Uptake of exogenous myristate by extraradical hyphae (ERH) of arbuscular mycorrhizal fungi (AMF) under symbiotic conditions in Exp. 1.

(a) Schematic illustration of the two-compartment AMF‒carrot hairy root co-cultivation system and the addition of myristate to the hyphae compartment (HC). Carrot hairy roots colonized by Rhizophagus irregularis, R. intraradices, or R. diaphanus were grown in the root compartment (RC). Twelve to fourteen weeks later, 0.1 mM of 13C1-labeled (13C1-Myr), non-labeled (12C-Myr) myristate, or ddH2O (Myr−, control) was added weekly to the HC with well-developed AMF extraradical hyphae (ERH) until harvest. (b) Branched absorbing structures (BAS) of R. irregularis, R. intraradices, and R. diaphanus were observed in the HC only when treated with exogenous myristate (Myr+). (c) 13C/12C ratio of the harvested ERH from HC or RC of R. irregularis, R. intraradices, and R. diaphanus when 13C1-labeled (13C1-Myr) or non-labeled (12C-Myr, background) myristate was added to HC. Two independent experimental trials were performed for R. irregularis. (d, e) Calculated 13C enrichment in ERH of R. irregularis, R. intraradices, and R. diaphanus in the HC and RC under 13C-Myr treatment. Black horizontal lines represent mean value from 2‒5 biological replicates. *, **, and *** indicate statistical significance between 13C1-labeled and non-labeled treatments at the 0.05, 0.01, and 0.001 probability levels (Mann-Whitney U-test), respectively. Different letters indicate significant difference at the 0.05 probability level (one-way ANOVA followed by Games-Howell’s post hoc test). Source data are provided as a Source Data file.

Effects of exogenous myristate on the expression of fungal fatty acid uptake and metabolism genes in the extraradical hyphae (ERH) and on root colonization by arbuscular mycorrhizal fungi (AMF) in Exp. 1.

(a, b) Normalized expression (relative to RiEF-1β) of AMF genes involved in (a) fatty acid uptake (RiFAT1 and RiFAT2) and (b) metabolism—β-oxidation (RiFAD1), palmitvaccenic acid synthesis (RiOLE1), and TCA cycle (RiCIT1)—in the ERH of R. irregularis collected from the hyphae compartment (HC) under myristate (Myr+) or ddH2O (Myr−) treatments (n = 4). (c−e) Percent root colonization by (c) R. irregularis (n = 3‒10), (d) R. intraradices (n = 9‒ 22), and (e) R. diaphanus (n = 6‒14) in carrot hairy roots from the root compartment (RC) when HC was treated with myristate (Myr+) or ddH2O (Myr−). Two independent experimental trials were performed for R. irregularis. Means ± SE, *, **, and *** indicate statistical significance between Myr+ and Myr− treatments at the 0.05, 0.01, and 0.001 probability levels, respectively. Source data are provided as a Source Data file.

Effects of exogenous myristate on the development of intraradical and extraradical structures of AMF (Rhizophagus irregularis) in the single-compartment AMF-carrot hairy root co-cultivation system (Exp. 2).

(a) Schematic illustration of the single-compartment R. irregularis-hairy root co-cultivation system and the addition (Myr+) or absence (Myr−) of myristate. (b) Spore germination rate, (c) extraradical hyphae (ERH) branching, and (d) ERH length in the R. irregularis-hairy root co-cultivation system with (Myr+) or without (Myr−) myristate treatment at 4 days post-inoculation (n = 11−12). (e) AMF percent root colonization in carrot hairy roots and (f) biomass of the AMF extraradical spore and hypha (per gram of medium) at 14 weeks post-inoculation (n = 3−6). (g) Dynamics of AMF spore density over time (per square centimeter, n = 3−5); and (h) AMF spore diameter at harvest with 0, 0.1, 0.5, 1, 5, or 10 mM myristate treatment under symbiotic conditions (n = 5, calculation from > 900 spores in each replicate). Means ± SE; different letters indicate significant differences at P < 0.05 level (One-way ANOVA followed by Tukey’s post hoc test); *** indicates statistical significance at the 0.001 probability level (Student’s t-test). Source data are provided as a Source Data file.

Environmental myristate levels and the effects of exogenous myristate on arbuscule abundance, as well as the intraradical and extraradical biomass of arbuscular mycorrhizal fungi (AMF, Rhizophagus irregularis) associated with Medicago sativa or Oryza sativa under low (LP) or high (HP) phosphorus conditions (Exp. 3).

(a) Schematic illustration of the field survey of environmental myristate levels and experimental design in the pot experiment (Exp. 3). (b) Myristate concentration in bulk soil, as well as in plant leaves, roots, leaf litter, and rhizosphere soil from the dominant species O. sativa, Axonopus compressus, and Morus alba in the paddy field, grassland, and woodland, respectively (n = 4). (c, d) Arbuscule abundance in M. sativa and O. sativa roots treated with 0.1 mM myristate (Myr+) or ddH2O (Myr−) (n = 10; vesicle and hyphal abundance are shown in Supporting Information Fig. S1); (e, f) AMF biomass, indicated by the AMF signature fatty acid (NLFA 16:1ω5), in AMF-colonized roots and rhizosphere soil of M. sativa and O. sativa treated with myristate (Myr+) or ddH2O (Myr−) (n = 5). Means ± SE; different letters indicate significant differences at P < 0.05 level (b: Student’s t-test, c−f: one-way ANOVA followed by Tukey’s post hoc test). Source data are provided as a Source Data file.

Effects of exogenous myristate on symbiotic carbon (C) allocation from Medicago sativa and Oryza sativa to arbuscular mycorrhizal fungi (AMF, Rhizophagus irregularis) and on the expression profiles of mycorrhizal C transportation marker genes.

(a, b) Plant-derived 13C flow to the AMF signature fatty acid (NLFA 16:1ω5) in AMF-colonized roots of M. sativa and O. sativa treated with 0.1 mM myristate (Myr+) or ddH2O (Myr−) under low (LP) and high (HP) phosphorus conditions (n = 5). (c−h) Normalized expression of plant and fungal (underlined) marker genes for mycorrhizal C transport in AMF-colonized (AM) and non-colonized (NM) roots treated with 0.1 mM myristate (Myr+) or ddH2O (Myr−) (n = 3). The mycorrhizal C transportation genes include the plant symbiotic fatty acid transporter STR2 (c, d), the fungal fatty acid transporters (RiFAT1, RiFAT2; e, f), and the monosaccharide transporter (RiMST2; g, h). Normalized expression of M. sativa, O. sativa, and AMF genes was calculated relative to MsACT2, OsCyc2, and RiEF-1β, respectively; n.d., not detected. Two-way ANOVA in each histogram applies only to AM roots. Means ± SE; * and ** indicate statistical significance at the 0.05 and 0.01 probability levels, respectively. Source data are provided as a Source Data file.

Effects of exogenous myristate on plant phosphorus (P) concentration, mycorrhizal P response, and expression of plant and fungal (underlined) marker genes for the mycorrhizal P pathway in Medicago sativa and Oryza sativa.

(a, b) Plant P concentration (n = 8−10); (c, d) mycorrhizal P response (n = 8−10); and (e, f) relative expression of MsPT8, OsPT11, and RiPT in M. sativa and O. sativa roots under low (LP) and high (HP) phosphorus conditions with 0.1 mM myristate (Myr+) or ddH2O (Myr−) treatments (n = 3). AM, inoculated with Rhizophagus irregularis; NM, non-inoculated; n.d., not detected. Normalized expression of M. sativa, O. sativa, and AMF genes was calculated relative to MsACT2, OsCyc2, and RiEF-1β, respectively. Two-way ANOVA in each histogram (c−f) applies only to AM roots. Means ± SE; different letters indicate significant differences at P < 0.05 level (one-way ANOVA followed by Tukey’s post hoc test). * and *** indicate statistical significance at the 0.05 and 0.001 probability levels (Student’s t-test), respectively. Source data are provided as a Source Data file.

Effects of exogenous myristate on the global transcriptome and putative AM-activated defense genes in Medicago sativa and Oryza sativa roots inoculated with arbuscular mycorrhizal fungi (AMF, Rhizophagus irregularis) under low (LP) and high (HP) phosphorus conditions.

(a, b) Significantly enriched gene ontology (GO) terms (Padj < 0.05) for differentially expressed genes (DEGs) between myristate (Myr+) and ddH2O (Myr−) treatments in RNA-seq. Underlined terms denote stress- or defense-related GO categories, with the expression profile of their component genes provided in Supporting Information (Fig. S4). (c, d) Quantitative real-time PCR analysis of the AM-activated defense gene expression—reactive oxygen species (ROS) balance (MsAOX3), stress responses (MsUGT85A, MsPRXP7, and OsPRX39), and defense responses (OsCHT1 and OsTGA2) — between Myr+ and Myr− treatments under LP and HP conditions (n = 3). Means ± SE; * indicates statistical significance between Myr+ and Myr− treatments at the 0.05 probability level (Student’s t-test). Source data are provided as a Source Data file.

A proposed model for direct uptake of exogenous myristate by symbiotic arbuscular mycorrhizal fungi (AMF) and its regulatory effects on the arbuscular mycorrhizal symbiosis.

Symbiotic AMF can uptake exogenous myristate, possibly through the fungal fatty acid transporters RiFAT2 in the extraradical hyphae (ERH), as indicated by 13C enrichment and transcriptional activation of fatty acid transport and metabolism genes in AMF extraradical hyphae. Exogenous myristate increased both intraradical and extraradical fungal biomass, possibly linked to the suppression of AM-activated defense responses in host roots. Unexpectedly, as a non-symbiotic carbon source, exogenous myristate disrupted the carbon (C)-phosphorus (P) exchange in AM symbiosis, resulting in reduced mycorrhizal P benefits for plants and decreased allocation of symbiotic C to AMF. Question mark or dashed arrow indicates putative or unclear mechanisms. SA, salicylic acid; ROS, reactive oxygen species; PR, pathogenesis-related; NLFA 16:1ω5, neutral lipid fatty acid 16:1ω5 (AMF marker).