The Acyl-ACP Thioesterase FatM from <em>Lotus japonicus</em> is involved in lipid transfer during Arbuscular Mycorrhiza Symbiosis

Abstract

Arbuscular mycorrhiza colonization of roots is an ancient symbiosis that presumably was involved in the conquest of the land by early plants. Its main benefit for plants is the increased access to soil mineral nutrients beyond the root depletion zone. Arbuscular mycorrhiza fungi follow an obligate biotrophic lifestyle. Metabolites derived from photosynthesis of the host plant represent the sole source of reduced carbon for AMF. During symbiotic growth, AMF colonize the root and penetrate the cortex cells where the highly branched hyphae form arbuscules and where metabolites are exchanged between host and symbiont. During post-symbiotic growth, the fungus forms lipid-storage structures called vesicles and eventually grows out of the root into the soil where it forms spores again that are filled with neutral storage lipids. Lipids in R. irregularis contain fatty acids with unusual double bond positions, i. e. 16:1 Δ11/ω5 that are specific for AMF and primarily accumulate in neutral lipids but also in membrane lipids. The genomes of multiple AMF, such as the model fungus Rhziophagus irregularis, lack type-I cytosolic fatty acid synthase genes (FAS) that are required for the synthesis of fatty acids. The absence of de novo fatty acid synthesis from AMF therefore led to the hypothesis that next to hexoses, fatty acids serve as an additional source of reduced carbon. <br /> AMF-host co-evolution has shaped host plant genomes to contain conserved genes exclusively dedicated to AMF symbiosis. One of these genes is the acyl-ACP Thioesterase M (FatM). Fat enzymes terminate fatty acid elongation during de novo synthesis by hydrolysis of the thioester and thereby liberate free fatty acids for lipid metabolism. The importance of FatM for AMF symbiosis was investigated in the host plant Lotus japonicus during symbiosis with R. irregularis. For this purpose, mycorrhiza-colonization of fatm transposon-insertion mutants was characterized, 13C-labeling of fatty acids during symbiosis was conducted and the recombinant FatM enzyme was studied in vitro. <br /> Consistent with an essential role in AMF symbiosis, FatM gene expression was only detectable in colonized roots. Reduction of FatM gene expression in fatm mutants led to impaired mycorrhiza symbiosis. Fungal root colonization was reduced and R. irregularis exhibited a defect in storage lipid accumulation. Furthermore, arbuscule branching was affected in fatm mutants and as a consequence, symbiotic phosphate supply was decreased. The fatty acids 16:0, 18:0 and 18:1Δ9 are the three main products of plant fatty acid de novo synthesis and R. irregularis mainly accumulates 16:0 and 16:1ω5. Recombinant expression and in vitro enzyme assays of FatM revealed that 16:0-ACP is the preferred substrate. Therefore, plastids in arbuscocytes are reprogrammed by induction of FatM to increase 16:0 fatty acid synthesis, which subsequently accumulate in R. irregularis. Quantification of β-monoacylglycerols (MAG) revealed that 16:0 β-MAG shows a mycorrhiza-dependent profile, i. e. it accumulated upon colonization in the WT as well as in ERM (hyphae and spores) but failed to accumulate to the same extent in colonized fatm mutants. 16:0 β-MAG synthesis and secretion to the PAS might therefore represent the major mechanism for 16:0 fatty acid transfer to the fungus. Labeling of fatty acids during symbiosis with 13C2-acetate demonstrated that FatM is essential for accumulation of the mycorrhiza-signature fatty acids 16:1ω5 and 20:3, as a decrease of 13C over-excess label was observed in colonized fatm mutants, which was independent of decreased fungal biomass. <br /> Similar to other oleaginous fungi, AMF contain multiple unsaturated long-chain fatty acids. The enzymes responsible for desaturation of these mycorrhiza-marker fatty acids were previously unknown. Two genes with homology to the yeast acyl-CoA desaturase OLE1 are present in R. irregularis, termed RiOLE1 and RiOLE1-LIKE. Both can act on 16:0 and 18:0 fatty acids to produce a Δ9 (RiOLE1) or the mycorrhiza-specific ω5/Δ11 (RiOLE1-LIKE) double bond, as revealed by heterologous expression in yeast and N. benthamiana. Expression also changed the acyl-CoA pool composition, which was enriched in 18:1 or 16:1, depending on the heterologous expression of RiOLE1 or RiOLE1-LIKE, respectively. Based on the high sequence similarity with ScOLE1, RiOLE1 and RiOLE1-LIKE are most likely acyl-CoA desaturases. <br /> The experiments presented here show that AMF reprogram fatty acid and lipid synthesis towards the production of 16:0 fatty acids via FatM that are then transferred to the fungus for desaturation and incorporation into lipids. In future, knowledge on symbiotic nutrient exchange can help to understand and improve the benefits of mycorrhiza symbiosis for crop plant nutrition and plant breeding.