Mutualistic interactions between free-living algae and fungi are widespread in nature and are hypothesized to have facilitated the evolution of land plants and lichens. In all known algal-fungal mutualisms, including lichens, algal cells remain external to fungal cells. Here, we report on an algal–fungal interaction in which Nannochloropsis oceanica algal cells become internalized within the hyphae of the fungus Mortierella elongata. This apparent symbiosis begins with close physical contact and nutrient exchange, including carbon and nitrogen transfer between fungal and algal cells as demonstrated by isotope tracer experiments. This mutualism appears to be stable, as both partners remain physiologically active over months of co-cultivation, leading to the eventual internalization of photosynthetic algal cells, which persist to function, grow and divide within fungal hyphae. Nannochloropsis and Mortierella are biotechnologically important species for lipids and biofuel production, with available genomes and molecular tool kits. Based on the current observations, they provide unique opportunities for studying fungal-algal mutualisms including mechanisms leading to endosymbiosis.

Photosynthetic microalgae are considered a viable and sustainable resource for biofuel feedstocks, because they can produce higher biomass per land area than plants and can be grown on non-arable land. Among many microalgae considered for biofuel production, Nannochloropsis oceanica (CCMP1779) is particularly promising, because following nutrient deprivation it produces very high amounts of triacylglycerols (TAG). The committed step in TAG synthesis is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT). Remarkably, a total of 13 putative DGAT-encoding genes have been previously identified in CCMP1779 but most have not yet been studied in detail. Based on their expression profile, six out of 12 type-2 DGAT-encoding genes (NoDGTT1-NoDGTT6) were chosen for their possible role in TAG biosynthesis and the respective cDNAs were expressed in a TAG synthesis-deficient mutant of yeast. Yeast expressing NoDGTT5 accumulated TAG to the highest level. Over-expression of NoDGTT5 in CCMP1779 grown in N-replete medium resulted in levels of TAG normally observed only after N deprivation. Reduced growth rates accompanied NoDGTT5 over-expression in CCMP1779. Constitutive expression of NoDGTT5 in Arabidopsis thaliana was accompanied by increased TAG content in seeds and leaves. A broad substrate specificity for NoDGTT5 was revealed, with preference for unsaturated acyl groups. Furthermore, NoDGTT5 was able to successfully rescue the Arabidopsis tag1-1 mutant by restoring the TAG content in seeds. Taken together, our results identified NoDGTT5 as the most promising gene for the engineering of TAG synthesis in multiple hosts among the 13 DGAT-encoding genes of N. oceanica CCMP1779. Consequently, this study demonstrates the potential of NoDGTT5 as a tool for enhancing the energy density in biomass by increasing TAG content in transgenic crops used for biofuel production.

The marine microalgae Nannochloropsis oceanica (CCMP1779) is a prolific producer of oil and is considered a viable and sustainable resource for biofuel feedstocks. Nitrogen (N) availability has a strong impact on the physiological status and metabolism of microalgal cells, but the exact nature of this response is poorly understood. To fill this gap we performed transcriptomic profiling combined with cellular and molecular analyses of N. oceanica CCMP1779 during the transition from quiescence to autotrophy. N deprivation-induced quiescence was accompanied by a strong reorganization of the photosynthetic apparatus and changes in the lipid homeostasis, leading to accumulation of triacylglycerol. Cell cycle activation and re-establishment of photosynthetic activity observed in response to resupply of the growth medium with N were accompanied by a rapid degradation of triacylglycerol stored in lipid droplets (LDs). Besides observing LD translocation into vacuoles, we also provide evidence for direct interaction between the LD surface protein (NoLDSP) and AUTOPHAGY-RELATED8 (NoATG8) protein and show a role of microlipophagy in LD turnover in N. oceanica CCMP1779. This knowledge is crucial not only for understanding the fundamental mechanisms controlling the cellular energy homeostasis in microalgal cells but also for development of efficient strategies to achieve higher algal biomass and better microalgal lipid productivity.