A combination of chitooligosaccharide and lipochitooligosaccharide recognition promotes arbuscular mycorrhizal associations in Medicago truncatula

Abstract

Plants associate with beneficial arbuscular mycorrhizal fungi facilitating nutrient acquisition. Arbuscular mycorrhizal fungi produce chitooligosaccharides (COs) and lipo-chitooligosaccharides (LCOs), that promote symbiosis signalling with resultant oscillations in nuclear-associated calcium. The activation of symbiosis signalling must be balanced with activation of immunity signalling, which in fungal interactions is promoted by COs resulting from the chitinaceous fungal cell wall. Here we demonstrate that COs ranging from CO4-CO8 can induce symbiosis signalling in Medicago truncatula. CO perception is a function of the receptor-like kinases MtCERK1 and LYR4, that activate both immunity and symbiosis signalling. A combination of LCOs and COs act synergistically to enhance symbiosis signalling and suppress immunity signalling and receptors involved in both CO and LCO perception are necessary for mycorrhizal establishment. We conclude that LCOs, when present in a mix with COs, drive a symbiotic outcome and this mix of signals is essential for arbuscular mycorrhizal establishment.

Fig. 1

COs and LCOs activate symbiotic calcium oscillations. a Representative traces of 10⁻⁸ M CO8, 10⁻⁸ M CO4, 0.8 mg/ml PGN, 10⁻⁸ M NS-LCO and 10⁻⁹ M SmLCO-induced calcium oscillations in M. truncatula trichoblasts on lateral roots. The y-axis represents the ratio of YFP to CFP in arbitrary units. Numbers denote responsive cells relative to total cells analysed. b CO8 and PGN-induced calcium oscillations were restricted to the nuclear region (arrows indicate the location of the nucleus). Images were taken every five seconds covering a single calcium transient. c Dose response curves for CO and LCO induction of calcium oscillations in M. truncatula lateral root trichoblasts. SmLCO: sulphated LCO produced by S. meliloti. NS-LCO: non-sulphated LCO produced by R. irregularis

Fig. 2

CO8 and PGN activate calcium oscillations dependent on DMI1, DMI2, MtCERK1 and LYR4. Representative calcium traces of M. truncatula trichoblasts of lateral roots responding to 10⁻⁸ M CO8 (a) and 0.8 mg/ml PGN (b) in wild type and different mutants. The traces denote the ratio of YFP to CFP in arbitrary units. Numbers indicate cells responding compared to total cells analyzed

Fig. 3

LYR4 and MtCERK1 bind to COs and PGN. a LYR4 and MtCERK1 binding with streptavidin beads fused to either CO4 or CO8. CK: streptavidin beads alone. b MtCERK1 binding with CO4 beads can be eluted by addition of CO4 and CO8. LYR4 binding with CO8 beads can be eluted with CO8, but not CO4. c LYR4 and MtCERK1, but not DMI2, bind to insoluble PGN and d bound proteins can by eluted by CO8 and the soluble fraction of PGN, but not SmLCO. The protein expression level of LYR4 and MtCERK1 used in the binding assay is shown as input

Fig. 4

MtCERK1 and LYR4 are required for CO8 and PGN-induced gene expression. a A heat map of genes induced by flg22, CO4, CO8 and SmLCO (6 h treatment). The expression of these genes in mutants and during mycorrhizal colonization is shown. qRT-PCR validation of representative symbiotic genes (b and c) and defense genes (d and e) induced by PGN, CO8, SmLCO and flg22. M. truncatula wild type and mutant roots were treated with 0.4 mg/ml PGN, 10⁻⁷ M CO8, 10⁻⁸ M SmLCO and 10⁻⁷ M flg22 using comparable conditions to the material used in the RNA-seq analysis. This experiment was repeated three times with similar results (mean ± s.e.m.; n = 3; significant difference relative to wild type by Student’s t-test, ***P < 0.001; **P < 0.05)

Fig. 5

A combination of CO8 and SmLCO can promote symbiosis and inhibit immunity signalling. a M. truncatula wild-type roots were pretreated with 10⁻⁸ M SmLCO for different time points (minutes) before incubation with 10⁻⁶ M CO8 for induction of ROS. The significant groupings were calculated with a Mann–Whitney Rank Sum Test (mean ± s.e.m., n = 6; P < 0.05). This experiment was repeated twice with similar results. b MAPK activation in wild type and nfp roots induced by 10⁻⁶ M CO8 with or without 30 min pretreatment of 10⁻⁸ M SmLCO. c Equivalent treatment as b for qRT-PCR detection of gene expression testing immunity reporters PR10 and a Chitinase and symbiosis reporters HA1 and Vapyrin in response to CO8 and PGN, with or without SmLCO. Relative fold change compared to individual water treatments are shown. Letters denote statistically significant groupings calculated with Mann–Whitney Rank Sum Test (mean ± s.e.m., n = 3; P < 0.05), this experiment was repeated twice with similar results

Fig. 6

NFP but not other symbiosis signalling components are required for LCO suppression of plant immunity. a M. truncatula wild-type and mutant roots were pre-treated with 10⁻⁸ M SmLCO for 30 min before challenging with CO8 for ROS induction. This experiment was repeated three times with similar results. b M. truncatula roots with or without SmLCO treatments were inoculated with P. palmivora and the lesion size quantified relative to root length or c quantity of pathogen measured using amplification of P. palmivora EF1a relative to M. truncatula H2A. These experiments were repeated twice with similar results. Letters denote statistically significant groupings calculated with Mann-Whitney Rank Sum Test (mean ± s.e.m., n = 6 for a, n = 30 for b and n = 3 for c, P < 0.05)

Fig. 7

MtCERK1 and NFP are required for arbuscular mycorrhizal colonization. a R. irregularis colonization shown as percent root length colonization assessed at 3 and 5 weeks post inoculation. b Individual fungal structures were quantified at 3 weeks post inoculation. This experiment was repeated three times with similar results. c Representative images of arbuscules in wild type and mutant plants. Scale bar = 10 µm. a, b Letters denote statistically significant groupings calculated with Mann–Whitney Rank Sum Test (mean ± s.e.m., n = 10; P < 0.05). d A model for COs/PGN and LCO recognition during symbiosis. The LYR4/CERK1 receptor complex responds to COs and PGN with activation of immunity and symbiosis signalling. In contrast, the LCO receptor activates symbiosis signalling. Activation of symbiosis signalling by LCOs suppresses immunity signalling. Note the co-receptor DMI2 is necessary for COs/PGN and LCO promotion of symbiosis signalling, but is not required for COs/PGN induction of immunity signalling