A role for the mevalonate pathway in early plant symbiotic signaling

Abstract

Rhizobia and arbuscular mycorrhizal fungi produce signals that are perceived by host legume receptors at the plasma membrane and trigger sustained oscillations of the nuclear and perinuclear Ca(2+) concentration (Ca(2+) spiking), which in turn leads to gene expression and downstream symbiotic responses. The activation of Ca(2+) spiking requires the plasma membrane-localized receptor-like kinase Does not Make Infections 2 (DMI2) as well as the nuclear cation channel DMI1. A key enzyme regulating the mevalonate (MVA) pathway, 3-Hydroxy-3-Methylglutaryl CoA Reductase 1 (HMGR1), interacts with DMI2 and is required for the legume-rhizobium symbiosis. Here, we show that HMGR1 is required to initiate Ca(2+) spiking and symbiotic gene expression in Medicago truncatula roots in response to rhizobial and arbuscular mycorrhizal fungal signals. Furthermore, MVA, the direct product of HMGR1 activity, is sufficient to induce nuclear-associated Ca(2+) spiking and symbiotic gene expression in both wild-type plants and dmi2 mutants, but interestingly not in dmi1 mutants. Finally, MVA induced Ca(2+) spiking in Human Embryonic Kidney 293 cells expressing DMI1. This demonstrates that the nuclear cation channel DMI1 is sufficient to support MVA-induced Ca(2+) spiking in this heterologous system.

Keywords: HMG-CoA reductase; arbuscular mycorrhization; calcium signaling; legume nodulation; mevalonate.

Fig. S1.

Enzymatic analysis of MtHMGR1 activity. To evaluate the catalytic activity of M. truncatula HMGR1, assays were performed by measuring the oxidation of NADPH in the presence of the substrate HMG-CoA. (A) There is no detectible change in the oxidation of NADPH in the absence of MtHMGR1. (B) However, in the presence of HMGR1, addition of HMG-CoA results in a progressive oxidation of NADPH. (C) Addition of the competitive inhibitor lovastatin (50 µM) to the reaction mixture inhibits HMGR1 activity, confirming that MtHMGR1 has HMG-CoA reductase activity. Arrows indicate the addition of HMG-CoA to initiate the reaction, and the oxidation of NADPH was determined by measuring the absorbance at 340 nm. (D) The double reciprocal Lineweaver–Burk plot was used to calculate the V_max as 3.62 ± 0.37 µmoles/min/mg of protein and K_m as 38.88 ± 4.41 µM. (E) Confirmation of the production of MVA in HMGR1 ezymatic assay as determined by ion chromatogram obtained through ultra gas chromatograph coupled to a quadrupole/orbitrap mass spectrometer. MVA production in the HMGR1 enzymatic assay was 5 times greater than the control.

Fig. S2.

Nod factor- and MVA-induced ENOD11 expression in M. truncatula wild-type, HMGR1-RNAi transgenic roots, and symbiotic defective mutants. (A) Control M. truncatula roots expressing the cytoplasmic cameleon YC3.6 show intense Nod factor-induced ENOD11-GUS expression after 12 h of treatment. (B) Silencing HMGR1 significantly reduces Nod factor-induced ENOD11 expression. (A and B scale bar, 5 mm.) (C) Quantitative RT-PCR confirms a major reduction (approx. eightfold) in Nod factor-induced ENOD11 induction in roots expressing the HMGR1-RNAi cassette. (D and E) ENOD11-GUS expression induced in wild-type M. truncatula plants after 24 h of treatment with 10 nM Nod factors (D) and 100 µM MVA (E). (F–I) Similarly, 100 µM MVA elicits ENOD11-GUS expression in the nfp-2 mutant (F), the dmi2 mutant background (G), but not in dmi1 (H) or dmi3 (I) mutants, thus showing that MVA acts downstream of DMI2 and upstream of DMI1 during symbiotic signaling. (D–I scale bar, 2 mm.)

Fig. S3.

Role of MVA pathway and HMGR1 in root hair growth in M. truncatula. (A) Silencing HMGR1 did not affect root hair growth. (B) The effect of lovastatin (0.5 µM and 1 µM) on root hair growth in M. truncatula. Application of lovastatin at 0.5 µM concentration, which inhibited nodulation (1), did not affect root hair growth.

Fig. 1.

Effect of silencing M. truncatula HMGR1 on nuclear-associated Ca²⁺ spiking in root epidermal cells. (A) Nod factor-induced Ca²⁺ spiking in M. truncatula root hair cells expressing YC3.6. (B) Absence of Nod factor-induced Ca²⁺ spiking in a nfp mutant expressing YC3.6. (C) Silencing HMGR1 abolishes Nod factor-induced Ca²⁺ spiking in M. truncatula root hair cells. Arrows indicate the addition of Nod factors.

Fig. S4.

Effect of silencing HMGR1 on AM GSE-induced nuclear-associated Ca²⁺ spiking. (A) R. irregularis GSE activates Ca²⁺ spiking in wild-type M. truncatula roots expressing the YC3.6 cameleon. (B) Absence of GSE-induced Ca²⁺ spiking in HMGR1-RNAi M. truncatula roots expressing YC3.6.

Fig. 2.

MVA-induced nuclear-associated Ca²⁺ spiking in legumes and nonlegumes. (A) Exogenous application of 100 µM MVA triggers Ca²⁺ spiking in M. truncatula root hair cells expressing YC3.6. (B) MVA restores Ca²⁺ spiking in roots silenced for HMGR1. (C) MVA induces nuclear Ca²⁺ spiking in atrichoblast cells of a M. truncatula root organ culture expressing nuclear-targeted YC2.1. (D) MVA-induced nuclear spiking in root hairs of L. japonicus expressing NLS-YC3.6. (E) Atrichoblasts of carrot root organ cultures expressing nuclear-targeted YC2.1 also respond to MVA. (F) In contrast, MVA does not elicit nuclear-associated Ca²⁺ spiking in trichoblast cells of Arabidopsis expressing YC3.6, which is unable to form endosymbiotic associations with either rhizobia or AM fungi.

Fig. S5.

Investigating the effect of downstream products of the MVA pathway on nuclear-associated Ca²⁺ spiking that is not due to acidification of the cytoplasm. (A) MVA 5-phosphate (100 µM) and (B) MVA 5-pyrophosphate (100 µM) induce Ca²⁺ spiking in the root hairs of M. truncatula expressing YC3.6. (C) In contrast, isopentenyl pyrophosphate (100 µM) does not induce Ca²⁺ spiking. (D) Sodium propionate (100 µM), used here to mimic cytoplasmic acidification, does not trigger Ca²⁺ spiking in root hairs of YC3.6-expressing roots.

Fig. 3.

Analyses of MVA-induced nuclear-associated Ca²⁺ spiking responses in symbiosis-defective mutants of M. truncatula. (A) MVA-induced Ca²⁺ spiking was not observed in an nfp mutant, which acts upstream of DMI2. (B) MVA activates Ca²⁺ spiking in the dmi2 mutant expressing YC3.6, (C) but not in the dmi1 mutant. (D) As expected, MVA-elicited Ca²⁺ spiking is not modified in the dmi3 mutant, as DMI3 acts downstream of the Ca²⁺ spiking machinery.

Fig. S6.

Analyses of MVA-induced ENOD11 expression and Ca²⁺ spiking in nfp mutants (nfp-1 and nfp-2). (A) MVA-induced ENOD11 expression in the trichoblast and atrichoblast cells of wild type. (B) MVA-induced ENOD11 expression in the trichoblast and atrichoblast cells of nfp-2. (C) Representative trichoblast cell expressing YC3.6 used for Ca²⁺ spiking analyses. (D) MVA failed to induce nuclear Ca²⁺ spiking in trichoblast cells of both nfp mutants. (E) Representative atrichoblast cells expressing YC3.6 used for Ca²⁺ spiking analyses. (F) MVA failed to induce Ca²⁺ spiking in atrichoblast cells of both nfp mutants. (Scale bar, 10 µm.) The small circle in panels C and E indicates the region of interest used for obtaining FRET and CFP channel intensities to analyze nuclear Ca²⁺ spiking.

Fig. 4.

MVA-induced Ca²⁺ spiking in HEK-293 cells expressing DMI1. (A and B) Absence of Ca²⁺ spiking in HEK-293 cells expressing the vector control pIRES2-YC3.6 in the absence (A) or presence of exogenous MVA (100 µM) (B). (C and D) Ca²⁺ spiking is only observed when MVA is added to HEK-293 cells expressing the pIRES2-YC3.6::DMI1 vector.

Fig. 5.

Model illustrating the proposed role of MVA within the common symbiosis pathway. Nod and Myc factors are perceived at the plasma membrane by a complex including the receptor-like kinase DMI2, interacting with either the Nod factor receptor component NFP or the so far unidentified Myc factor receptor. Based on our observations, we propose that HMGR1—which is known to interact with DMI2—generates MVA as a second messenger, transducing the signal from the plasma membrane to the nuclear compartment where DMI1, the nuclear envelope-localized cation channel, is required for the initiation of nuclear Ca²⁺ spiking. This Ca²⁺ response is then decoded by the Ca²⁺ and calmodulin-dependent kinase DMI3, which in turn leads to downstream endosymbiosis-related gene activation. In our experiments, the exogenous application of MVA is sufficient to activate the common symbiosis pathway and trigger nuclear Ca²⁺ spiking in the absence of receptor activation.