Supporting Information Legends
Figure S1. Lipid and fatty acid pattern of R. irregularis ERM. Lipids were extracted from
ERM and fractionated by SPE. (a) Nonpolar lipid fractions were separated by thin layer
chromatography (TLC) using a nonpolar solvent system. 1,100 % hexane fraction; 2,
hexane/diethylether (95:5, v/v) fraction, (1/10 of the lipid); 3, diethylether fraction. (b) Polar
lipids were separated by TLC in a polar solvent system. 4, acetone-isopropanol (1:1, v/v)
fraction (glycolipids); 5, methanol fraction phospholipids). Lipids were stained with iodine
vapor. (c) Fatty acid pattern of the nonpolar (chloroform) lipid fraction; (d) fatty acid
composition of the polar (methanol) lipid fraction. Fatty acid methyl esters were measured by
GC-MS. 15:0 (internal standard). Two isomers of 18:1 are found in R. irregularis, 18:1ω7
and 18:1ω9 (Olsson & Johansen, 2000). ASG, acylated sterol glycosides; DAG,
diacylglycerol; FFA, free fatty acids; FS, free sterol; GlcCer, glycosylceramide; MAG,
monoacyldiacylglycerol; PA, phosphatidic acid; PC, phosphatidylcholine; PE,
phosphatidylethanolamine; PI, phosphatidylinositol; PS, phosphatidylserine; SE, sterol ester;
SG, sterol glycoside; TAG, triacylglycerol.
Figure S2. Diacylglycerol content in R. irregularis and L. japonicus. DAG was analyzed by
Q-TOF MS/MS in the nonpolar lipid fraction of extraradical mycelium (ERM), and in mockinfected (-P mock) and mycorrhized (-P myc) roots of L. japonicus grown with low
phosphate. Data are means and SD of at least 3 measurements and were confirmed by a
second independent experiment. Values significantly different to -P mock are indicated with
an asterisk (Student's t test, p < 0.05).
Figure S3. Fatty acid synthesis and degradation in R. irregularis. (a) Possible pathways for
de novo fatty acid synthesis in the cytosol. (b) Pathway for mitochondrial fatty acid synthesis
(type II). (c) β-oxidation (peroxisomal). Rattus norvegicus (Rn), Laccaria bicolor (Lb),
Ustilago maydis (Um), Saccharomyces cerevisiae (Sc), Aspergillus nidulans (An) or
Cryptococcus neoformans (Cn) sequences that resulted in the identification of R. irregularis
sequences with high similarity (tblastn score < E-5 with the MIRAv1 or MIRAv2 database)
are shown. Strikethrough indicates that no corresponding R. irregularis sequence was found.
Figure S4. Fatty acid elongation and desaturation (Endoplasmic Reticulum). Possible
pathway for the synthesis of long chain and very long chain, unsaturated fatty acids in R.
irregularis. Saccharomyces cerevisiae (Sc) or Mortierella alpina (Ma) sequences that
resulted in the identification of R. irregularis sequences with high similarity (tblastn score <
E-5 with the MIRAv1 or MIRAv2 database) are shown. Red arrows indicate upregulation (↑)
of R. irregularis gene expression as shown by RT-PCR (Figure 3). Note that double bond
positions in C20 fatty acids of R. irregularis have not been experimentally determined.
16:1(ω5) = 16:1(Δ11); 16:1(ω7) = 16:1(Δ9); 18:1(ω7) = 18:1(Δ11); 18:1(ω9) = 18:1(Δ9).
Figure S5. Molecular species composition of (a) PE, (b) PI and (c) PS. Phospholipids were
quantified in ERM and mock-infected (-P mock) and mycorrhized (-P myc) roots of L.
japonicus grown with low phosphate. Data represent means and SD of 4 replicates and were
confirmed by a second independent experiment. Values significantly different to -P mock are
indicated with an asterisk (Student's t test, p < 0.05). Note that 40:7, 41:2 and 42:9 PS could
not be quantified in roots due to the presence of contaminants with the same mass to charge
ratio (m/z).
Figure S6. Glycerolipid metabolism in R. irregularis. Possible pathways for phospholipid
and triacylglycerol metabolism. Saccharomyces cerevisiae (Sc) sequences that resulted in the
identification of R. irregularis sequences with high similarity (tblastn score < E-5 with the
MIRAv1 or MIRAv2 database) are shown. CDP, cytidinediphosphate; Cho, choline; CL,
cardiolipin; DAG, diacylglycerol; DMPE, dimethylphosphatidylethanolamine; Etn,
ethanolamine; Gro, glycerol; MMPE, monomethylphosphatidylethanolamine; P, phosphate;
PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG,
phosphatidylglycerol; PS, phosphatidylserine; TAG, triacylglycerol.
Figure S7. Free sterols and sterol glucosides in R. irregularis and L. japonicus. (a) Free
sterol (FS) and (b) sterol glucoside (SG) molecular species composition in extraradical
mycelium (ERM) and in mock-infected and mycorrhized L. japonicus roots grown with low
phosphate. Data show means and SDs of 3 measurements. Values significantly different to -P
mock are indicated with an asterisk (Student's t test, p < 0.05). The following sterols were not
distinguished during Q-TOF MS/MS analysis: Ergostadienol, 24-methyldesmosterol, 24methylene-cholesterol; stigmasterol, isofucosterol, 24-ethylidene-cholesterol, 24Ethylcholesta-5,22-dienol; lanosterol, cycloartenol; sitosterol, 24-ethylcholesterol; 24methylcholesterol, campesterol (Fontaine et al., 2004).
Figure S8. Sterol metabolism in R. irregularis. Possible pathways for (a) sterol synthesis and
and for (b) sterol glycoside/sterol ester synthesis. Saccharomyces cerevisiae (Sc) or
Arabidopsis thaliana (At) sequences that resulted in the identification of R. irregularis
sequences with high similarity (tblastn score < E-5 with the MIRAv1 or MIRAv2 database)
are shown. Strikethrough indicates that no R. irregularis sequence was found by tblastn
search with the respective protein.
Figure S9. Sphingolipid metabolism in R. irregularis. Possible pathways for the synthesis of
sphingolipids. Saccharomyces cerevisiae (Sc) or Pichia pastoris (Pp) sequences that resulted
in the identification of R. irregularis sequences with high similarity (tblastn score < E-5 with
the MIRAv1 or MIRAv2 database) are shown. Note that the positions of double bonds and of
the methyl group in the sphingobase were not experimentally determined. d18:0, sphinganine;
t18:0, phytosphingosine; d18:1Δ4, sphingosine; d18:2Δ4,8, sphingadienine; d18:2Δ4,8,9-me,
methyl-sphingadienine.
Table S1. Genes involved in lipid biosynthesis in R. irregularis. Protein sequences from
Saccharomyces cerevisiae (Sc), Rattus novergicus (Rn), Aspergillus nidulans (An), Laccaria
bicolor (Lc), Ustilago maydis (Um), Cryptococcus neoformans (Cn) and Mortierella alpina
(Ma) were employed to search the R. irregularis nonredundant virtual transcript (NRVT)
databases MIRA v1 and MIRA v2 (Tisserant et al., 2012) by tblastn. R. irregularis NRVTs
sequences with significant E values (> 1E-5) were retrieved, and the ratios of expression of
intraradical (IRM) to extraradical mycelium (ERM) are shown.
Table S2. Oligonucleotides used for RT-PCR.