Andreas Keymer

Priya Pimprikar

Vera Wewer

Claudia Huber

Mathias Brands

Simone L Bucerius

Pierre-Marc Delaux

Verena Klingl

Edda Von Ro ¨penack-Lahaye

Trevor L Wang

Wolfgang Eisenreich

Peter Do ¨rmann

Martin Parniske

Caroline Gutjahr
email: caroline.gutjahr@lmu.de

Lipid transfer from plants to arbuscular mycorrhiza fungi

Arbuscular mycorrhiza (AM) symbioses contribute to global carbon cycles as plant hosts divert up to 20% of photosynthate to the obligate biotrophic fungi. Previous studies suggested carbohydrates as the only form of carbon transferred to the fungi. However, de novo fatty acid (FA) synthesis has not been observed in AM fungi in absence of the plant. In a forward genetic approach, we identified two Lotus japonicus mutants defective in AM-specific paralogs of lipid biosynthesis genes (KASI and GPAT6). These mutants perturb fungal development and accumulation of emblematic fungal 16:1w5 FAs. Using isotopolog profiling we demonstrate that 13 C patterns of fungal FAs recapitulate those of wild-type hosts, indicating cross-kingdom lipid transfer from plants to fungi. This transfer of labelled FAs was not observed for the AM-specific lipid biosynthesis mutants. Thus, growth and development of beneficial AM fungi is not only fueled by sugars but depends on lipid transfer from plant hosts.

Introduction

Arbuscular mycorrhiza (AM) is a widespread symbiosis between most land plants and fungi of the Glomeromycota [START_REF] Smith | Mycorrhizal Symbiosis[END_REF]. The fungi provide mineral nutrients to the plant. These nutrients are taken up from the soil and released inside root cortex cells at highly branched hyphal structures, the arbuscules [START_REF] Javot | A Medicago truncatula phosphate transporter indispensable for the arbuscular mycorrhizal symbiosis[END_REF]. For efficient soil exploration, arbuscular mycorrhiza fungi (AMF) develop extended extraradical hyphal networks. Their growth requires a large amount of energy and carbon building blocks, which are transported mostly as lipid droplets and glycogen to the growing hyphal tips [START_REF] Bago | Translocation and utilization of fungal storage lipid in the arbuscular mycorrhizal symbiosis[END_REF][START_REF] Bago | Carbon export from arbuscular mycorrhizal roots involves the translocation of carbohydrate as well as lipid[END_REF]. AMF are obligate biotrophs, as they depend on carbon supply by their host [START_REF] Smith | Mycorrhizal Symbiosis[END_REF]. In the past, detailed 13 C-labeled tracerbased NMR studies demonstrated that hexose sugars are a major vehicle for carbon transfer from plants to fungi [START_REF] Shachar-Hill | Partitioning of intermediary carbon metabolism in vesicular-arbuscular mycorrhizal leek[END_REF]. In addition, a fungal hexose transporter, with high transport activity for glucose is required for arbuscule development and quantitative root colonization as shown by host induced gene silencing [START_REF] Helber | A versatile monosaccharide transporter that operates in the arbuscular mycorrhizal fungus Glomus sp is crucial for the symbiotic relationship with plants[END_REF], indicating the importance of hexose transfer for intra-radical fungal development.

AMF store carbon mainly in the form of lipids [START_REF] ´panier | Dependence of arbuscularmycorrhizal fungi on their plant host for palmitic acid synthesis[END_REF]. The predominant storage form is triacylglycerol (TAG) and the major proportion of FAs found in AMF is composed of 16:0 (palmitic acid), and of 16:1w5 (palmitvaccenic acid). The latter is specific to AM fungi and certain demands of arbscocytes for metabolites including lipids. The importance of plant lipid biosynthesis for arbuscule development has been demonstrated by Medicago truncatula mutants in AM-specific paralogs of two lipid biosynthesis genes FatM and REDUCED ARBUSCULAR MYCORRHIZA2 (RAM2) [START_REF] Wang | A common signaling process that promotes mycorrhizal and oomycete colonization of plants[END_REF][START_REF] Bravo | Arbuscular mycorrhiza-specific enzymes FatM and RAM2 fine-tune lipid biosynthesis to promote development of arbuscular mycorrhiza[END_REF]. FatM encodes an ACP-thioesterase, which terminates fatty acid chain elongation in the plastid by cleaving the ACP off the acyl group releasing free FAs and soluble ACP [START_REF] Jones | Palmitoyl-acyl carrier protein (ACP) thioesterase and the evolutionary origin of plant acyl-ACP thioesterases[END_REF]. RAM2 encodes a glycerol 3-phosphate acyl transferase (GPAT) and is most similar to Arabidopsis GPAT6. In Arabidopsis, GPAT6 acetylates the sn-2 position of glycerol-3-phosphate with an FA and cleaves the phosphate from lysophosphatidic acid, thereby producing sn-2-monoacylglycerol (ßMAG, [START_REF] Yang | A distinct type of glycerol-3-phosphate acyltransferase with sn-2 preference and phosphatase activity producing 2-monoacylglycerol[END_REF]. Mutations in both FatM and RAM2 impair arbuscule branching [START_REF] Wang | A common signaling process that promotes mycorrhizal and oomycete colonization of plants[END_REF][START_REF] Bravo | Arbuscular mycorrhiza-specific enzymes FatM and RAM2 fine-tune lipid biosynthesis to promote development of arbuscular mycorrhiza[END_REF]. In addition, arbuscule branching requires a complex of two half ABC transporters STR and STR2 [START_REF] Zhang | Two Medicago truncatula half-ABC transporters are essential for arbuscule development in arbuscular mycorrhizal symbiosis[END_REF][START_REF] Gutjahr | The half-size ABC transporters STR1 and STR2 are indispensable for mycorrhizal arbuscule formation in rice[END_REF]. The substrate of STR/STR2 is unknown but other members of the ABCG transporter family are implicated in lipid transport [START_REF] Wittenburg | Biliary cholesterol secretion by the twinned sterol half-transporters ABCG5 and ABCG8[END_REF][START_REF] Wang | Sequences in the nonconsensus nucleotidebinding domain of ABCG5/ABCG8 required for sterol transport[END_REF][START_REF] Fabre | The ABCG transporter PEC1/ABCG32 is required for the formation of the developing leaf cuticle in Arabidopsis[END_REF][START_REF] Hwang | Plant ABC transporters enable many unique aspects of a terrestrial plant's lifestyle[END_REF][START_REF] Lee | Crystal structure of the human sterol transporter ABCG5/ABCG8[END_REF]. Therefore, and due to its localization in the peri-arbuscular membrane [START_REF] Zhang | Two Medicago truncatula half-ABC transporters are essential for arbuscule development in arbuscular mycorrhizal symbiosis[END_REF] it was speculated that the STR/STR2 complex may transport lipids towards arbuscules [START_REF] Gutjahr | The half-size ABC transporters STR1 and STR2 are indispensable for mycorrhizal arbuscule formation in rice[END_REF][START_REF] Bravo | Arbuscular mycorrhiza-specific enzymes FatM and RAM2 fine-tune lipid biosynthesis to promote development of arbuscular mycorrhiza[END_REF]. Transcriptional activation of RAM2 and STR is controlled by the GRAS transcription factor REDUCED ARBUSCULAR MYCORRHIZA1 (RAM1) [START_REF] Gobbato | A GRAS-type transcription factor with a specific function in mycorrhizal signaling[END_REF][START_REF] Park | Hyphal Branching during Arbuscule Development Requires Reduced Arbuscular Mycorrhiza1[END_REF][START_REF] Pimprikar | A CCaMK-CYCLOPS-DELLA complex activates transcription of RAM1 to regulate Arbuscule branching[END_REF] and also in ram1 mutants, arbuscule branching is impaired [START_REF] Park | Hyphal Branching during Arbuscule Development Requires Reduced Arbuscular Mycorrhiza1[END_REF][START_REF] Xue | Network of GRAS transcription factors involved in the control of arbuscule development in Lotus japonicus[END_REF][START_REF] Pimprikar | A CCaMK-CYCLOPS-DELLA complex activates transcription of RAM1 to regulate Arbuscule branching[END_REF]. Thus, RAM1, FatM, RAM2 and STR/STR2 appear to form an AM-specific operational unit for lipid biosynthesis and transport in arbuscocytes. Consistently, they were found to be absent from genomes of plants that have lost the ability to form AM [START_REF] Delaux | Comparative phylogenomics uncovers the impact of symbiotic associations on host genome evolution[END_REF][START_REF] Favre | A novel bioinformatics pipeline to discover genes related to arbuscular mycorrhizal symbiosis based on their evolutionary conservation pattern among higher plants[END_REF][START_REF] Bravo | Genes conserved for arbuscular mycorrhizal symbiosis identified through phylogenomics[END_REF].

Here, we analyzed two Lotus japonicus mutants identified in a forward genetic screen, which are impaired in arbuscule branching [START_REF] Groth | Two Lotus japonicus symbiosis mutants impaired at distinct steps of arbuscule development[END_REF]. Positional cloning combined with genome resequencing revealed mutations in a novel AM-specific b-keto-acyl ACP synthase I (KASI) gene and in the L. japonicus ortholog of M. truncatula RAM2. KASI likely acts upstream of RAM2 in producing 16:0 FAs. The identity of the genes and the phenotypes led us to hypothesize that AMF may depend on delivery of 16:0 FAs from the plant host. Using a combination of microscopic mutant characterization, lipidomics and isotopolog profiling of 16:0 and 16:1w5 FAs in roots and extraradical fungal mycelium, we provide strong evidence for requirement of both genes for AM-specific lipid biosynthesis and cross-kingdom lipid transfer from plants to AMF.

Results

Two L. japonicus arbuscule-branching mutants are defective in lipidbiosynthesis genes

We previously identified two L. japonicus mutants disorganized arbuscules (dis-1, SL0154-N) and SL0181-N (red) deficient in arbuscule branching [START_REF] Groth | Two Lotus japonicus symbiosis mutants impaired at distinct steps of arbuscule development[END_REF] (Figure 1A-B). Both mutants also suffered from a reduction in root length colonization and blocked the formation of lipid-containing vesicles of the fungus Rhizophagus irregularis (Figure 1C-D). We identified the causative mutations with a combination of classical mapping and next generation sequencing (see Materials and methods). DIS encodes a b-keto-acyl ACP synthase I (KASI, Figure 1-figure supplements 1A-C and 2). KASI enzymes catalyze successive condensation reactions during fatty acyl chain elongation from C4:0-ACP to C16:0-ACP [START_REF] Li-Beisson | Acyl-lipid metabolism[END_REF]. SL0181-N carries one mutation (ram2-1) in the L. japonicus orthologue of the previously identified Medicago truncatula REDUCED ARBUSCU-LAR MYCORRHIZA2 (RAM2, Figure 1-figure supplements 3 and 4). Arabidopsis GPAT6 has been shown to produce ß-MAG with a preference for 16:0 FAs [START_REF] Yang | A land-plant-specific glycerol-3phosphate acyltransferase family in Arabidopsis: substrate specificity, sn-2 preference, and evolution[END_REF]. Therefore, we hypothesized that DIS and RAM2 act in the same biosynthetic pathway.

We identified additional allelic dis mutants by TILLING (Figure 1-figure supplement 1E, Supplementary file 1) [START_REF] Perry | A TILLING reverse genetics tool and a web-accessible collection of mutants of the legume Lotus japonicus[END_REF] and a ram2 mutant caused by a LORE1 insertion in the RAM2 gene (Figure 1-figure supplement 3B) [START_REF] Małolepszy | The LORE1 insertion mutant resource[END_REF]. Among the allelic dis mutants we chose dis-4 for further investigation because it suffers from a glycine replacement at the border of a conserved ß-sheet (Figure 1-figure supplement 2), which likely affects protein folding [START_REF] Perry | TILLING in Lotus japonicus identified large allelic series for symbiosis genes and revealed a Bias in functionally defective ethyl methanesulfonate alleles toward glycine replacements[END_REF]. Both allelic mutants dis-4 and ram2-2 phenocopied dis-1 and ram2-1, respectively. Furthermore, transgenic complementation of both dis-1 and ram2-1 with the wild-type versions of the mutated genes restored arbuscule-branching and wild-type-like levels of root length colonization and vesicle formation (Figure 1A-B). Taken together this confirmed identification of both causal mutations. Consistent with a role of both genes in arbuscule development GUS activity was predominantly detected in arbuscocytes (arbuscule-containing cells) in both wild-type and the corresponding mutant roots (Figure 2-figure supplement 1A-B).

To correlate promoter activity with the precise stage of arbuscule development we used nuclear localized YFP as a reporter. To visualize the fungus, the promoter:reporter cassette was co-transformed with a second expression cassette containing secreted mCherry fused to the SbtM1 promoter. This promoter drives expression in colonized cells, in cells neighboring apoplastically growing hyphae and in cells forming pre-penetration apparatuus (PPAs, cytoplasmic aggregations that assemble in cortex cells prior to arbuscule development) [START_REF] Genre | Prepenetration apparatus assembly precedes and predicts the colonization patterns of arbuscular mycorrhizal fungi within the root cortex of both Medicago truncatula and Daucus carota[END_REF][START_REF] Takeda | Apoplastic plant subtilases support arbuscular mycorrhiza development in Lotus japonicus[END_REF][START_REF] Takeda | Nuclear-localized and deregulated calcium-and calmodulin-dependent protein kinase activates rhizobial and mycorrhizal responses in Lotus japonicus[END_REF]. When expressed under the control of the SbtM1 promoter, secreted mCherry accumulates in the apoplast surrounding fungal structures and PPAs, thereby revealing the silhouette of these structures (Figure 2A-B, Videos 1-2). Nuclear localized YFP fluorescence indicated activity of both promoters in cells containing PPAs (c, Videos 1-2) and containing sparsely branched (d) or mature (e) arbuscules. Furthermore, we rarely detected YFP fluorescence in non-colonized cells in direct neighborhood of arbuscocytes, which were possibly preparing for PPA formation (a). However, YFP signal was absent from cells containing collapsed arbuscules (f), indicating that the promoters were active during arbuscule development and growth but inactive during arbuscule degeneration (Figure 2A-B). RAM2 promoter activity was strictly correlated with arbuscocytes, while the DIS promoter showed additional activity in cortical cells of non-colonized root segments (Figure 2A To examine, whether arbuscocyte-specific expression of DIS and RAM2 is sufficient for fungal development we complemented the dis-1 and ram2-1 mutants with the corresponding wild-type genes fused to the arbuscocyte-specific PT4 promoter [START_REF] Volpe | An AMinduced, MYB-family gene of Lotus japonicus (LjMAMI) affects root growth in an AM-independent manner[END_REF]. This restored arbusculebranching, vesicle formation as well as root length colonization in the mutants (Figure 2C-F), showing that arbuscocyte-specific expression of DIS and RAM2 suffices to support AM development. Thus, expression of lipid biosynthesis genes in arbuscocytes is not only important for arbuscule branching but also for vesicle formation and quantitative colonization.

The KASI family comprises three members in L. japonicus Growth and development of dis and ram2 mutants are not visibly affected (Figure 3-figure supplement 2), although they carry defects in important lipid biosynthesis genes. RAM2 is specific to AMcompetent plants [START_REF] Wang | A common signaling process that promotes mycorrhizal and oomycete colonization of plants[END_REF][START_REF] Delaux | Comparative phylogenomics uncovers the impact of symbiotic associations on host genome evolution[END_REF][START_REF] Favre | A novel bioinformatics pipeline to discover genes related to arbuscular mycorrhizal symbiosis based on their evolutionary conservation pattern among higher plants[END_REF][START_REF] Bravo | Genes conserved for arbuscular mycorrhizal symbiosis identified through phylogenomics[END_REF] and activated in an AM-dependent manner (Figure 2, Figure 3-figure supplement 1A) [START_REF] Gobbato | A GRAS-type transcription factor with a specific function in mycorrhizal signaling[END_REF][START_REF] Gobbato | RAM1 and RAM2 function and expression during arbuscular mycorrhizal symbiosis and Aphanomyces euteiches colonization[END_REF]. Plants contain an additional GPAT6 paralog, which likely fulfills the housekeeping function (Figure 1-figure supplement 4, [START_REF] Yang | A land-plant-specific glycerol-3phosphate acyltransferase family in Arabidopsis: substrate specificity, sn-2 preference, and evolution[END_REF][START_REF] Delaux | Algal ancestor of land plants was preadapted for symbiosis[END_REF]. To understand whether the same applies to DIS we searched the L. japonicus genome for additional KASI genes. We detected three paralogs KASI, DIS and DIS-LIKE (Figure 1-figure supplement 1D-E and Figure 1-figure supplement 2), of which only DIS was transcriptionally activated in AM roots (Figure 3-figure supplement 1A). Phylogenetic analysis revealed a split of seed plant KASI proteins into two different clades, called KASI and DIS (Figure 3). Members of the KASI clade, are presumably involved in housekeeping functions as this clade contains the product of the KASI single copy gene in Arabidopsis [START_REF] Wu | Arabidopsis b-ketoacyl-[acyl carrier protein] synthase i is crucial for fatty acid synthesis and plays a role in chloroplast division and embryo development[END_REF]. Members of the DIS clade are found specifically in AM-host dicotyledons and in a gymnosperm (Figure 3). As confirmed by synteny analysis (Figure 3figure supplement 3), DIS is absent from all eight analyzed non-host dicotyledon genomes, a phylogenetic pattern similar to other symbiosis genes [START_REF] Delaux | Comparative phylogenomics uncovers the impact of symbiotic associations on host genome evolution[END_REF][START_REF] Favre | A novel bioinformatics pipeline to discover genes related to arbuscular mycorrhizal symbiosis based on their evolutionary conservation pattern among higher plants[END_REF][START_REF] Bravo | Genes conserved for arbuscular mycorrhizal symbiosis identified through phylogenomics[END_REF]. The occurrence of DIS in Lupinus species, which lost AM competence but still form root nodule symbiosis, may be a relic from the AM competent ancestor. An apparently, Lotusspecific, and thus recent duplication of the DIS gene resulted in an 87% identical copy (DIS-LIKE) located directly adjacent to DIS in a tail-to-tail orientation (Figure 1 ). Nevertheless, because of its sequence similarity to DIS, we examined whether DIS-LIKE is also required for arbuscule formation using the dis-like-5 mutant, which suffers from a glycine replacement at position 180 at the border of a highly conserved b-sheet that likely affects protein function [START_REF] Perry | TILLING in Lotus japonicus identified large allelic series for symbiosis genes and revealed a Bias in functionally defective ethyl methanesulfonate alleles toward glycine replacements[END_REF] (Supplementary file 1, Figure 1-figure supplement 2). However, in roots of dis-like-5 AM and arbuscule development was indistinguishable from wild type (Figure 3figure supplement 1B). Therefore, DIS-LIKE might have lost its major role in arbuscule development after the duplication.

DIS functions like a canonical KASI in planta

We examined whether DIS can substitute the phylogenetically related housekeeping KASI. To this end we transgenically complemented an Arabidopsis kasI mutant [START_REF] Wu | Arabidopsis b-ketoacyl-[acyl carrier protein] synthase i is crucial for fatty acid synthesis and plays a role in chloroplast division and embryo development[END_REF] with Lotus DIS driven by the Arabidopsis KASI promoter. Arabidopsis kasI exhibits an altered FA profile and reduced rosette growth [START_REF] Wu | Arabidopsis b-ketoacyl-[acyl carrier protein] synthase i is crucial for fatty acid synthesis and plays a role in chloroplast division and embryo development[END_REF]. Complementation with DIS restored both wild-typelike rosette growth and FA accumulation. The kasI phenotypes persisted when the dis-1 mutant allele was transformed as a negative control (Figure 4C-E). In the reverse cross-species complementation AtKASI driven by the DIS promoter restored colonization, arbuscule branching and vesicle The AM-specific increase in 16:0 and 16:1v5 FA containing lipids is abolished in the dis mutant

To characterize the role of DIS in determining the lipid composition of non-colonized and colonized roots we quantified triacylglycerols (TAGs), diacylglycerols (DAGs), galactolipids and phospholipids in wild-type and dis-1. The lipid profile of colonized roots contains both plant and fungal lipids, however using the fungal marker FA 16:1w5 and previous data on fungus-specific lipids [START_REF] Wewer | Fatty acid synthesis and lipid metabolism in the obligate biotrophic fungus rhizophagus irregularis during mycorrhization of Lotus japonicus[END_REF], many fungal lipids can be clearly distinguished from plant lipids. The lipid profile of non-colonized roots was not affected by the dis-1 mutation. However, the strong and significant increase of 16:0 and 16:1 (most probably fungus-specific 16:1w5) containing TAGs, which is characteristic for colonization of wild-type roots [START_REF] Wewer | Fatty acid synthesis and lipid metabolism in the obligate biotrophic fungus rhizophagus irregularis during mycorrhization of Lotus japonicus[END_REF] was abolished in dis-1 (Figure 5A-D, Figure 5figure supplement 1B). Also, AM-and fungus-specific DAG and phospholipid molecular species were enhanced in colonized wild-type roots but not in colonized dis-1 roots (Figure 5-figure supplements 1A and 2). In contrast, galactolipids were not affected by root colonization or genotype (Figure 5-figure supplement 3). In summary, DIS affects the glycerolipid and phospholipid profile of colonized L. japonicus roots and does not interfere with lipid accumulation in the non-colonized state. Most lipids affected by the DIS mutation are fungus-specific and therefore reflect the amount of root colonization and of fungal lipid-containing vesicles. However, since the root lipid profile is hardly affected, absence of FA elongation by DIS was the cause of reduced lipid accumulation and root colonization.

RAM1, DIS, RAM2 and STR are required for accumulation of AM signature lipids

Similar to dis and ram2 L. japonicus mutants in the ABCG half-transporter STR and the GRAS protein RAM1 are affected in arbuscule branching [START_REF] Kojima | Isolation and phenotypic characterization of Lotus japonicus mutants specifically defective in arbuscular mycorrhizal formation[END_REF][START_REF] Pimprikar | A CCaMK-CYCLOPS-DELLA complex activates transcription of RAM1 to regulate Arbuscule branching[END_REF][START_REF] Xue | Network of GRAS transcription factors involved in the control of arbuscule development in Lotus japonicus[END_REF], quantitative root colonization and formation of lipid-containing fungal vesicles (Figure 5-figure supplement 4). Moreover, the AM-dependent transcriptional activation of DIS and KASIII, the latter of which is a single copy gene in L. japonicus and produces precursors for DIS-activity by catalyzing FA chain elongation from C2 to C4, was absent from ram1 mutants (Figure 6). In contrast, induction of the single copy gene KASII, which elongates fatty acyl chains from C16 to C18 was not hampered by RAM1 deficiency. Thus, RAM1 may play an important role in the regulation of lipid biosynthesis in arbuscocytes, since it also mediates expression of RAM2 and STR [START_REF] Gobbato | A GRAS-type transcription factor with a specific function in mycorrhizal signaling[END_REF][START_REF] Park | Hyphal Branching during Arbuscule Development Requires Reduced Arbuscular Mycorrhiza1[END_REF][START_REF] Pimprikar | A CCaMK-CYCLOPS-DELLA complex activates transcription of RAM1 to regulate Arbuscule branching[END_REF][START_REF] Luginbuehl | Fatty acids in arbuscular mycorrhizal fungi are synthesized by the host plant[END_REF].

We hypothesized that RAM1, DIS, RAM2 and STR form a specific operational unit for lipid biosynthesis and transport in arbuscocytes. Therefore, we directly compared their impact on the AM-specific root lipid profile and measured galactolipids, phospholipids, TAGs and also total and free fatty acids in colonized roots of ram1, dis, ram2, str mutants and wild-type in parallel. Consistent with our previous observation in dis-1, galactolipid accumulation was similar in colonized roots of wild-type and all mutants (Figure 5-figure supplement 3C-D). In contrast, total 16:0 FAs (FAMEs) as well as 16:1 and 18:1 (likely 18:1w7 FA of fungal origin) FAs were strongly reduced in all colonized mutants compared to the corresponding wild-type. Free FAs showed a similar pattern except for 18:1 FAs ). However, the two allelic ram2 mutants formed an exception. They specifically over-accumulated 16:0-16:0 FA-containing phospholipids in particular 32:0 PA and 32:0-PC but also to a smaller extend 32:0-PE and 32:0-PI (Figure 5-figure supplements 6-10). A similar pattern was observed for tri-16:0 TAGs (Figure 5F). This suggests that RAM2 acts downstream of DIS in a biosynthetic pathway and uses the 16:0 FAs synthesized by DIS in arbuscocytes as substrates. In the absence of functional RAM2 the FA products of DIS, are probably redirected into phospholipid biosynthesis and storage lipid biosynthesis via PA and PC [START_REF] Li-Beisson | Acyl-lipid metabolism[END_REF] leading to the observed higher accumulation of 16:0 FA containing lipid species in ram2 mutants. This higher accumulation of specific lipids did not correlate with colonization levels in ram2 mutants (Figure 5-figure supplement 4) confirming that reduced colonization levels are not the primary cause for altered lipid profiles in the colonized mutant roots. Instead, defective AM-specific lipid biosynthesis in the mutants more likely impairs fungal development.

The abundance of 16:0 ß-monoacyl-glycerol is reduced in all mutants

The first step in TAG and phospholipid production after FA biosynthesis is the esterification of FAs with glycerol by GPATs in the plastid or endoplasmic reticulum to produce a-MAGs (sn1/3-MAGs, [START_REF] Li-Beisson | Acyl-lipid metabolism[END_REF]. RAM2 is predicted to produce a different type of glycerolipid ß-MAG (sn2-MAG) with a preference towards 16:0 and 18:1 FAs [START_REF] Yang | A distinct type of glycerol-3-phosphate acyltransferase with sn-2 preference and phosphatase activity producing 2-monoacylglycerol[END_REF][START_REF] Wang | A common signaling process that promotes mycorrhizal and oomycete colonization of plants[END_REF][START_REF] Yang | A land-plant-specific glycerol-3phosphate acyltransferase family in Arabidopsis: substrate specificity, sn-2 preference, and evolution[END_REF]. To examine the role of RAM2 in MAG biosynthesis, we quantified a-MAG and ß-MAG species in colonized roots of wild-type and all mutants. The abundance of ß-MAGs was generally lower than that of a-MAGs (Figure 7). The amount of most a-MAG species did not differ among the genotypes. Only the fungus-specific 16:1 and 18:1w7 a-MAGs were reduced in all mutants reflecting the lower fungal biomass (Figure 7A). Fungus-specific ß-MAGs with 16:1 and 18:1w7 acyl groups were not detected and most ß-MAG molecular species accumulated to similar levels in all genotypes. Exclusively the levels of 16:0 ß-MAGs were significantly lower in all mutants as compared to the corresponding wild-type roots (Figure 7B). This supports a role of RAM2 in 16:0 ß-MAG synthesis during AM and a role of DIS in providing 16:0 FA precursors for RAM2 activity. A low accumulation, of 16:0 ß-MAGs in ram1 mutants is consistent with RAM1's role in regulating the FA and lipid biosynthesis genes (Figure 6) [START_REF] Gobbato | A GRAS-type transcription factor with a specific function in mycorrhizal signaling[END_REF][START_REF] Pimprikar | A CCaMK-CYCLOPS-DELLA complex activates transcription of RAM1 to regulate Arbuscule branching[END_REF]. In str 16:0 ß-MAGs likely did not accumulate because of reduced RAM2 expression in str roots due to low root length colonization and/or a regulatory feedback loop [START_REF] Bravo | Arbuscular mycorrhiza-specific enzymes FatM and RAM2 fine-tune lipid biosynthesis to promote development of arbuscular mycorrhiza[END_REF]. DIS, RAM2 and STR are required for transfer of 13 C label from plant to fungus

In plants, ß-MAGs serve as precursors for cutin polymers at the surface of aerial organs [START_REF] Yang | A land-plant-specific glycerol-3phosphate acyltransferase family in Arabidopsis: substrate specificity, sn-2 preference, and evolution[END_REF][START_REF] Yeats | The identification of cutin synthase: formation of the plant polyester cutin[END_REF]. For their use in membrane or storage lipid biosynthesis they first need to be isomerized to a-MAGs [START_REF] Li-Beisson | Acyl-lipid metabolism[END_REF]. The recruitment of a GPAT6 (RAM2) instead of a a-MAG-producing GPAT for AM-specific lipid synthesis supports the idea that RAM2-products are destined for something else than membrane biosynthesis of the host cell. Since AM fungal genomes lack genes encoding cytosolic FA synthase subunits [START_REF] Wewer | Fatty acid synthesis and lipid metabolism in the obligate biotrophic fungus rhizophagus irregularis during mycorrhization of Lotus japonicus[END_REF][START_REF] Ropars | Evidence for the sexual origin of heterokaryosis in arbuscular mycorrhizal fungi[END_REF]; [START_REF] Tang | A survey of the Gene repertoire of Gigaspora rosea unravels conserved features among Glomeromycota for Obligate Biotrophy[END_REF] we hypothesized that 16:0 ß-MAGs synthesized by DIS-and RAM2 are predominantly delivered to the fungus. To test this hypothesis, we examined lipid transfer by FA isotopolog profiling. Isotopologs are molecules that differ only in their isotopic composition. For isotopolog profiling an organism is fed with a heavy isotope labelled precursor metabolite. Subsequently the labelled isotopolog composition of metabolic products is analyzed. The resulting characteristic isotopolog pattern yields information about metabolic pathways and fluxes [START_REF] Ahmed | Isotopo' a database application for facile analysis and management of mass isotopomer data[END_REF].

G i f u d i s -1 r a m 1 -3 r a m 1 -4 d i s -4 r a m 2 -1 r a m 2 -2 W T -M G 2 0 s t r TAG [nmol/mg FW]
We could not detect fungus-specific 16:1w5 ß-MAGs in colonized roots (Figure 7B). Therefore, we reasoned that either a downstream metabolite of ß-MAG is transported to the fungus, or alternatively, ß-MAG is rapidly metabolized in the fungus prior to desaturation of the 16:0 acyl residue. Since the transported FA groups can be used by the fungus for synthesizing a number of different lipids, we focused on total 16:0 FA methyl esters (FAMEs, subsequently called FAs for simplicity) and 16:1w1 FAMEs as markers for lipid transfer. We fed L. japonicus wild-type, dis-1, ram2-1 and str with [U-13 C 6 ]glucose and then measured the isotopolog composition of 16:0 FAs and 16:1w5 FAs in L. japonicus roots and in associated extraradical fungal mycelium with spores. To generate sufficient hyphal material for our measurements the fungus was pre-grown on split Petri dishes in presence of a carrot hairy root system as nurse plant (Figure 8-figure supplement 1). Once the fungal mycelium had covered the plate, L. japonicus seedlings were added to the plate on the side opposing the carrot root. During the whole experiment, the fungus was simultaneously supported by the carrot hairy root and the L. japonicus seedling. Once the L. japonicus roots had been colonized, labelled glucose was added to the side containing L. japonicus. After an additional week, FAs were esterified and extracted from colonized L. japonicus roots and from the associated extraradical mycelium and the total amount of 13 C labelled 16:0 and 16:1w5 FAs as well as their isotopolog composition was determined. In L. japonicus wild-type 13 C-labeled 16:0 and 16:1w5 FAs were detected in colonized

Host plants determine the isotopolog pattern of fungal FAs

Remarkably, the isotopolog profile of 16:0 FAs was close to identical between colonized L. japonicus roots and the connected extraradical mycelium, for 11 independent samples of wild-type Gifu (Figure 8C

Relative fraction of isotopologs [%]

1 3 .0 2 1 2 .9 0 1 6 .5 9 2 0 .8 5 1 6 .6 7 2 7 .5 6 5 2 .6 8 3 1 .4 0 2 9 .6 3 5 9 .8 6 3 7 .4 3 3 5 .2 8 4 5 .8 2 2 4 .3 8 2 1 .9 2

M R M R M R R R R M R M R M R Lotus WT 2 0 .
M R M R M R R R R M R M R M R dis-1 2 6 .
M R M R M R R R R M R M R M R ram2-1 control AM o.e [%]
2 0 .0 9 1 6 .6 2 1 5 .5 3 1 0 .9 4 9 .2 0 1 3 .1 3 0 .0 3 0 .0 4 0 .2 4 0 .1 0 .1 2 0 .9 0 .0 8 0 .0 6 0 .1 3 In these root organ cultures, sugar uptake from the medium does not compete with photosynthesis, as in whole seedlings. Additionally, the carrot roots explore a larger surface of the Petri dish, increasing access to substances in the nutrient medium. Consequently, and likely because of increased uptake of labelled glucose from the medium, the isotopolog pattern of carrot ROCs differed from Lotus and was shifted towards more highly labeled 16:0 FA isotopologs. This fingerprint was again recapitulated in the extraradical fungal mycelium as well as in fungus-specific 16:1w5 FAs inside and outside the root for 10 independent samples (Figure 8C, Figure 8-figure supplement 4). These data provide strong support for direct transfer of a 16:0 FA containing lipid from plants to AMF (Figure 9).

o.e [%] o.e [%] AM R R R R M R M R M R M R M R M D carrot (

Discussion

Here we identified DIS and RAM2, two AM-specific paralogs of the lipid biosynthesis genes KASI and GPAT6 using forward genetics in Lotus japonicus. The dis and ram2 mutants enabled us to demonstrate lipid transfer from plants to AMF using isotopolog profiling.

During AM symbiosis, an array of lipid biosynthesis genes is induced in arbuscocytes (Gaude et al., 2012a(Gaude et al., , 2012b)), indicating a large demand for lipids in these cells. Indeed, two genes encoding lipid biosynthesis enzymes, the thioesterase FatM and the GPAT6 RAM2, have previously been shown to be required for arbuscule branching in M. truncatula [START_REF] Wang | A common signaling process that promotes mycorrhizal and oomycete colonization of plants[END_REF][START_REF] Bravo | Arbuscular mycorrhiza-specific enzymes FatM and RAM2 fine-tune lipid biosynthesis to promote development of arbuscular mycorrhiza[END_REF][START_REF] Jiang | Plants transfer lipids to sustain colonization by mutualistic mycorrhizal and parasitic fungi[END_REF]. Both enzymes have a substrate preference for 16:0 FAs [START_REF] Salas | Characterization of substrate specificity of plant FatA and FatB acyl-ACP thioesterases[END_REF][START_REF] Yang | A land-plant-specific glycerol-3phosphate acyltransferase family in Arabidopsis: substrate specificity, sn-2 preference, and evolution[END_REF][START_REF] Bravo | Arbuscular mycorrhiza-specific enzymes FatM and RAM2 fine-tune lipid biosynthesis to promote development of arbuscular mycorrhiza[END_REF] and, consistent with this, we and others observed that colonized ram2 mutant roots over-accumulate 16:0 FA containing phospholipids and TAGs (Figure 7, [START_REF] Bravo | Arbuscular mycorrhiza-specific enzymes FatM and RAM2 fine-tune lipid biosynthesis to promote development of arbuscular mycorrhiza[END_REF]), indicating re-channeling of superfluous 16:0 FAs in the absence of RAM2 function and placing RAM2 downstream of FatM (Figure 9).

Our discovery of DIS, a novel and AM-specific KASI gene, now provides evidence for the enzyme which synthesizes these 16:0 FAs in arbuscocytes. The arbuscule phenotype, as well as the lipid profile of colonized dis mutants is very similar to fatm and ram2 mutants except for the accumulation of 16:0 FA-containing lipids in ram2 (Figure 1, Figure 5 and all figure supplements), consistent with the predicted function. Together, this strongly suggests that DIS, FatM and RAM2 act in the same lipid biosynthesis pathway, which is specifically and cell-autonomously induced when a resting root cortex cell differentiates into an arbuscocyte (Figure 2A-B, Figure 9, [START_REF] Bravo | Arbuscular mycorrhiza-specific enzymes FatM and RAM2 fine-tune lipid biosynthesis to promote development of arbuscular mycorrhiza[END_REF]). Interestingly, DIS was exclusively found in genomes of AM-competent dicotyledons and a gymnosperm (Figure 3). This implies that DIS has been lost at the split of the mono-from dicotyledons. Despite the phylogenetic divergence, DIS and the single copy housekeeping KASI gene of Arabidopsis are interchangeable (Figure 5). Therefore, the specificity of DIS to function in AM symbiosis is probably encoded in its promoter (Figure 2). In monocotyledons, the promoter of the housekeeping KASI gene may have acquired additional regulatory elements, sufficient for arbuscocyte-specific activation, thus making DIS dispensable.

We provide several pieces of complementary evidence that lipids synthesized by DIS and RAM2 in the arbuscocyte are transferred from plants to AMF and are required for fungal development. We fed host plants with [U-13 C 6 ]glucose and subsequently determined the isotopolog profile of freshly synthetized 16:0 and 16:1w5 FAs in roots and associated fungal extraradical mycelia (Figure 8). This showed that: (1) AMF were unable to incorporate 13 C into FAs when fed with [U-13 C 6 ]glucose in absence of the host plant. ( 2) When associated with a wild-type host, the fungal extraradical mycelium accumulated 13 C labelled 16:0 FAs and the isotopolog profile of these 16:0 FAs was almost identical with the host profile. (3) The 16:0 FA isotopolog fingerprint differed strongly between two different wild-type plant systems (Lotus seedling and carrot hairy root) but for each of them the fungal mycelium recapitulated the isotopolog profile. Therefore clearly, the plant dominates the profile of the fungus, because it is impossible that the fungus by itself generates the same FA isotopolog pattern as the plant -especially in the absence of cytosolic FA synthase. Therefore, this result provides compelling evidence for interkingdom transfer of 16:0 FAs from plants to AMF. (4) In agreement, the isotopolog profile of fungus-specific 16:1 w5 FAs inside and outside the root also resembled the plant 16:0 FA profile. ( 5) Colonized dis and ram2 mutant roots resembled the 16:0 FA isotopolog profile of L. japonicus wild-type roots. However, the 16:0 FA profile of the fungal extraradical mycelium and the 16:1w5 FA profile inside the roots showed a very different pattern, consistent with very low transport of labelled FAs to the fungus when associated with the mutants. ( 6) DIS and RAM2 are specifically required for the synthesis of 16:0 ß-MAG (Figure 7) and the predominant FA chain length found in AM fungi is precisely 16. ( 7) dis and ram2 roots do not allow the formation of lipid-containing fungal vesicles and accumulate very low levels of fungal signature lipids (Figure 5 and figure supplements). Together this strongly supports the idea that DIS and RAM2 are required to provide lipids for transfer to the fungus. Consequently, in the mutants, the fungus is deprived of lipids.

The L. japonicus mutants were originally identified due to their defective arbuscule branching [START_REF] Groth | Two Lotus japonicus symbiosis mutants impaired at distinct steps of arbuscule development[END_REF]. The promoters of DIS and RAM2 are active in arbusocytes and already during PPA formation, the earliest visible stage of arbuscocyte development. Together with the stunted arbuscule phenotype of dis, ram2 and fatm mutants (Figure 1 [START_REF] Bravo | Arbuscular mycorrhiza-specific enzymes FatM and RAM2 fine-tune lipid biosynthesis to promote development of arbuscular mycorrhiza[END_REF]) this suggests that plant lipids are needed for arbuscule growth, probably to provide material for the extensive plasma-membrane of the highly branched fungal structure. It also indicates that the arbuscule dictates development of the AMF as a whole, since lipid uptake at the arbuscule is required for vesicle formation, full exploration of the root and development of extraradical mycelia and spores. Defective arbuscule development was also observed for the different and phylogenetically distantly related AMF Gigaspora rosea [START_REF] Groth | Two Lotus japonicus symbiosis mutants impaired at distinct steps of arbuscule development[END_REF], which similar to R. irregularis lacks genes encoding cytosolic FA synthase from their genomes [START_REF] Wewer | Fatty acid synthesis and lipid metabolism in the obligate biotrophic fungus rhizophagus irregularis during mycorrhization of Lotus japonicus[END_REF][START_REF] Tang | A survey of the Gene repertoire of Gigaspora rosea unravels conserved features among Glomeromycota for Obligate Biotrophy[END_REF]. Hence the dependence on plant lipids delivered at the arbuscule is likely a common phenomenon among AMF and a hallmark of AMF obligate biotrophy.

Despite the obvious central importance of lipid uptake by the arbuscule, the fungus can initially colonize the mutant roots with a low amount of intraradical hyphae and stunted arbuscules (Figure 1, Figure 5-figure supplement 4). The construction of membranes for this initial colonization may be supported by the large amounts of lipids stored in AMF spores. This would be consistent with the frequent observation that in wild-type roots, at initial stages of root colonization, AMF form from the plant cell to the fungus. The PAM-localized ABCG transporter STR/STR2 is a hypothetical candidate for lipid transport across the PAM. Desaturation of 16:0 FAs by fungal enzymes [START_REF] Wewer | Fatty acid synthesis and lipid metabolism in the obligate biotrophic fungus rhizophagus irregularis during mycorrhization of Lotus japonicus[END_REF] leads to accumulation of lipids containing specific 16:1w5 FAs. Mal-CoA, Malonyl-Coenzyme A; FA, fatty acid; KAS, b-keto-acyl ACP synthase; GPAT, Glycerol-3-phosphate acyl transferase; PAM, periarbuscular membrane; LPA, lysophosphatic acid; MAG, monoacylglycerol; DAG, diacylglycerol; TAG, triacylglycerol; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; CDP-DAG, cytidine diphosphate diacylglycerol; PG, phosphatidylglycerol; PI, phosphatidylinositol. DOI: 10.7554/eLife.29107.043 arbuscules immediately after reaching the inner cortex and before colonizing longer distances, possibly as a strategy to accquire lipids quickly after the reserves in the spore have been depleted. Alternatively, it is possible that plant housekeeping enzymes provide lipids to intraradical hyphae before arbuscule formation. Activity of the housekeeping KASI may also be responsible for slightly higher colonization levels observed for dis in some experiments as compared to other mutants.

It has recently been reported that photosynthetic wild-type nurse plants can restore arbusculebranching in Medicago ram2 and str mutants [START_REF] Jiang | Plants transfer lipids to sustain colonization by mutualistic mycorrhizal and parasitic fungi[END_REF][START_REF] Luginbuehl | Fatty acids in arbuscular mycorrhizal fungi are synthesized by the host plant[END_REF], suggesting that lipids can be supplied to arbuscules via the extraradical hyphal network and intraradical hyphae through this route support arbuscule fine-branching. Based on four observations, we favor an alternative szenario, in which lipids need to be provided cell-autonomously by the arbuscocyte to support arbuscule fine-branching. However, we cannot exclude that our observations differ from the reported observations due to growth conditions or plant species. (1) Presence of nurse carrot hairy roots did not restore arbuscule branching in dis, ram2 and str (Figure 8-figure supplement 1C-F).

(2) dis and ram2 were found in a forward genetics screen based on their stunted arbuscule phenotype. In this screen, the fungal inoculum was provided via chive nurse plants [START_REF] Groth | Two Lotus japonicus symbiosis mutants impaired at distinct steps of arbuscule development[END_REF]. (3) Map-based cloning of Lotus dis, ram2 and str [START_REF] Kojima | Isolation and phenotypic characterization of Lotus japonicus mutants specifically defective in arbuscular mycorrhizal formation[END_REF] was performed with segregating mutant populations grown in the same pot, in which the wild-type and heterozygeous siblings acted as nurse plants on the homozygeous mutants. In this system, the stunted arbuscule phenotype was easily observable. (4) Arbuscule branching in a rice str mutant was not restored by wild type nurse plants [START_REF] Gutjahr | The half-size ABC transporters STR1 and STR2 are indispensable for mycorrhizal arbuscule formation in rice[END_REF].

It still remains to be shown, which types of lipids are transported from the plant arbuscocyte to the fungal arbuscule and how. RAM2 is the most downstream acting enzyme in arbuscocyte-specific lipid biosynthesis known to date (Figure 9). It is predicted to synthesize ß-MAG and we and others have shown that 16:0 ß-MAGs are indeed reduced in colonized roots of dis, fatm and ram2 mutants, providing evidence that this is likely the case (Figure 7, [START_REF] Bravo | Arbuscular mycorrhiza-specific enzymes FatM and RAM2 fine-tune lipid biosynthesis to promote development of arbuscular mycorrhiza[END_REF]). Although, we cannot exclude that a downstream metabolite of 16:0 ß-MAG is transported to the fungus, 16:0 ß-MAG as transport vehicle for 16:0 FAs to the fungus is a good candidate because conceptually this molecule may bear certain advantages. It has been shown in Arabidopsis that ß-MAGs are not used for plant storage or membrane lipid biosynthesis but rather as pre-cursors for cuticle formation [START_REF] Li | Monoacylglycerols are components of root waxes and can be produced in the aerial cuticle by ectopic expression of a suberin-associated acyltransferase[END_REF]. The production of ß-MAGs could therefore, be a way, to withdraw FAs from the plants own metabolism to make them available to the fungus. In addition, ß-MAGs are small and amphiphilic and could diffuse across the short distance of the hydrophilic apoplastic space between plant and fungal membrane. At the newly growing arbuscule branches the distance between the plant and fungal membrane is indeed very small and has been measured to be 80-100 nm on TEM images of high-pressure freeze-substituted samples [START_REF] Bonfante | At the interface between mycorrhizal fungi and plants: the structural organization of cell wall, plasma membrane and cytoskeleton[END_REF]. However, we could not detect fungusspecific 16:1w5 ß-MAGs in colonized roots. This could mean that the fungus metabolizes them before desaturation of the 16:0 FAs to synthesize membrane and storage lipids. Alternatively, ß-MAGs may not be taken up by the fungus. ß-MAGs are known to isomerize to a-MAGs in acid or basic conditions [START_REF] Iqbal | Intestinal lipid absorption[END_REF]. It is therefore, possible that they isomerize in the acidic periarbuscular space [START_REF] Guttenberger | Arbuscules of vesicular-arbuscular mycorrhizal fungi inhabit an acidic compartment within plant roots[END_REF] before being taken up by the arbuscule.

How are MAGs transported across the peri-arbuscular membrane? Good candidates for MAG transporters are the ABCG half transporters STR and STR2. Similar ABCG transporters have been implicated genetically in cuticle formation, which also requires ß-MAGs [START_REF] Pighin | Plant cuticular lipid export requires an ABC transporter[END_REF][START_REF] Panikashvili | The Arabidopsis ABCG13 transporter is required for flower cuticle secretion and patterning of the petal epidermis[END_REF][START_REF] Yeats | The identification of cutin synthase: formation of the plant polyester cutin[END_REF]. The half ABCG transporters STR and STR2 are both independently required for arbuscule branching and they need to interact to form a full transporter [START_REF] Zhang | Two Medicago truncatula half-ABC transporters are essential for arbuscule development in arbuscular mycorrhizal symbiosis[END_REF]. We found that colonized roots of a L. japonicus str mutant, did not allow the formation of fungal vesicles and had the same lipid profile as dis and ram2 (Figure 5 and figure supplements). Furthermore, our 13 C labelling experiment demonstrated that str mutants do not transfer lipids to the fungus (Figure 8-figure supplement 2). Although these are encouraging indications, strong evidence for the role of STR in lipid transport across the periarbuscular membrane is still lacking and the substrate of STR remains to be determined. Therefore, currently, it cannot be excluded that mutation of str has an indirect effect on lipid transport and alternative mechanisms for example lipid translocation via vesicle fission and fusion are possible. Nevertheless, also in AMF, several ABC transporter genes are expressed in planta [START_REF] Tisserant | The transcriptome of the arbuscular mycorrhizal fungus Glomus intraradices (DAOM 197198) reveals functional tradeoffs in an obligate symbiont[END_REF][START_REF] Tang | A survey of the Gene repertoire of Gigaspora rosea unravels conserved features among Glomeromycota for Obligate Biotrophy[END_REF]. They are not characterized, but if lipid transport via ABC transporters instead of other mechanisms would play a role, some of them could be involved in uptake of lipids into the fungal cytoplasm.

We found that mutants in the GRAS gene RAM1 are impaired in AM-specific lipid accumulation in colonized roots and in AM-mediated activation of DIS and the single copy gene KASIII (Figure 6), in addition to FatM, RAM2 and STR [START_REF] Wang | A common signaling process that promotes mycorrhizal and oomycete colonization of plants[END_REF][START_REF] Park | Hyphal Branching during Arbuscule Development Requires Reduced Arbuscular Mycorrhiza1[END_REF][START_REF] Pimprikar | A CCaMK-CYCLOPS-DELLA complex activates transcription of RAM1 to regulate Arbuscule branching[END_REF][START_REF] Luginbuehl | Fatty acids in arbuscular mycorrhizal fungi are synthesized by the host plant[END_REF]. This suggests that plants have evolved an AM-specific regulatory module for lipid production in arbuscocytes and delivery to the fungus. It remains to be shown, whether RAM1 regulates lipid biosynthesis genes directly and how this occurs mechanistically.

Our finding that plants transfer lipids to AMF completely changes the previous view that the fungus receives only sugars from the plant [START_REF] Pfeffer | Carbon uptake and the metabolism and transport of lipids in an arbuscular mycorrhiza[END_REF][START_REF] ´panier | Dependence of arbuscularmycorrhizal fungi on their plant host for palmitic acid synthesis[END_REF]. It will now be interesting to determine the relative contributions of sugar and lipid transfer to AMF, and whether this may be a determinant of variation in root length colonization and extraradical mycelium formation depending on the plant-fungal genotype combination [START_REF] Sawers | Phosphorus acquisition efficiency in arbuscular mycorrhizal maize is correlated with the abundance of root-external hyphae and the accumulation of transcripts encoding PHT1 phosphate transporters[END_REF]. An interesting question refers to why AMF have lost the genes encoding cytosolic FA synthase to depend on the lipid biosynthesis machinery of the host. FA biosynthesis consumes more energy than biosynthesis of carbohydrates and organic carbon provided by the plant needs to be transported in fungal hyphae over long distances from the inside of the root to the extremities of the extraradical mycelium. Therefore, it is conceivable that supply of plant lipids to the fungus plus fungal lipid transport is more energy efficient for the symbiosis as a whole than fungal carbohydrate transport plus fungal lipid biosynthesis. Hence, inter-organismic lipid transfer followed by loss of fungal FA biosynthesis genes may have been selected for during evolution because it likely optimized the symbiosis for most rapid proliferation of extraradical mycelium, thus ensuring efficient mineral nutrient acquisition from the soil for supporting the plant host. Lipid transfer across kingdoms has also been observed in human parasites or symbiotic bacteria of insects [START_REF] Caffaro | Evidence for host cells as the Major contributor of lipids in the intravacuolar network of Toxoplasma-infected cells[END_REF][START_REF] Elwell | Chlamydia trachomatis co-opts GBF1 and CERT to acquire host sphingomyelin for distinct roles during intracellular development[END_REF][START_REF] Herren | Insect endosymbiont proliferation is limited by lipid availability[END_REF]. It will be interesting to learn whether this is a more widespread phenomenon among biotrophic inter-organismic interactions.

Materials and methods

Plant growth and inoculation with AM fungi

Lotus japonicus ecotype and ecotype MG-20 wild-type and str mutant (kindly provided by Tomoko Kojima (NARO, Tochigi, Japan) seeds were scarified and surface sterilized with 1% NaClO. Imbibed seeds were germinated on 0.8% Bacto Agar (Difco) at 24 ˚C for 10-14 days. Seedlings were cultivated in pots containing sand/vermiculite (2/1 vol.) as substrate. For colonization with Rhizophagus irregularis roots were inoculated with 500 spores (SYMPLANTA, Munich, Germany or Agronutrition, Toulouse, France) per plant. Plants were harvested 5 weeks post inoculation (wpi); except for dis-1 complementation in Figure 1A, which was harvested at 4 wpi. Arabidopsis thaliana seeds of Col-0 wild-type, kasI mutant in the Col-0 background and the transgenically complemented kasI mutant were surface sterilized with 70% EtOH +0.05% Tween 20% and 100% EtOH, germinated on MS-Medium for 48 hr at 4 ˚C in the dark followed by 5-6 days at 22 ˚C (8 hr light/dark).

Identification of DIS by map-based cloning and next generation sequencing

The L. japonicus dis mutant (line SL0154, [START_REF] Groth | Two Lotus japonicus symbiosis mutants impaired at distinct steps of arbuscule development[END_REF]) resulting from an EMS mutagenesis program [START_REF] Perry | A TILLING reverse genetics tool and a web-accessible collection of mutants of the legume Lotus japonicus[END_REF][START_REF] Perry | TILLING in Lotus japonicus identified large allelic series for symbiosis genes and revealed a Bias in functionally defective ethyl methanesulfonate alleles toward glycine replacements[END_REF] was backcrossed to ecotype Gifu wild-type and outcrossed to the polymorphic mapping parent ecotype MG-20. The dis locus segregated as a recessive monogenic trait and was previously found to be linked to marker TM2249 on chromosome 4 [START_REF] Groth | Two Lotus japonicus symbiosis mutants impaired at distinct steps of arbuscule development[END_REF]. We confirmed the monogenic segregation as 26 of 110 individuals originating from the cross to MG-20 (c 2 : P(3:1)=0.74) and 32 of 119 individuals originating from the cross to Gifu (c 2 : P(3:1) =0.63) exhibited the mutant phenotype. To identify SL0154-specific mutations linked to the dis locus, we employed a genome re-sequencing strategy. Nuclear DNA of Gifu wild-type and the SL0154 mutant was subjected to paired end sequencing (2 Â 100 bp) of a 300-500 bp insert library, on an Illumina Hi-Seq 2000 instrument resulting in between 16.7 and 19.5 Gigabases per sample, equivalent to roughly 35-41 fold coverage assuming a genome size of 470 Megabases. Reads were mapped to the reference genome of MG-20 v2.5 [START_REF] Sato | Genome structure of the legume, Lotus japonicus[END_REF] and single nucleotide polymorphisms identified using CLC genomics workbench (CLC bio, Aarhus, Denmark). SL0154-specific SNPs were identified by subtracting Gifu/MG-20 from SL0154/MG-20 polymorphisms. 19 potentially EMS induced (11x G->A, 8x C->T) SNPs called consistently in all mapped reads from SL0514 but not in Gifu were identified between the markers TM0046/TM1545, the initial dis target region (Figure 1-figure supplement 1A. In a screen for recombination events flanking the dis locus, 63 mutants out of 254 total F2 individuals of a cross MG-20 x SL0154 were genotyped with markers flanking the dis locus (Figure 1-figure supplement 1B). Interrogating recombinant individuals with additional markers in the region narrowed down the target interval between TM2249 and BM2170 (2 cM according to markers; ca. 650 kb). In this interval, 3 SL0154-specific SNPs with typical EMS signature (G to A transition) remained, of which one was predicted to be located in exon 3 of CM0004.1640.r2 (reference position 40381558 in L. japonicus genome version 2.5; http://www. kazusa.or.jp/lotus/), a gene annotated as ketoacyl-(acyl carrier protein) synthase. This co-segregation together with phenotyping of one additional mutant allele obtained through TILLING (Supplementary file 1, [START_REF] Perry | A TILLING reverse genetics tool and a web-accessible collection of mutants of the legume Lotus japonicus[END_REF][START_REF] Perry | TILLING in Lotus japonicus identified large allelic series for symbiosis genes and revealed a Bias in functionally defective ethyl methanesulfonate alleles toward glycine replacements[END_REF]) as well as transgenic complementation (Figure 1A)) confirmed the identification of the mutation causing the dis phenotype of the SL0514 line. The two remaining mutations in the target region were located in a predicted intron of chr4. CM0004.1570.r2.a, a cyclin-like F-box protein (reference position: 40356684) and in a predicted intergenic region (reference position: 40364479). Untranslated regions of DIS and DIS-LIKE were determined using the Ambion FirstChoice RLM RACE kit according to manufacturer's instructions (http://www.ambion.de/). DIS sequence information can be found under the NCBI accession number KX880396.

Identification of RAM2 by map-based cloning and Sanger sequencing

The L. japonicus Gifu mutant reduced and degenerate arbuscules (red, line SL0181-N) resulting from an EMS mutagenesis [START_REF] Perry | A TILLING reverse genetics tool and a web-accessible collection of mutants of the legume Lotus japonicus[END_REF][START_REF] Perry | TILLING in Lotus japonicus identified large allelic series for symbiosis genes and revealed a Bias in functionally defective ethyl methanesulfonate alleles toward glycine replacements[END_REF] was outcrossed to the ecotype MG-20 and previously reported to segregate for two mutations, one on chromosome 1 and one on chromosome 6 [START_REF] Groth | Two Lotus japonicus symbiosis mutants impaired at distinct steps of arbuscule development[END_REF]. They were separated by segregation and the mutation on chromosome 1 was previously found in the GRAS transcription factor gene REDUCED ARBUSCULAR MYCORRHIZA 1 (RAM1) [START_REF] Pimprikar | A CCaMK-CYCLOPS-DELLA complex activates transcription of RAM1 to regulate Arbuscule branching[END_REF]. A plant from the F2 population, which showed wild-type phenotype but was heterozygous for the candidate interval on chromosome 6 and homozygous Gifu for the candidate interval on chromosome 1 was selfed for producing an F3. The F3 generation segregated for only one mutation as 38 out of 132 individuals exhibited the mutant phenotype (c 2 : P(3:1)=0.68). A plant from the F3 population, which displayed wild-type phenotype but was heterozygous for the candidate interval on chromosome 6 was selfed for producing an F4. The F4 generation also segregated for only one mutation as 17 out of 87 individuals exhibited the mutant phenotype (c 2 : P(3:1) =0.76). To identify the mutation on chromosome 6 linked to the previously identified interval [START_REF] Groth | Two Lotus japonicus symbiosis mutants impaired at distinct steps of arbuscule development[END_REF], we employed additional markers for fine mapping in F3 segregating and F4 mutant populations. This positioned the causative mutation between TM0082 and TM0302 (Figure 1-figure supplement 3A). Due to a suppression of recombination in this interval we could not get closer to the mutation and also next generation sequencing (see [START_REF] Pimprikar | A CCaMK-CYCLOPS-DELLA complex activates transcription of RAM1 to regulate Arbuscule branching[END_REF] for the methodology) failed to identify a causative mutation. The Medicago truncatula ram2 mutant displays stunted arbuscules similar to our mutant [START_REF] Wang | A common signaling process that promotes mycorrhizal and oomycete colonization of plants[END_REF]. L. japonicus RAM2 had not been linked to any chromosome but was placed on chromosome 0, which prevented identification of a RAM2 mutation in the target interval on chromosome 6. Therefore, we sequenced the RAM2 gene by Sanger sequencing. Indeed, mutants with stunted arbuscule phenotype in the F3 and F4 generation carried an EMS mutation at base 1663 from G to A leading to amino acid change from Glycine to Glutamic acid, which co-segregated with the mutant phenotype (Figure 1-figure supplement 3B-C). An additional allelic mutant ram2-2 (Figure 1-figure supplement 3B) caused by a LORE1 retrotransposon insertion [START_REF] Małolepszy | The LORE1 insertion mutant resource[END_REF] and transgenic complementation with the wildtype RAM2 gene confirmed that the causative mutation affects RAM2 (Figure 1B). Untranslated regions of RAM2 were determined using the Ambion FirstChoice(R) RLM RACE kit according to manufacturer's instructions (http://www.ambion.de/). A 1345 bp long sequence upstream of ATG was available from the http://www.kazusa.or.jp/lotus/blast.html. To enable cloning a 2275 bp promoter fragment upstream of ATG of RAM2 the remaining upstream sequence of 1047 bp was determined by primer walking on TAC Lj T46c08. L. japonicus RAM2 sequence information can be found under the NCBI accession number KX823334 and the promoter sequence under the number KX823335.

Sequence alignement and phylogeny L. japonicus KASI, DIS, DIS-LIKE, RAM2, Lj1g3v2301880.1 (GPAT6) protein sequences were retrieved from Lotus genome V2.5 and V3.0 respectively (http://www.kazusa.or.jp/lotus/) and A. thaliana KASI, E. coli KASI, E. coli KASII, M. truncatula RAM2 and Medtr7g067380 (GPAT6) were obtained from NCBI (http://www.ncbi.nlm.nih.gov). The sequences from L. japonicus were confirmed with a genome generated by next generation sequencing in house. Protein alignment for DIS was performed by CLC Main Workbench (CLC bio, Aarhus, Denmark). The Target Peptide was predicted using TargetP 1.0 Server (www.cbs.dtu.dk/services/TargetP-1.0/). RAM2 Protein alignment was performed by MEGA7 using ClustalW. The percentage identity matrix was obtained by Clustal Omega (http://www.ebi.ac.uk/Tools/msa/clustalo/).

To collect sequences for phylogeny construction corresponding to potential DIS orthologs, Lotus DIS and KASI (outgroup) protein sequences were searched in genome and transcriptome datasets using BLASTp and tBLASTn respectively. The list of species and the databases used are indicated in Figure 3-source data 1. Hits with an e-value >10 À50 were selected for the phylogenetic analysis. Collected sequences were aligned using MAFFT (http://mafft.cbrc.jp/alignment/server/) and the alignment manually checked with Bioedit. Phylogenetic trees were generated by Neighbor-joining implemented in MEGA5 [START_REF] Tamura | MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods[END_REF]. Partial gap deletion (95%) was used together with the JTT substitution model. Bootstrap values were calculated using 500 replicates.

Synteny analysis

A ~200 kb sized region in the L. japonicus genome containing the DIS locus (CM00041640.r2.a) was compared to the syntenic region in A. thaliana (Col-0) using CoGe Gevo (https://genomevolution. org/CoGe/GEvo.pl - [START_REF] Lyons | Finding and comparing syntenic regions among Arabidopsis and the outgroups papaya, poplar, and grape: coge with rosids[END_REF] as described in [START_REF] Delaux | Comparative phylogenomics uncovers the impact of symbiotic associations on host genome evolution[END_REF]. Loci encompassing DIS orthologs from Medicago truncatula, Populus trichocarpa, Carica papaya, Phaseolus vulgaris and Solanum lycopersicum were added as controls.

AM staining and quantification

Rhizophagus irregularis in colonized L. japonicus roots was stained with acid ink [START_REF] Vierheilig | Ink and vinegar, a simple staining technique for arbuscularmycorrhizal fungi[END_REF]. Root length colonization was quantified using a modified gridline intersect method [START_REF] Mcgonigle | A new method which gives an objective measure of colonization of roots by vesicular-arbuscular mycorrhizal fungi[END_REF]. For confocal laser scanning microscopy (CLSM) fungal structures were stained with 1 mg WGA Alexa Fluor 488 (Molecular Probes, http://www.lifetechnologies.com/) [START_REF] Panchuk-Voloshina | Alexa dyes, a series of new fluorescent dyes that yield exceptionally bright, photostable conjugates[END_REF].

Microscopy

For quantification of AM colonization in L. japonicus roots a light microscope (Leica) with a 20x magnification was used. For observation of GUS-staining in L. japonicus hairy roots an inverted microscope (Leica DMI6000 B) was used with 10x and 20x magnification. Transformed roots were screened by stereomicroscope (Leica MZ16 FA) using an mCherry fluorescent transformation marker or the pSbtM1:mCherry marker for fungal colonization (for Figure 2A andB). Confocal microscopy (Leica SP5) for WGA-AlexaFluor488 detection using 20x and 63x magnification was performed as described [START_REF] Groth | NENA, a Lotus japonicus homolog of Sec13, is required for rhizodermal infection by arbuscular mycorrhiza fungi and rhizobia but dispensable for cortical endosymbiotic development[END_REF]. Transgenic roots showing mCherry fluorescence signal due to SbtM1 promoter activity linked with fungal colonization were cut into pieces immediately after harvesting. The living root pieces were placed on a glass slide with a drop of water, covered by a cover slip and immediately subjected to imaging. Sequential scanning for the YFP and RFP signal was carried out simultaneously with bright field image acquisition. YFP was excited with the argon ion laser 514 nm and the emitted fluorescence was detected from 525 to 575 nm; RFP was excited with the Diode-Pumped Solid State laser at 561 nm and the emitted fluorescence was detected from 580 to 623 nm. Images were acquired using LAS AF software. Several z-optical sections were made per area of interest and assembled to a z-stack using Fiji. The z-stack movies and 3D projections were produced using the 3D viewer function in Fiji [START_REF] Schindelin | Fiji: an open-source platform for biological-image analysis[END_REF].

Extraction and purification of phospho-and glycoglycerolipids and triacylglycerols

Approximately 50-100 mg of root or leaf material was harvested, weighed and immediately frozen in liquid nitrogen to avoid lipid degradation. The frozen samples were ground to a fine powder before extraction with organic solvents. Total lipids were extracted as described previously [START_REF] Wewer | Quantification of sterol lipids in plants by quadrupole time-of-flight mass spectrometry[END_REF][START_REF] Wewer | Fatty acid synthesis and lipid metabolism in the obligate biotrophic fungus rhizophagus irregularis during mycorrhization of Lotus japonicus[END_REF]. Briefly, 1 mL chloroform/methanol/formic acid (1:1:0.1, v/v/v) was added and the sample was shaken vigorously. At this point the internal standards for TAG and fatty acid analysis were added. Phase separation was achieved after addition of 0.5 mL 1M KCl/0.2 M H 3 PO 4 and subsequent centrifugation at 4000 rpm for 5 min. The lipid-containing chloroform phase was transferred to a fresh glass tube and the sample was re-extracted twice with chloroform. The combined chloroform phases were dried under a stream of air and lipids were re-dissolved in 1 mL chloroform to yield the total lipid extract.

For phospho-and glycerolipid analysis 20 ml of the total lipid extract were mixed with 20 ml of the internal standard mix and 160 ml of methanol/chloroform/300 mM ammonium acetate (665:300:35, v/v/v) [START_REF] Welti | Profiling membrane lipids in plant stress responses. role of phospholipase D alpha in freezing-induced lipid changes in Arabidopsis[END_REF]. For triacylglycerol analysis 500 ml of the total lipid extract were purified by solid phase extraction on Strata silica columns (1 ml bed volume; Phenomenex) as described [START_REF] Wewer | Quantification of sterol lipids in plants by quadrupole time-of-flight mass spectrometry[END_REF]. TAGs were eluted from the silica material with chloroform, dried under a stream of air and re-dissolved in 1 mL methanol/chloroform/300 mM ammonium acetate (665:300:35, v/v/v).

Extraction and purification of free fatty acids and monoacylglycerol (MAG)

Total lipids were extracted into chloroform and dried as described above. 15-0 FA and a mixture of 15-0 a-MAG and b-MAG were added as internal standard before the extraction. Dried extracts were resuspended in 1 ml n-hexane and applied to silica columns for solid-phase extraction with a n-hexane:diethylether gradient. Free fatty acids were eluted with a mixture of 92:8 (v/v) n-hexane: diethylether as described bevore [START_REF] Gasulla | The role of lipid metabolism in the acquisition of desiccation tolerance in Craterostigma plantagineum: a comparative approach[END_REF] and pure diethylether were used for elution of MAG.

Analysis of total fatty acids and free fatty acids by GC-FID

For measurement of total fatty acids, 100 ml of the total lipid extract were used. For measurement of free fatty acids, the SPE-fraction containing free fatty acids was used. Fatty acid methyl esters (FAMEs) were generated from acyl groups of total lipids and free fatty acids by addition of 1 mL 1N methanolic HCL (Sigma) to dried extracts and incubation at 80 ˚C for 30 min (Browse et al., 1986).

Subsequently, FAMEs were extracted by addition of 1 mL n-hexane and 1 mL of 0.9% (w/v) NaCl and analyzed on a gas chromatograph with flame-ionization detector (GC-FID, Agilent 7890A PlusGC). FAMEs were separated on an SP 2380 fused silica GC column (Supelco, 30 mx 0.53 mm, 0.20 mm film) as described [START_REF] Wewer | Analysis and quantification of plant membrane lipids by thin-layer chromatography and gas chromatography[END_REF], with a temperature -gradient starting at 100 ˚C, increased to 160 ˚C with 25 ˚C/min, then to 220 ˚C with10 ˚C/min and reduced to 100 ˚C with 25 ˚C/ min. FAMEs were quantified in relation to the internal standard pentadecanoic acid (15:0).

For MAG measurement, dried diethylether fractions were resuspended in 4:1 (v/v %) pyridine:N-Methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA), incubated at 80 ˚C for 30 min, dried and re-suspended in hexane prior to application on an Agilent 7890A Plus gas chromatography-mass spectrometer. MAGs were quantified by extracted ion monitoring, using [M+ -103] for a-MAGs and [M+ -161] for b-MAGs as previously reported for 16:0 MAG [START_REF] Destaillats | Identification of monoacylglycerol regio-isomers by gas chromatography-mass spectrometry[END_REF] and 24:0 MAG [START_REF] Li | Monoacylglycerols are components of root waxes and can be produced in the aerial cuticle by ectopic expression of a suberin-associated acyltransferase[END_REF].

Quantification of glycerolipids by Q-TOF MS/MS

Phosphoglycerolipids (PC, PE, PG, PI, PS), glycoglycerolipids (MGDG, DGDG, SQDG) and triacylglycerol (TAG) were analyzed in positive mode by direct infusion nanospray Q-TOF MS/MS on an Agilent 6530 Q-TOF instrument as described previously [START_REF] Lippold | Fatty acid phytyl ester synthesis in chloroplasts of Arabidopsis[END_REF][START_REF] Gasulla | The role of lipid metabolism in the acquisition of desiccation tolerance in Craterostigma plantagineum: a comparative approach[END_REF]. A continuous flow of 1 ml/min methanol/chloroform/300 mM ammonium acetate (665:300:35, v/v/v) [START_REF] Welti | Profiling membrane lipids in plant stress responses. role of phospholipase D alpha in freezing-induced lipid changes in Arabidopsis[END_REF] was achieved using a nanospray infusion ion source (HPLC/chip MS 1200 with infusion chip). Data are displayed as X:Y, where X gives the number of C atoms of the fatty acid chain and Y the amount of desaturated carbo-carbon bonds inside that fatty acid chain.

Internal standards

Internal standards for phospho-and glycoglycerolipid analysis were prepared as described previously [START_REF] Gasulla | The role of lipid metabolism in the acquisition of desiccation tolerance in Craterostigma plantagineum: a comparative approach[END_REF][START_REF] Wewer | Fatty acid synthesis and lipid metabolism in the obligate biotrophic fungus rhizophagus irregularis during mycorrhization of Lotus japonicus[END_REF]. The following standards were dissolved in 20 ml of chloroform/methanol (2:1, v/v): 0.2 nmol of each di14:0-PC, di20:0-PC, di14:0-PE, di20:0-PE, di14:0 PG, di20:0 PG, di14:0 PA and di20:0 PA; 0.03 nmol of di14:0-PS and di20:0-PS; 0.3 nmol of 34:0-PI; 0.15 nmol of 34:0-MGDG, 0.10 nmol of 36:0-MGDG; 0.2 nmol of 34:0-DGDG, 0.39 nmol of 36:0 DGDG and 0.4 nmol of 34:0 SQDG. 1 nmol each of tridecanoin (tri-10:0) and triundecenoin (tri-11:1), and 2 nmol each of triarachidin (tri-20:0) and trierucin (tri22:1) were used as internal standards for TAG quantification [START_REF] Lippold | Fatty acid phytyl ester synthesis in chloroplasts of Arabidopsis[END_REF]. For quantification of total fatty acids and free fatty acids 5 mg of pentadecanoic acid (FA 15:0) was added to the samples [START_REF] Wewer | Analysis and quantification of plant membrane lipids by thin-layer chromatography and gas chromatography[END_REF].

Cultivation and 13 C-Labeling of L. japonicus and Daucus carota hairy roots

The method for cultivation and stable isotope labelling of Lotus japonicus and Daucus carota hairy roots as well as for isotopolog profiling are described in more detail at Bio-protocol [START_REF] Keymer | Tracking Lipid Transfer by Fatty Acid Isotopolog Profiling from Host Plants to Arbuscular Mycorrhiza Fungi[END_REF]. To determine lipid transfer from L. japonicus to the fungus we used the carrot root organ culture system [START_REF] Be ´card | Early events of vesicular-arbuscular mycorrhiza formation on Ri T-DNA transformed roots[END_REF] to obtain sufficient amounts of fungal material for isotopolog profiling. (On petri dishes this was not possible with L. japonicus and in particular the lipid mutants alone). One compartment (carrot compartment) of the 2-compartmented petri dish system [START_REF] ´panier | Dependence of arbuscularmycorrhizal fungi on their plant host for palmitic acid synthesis[END_REF] was filled with MSR-medium (3% gelrite) containing 10% sucrose to support the shoot-less carrot root, and the other compartment (Lotus compartment) was filled with MSRmedium (3% gelrite) without sucrose. Ri T-DNA transformed Daucus carota hairy roots were placed in the carrot compartment. 1 week later, roots were inoculated with R. irregularis. Petri dishes were incubated at constant darkness and 30 ˚C. Within 5 weeks R. irregularis colonized the carrot roots and its extraradical mycelium spread over both compartments of the petri dish and formed spores. At this stage two 2 week old L. japonicus seedlings (WT, dis-1, ram2-1) were placed into the Lotus compartment (Figure 8-figure supplement 1).

The plates were incubated at 24 ˚C (16 hr light/8 hr dark). To keep the fungus and root in the dark the petri dishes were covered with black paper. 3 weeks after Lotus seedlings were placed into the petri dish [U-13 C 6 ]glucose (100 mg diluted in 2 ml MSR-medium) (Sigma-Aldrich) was added to the Lotus compartment. Therefore, only Lotus roots but not the carrot roots took up label. For transfer experiments with carrot roots no Lotus plant was placed into the Lotus compartment and the [U-13 C 6 ]glucose was added to the carrot compartment. 1 week after addition of [U-13 C 6 ]glucose the roots were harvested. The extraradical mycelium was extracted from the agar using citrate buffer pH 6 and subsequent filtration, after which it was immediately shock-frozen in liquid nitrogen.

Isotopolog profiling of 13 C-labelled 16:0 and 16:1v5 fatty acids

Root and fungal samples were freeze dried and subsequently derivatised with 500 ml MeOH containing 3 M HCl (Sigma-Aldrich) at 80 ˚C for 20 hr. MeOH/HCL was removed under a gentle stream of nitrogen and the methyl esters of the fatty acids were solved in 100 ml dry hexane. Gas chromatography mass spectrometry was performed on a GC-QP 2010 plus (Shimadzu, Duisburg, Germany) equipped with a fused silica capillary column (equity TM-5; 30 m by 0.25 mm,0.25-m m film thickness; Supelco, Bellafonte, PA). The mass detector worked in electron ionization (EI) mode at 70 eV. An aliquot of the solution was injected in split mode (1:5) at an injector and interface temperature of 260 ˚C. The column was held at 170 ˚C for 3 min and then developed with a temperature gradient of 2 ˚C/min to a temperature of 192 ˚C followed by a temperature gradient of 30 ˚C/ min to a final temperature of 300 ˚C. Samples were analyzed in SIM mode (m/z values 267 to 288) at least three times. Retention times for fatty acids 16:1w5 (unlabeled m/z 268) and 16:0 (unlabeled m/ z 270) are 12.87 min and 13.20 min, respectively. Data were collected with LabSolution software (Shimadzu, Duisburg, Germany). The overall 13 C enrichment and the isotopolog compositions were calculated according to [START_REF] Lee | Mass isotopomer analysis: theoretical and practical considerations[END_REF] and [START_REF] Ahmed | Isotopo' a database application for facile analysis and management of mass isotopomer data[END_REF]. The software package is open source and can be downloaded by the following link: http://www.tr34.uni-wuerzburg.de/software_ developments/isotopo/.

Four independent labeling experiments were performed. Overall excess (o.e.) is an average value of 13 C atoms incorporated into 16:0/16:1w5 fatty acids.

Figure 1 .

 1 Figure 1. DIS and RAM2 are required for arbuscule branching and vesicle formation. Arbuscule phenotype and complementation of dis (A) and ram2 (B) mutants. The fungus was stained with wheat-germ agglutinin (WGA)-AlexaFluor488. (C-D) Percent root length colonization of dis (C) and ram2 (D) mutants as compared to wild-type. Different letters indicate significant differences among treatments (ANOVA; posthoc Tukey). (C): n = 13; p 0.1, F 2,10 = 8.068 (total & int. hyphae); p 0.001 F 2,10 = 124.5 (arbuscules); p 0.001, F 2,10 = 299.1 (vesicles) (D): n = 15; p 0.1, F 2,12 = 10.18 (total & int. hyphae); p 0.001 F 2,12 = 57.86 (arbuscules); p 0.001, F 2,12 = 72.37 (vesicles). (A-D) Plants were inoculated with R. irregularis and harvested at 5 weeks post inoculation (wpi). DOI: 10.7554/eLife.29107.003 The following figure supplements are available for figure 1:

  -B, Figure 2figure supplement 1C-D, Videos 3-6).

Figure 2 .

 2 Figure 2. Arbuscocyte-specific expression of DIS and RAM2 is sufficient for arbuscule branching. Promoter activity indicated by nuclear localized yellow fluorescence in colonized transgenic L. japonicus wild-type roots transformed with constructs containing a 1.5 kb promoter fragment of DIS (A) or a 2.275 kb promoter fragment of RAM2 (B) fused to NLS-YFP. (A-B) Red fluorescence resulting from expression of pSbtM1:SP-mCherry labels the apoplastic space surrounding pre-penetration apparatuus (PPAs) and fungal structures, thereby evidencing the silhouette of these structures. a Figure 2 continued on next page

Figure supplement 1 .

 1 Figure supplement 1. DIS and RAM2 promoter activity in wild type and dis and ram2 mutants. DOI: 10.7554/eLife.29107.009

Figure 3 .

 3 Figure 3. Phylogenetic tree of KASI proteins in land plants. Protein sequences were aligned using MAFFT. Phylogenetic trees were generated by neighbor-joining implemented in MEGA5 (Tamura et al., 2011). Partial gap Figure 3 continued on next page

Figure 3

 3 Figure 3 continued deletion (95%) was used together with the JTT substitution model. Bootstrap values were calculated using 500 replicates. DIS likely originated before the angiosperm divergence (red star). DOI: 10.7554/eLife.29107.016 The following source data and figure supplements are available for figure 3: Source data 1. Accession numbers for protein sequences used in the phyologenic tree. DOI: 10.7554/eLife.29107.017 Figure supplement 1. Transcript accumulation of KASI and RAM2 genes. DOI: 10.7554/eLife.29107.018 Figure supplement 2. Shoot phenotypes of dis and ram2 mutants. DOI: 10.7554/eLife.29107.019 Figure supplement 3. Genomic comparison of the DIS locus in host and non-host species. DOI: 10.7554/eLife.29107.020

Figure 4 .

 4 Figure 4. DIS function is equivalent to a canonical KASI. (A) Microscopic AM phenotype of transgenic dis-1 mutant and wild-type hairy roots transformed with either an empty vector (EV) or the Arabidopsis KASI gene fused to the L. japonicus DIS promoter. White arrowheads indicate arbuscules. (B) Quantification of AM colonization in transgenic roots of dis-1 transformed with EV (open circles), dis-1 transformed with pDIS-AtKASI (grey circles) and wild-type transformed with EV (black squares). int. hyphae, intraradical hyphae. Different letters indicate significant differences (ANOVA; posthoc Tukey; n = 15; p 0.001) among genotypes for each fungal structure separately. F 2,12 = 0.809 (total and intraradical hyphae), F 2,12 = 43.65 (arbuscules), F 2,12 = 0.0568 (vesicles). (C) Rosettes of Arabidopsis, kasI mutant, Col-0 wild-type plants and kasI mutant plants transformed either Figure 4 continued on next page

Figure 4

 4 Figure 4 continued with the native AtKASI gene, the dis-1 mutant or the DIS wild-type gene driven by the Arabidopsis KASI promoter at 31 days post planting. (D) Rosette fresh weight of kasI mutant, Col-0 wild-type plants, one transgenic pAtKASI:AtKASI complementation line (Wu and Xue, 2010) and two independent transgenic lines each of kasI mutant plants transformed either with the dis-1 mutant or the DIS wild-type gene driven by the Arabidopsis KASI promoter at 31 days post planting. Different letters indicate significant differences (ANOVA; posthoc Tukey; n = 70; p 0.001; F 6,63 = 34.06) among genotypes. (E) Q-TOF MS/MS analysis of absolute amount of digalactosyldiacylglycerols (DGDG) containing acyl chains of 16:x + 18:x(34:x DGDG) or di18:x(36:x DGDG) derived from total leaf lipids of the different Arabidopsis lines. Different letters indicate significant differences (ANOVA; posthoc Tukey; n = 32; (p 0.05, F 6,25 = 14.48 (36:6)). (F) Subcellular localization of DIS in transiently transformed Nicotiana benthamiana leaves. Free RFP localizes to the nucleus and cytoplasm (upper panel). RFP fused to DIS co-localizes with the Arabidopsis light harvesting complex protein AtLHCB1.3-GFP in chloroplasts (lower panel). (G) Subcellular localization in plastids of DIS-YFP expressed under the control of the L. japonicus Ubiquitin promoter in R. irregularis colonized (upper panel) and non-colonized (lower panel) L. japonicus root cortex cells. BF, bright field; IH, intercellular hypha; A, arbuscule. DOI: 10.7554/eLife.29107.021

Figure 5 .

 5 Figure 5. Lack of characteristic accumulation of triacylglycerols in AM-defective mutants. (A-D) Quantitative accumulation of (A) total triacylglycerols, (B) tri16:0-triacylglycerol (C) tri16:x-triacylglycerols and (D) of triacylglycerols harbouring 16:x and 18:x FA-chains in non-colonized and R. irregularis colonized wild-type and dis-1 roots. Different letters indicate significant differences (ANOVA; posthoc Tukey) (A): n = 18; p 0.001; F 3,14 = 68.16. (B): n = 18; p 0.001; F 3,14 = 68.48. (C): n = 19; p 0.01, F 3,15 = 7.851 (16:1-16:1-16:1); p 0.001, F 3,15 = 14.52 (16:0-16:1-16:1); p 0.001, F 3,15 = 39.22 (16:0-16:0-16:1). (D): n = 19; p 0.001, F 3,15 = 12.15 (48:x), F 3,15 = 15.56 (50:x); p 0.01, F 3,15 = 22.93 (54:x). (E-G) Quantitative accumulation of (E) total triacylglycerols, (F) tri16:0-triacylglycerols, (G) tri16:x-triacylglycerols and (H) of triacylglycerols harbouring 16:x and 18:x FA-chains in colonized roots of L. japonicus wild-type Gifu, wild-type MG-20 and arbuscule-defective mutants. Different letters indicate significant differences (ANOVA; posthoc Tukey).Figure 5 continued on next page

  Figure 5. Lack of characteristic accumulation of triacylglycerols in AM-defective mutants. (A-D) Quantitative accumulation of (A) total triacylglycerols, (B) tri16:0-triacylglycerol (C) tri16:x-triacylglycerols and (D) of triacylglycerols harbouring 16:x and 18:x FA-chains in non-colonized and R. irregularis colonized wild-type and dis-1 roots. Different letters indicate significant differences (ANOVA; posthoc Tukey) (A): n = 18; p 0.001; F 3,14 = 68.16. (B): n = 18; p 0.001; F 3,14 = 68.48. (C): n = 19; p 0.01, F 3,15 = 7.851 (16:1-16:1-16:1); p 0.001, F 3,15 = 14.52 (16:0-16:1-16:1); p 0.001, F 3,15 = 39.22 (16:0-16:0-16:1). (D): n = 19; p 0.001, F 3,15 = 12.15 (48:x), F 3,15 = 15.56 (50:x); p 0.01, F 3,15 = 22.93 (54:x). (E-G) Quantitative accumulation of (E) total triacylglycerols, (F) tri16:0-triacylglycerols, (G) tri16:x-triacylglycerols and (H) of triacylglycerols harbouring 16:x and 18:x FA-chains in colonized roots of L. japonicus wild-type Gifu, wild-type MG-20 and arbuscule-defective mutants. Different letters indicate significant differences (ANOVA; posthoc Tukey).Figure 5 continued on next page

  Figure 5 continued (E): n = 40; p 0.001; F 8,31 = 38.42. (F) Left: absolute tri16:0 TAG content: n = 40; p 0.001; F 8,31 = 19.05. Right: tri16:0 TAG proportion among all TAGs, n = 40; p 0.001; F 8,31 = 14.21. (G): p 0.001; n = 41, F 8,32 = 86.16 (16:1-16:1-16:1); n = 39, F 8,30 = 24.16 (16:0-16:1-16:1); n = 40, F 8,31 = 17.67 (16:0-16:0-16:1). (H): n = 40; p 0.001, F 8,31 = 39.26 (48:x), F 8,31 = 28.93 (50:x); p 0.01, F 8,31 = 19.78 (52:x); p 0.05, F 8,31 = 13.77 (54:x). (A-H) Bars represent means ±standard deviation (SD) of 3-5 biological replicates. DOI: 10.7554/eLife.29107.022 The following source data and figure supplements are available for figure 5: Source data 1. Raw data for lipid profiles in Figure 5 and Figure 5-figure supplements 1-3 and 5-11. DOI: 10.7554/eLife.29107.023 Figure supplement 1. Diacylglycerol (DAG) and triacylglycerol (TAG) profiles of L. japonicus WT and dis-1 control and AM roots. DOI: 10.7554/eLife.29107.024 Figure supplement 2. Profiles of phospholipids in non-colonized and colonized L. japonicus WT Gifu and dis-1 roots. DOI: 10.7554/eLife.29107.025 Figure supplement 3. MGDG and DGDG profiles do not differ among L. japonicus wild-type and mutant roots. DOI: 10.7554/eLife.29107.026 Figure supplement 4. All arbuscule-deficient mutants show reduced root length colonization. DOI: 10.7554/eLife.29107.027 Figure supplement 5. Total fatty acid and free fatty acid profiles of colonized L. japonicus WT and AM-defective mutant roots. DOI: 10.7554/eLife.29107.028 Figure supplement 6. Triacylglycerol (TAG) profiles of colonized L. japonicus WT and AM-defective mutant roots. DOI: 10.7554/eLife.29107.029 Figure supplement 7. Phosphatidic acid (PA) profiles in L. japonicus WT and AM-defective mutants. DOI: 10.7554/eLife.29107.030 Figure supplement 8. Profile of phosphatidylcholines (PC) in L. japonicus WT and AM-defective mutants. DOI: 10.7554/eLife.29107.031 Figure supplement 9. Phosphatidylethanolamine (PE) profile in L. japonicus WT and AM-defective mutants. DOI: 10.7554/eLife.29107.032 Figure supplement 10. Phosphatidylinositol (PI) profile in L. japonicus WT and AM-defective mutants. DOI: 10.7554/eLife.29107.033 Figure supplement 11. Phosphatidylserine (PS) profile in L. japonicus WT and AM-defective mutants. DOI: 10.7554/eLife.29107.034

Figure 6 .Figure 7 .

 67 Figure 6. Loss of RAM1 affects AM-dependent induction of KASIII and DIS. (A) RAM1 effects on AM-dependent induction of KASIII and DIS, which catalyze 16:0 FA biosynthesis, and absence of effects on KASII. According to BLAST analysis via Kazusa (http://www.kazusa.or.jp/lotus/) and NCBI (http://www.ncbi.nlm.nih.gov/) KASIII and KASII are single copy genes in L.japonicus. Transcript accumulation of KASIII, DIS and KASII in non-colonized (open circles) and colonized (black circles) roots of Gifu WT, ram1-3 and ram1-4. Different letters indicate different statistical groups (ANOVA; posthoc Tukey; p 0.001; n = 23 F 5,12 = 65.04(KASIII); n = 24 F 5,18 = 54.42 (DIS); n = 18 F 5,12 = 33.11 (KASII)). Transcript accumulation was determined by qRT-PCR and the housekeeping gene Ubiquitin10 was used for normalization. AM plants were inoculated with R. irregularis and harvested 5 wpi. DOI: 10.7554/eLife.29107.035

Figure 8 .

 8 Figure 8. Isotopolog profiling indicates lipid transfer from plant to fungus. (A-B) Overall excess (o.e.) 13 C over air concentration in 16:0 FAs (A) and in 16:1w5 FAs (B) detected in non-colonized (only 16:0 FAs) and colonized carrot, L. japonicus wild-type, dis-1, ram2-1 roots and in the extraradical mycelium of R. irregularis. P values were generated by ANOVA using the Dunnett Test for multiple comparisons to L. japonicus wild-type (n = 29 (16:0 control roots); n = 33 (16:0 root AM); n = 39 (16:0 extraradical mycelium); n = 33 (16:1w5 root AM); n = 39 (16:1w5 extraradical mycelium), ***p<0.001, Figure 8 continued on next page

Figure 9 .

 9 Figure 8 continued **p<0.01, *p<0.05). (C) Relative fraction of 13 C isotopologs for 16:0 FAs of three replicates of carrot, L. japonicus WT Gifu, dis-1, ram2-1 in control roots (upper panel) and AM roots and each of the associated R. irregularis extraradical mycelia with spores (middle panel) and 16:1w5 FAs in AM roots and extraradical mycelia with spores (lower panel). Individual bars and double bars indicate individual samples. Values from roots are indicated by 'R' and from fungal extraradical mycelia with spores by 'M'. For carrot and L. japonicus WT the 13 C labelling pattern of 16:0 and 16:1w5 FAs in the plant is recapitulated in the fungal extraradical mycelium. Extraradical mycelium associated with dis-1 and ram2-1 does not mirror these patterns. Compare bars for AM roots and extraradical mycelium side by side. Black numbers indicate 13 C o. e. for individual samples. Colors indicate 13 C-isotopologs carrying one, two, three, etc. 13 C-atoms (M + 1, M + 2, M + 3, etc.). (D) Schematic and simplified illustration of carbon flow and 12 C vs. 13 C-carbon contribution to plant lipid metabolism and transport to the fungus in the two-compartment cultivation setup used for isotope labelling. Carbohydrate metabolism and transport is omitted for simplicity. ERM, extraradical mycelium. DOI: 10.7554/eLife.29107.037 The following source data and figure supplements are available for figure 8: Source data 1. Raw data for isotopolog profiles in Figure 8 and Figure 8-figure supplements 2,4. DOI: 10.7554/eLife.29107.038 Figure supplement 1. Two-compartment cultivation setup used for labelling experiments. DOI: 10.7554/eLife.29107.039 Figure supplement 2. Isotopolog profiles of wild-type MG20 and str. DOI: 10.7554/eLife.29107.040 Figure supplement 3. Proportion of 16:0 and 16:1w5 FA containing only non-labelled 12 C in plant and fungal tissue. DOI: 10.7554/eLife.29107.041 Figure supplement 4. Isotopolog profiles of additional samples. DOI: 10.7554/eLife.29107.042

  Figure 9 continued

  Transcript levels of both DIS and RAM2 increased in colonized roots (Figure3-figure supplement 1A). To analyze the spatial activity pattern of the DIS and RAM2 promoters during colonization we fused 1.5 kb for DIS and 2.275 kb for RAM2 upstream of the translational start site to the uidA gene.

	DIS and RAM2 expression in arbuscocytes is sufficient for arbuscule
	development
	DOI: 10.7554/eLife.29107.005
	Figure supplement 3. Identification of mutation in the RAM2 gene.
	DOI: 10.7554/eLife.29107.006
	Figure supplement 4. Protein sequence alignment of L.
	DOI: 10.7554/eLife.29107.007

2,12 = 10.18 (total & int. hyphae); p 0.001 F 2,12 = 57.86 (arbuscules); p 0.001, F 2,12 = 72.37 (vesicles). (A-D) Plants were inoculated with R. irregularis and harvested at 5 weeks post inoculation (wpi). DOI: 10.7554/eLife.29107.003

The following figure supplements are available for figure

1

: Figure supplement 1. Identification of the dis mutation. DOI: 10.7554/eLife.29107.004 Figure supplement 2. Protein sequence alignment of L. japonicus DIS with other KASI proteins.

  Colonized root, b non-colonized part of colonized root, c PPAs, (white arrow heads indicate the silhouette of fungal intraradical hyphae) d small arbuscules, e fully developed arbuscules f collapsed arbuscules. Merged confocal and bright field images of whole mount roots are shown. (C-D) Transgenic complementation of dis-1 (C) and ram2-1 (D) hairy roots with the respective wild-type gene driven by the PT4 promoter. The mutant gene was used as negative control. White arrowheads indicate arbuscules. (E-F) Quantification of AM colonization in transgenic roots shown in (C-D).

	Figure 2 continued
	Different letters indicate significant differences (ANOVA; posthoc Tukey; n = 15; p 0.001) among genotypes for each fungal structure separately. Int.
	hyphae, intraradical hyphae. (E): F 2,12 = 26.53 (total), F 2,12 = 46.97 (arbuscules), F 2,12 = 27.42 (vesicles). (F) F 2,12 = 341.5 (total), F 2,12 = 146.3 (arbuscules),
	F 2,12 = 35.86 (vesicles).
	DOI: 10.7554/eLife.29107.008
	The following figure supplement is available for figure 2:

Keymer et al. eLife 2017;6:e29107. DOI: 10.7554/eLife.29107

Research articlePlant Biology

Acknowledgements

We are grateful to Tomoko Kojima (NARO, Tochigi, Japan) for providing Lotus japonicus str mutant seeds and to LotusBase for providing LORE1 insertion lines. We thank Julia Mayer for help with setting up carrot hairy root systems, Samy Carbonnel, David Chiasson, Michael Paries and Jose ´Antonio Villae ´cija-Aguilar for contributing a golden gate module.

Additional information

Funding

Funder

Grant reference number

Data availability

Lunularia cruciata: For this species, the raw RNAseq reads have been previously deposited to NCBI under the accession number SRR1027885. It is annotated with Rhizophagus irregularis (10% of sequences) as the transcriptome was partly prepared from Lunularia plant tissue colonized by the fungus Rhizophagus irregularis. The corresponding Lunularia transcriptomic assembly is available at www.polebio.lrsv.ups-tlse.fr/Luc_v1/

Statistics

All statistical analyses (Source code 1) were performed and all boxplots were generated in R (www. r-project.org).

Plasmid generation

Genes and promoter regions were amplified using Phusion PCR according to standard protocols and using primers indicated in Supplementary file 2. Plasmids were constructed as indicated in Supplementary file 3. For localization of DIS in L. japonicus hairy roots the LIII tricolor plasmid [START_REF] Binder | A modular plasmid assembly kit for multigene expression, gene silencing and silencing rescue in plants[END_REF] was used. The plasmid containing 35S:RFP for localization of free RFP in Nicotiana benthamiana leaves was taken from [START_REF] Yano | CYCLOPS, a mediator of symbiotic intracellular accommodation[END_REF].

Induction of transgenic hairy roots in L. japonicus Hypocotyls of L. japonicus were transformed with plasmids shown in Supplementary file 3 for hairy root induction using transgenic Agrobacterium rhizogenes AR1193 as described [START_REF] Takeda | Apoplastic plant subtilases support arbuscular mycorrhiza development in Lotus japonicus[END_REF].

Floral dipping and rosette growth assay of Arabidopsis thaliana

Five plants per pot were sown. One week before transformation the primary bolt was cut off to induce growth of secondary floral bolts. 5 ml LB culture of A. tumefaciens transformed with a binary vector was incubated at 28 ˚C, 300 rpm over night. 500 ml of the preculture was added to 250 ml LB medium with appropriate antibiotics. This culture was incubated again at 28 ˚C, 300 rpm over night until an OD600 of 1.5 was reached. Plants were watered and covered by plastic bags the day before the dipping to ensure high humidity. The cells were harvested by centrifugation (10 min, 5000 rpm) and resuspended in infiltration medium (0.5 x MS medium, 5% sucrose). The resuspended cell culture was transferred to a box and Silwet L-77 was added (75 ml to 250 ml medium). The floral bolts of the plants were dipped into the medium for 5 s and put back into plastic bags and left in horizontal position for one night. After that, plants were turned upright, bags were opened and mature siliques were harvested. For rosette growth assays T3 plants were used. 31 days post sowing the rosettes were photographed and then cut and dried in an oven at 65 ˚C for the determination of rosette dry weight.

Spatial analysis of promoter activity

For promoter:GUS analysis L. japonicus hairy roots transformed with plasmids containing the DIS and RAM2 promoter fused to the uidA gene and colonized by R. irregularis were subjected to GUS staining as described [START_REF] Takeda | Apoplastic plant subtilases support arbuscular mycorrhiza development in Lotus japonicus[END_REF]. To correlate DIS and RAM2 promoter activity precisely with the stage of arbuscule development two expression cassettes were combined in the same golden gate plasmid for simultaneous visualization of arbuscule stages and promoter activity. The fungal silhouette including all stages of arbuscule development and pre-penetration apparatuus were made visible by expressing secretion peptide coupled mCherry under the control of the SbtM1 promoter region comprising 704 bp upstream of the SbtM1 gene [START_REF] Takeda | Apoplastic plant subtilases support arbuscular mycorrhiza development in Lotus japonicus[END_REF]. Promoter activity was visualized using a YFP reporter fused to a nuclear localization signal (NLS).

Transient transformation of N. benthamiana leaves N. benthamiana leaves were transiently transformed by infiltration of transgenic A. tumefaciens AGL1 as described [START_REF] Yano | CYCLOPS, a mediator of symbiotic intracellular accommodation[END_REF].

Real time qRT-PCR

For analysis of transcript levels, plant tissues were rapidly shock frozen in liquid nitrogen. RNA was extracted using the Spectrum Plant Total RNA Kit (www.sigmaaldrich.com). The RNA was treated with Invitrogen DNAse I amp. grade (www.invitrogen.com) and tested for purity by PCR. cDNA synthesis was performed with 500 ng RNA using the Superscript III kit (www.invitrogen.com). qRT-PCR was performed with GoTaq G2 DNA polymerase (Promega), 5 x colorless GoTaq Buffer (Promega) and SYBR Green I (Invitrogen S7563, 10.000x concentrated, 500 ml) -diluted to 100x in DMSO. Primers (Supplementary file 2) were designed with primer3 (58). The qPCR reaction was run on an iCycler (Biorad, www.bio-rad.com/) according to manufacturer's instructions. Thermal cycler conditions were: 95 ˚C 2 min, 45 cycles of 95 ˚C 30 s, 60 ˚C/62 ˚C 30 s and 72 ˚C 20 s followed by dissociation curve analysis. Expression levels were calculated according to the DDCt method [START_REF] Rozen | Primer3 on the WWW for general users and for biologist programmers[END_REF]. For each genotype and treatment three to four biological replicates were tested and each sample was represented by two to three technical replicates. . Supplementary file 4. Accession numbers for protein sequences used in the phyologenic tree (Figure 3). DOI: 10.7554/eLife.29107.048