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The exudate from an arbuscular mycorrhizal fungus induces nitric oxide accumulation in Medicago truncatula roots

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Abstract

Nitric oxide (NO) is a signaling molecule involved in plant responses to abiotic and biotic stresses. While there is evidence for NO accumulation during legume nodulation, almost no information exists for arbuscular mycorrhizas (AM). Here, we investigated the occurrence of NO in the early stages of Medicago truncatula–Gigaspora margarita interaction, focusing on the plant response to fungal diffusible molecules. NO was visualized in root organ cultures and seedlings by confocal microscopy using the specific probe 4,5-diaminofluorescein diacetate. Five-minute treatment with the fungal exudate was sufficient to induce significant NO accumulation. The specificity of this response to AM fungi was confirmed by the lack of response in the AM nonhost Arabidopsis thaliana and by analyzing mutants impaired in mycorrhizal capacities. NO buildup resulted to be partially dependent on DMI1, DMI2, and DMI3 functions within the so-called common symbiotic signaling pathway which is shared between AM and nodulation. Significantly, NO accumulation was not induced by the application of purified Nod factor, while lipopolysaccharides from Escherichia coli, known to elicit defense-related NO production in plants, induced a significantly different response pattern. A slight upregulation of a nitrate reductase (NR) gene and the reduction of NO accumulation when the enzyme is inhibited by tungstate suggest NR as a possible source of NO. Genetic and cellular evidence, therefore, suggests that NO accumulation is a novel component in the signaling pathway that leads to AM symbiosis.

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Acknowledgements

We are grateful to Mireille Chabaud and David Barker for kindly providing root organ cultures and seeds of M. truncatula and for fruitful discussion and to Allan Downie for the Nod factor. The research was supported by a university grant (60%) to L.L. and by a grant from the SOILSINK Project (FISR) and the project Converging Technologies-BioBITs, funded by CIPE-Regione Piemonte to P.B.

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  1. Dipartimento di Biologia Vegetale, Università degli Studi di Torino, Viale Mattioli 25, 10125, Turin, Italy

    Cristina Calcagno, Mara Novero, Andrea Genre, Paola Bonfante & Luisa Lanfranco

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  1. Cristina Calcagno
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  2. Mara Novero
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  3. Andrea Genre
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Correspondence to Luisa Lanfranco.

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Figure S1

Histochemical analysis of transgenic M. truncatula roots carrying the MtENOD11 pro-GUS fusion construct treated with fungal exudate (a and c) and sterile water (b and d). Fungal exudate treatment induced GUS activity in patches of cortical cells in the young differentiated zone of lateral roots (a) and occasionally in epidermal cells and root hairs (c). In the corresponding zone of control roots treated with sterile water, GUS staining was limited to the vascular tissues (b, d). Bars = 700 μm (a and b), 60 μm (c and d). v vascular tissues, c cortex, e epidermis. (JPEG 304 kb) (JPEG 304 kb)

Figure S2

Root meristems (a) and emergences of lateral roots (b) of a nonmycorrhizal root after DAF-2DA staining, and root meristems (c) and emergences of lateral roots (d) of a nonmycorrhizal root after treatment with the NO scavenger cPTIO and DAF-2DA staining. Bars = 700 μm (a), 80 μm (b), 800 μm (c) and 100 μm (d). (JPEG 243 kb) (JPEG 243 kb)

Table S1

Mean and standard deviation values for data presented in Fig. 3 (DOC 27 kb) (DOC 27 kb)

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Calcagno, C., Novero, M., Genre, A. et al. The exudate from an arbuscular mycorrhizal fungus induces nitric oxide accumulation in Medicago truncatula roots. Mycorrhiza 22, 259–269 (2012). https://doi.org/10.1007/s00572-011-0400-4

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