhost Arabidopsis (Béguiristain et al.,1995; Reboutier et al., 2002). Hormones, mainly indole-3-acetic acid (IAA), have also been often implicated in symbiotic interactions (Barker and Tagu, 2000; Martin et al., 2001; Sirrenberg et al., 2007; Contreras-Cornejo et al., 2009). Most studies involving hormones have focused at a late stage of interaction, when ECMs were developing or already formed (for review, see Barker and Tagu, 2000). Indeed IAA-overproducing mutants of the ectomycorrhizal fungus Hebeloma cylindrosporum form significantly more mycorrhizas with the host Pinus pinaster than the wild type (Gay et al., 1994). Similarly applying exogenous IAA to the ectomycorrhizal system Piloderma croceum/Quercus robur also resulted in a more intense ECM colonization of the host compared to controls with no additional IAA (Herrmann et al., 2004).Truffles form ECM with a variety of hosts such as oaks, hazels, but also some shrubs (i.e. Cistus). Under laboratory conditions, the establishment of the symbiotic phase takes 2 to 3 months (Sisti et al., 1998; Miozzi et al., 2005). Strains of the same truffle species vary in their capacity to colonize a single host (Giomaro et al., 2000). Volatile organic compounds have been implicated in the signaling between truffle and plants. Splivallo et al. (2007) discussed the phytotoxic activity of fruiting body volatiles and Menotta et al. (2004) highlighted their potential role as mycorrhization signals. Fruiting bodies volatiles shortened primary roots of plants (Splivallo et al., 2007) while the effect of mycelial metabolites has not been addressed.Our aim here was to investigate how truffle mycelia modify plant root architecture. We focus our effort on an early stage of interaction (10 d) to explain changes in root morphology prior to ECM formation and highlight the action of diffusible signals on the host plant Cistus incanus and the nonhost Arabidopsis. Using Arabidopsis mutants, we did not aim to investigate the mechanism behind the IAA/ethylene cross talk but rather to show that the fungal metabolites are perceived in planta through both auxin and ethylene signaling pathways. We illustrate how truffle metabolites modify the auxin response of the root meristem using the auxin reporter line DR5∷GFP. We further demonstrate that both hormones are produced by truffles at concentrations that fully explain the root phenotypic responses of the host and nonhost plants. Last, we elucidate ethylene biosynthesis in truffles.