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. 2003 Mar;131(3):952-62.
doi: 10.1104/pp.011882.

A diffusible factor from arbuscular mycorrhizal fungi induces symbiosis-specific MtENOD11 expression in roots of Medicago truncatula

Sonja Kosuta  1 , Mireille Chabaud, Géraldine Lougnon, Clare Gough, Jean Dénarié, David G Barker, Guillaume Bécard
Affiliations

A diffusible factor from arbuscular mycorrhizal fungi induces symbiosis-specific MtENOD11 expression in roots of Medicago truncatula

Sonja Kosuta et al. Plant Physiol. 2003 Mar.

Abstract

Using dual cultures of arbuscular mycorrhizal (AM) fungi and Medicago truncatula separated by a physical barrier, we demonstrate that hyphae from germinating spores produce a diffusible factor that is perceived by roots in the absence of direct physical contact. This AM factor elicits expression of the Nod factor-inducible gene MtENOD11, visualized using a pMtENOD11-gusA reporter. Transgene induction occurs primarily in the root cortex, with expression stretching from the zone of root hair emergence to the region of mature root hairs. All AM fungi tested (Gigaspora rosea, Gigaspora gigantea, Gigaspora margarita, and Glomus intraradices) elicit a similar response, whereas pathogenic fungi such as Phythophthora medicaginis, Phoma medicaginis var pinodella and Fusarium solani f.sp. phaseoli do not, suggesting that the observed root response is specific to AM fungi. Finally, pMtENOD11-gusA induction in response to the diffusible AM fungal factor is also observed with all three M. truncatula Nod(-)/Myc(-) mutants (dmi1, dmi2, and dmi3), whereas the same mutants are blocked in their response to Nod factor. This positive response of the Nod(-)/Myc(-) mutants to the diffusible AM fungal factor and the different cellular localization of pMtENOD11-gusA expression in response to Nod factor versus AM factor suggest that signal transduction occurs via different pathways and that expression of MtENOD11 is differently regulated by the two diffusible factors.

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Figures

Figure 1
Figure 1
Schematic representation of the membrane-separated coculture of AM fungi and M. truncatula. a, Excised (Ri T-DNA-transformed) roots; b, seedlings. c, Detail of the corridor containing dense hyphal growth and ramifications, in which MtENOD11 induction occurs. 1, Spores; 2, location of membrane; 3, primary root; 4, secondary root; 5, tertiary root; 6, negatively geotropic germ tubes; 7, corridor containing hyphal ramifications.
Figure 2
Figure 2
Root pMtENOD11-gusA induction in membrane-separated coculture. a, b, d, f, and g, Ri T-DNA-transformed roots membrane-separated from G. rosea. c, e, and h, Control roots cultured with membrane separation but without fungus. a, GUS activity colocalizes with hyphal ramifications on other side of membrane (white arrowheads), bar = 0.2 cm. b, MtENOD11 induction occurs mainly in tertiary Ri T-DNA-transformed roots; c, only constitutive expression (see also e) is observed in control roots, bars = 2 cm. d, AM factor-induced GUS activity stretches from 0.1 cm behind the root cap as far as the zone of mature root hair growth, bar = 0.2 cm; e, in control roots, only constitutive expression is present in root caps and at the base of lateral roots, bar = 0.1 cm. f, Detail of pMtENOD11-gusA induction in epidermal cells including root hairs, bar = 50 μm. g and h, Seventy-five-micrometer-thick transverse sections of AM-inoculated (g) and control (h) roots, bars = 150 μm. i, Membrane-separated coculture of G. rosea with whole plants; j, control plants. i, pMtENOD11-gusA is induced in young lateral roots, bar = 0.3 cm; j, only background GUS activity is seen in vascular tissues in control roots, bar = 0.3 cm. k and l, pMtENOD11-gusA induction in membrane-separated coculture with other fungi. Induction occurs in Ri T-DNA-transformed roots cocultured with G. margarita (k), but not with Phy. medicaginis (l), bars = 0.6 cm. m, pMtENOD11-gusA induction in membrane-separated coculture of G. rosea and with Ri T-DNA-transformed roots derived from the M. truncatula Myc− mutant dmi2-2, bar = 0.7 cm.
Figure 3
Figure 3
PCR analysis of potential fungal contamination. Products from plant and fungal DNA amplified with universal primers ITS1/4 (odd lanes) and fungal specific primers ITS1F/4 (even lanes). Lanes 1 and 2, DNA of M. truncatula control roots; Lanes 3 and 4, DNA from roots harvested from a membrane-separated coculture of M. truncatula and G. rosea at 10 dai; Lanes 5 and 6, DNA of M. truncatula roots colonized by G. rosea; Lanes 7 and 8, DNA from G. rosea spores; and Lanes 9 and 10, water control. Note that the band (550 bp) amplified by fungal primers in spores (lane 8) and colonized roots (lane 6; arrowheads), could not be detected in roots separated from AM fungi by a membrane (lane 4). Two faint non-specific bands (600 and 680 bp) can be seen in lanes 2 and 4 corresponding to root DNA.
Figure 4
Figure 4
pMtENOD11-gusA induction in M. truncatula Nod−/Myc− dmi mutants. Percentage of Ri T-DNA-transformed roots of M. truncatula wild-type and dmi mutants that show induction of pMtENOD11-gusA after 10 d of membrane-separated coculture with G. rosea (+AM) and without (−AM). Roots were counted in the “corridor” of dense fungal growth and hyphal ramifications. Data from three independent experiments were pooled and treated as subsets (blocks) of a single data set for statistical analysis. Means ± se labeled with a different letter (a and b) are significantly different according to analysis of variance followed by Tukey's test (P < 0.05, n = 7).
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