www.sciencemag.org/cgi/content/full/science.aan0081/DC1

Supplementary Materials for

Fatty acids in arbuscular mycorrhizal fungi are synthesized by the host

plant

Leonie H. Luginbuehl, Guillaume N. Menard, Smita Kurup, Harrie Van Erp, Guru V.

Radhakrishnan, Andrew Breakspear, Giles E. D. Oldroyd,* Peter J. Eastmond*

*Corresponding author. Email: giles.oldroyd@jic.ac.uk (G.E.D.O.); peter.eastmond@rothamsted.ac.uk

(P.J.E.)

Published 8 June 2017 on Science First Release

DOI: 10.1126/science.aan0081

This PDF file includes:

Materials and Methods

Figs. S1 to S12

Captions for Tables S1 and S2

References

Other Supplementary Material for this manuscript includes the following:

(available at www.sciencemag.org/cgi/content/full/science.aan0081/DC1)

Tables S1 and S2

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Materials and Methods

Biological materials and growth conditions The Medicago truncatula ram1 and ram2 mutants used in this study have been described previously (2, 4). The M. truncatula plastidic acetyl-CoA synthetase (ACS; Medtr7g084850) mutants acs-1 (NF8047) and acs-2 (NF17886) were obtained from the Samuel Roberts Noble Foundation Tnt1 database (http://medicago-mutant.noble.org/mutant/database.php). The wild type background for ram1 and ram2 is Jemalong A17 and for the Tnt1 mutants it is R108. The genotypes of the Tnt1 mutants were confirmed by performing PCR on genomic DNA using primers listed in Table S1. Seeds were scarified, surface sterilized with 10% (v/v) bleach solution and germinated on agar plates. For mycorrhization experiments, seedlings were transplanted into a sterile mixture of terragreen and sand (1:1 v/v) containing an inoculum of ~400 Rhizophagus irregularis spores per plant (Symplanta) for ‘nurse plant’ and radiolabelling experiments or 10% of the mycorrhizal inoculum Solrize Pro (Agrauxine, France) for the production of root material for RNA-sequencing. For ‘nurse plant’ experiments, the nurse and test plants were planted together in the same container separated by a 125 µm nylon mesh to segregate the two root systems but allow hyphae to spread between them. Plants were grown in a controlled environment chamber set to a 16 h light (22ºC)/8 h dark (18ºC) period (photon flux density = 250 µmol m2 s-1).

Microscopy Mycorrhizal roots were washed with water and treated with 10 % (w/v) KOH for 6 min at 95oC followed by 3 min in ink essentially as described by (26). Root length colonization was quantified using the grid line intersect method described by (27) and imaged under an Olympus SZX7 light microscope. Mycorrhizal roots were also stained using WGA-AlexaFluor 488 (Molecular probes). The roots were placed in 50% (v/v) ethanol for more than 4 hours. The roots were then transferred to 20 % (w/v) KOH for ~3 days, followed by 0.1M HCl for ~2 hours. The roots were then rinsed and immersed in WGA-AlexaFluor 488 staining solution (0.2 µg ml-1 in PBS) in the dark for 6 hours. For staining of lipid droplets, the roots were placed in 20 % (w/v) KOH for ~3 days then rinsed and immersed in 10 µg ml-1 WGA-AlexaFluor 488 in PBS plus 0.01 % v/v Tween 20 and incubated in the dark for 16 hours at 37oC. The roots were then rinsed and immersed in 10 µg ml-1 Nile Red (Sigma-Aldrich) in PBS for 10 min in the dark and then rinsed. The roots were imaged with an Olympus FV1000, Zeiss LSM780 or Leica DM6000 microscope. Confocal microscopy was performed using standard settings for WGA-AlexaFluor 488 and Nile Red.

Gene expression analysis For RNA sequencing of mycorrhized and non-mycorrhized roots, 4-5 root systems were pooled to obtain 1 biological replicate, and 4 biological replicates per treatment and genotype were analyzed. For qRT-PCR analysis of GFP- and RAM1-overexpression lines, 6 root systems were pooled for 1 biological replicate, and 3 biological replicates per line were analyzed. RNA was extracted from 100 mg of root tissue using the RNeasy plant mini kit (Qiagen) according to the manufacturer’s instructions. To remove genomic DNA, the RNase free DNase kit (Qiagen) was used according to the manufacturer’s instructions. Reverse transcription was carried out with 1 μg of RNA using the iScript cDNA synthesis

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kit (BioRad) or SuperScript IV First-Strand Synthesis System (ThermoFisher Scientific) according to the manufacturer’s instructions. Quantitative PCRs were performed using a C1000 touch thermal cycler and the SYBR Green PCR mix (BioRad). Data were analyzed using the 2-ΔΔCt method (28) with Ubiquitin or Elongation factor 1 alpha (EF1) as reference genes. The primer pairs used for gene expression analysis are listed in Table S1. RNA sequencing was performed by IMGM Laboratories (Martinsried, Germany). RNA sequencing libraries were prepared with the Illumina TruSeq® Stranded mRNA HT kit and sequencing of the libraries was performed on the Illumina NextSeq500 next generation sequencing system using the high output mode with 1 x 75 bp single-end read chemistry. The resulting reads were quality controlled and mapped against the most recent version of the M. truncatula reference genome (Mtv4.0). Differentially expressed genes (DEGs) were identified by pair-wise comparisons of expression levels (total exon reads) using the CLC Genomics Workbench tool ‘empirical analysis of digital gene expression’ (EDGE). For further analyses, only DEGs with a fold change larger than 1.5 and a false discovery rate (FDR)-corrected p-value smaller than 0.05 were considered.

Cloning and hairy root transformation RAM2 and ACS were amplified from root cDNA using primer pairs listed in Table S1. The products were cloned using a TOPO TA Cloning Kit and transferred into pK7WG2R by Gateway LR reactions (Invitrogen). The cDNA sequences of Umbellularia californica acyl-ACP thioesterase (UcFATB; GenBank accession: M94159), GFP, and M. truncatula RAM1, and the promoter sequences of RAM2, FatM, WRI5s, and ABCG3 were domesticated for use in the Golden Gate modular cloning system (29), synthesized and then inserted into pMS (GeneArt). The binary vector pL2B-10932 was subsequently assembled containing the constitutively active Lotus japonicus Ubiquitin promoter driving expression of the cDNA sequences of UcFATB, GFP, or RAM1 and a DsRed cassette for selection of transgenic roots, or containing the promoter sequences of RAM2, FatM, WRI5s, and ABCG3 driving the expression of GUS. A second binary vector pL2B-10939 containing only the DsRed cassette was constructed to provide an empty vector control (EVC). All vectors were introduced into Agrobacterium rhizogenes strain AR1193, and used to transform M. truncatula following the protocol described (30). Three weeks later, untransformed roots were removed and composite plants were harvested for RNA-extraction or transferred to growth medium for inoculation.

Histochemical GUS staining To visualize GUS activity, M. truncatula hairy roots were washed with distilled water and incubated in GUS staining solution (50 mM phosphate buffer pH 7.2, 0.5 mM K3Fe(CN)6, 0.5 mM K4Fe(CN)6, 50 mM EDTA, 1% (v/v) Triton-X and 2 mM X-Gluc (5-bromo-4-chloro-3-indolyl-beta-D-glucuronide)) in the dark at 37°C for 6 h or overnight. To test whether GUS activity correlates with mycorrhizal infection structures, GUS-stained roots were additionally stained with Alexa Fluor 488 WGA as described above. Images were obtained using a Leica DM6000 microscope.

Transient expression in Nicotiana benthamiana leaves The cDNA sequences of Arabidopsis thaliana WRI1 (AtWRI1), M. truncatula WRI5a, WRI5b and WRI5c were domesticated for use in the Golden Gate modular cloning system

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(29), synthesized and then inserted into pMS (GeneArt). The binary vector pL2B-10932 was subsequently assembled containing the constitutively active L. japonicus Ubiquitin promoter driving expression of the cDNA sequences and a p19 expression cassette. Leaves of three-week old N. benthamiana plants were transformed by inoculation with Agrobacterium tumefaciens strain GV3101 carrying the appropriate binary vector as described previously (31). Leaf discs with a diameter of 1.5 cm were collected 5 days post infiltration and frozen at -80°C. The disks were freeze-dried, weighed and their TAG content was determined following the procedure described in (32).

RAM2 in vitro expression and biochemical characterization To perform enzymatic analysis on the RAM2 protein the cDNA was amplified by PCR and ligated into the RTS pIVEX wheat germ His-6-tag vector pIVEX 1.3WG. Translation was achieved by using the RTS 100 Wheat Germ CECF Kit (5 PRIME) according to the manufacturer’s instructions. Liposomes (10 mg ml−1) were prepared from acetone-washed soybean lethicin (l-α-phosphatidylcholine) and added at 50 μg per reaction. The reaction mixtures with translated protein were stored at −80°C prior to assays. Glycerol-3-phosphate acyltransferase activity was determined by quantifying the amount of [U-14C]glycerol-3-phosphate substrate incorporated into glycerolipid products following the methods described by (12). Conventional fatty acyl-CoA ester substrates and ω-hydroxy fatty acids were obtained from Sigma-Aldrich. ω-hydroxy fatty acyl-CoAs were synthesized and their purity verified using the methods described by (12).

Radioisotope labelling experiments For 14C-labelling experiments designed to trace carbon flow from the plant into fungal lipids, intact wild-type, mutant or hairy root transformed plants were grown in a sterile mixture of terragreen and sand (1:1 v/v) containing an inoculum of ~400 R. irregularis spores per plant (Symplanta). For two compartment ‘nurse plant’ experiments, the nurse and test plants were planted together at a ratio of three to one in the same container separated by a 125 µm nylon mesh to segregate the two root systems but allow hyphae to spread between them and plants were watered every two days with sterile ‘M medium’ (33). In the two compartment system radiolabeling was initiated at 35 days post inoculation (dpi) by injecting 2 μCi of [U-14C]sucrose into the leaves of test plants using a 1 µl Hamilton syringe. In single compartment experiments, 5 μCi of [1-14C]acetate or [U-14C]sucrose were added directly to the growth medium at 35 dpi. In both cases at 77 dpi the growth medium surrounded the intact plant root systems was removed and spores were purified from it by wet sieving (34) and spore numbers were determined by light microscopy. Experiments were also performed on extraradicular mycelium (ERM) and colonized root systems of 14C-labelled plants collected at 42 dpi. The ERM was separated using the technique described in (35). Total lipids were extracted from the spores or colonized root systems and the triacylglycerol (TAG) was purified by thin layer chromatography (TLC, 32). Fatty acid methyl esters (FAMEs) were prepared from the TAG using direct transesterification and their quantity and molecular identity was determined by gas chromatography (GC, 32). The 14C content was measured by liquid scintillation counting and the specific activity of the total FAMEs was calculated and compared between treatments. Where analysis was performed on TAG from colonized root systems, FAMEs were separated by argenation TLC using the method developed by (36).

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The methyl ester of the unique arbuscular mycorrhizal fungal marker fatty acid 11-cis-hexadecenoic acid (16:1Δ11c) was identified by co-migration with a standard, quantified by GC, and the unusual position of the double bond was confirmed by permanganate-periodate oxidation and TLC analysis of the products (37). Short-term [1-14C]acetate and [U-14C]sucrose labelling of total fatty acids in roots and acetyl-CoA synthetase assays were performed following the methods described by (24).

Phylogenetic analyses The homologs of the WRINKLED and ABCG families were obtained using BLAST from the predicted proteomes of Amborella trichopoda, A. thaliana, Oryza sativa, M. truncatula, Marchantia polymorpha, Physcomitrella patens, Selaginella moellendorffii (from phytozome.net) and L. japonicus (from kazusa.or.jp). The BLAST hits were aligned using MAFFT v7.305 (L-INS-i; refinement 1000 iterations; BLOSUM62 for alignment scoring). The evolutionary model that fits the data best was selected using MEGA v7 (38). For both the WRINKLED and ABCG alignments, the JTT model was the highest scoring. The phylogenetic trees were constructed using MEGA employing the JTT model after removing regions of the alignment that had gaps in >75% of the sequences in the alignment. The trees shown are the consensus trees constructed from 100 bootstrap replicates.

Bioinformatic analyses To produce heat maps showing fold changes of differentially expressed genes, the web-based tool GENE-E (now called Morpheus, https://software.broadinstitute.org/GENE-E/index.html) was used. Hierarchical clustering of genes was performed using a Euclidean distance metric. To identify significantly enriched gene ontology (GO) terms of RAM1-dependent genes identified by clustering, a singular enrichment analysis was performed using the web-based tool AgriGO (39). M. truncatula gene identifiers (Mtv4.0) were used for the input sample list, and the analysis was performed using the whole M. truncatula genome (Mtv4.0) as background. To test for significance, Fisher’s exact test was used, and only GO terms with an FDR-corrected p-value smaller than 0.05 were considered (calculated using the multi-test method Yekutieli).

Statistical analysis For all experiments that generated quantitative data, 3 to 12 biological replicates were used and the mean values are presented with bars representing the SEM. Statistical analyses were performed on the data using either unpaired Student’s t-test or one-way analysis of variance (one-way ANOVA). For ANOVA, following significant (P < 0.05) F-test results, means of interest were compared using the appropriate least significant difference (LSD) value at the 5% (P = 0.05) level of significance or higher, on the corresponding degrees of freedom (df). The GenStat (2011, 14th edition; ©VSN International Ltd, Hemel Hempstead, UK) statistical system was used for these analyses.

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Figure S1: RAM1-dependent genes are enriched for lipid and carbohydrate metabolic processes. (A) Heat map showing the log10 fold changes (FC) of genes that were induced in mycorrhized versus non-mycorrhized wild-type (WT) roots at 8 dpi, 13 dpi, and/or 27 dpi and were dependent on RAM1 for their induction during at least one of these time points. Cluster analysis showed that out of 1092 genes analyzed in total, 768 were not induced in ram1 at any of the three time points tested. (B) GO term analysis of genes that were consistently dependent on RAM1 for their induction during mycorrhization. Significantly enriched terms of biological processes with an FDR-corrected p-value <0.05 are shown. (C) Heat map showing the log10 FC of selected lipid-related genes that are dependent on RAM1 for their induction during mycorrhizal colonization. The upper group are homologues of genes that are regulated by WRI transcription factors in A. thaliana (7, 8). The lower pair of ABCG transporters (STR and STR2, 40) are partially dependent on RAM1 for their induction during mycorrhizal colonization. For both (A) and (C), a 1.5 fold cut-off was used for fold changes.

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Figure S2: Validation of RNA-seq gene expression analysis by qRT-PCR. Quantification of transcript levels of RAM1-dependent genes identified by RNA-seq in non-mycorrhized and mycorrhized wild-type (WT) and ram1 roots at 8 dpi, 13 dpi, and 27 dpi. Expression levels of (A) PT4, (B) RAM2, (C) FatM, and (D) ABCG3 are shown. Expression levels were measured by qRT-PCR and normalized to Ubiquitin expression. Values are the mean of 4 biological replicates ± SEM. Asterisks indicate significant differences between expression levels in mycorrhized and non-mycorrhized roots of the same genotype at the corresponding time point (Student’s t-test, **, P < 0.01; ***, P < 0.001, n.s., no statistical difference between mycorrhized and non-mycorrhized roots).

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Figure S3: RAM1 is required for the upregulation of ABCG transporters with homology to A. thaliana lipid transporters. A phylogenetic tree of the ABCG family genes was constructed using homologs from A. trichopoda, A. thaliana, O. sativa, M. truncatula, M. polymorpha, P. patens, S. moellendorffii and L. japonicus. The tree was constructed using MEGA employing the JTT model of evolution from 100 bootstrap replicates. The two RAM1-dependent ABCG transporters ABCG3 and ABCG4 are indicated. STR and STR2, which are partially dependent on RAM1 for their induction during mycorrhizal colonization, are also indicated (40). A. thaliana ABCG transporters involved in wax, cutin, and/or suberin deposition, AtABCG2, AtABCG6, AtABCG11, AtABCG12, AtABCG13 and AtABCG20 are marked (6, 41).

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Figure S4: Several WRINKLED homologs are transcriptionally induced during colonization with arbuscular mycorrhizal fungi. (A) A phylogenetic tree of the WRI family genes was constructed using homologs from A. trichopoda, A. thaliana, O. sativa, M. truncatula, M. polymorpha, P. patens, S. moellendorffii and L. japonicus. The tree was constructed using MEGA employing the JTT model of evolution from 100 bootstrap replicates and rooted using the clade containing AP2-domain proteins from A. thaliana that do not belong to the WRI family. Each subclade of the WRI family that contains a representative in the basal angiosperm A. trichopoda is highlighted as a separate WRI subfamily. Filled circles indicate transcriptional induction in a RAM1-dependent manner; empty circles indicate transcriptional induction in a partial RAM1-dependent manner. (B) Heat map showing the log10 fold changes (FC) of five WRI homologs in wild type (WT) and ram1. A 1.5 fold cut-off was used for fold changes. (C) Quantification of TAG in N. benthamiana leaves overexpressing AtWRI1, WRI5a, WRI5b or WRI5c. GFP was used as a negative control. Statistical comparison to pUBI::GFP. Values are the mean of 3 biological replicates ± SEM (LSD-test; ***, P < 0.001, where F-test P < 0.05).

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Figure S5: RAM1 is sufficient for the activation of several arbuscule-associated genes. (A) Localization of WRI5a and WRI5c gene expression assessed using promoter-GUS fusions in mycorrhized M. truncatula roots. Fungal structures were visualized by staining roots with Alexa Fluor 488 WGA. Arrowheads indicate cells containing arbuscules. Scale bar = 150 µm. (B) Quantification of transcript levels of RAM1 and different putative RAM1 targets (as indicated, including the H+-ATPase HA1; 42, 43) in M. truncatula roots overexpressing GFP (pUBI::GFP) or RAM1 (pUBI::RAM1) in the absence of mycorrhizal fungi. Expression levels were measured by qRT-PCR and normalized to Ubiquitin. Statistical comparison to pUBI::GFP. Values are the mean of 3 biological replicates ± SEM (Student’s t-test; *, P < 0.05; **, P < 0.01, ***, P < 0.001).

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Figure S6: Substrate specificity of RAM2. (A) Cartoon of the sn-2 glycerol-3-phosphate (G3P) acyltransferase and sn-2 lysophosphatidic acid (2-LPA) phosphatase reactions catalysed by RAM2. (B) Acyl specificity of in vitro translated RAM2 using a range of fatty acyl-CoAs of different carbon chain length and G3P as substrates. (C) Regiospecificity of monoacylglycerol (MAG) produced using palmitoyl-CoA (C16:0) and G3P as substrates. Statistical comparison to sn-2. 16:0-OH and 18:1-OH are omega hydroxy palmitoyl and oleoyl-CoA, respectively. Values are the mean quantity of product (± SEM) formed in 10 min assays carried out on 4 independent batches of in vitro translated RAM2 (Student’s t-test; ***, P < 0.001).

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Figure S7: Sucrose labelling of fatty acyl groups in fungal spores. (A) Diagram illustrating the experimental design used to obtain the data presented in Fig. 3. (B) Representative images of arbuscules (marked with arrowhead) in colonized roots of test plant at 42 dpi. Test plants include ram2, ram2 complemented by pUBI-RAM2 (ram2 + RAM2) or ram2 transformed with an empty vector control (ram2 + EVC). Fungal structures were visualized by staining with Alexa Fluor 488 WGA and imaged using confocal microscopy. Scale bar = 10 µm. (C) Quantification of arbuscule length at 42 dpi. Thirty arbuscules were scored for each biological replicate. (D) Number of spores obtained from test plant compartment for lipid analysis at 77 dpi. Statistical comparison to ram2/ram2 in (C) and left panel of (D), and to WT / ram2 + EVC in right panel of (D). Values are the mean of 12 biological replicates ± SEM (LSD-test; ***, P < 0.001, where F-test P < 0.05 and Student’s t-test; *, P < 0.05; ***, P < 0.001).

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Figure S8: Sucrose labelling of fatty acyl groups in the extraradicular mycelium (ERM) and roots. (A) Diagram illustrating the experimental design used for the experiment. (B) The incorporation of [14C]sucrose into fatty acyl groups in triacylglycerol (TAG) extracted from ERM and colonized roots of the test plant. Test plants include ram2, ram2 complemented by pUBI-RAM2 (ram2 + RAM2) or ram2 transformed with an empty vector control (ram2 + EVC). [14C]Sucrose was infiltrated into the leaves of test plants grown in the presence of a nurse plant at 35 dpi and ERM and roots were extracted and analyzed at 42 dpi. Values are expressed relative to WT (left panel) and ram2 + RAM2 (right panel) which are normalized to 1 and corresponding statistical comparisons made. In the case of roots, the labelling of 11-cis-hexadecenoic acid (16:1Δ11c) in TAG was used, since this is a specific fungal marker fatty acid that is very abundant in fungal TAG, but not present in plants (19-21). (C) Level of mycorrhizal colonization in test plants of (B). Values are the mean of 12 biological replicates ± SEM (Student’s t-test; *, P < 0.05; ***, P < 0.001).

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Figure S9: Lipid droplet abundance and triacylglycerol (TAG) content in mycorrhized ram2 roots. (A-D) Representative confocal microscopy images of Alexa Fluor 488 WGA stained fungal hyphae and Nile Red stained lipid droplets in ‘test plant’ roots (listed second) of 42 dpi colonized plants grown using the experimental design depicted in Fig. S8A. Scale bar = 50 µm. In ram2/ram2 (A) arbuscules fail to develop fully in the test plant (4) and few lipid droplets are detected. In WT/WT (B) many lipid droplets (marked with an arrowhead) are visible in hyphae within regions of wild type test plant where arbuscules are fully developed (44). In WT/ram2 (C) few lipid droplets are found near developed arbuscules in colonized ram2 roots where WT is the nurse. Panel (D) is included to show that lipid droplets can be detected in hyphae of WT/ram2 that are not near arbuscules. (E) Number of lipid droplets per mm of hyphal length in z-stacks of 50 µm thickness within regions surrounding developed arbuscules (WT/WT and WT/ram2) and stunted arbuscules (ram2/ram2). Values are the mean of 10 z-stacks from 4 biological replicates ± SEM. (F) Total TAG content as % of dry weight (DW) of colonized (+AM) and uncolonized (-AM) roots. Values are the mean of 12 biological replicates ± SEM. In (E) statistical comparison is to ram2/ram2 (LSD-test; ***, P < 0.001, *, P < 0.05, where F-test P < 0.05) and in (F) statistical comparison is to -AM (Student’s t-test; ***, P < 0.001).

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Figure S10: Isolation and characterization of M. truncatula acs mutants. (A) Gene model showing the positions of Tnt1 retrotransposon insertion sites in ACS (Medtr7g084850). (B) RT-PCR performed on RNA from roots of acs mutants. Primer positions are shown in (A). EF1 is included as a positive control. (C) ACS activity and (D) rate of [14C]acetate incorporation into fatty acyl groups in root lipids of acs mutants, wild type (WT) and acs complemented roots (pUBI::ACS). EVC: empty vector transformed control. Values in (C) and (D) are expressed relative to WT (left panel) and acs-1 + ACS (right panel) which are normalized to 1 and corresponding statistical comparisons made. (E) Total dry weight of eight-week-old acs plants. Statistical comparison to WT. Values are the mean of 8 biological replicates ± SEM (LSD-test; ***, P < 0.001, where F-test P < 0.05 and Student’s t-test; ***, P < 0.001).

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Figure S11: Mycorrhization and spore production of acs mutants and plants expressing lauroyl-ACP thioesterase (UcFATB). Quantification of (A) mycorrhizal colonization at 42 dpi and (B) spore production at 77 dpi in acs mutants. acs-1 was transformed with pUBI-ACS (ACS) or empty vector control (EVC). Quantification of (C) mycorrhizal colonization at 42 dpi and (D) spore production at 77 dpi in plants expressing UcFATB. In (A) and (C) the closed bars are total root length colonization and the open bars are developed arbuscules. Values are the mean of 12 biological replicates ± SEM.

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Figure S12: Expression of lauroyl-ACP thioesterase leads to accumulation of lauroyl groups in the lipids of M. truncatula roots. (A) RT-PCR performed on RNA from roots of wild type (WT), empty vector control (EVC) and pUBI::UcFatB overexpressing roots. EF1a was used as a positive control. (B) Lauroyl groups (C12:0) in WT, EVC and UcFatB root lipids expressed as mol% of total fatty acyl groups. Statistical comparison is with EVC. Values are the mean of 8 biological replicates ± SEM (Student’s t-test; ***, P < 0.001).

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Tables S1 and S2 (separate file)

Table S1: Primer sequences used in this study Table S2: RAM1-dependent mycorrhizal-induced genes

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