Glycerol-3-Phosphate Acyltransferase Contributes to TriacylglycerolBiosynthesis, Lipid Droplet Formation, and Host Invasion inMetarhizium robertsii

Qiang Gao, Yanfang Shang, Wei Huang, Chengshu Wang

Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese Academyof Sciences, Shanghai, China

Enzymes involved in the triacylglycerol (TAG) biosynthesis have been well studied in the model organisms of yeasts and animals.Among these, the isoforms of glycerol-3-phosphate acyltransferase (GPAT) redundantly catalyze the first and rate-limiting stepin glycerolipid synthesis. Here, we report the functions of mrGAT, a GPAT ortholog, in an insect-pathogenic fungus, Metarhiz-ium robertsii. Unlike in yeasts and animals, a single copy of the mrGAT gene is present in the fungal genome and the gene dele-tion mutant is viable. Compared to the wild type and the gene-rescued mutant, the �mrGAT mutant demonstrated reduced abil-ities to produce conidia and synthesize TAG, glycerol, and total lipids. More importantly, we found that mrGAT is localized tothe endoplasmic reticulum and directly linked to the formation of lipid droplets (LDs) in fungal cells. Insect bioassay resultsshowed that mrGAT is required for full fungal virulence by aiding fungal penetration of host cuticles. Data from this study notonly advance our understanding of GPAT functions in fungi but also suggest that filamentous fungi such as M. robertsii canserve as a good model to elucidate the role of the glycerol phosphate pathway in fungal physiology, particularly to determine themechanistic connection of GPAT to LD formation.

The glycerol phosphate pathway is the major pathway for tri-glyceride biosynthesis in various organisms ranging from bac-

teria to animals to provide the crucial energy molecules as well asserve as a repository for biosynthesis of fatty acids and phospho-lipids (1). Among the various enzymes involved in this pathway,glycerol-3-phosphate acyltransferase (GPAT) is the first enzymeand catalyzes the acylation of glycerol 3-phosphate (G3P) thatresults in lysophosphatidic acid (LPA), which is the precursor forthe biosynthesis of phosphatidic acid, diacylglycerol (DAG), andtriacylglycerol (TAG) (2, 3). Different isoforms of GPAT havebeen characterized in yeast, plants, and animals. For example, tworedundant GPATs, i.e., GAT1 (GPT2p) and GAT2 (SCT1p), havebeen identified in the budding yeast Saccharomyces cerevisiae,three copies each in Drosophila melanogaster and Caenorhabditiselegans, four copies each in humans and mouse, and eight copiesin the plant Arabidopsis thaliana (3, 4). While most GPATs acylatethe sn-1 position of G3P to produce LPA, the GPAT4 and GPAT6isoforms in A. thaliana are involved in cutin biosynthesis andpredominantly esterify acyl groups at the sn-2 position of G3P toproduce sn-2 monoacylglycerol (5). In animals, a peroxisomaldihydroxyacetone-phosphate acyltransferase (DHAPAT) canprovide an alternate route for LPA production by acylation ofdihydroxyacetone phosphate (DHAP) and the subsequent reduc-tion of 1-acyl-DHAP to LPA (6). Yeast GAT1 can also convertDHAP into 1-acyl DHAP (7, 8), and the latter can be furtherreduced to LPA by 1-acyldihydroxyacetone-phosphate reductase(AYR1) in yeast (9). A fungus-like DHAPAT is still unknown.

Mammalian GPAT1 and GPAT2 are localized on the outermembrane of mitochondria (MT), while GPAT3 and GPAT4 aretargeted to the endoplasmic reticulum (ER) (3, 10). A recent studyshowed that D. melanogaster and mammalian GPAT4 isoformscould be relocalized from the ER to the surface of nascent lipiddroplets (LDs), where they mediate LD growth (4). Similarly,GPAT isoforms ACL-4 and ACL-5 from C. elegans are also local-

ized on the ER membrane. However, ACL-6 is an MT-type GPAT,which is required to control MT fusion and nematode oogenesis(10). Yeast GAT1 and GAT2, also called microsomal GPATs, areER-type GPATs (11) and contribute to polarized cell growth (12).Deletion of either gene does not affect yeast cell growth; however,the double deletion mutants are not viable (8). The GPAT ho-molog has not been characterized thus far in filamentous fungi.

The ubiquitous insect-pathogenic fungus Metarhizium robert-sii is a biocontrol agent used worldwide to control different insectpests (13, 14). Similar to plant pathogens, insect pathogens such asM. robertsii infect hosts by penetrating host cuticles (15), which ismediated by high concentrations of glycerol within the infectionstructures appressoria (16, 17). It has been shown that high con-centrations of glycerol in the appressoria of the plant pathogenMagnaporthe oryzae are largely lipolytic products that originatedfrom stored TAG (ca. 44% of LD components [18]) rather thanfrom carbohydrate sources by on-site biosynthesis (19). It is pos-sible that the GPAT(s) in fungal pathogens also contributes tofungal virulence. To investigate this, we studied the functionalcharacteristics of a GPAT from the entomopathogenic fungus M.robertsii, designated mrGAT (MAA_02162) (20). Our data indi-cate the presence of a single copy of mrGAT in the genome of M.robertsii, and the gene deletion mutant, although viable, has sig-nificantly impaired TAG accumulation, LD formation, and viru-

Received 28 August 2013 Accepted 24 September 2013

Published ahead of print 27 September 2013

Address correspondence to Chengshu Wang, cswang@sibs.ac.cn.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.02905-13.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

doi:10.1128/AEM.02905-13

7646 aem.asm.org Applied and Environmental Microbiology p. 7646 –7653 December 2013 Volume 79 Number 24

lence against insect hosts. We also found a single copy of a humanDHAPAT (NP_055051)-like protein gene present in the genomeof M. robertsii (MAA_02767; 25% identity with human DHAPAT;designated mrDHAPAT). Deletion of this gene did not result inany obvious phenotypic and physiological changes in the mutantscompared with the wild-type (WT) strain.

MATERIALS AND METHODSStrains and culture conditions. To collect conidia for the experiments,the WT strain ARSEF2575 of M. robertsii was grown on potato dextroseagar (PDA; Difco) at 25°C for 20 days. Spore germination and appresso-rium induction assays were conducted using locust (Schistocerca gregaria)hind wings or the minimal medium (MM) (NaNO3, 6 g liter�1; KCl, 0.52g liter�1; MgSO4 · 7H2O, 0.52 g liter�1; KH2PO4, 0.25 g liter�1) amendedwith 1% glycerol as the sole carbon resource (MMGly) (17). For genomicDNA and RNA extractions, fungal spores were cultured in Sabourauddextrose broth (SDB; Difco) at 25°C and 200 rpm for 3 days in a rotaryshaker.

Phylogenetic analysis. To determine the phylogeny of mrGAT acrossfungal lineages, the homologs of mrGAT were retrieved from selectedfungal pathogens and saprophytes of ascomycetes, basidiomycetes, mi-crosporidia, chytrids, and zygomycetes with well-annotated genomes us-ing BLASTP searches with a cutoff E value of �1e�100. Sequence align-ment was conducted using ClustalX 2.0, and a neighbor-joining tree wasgenerated using MEGA 5.2 (21) with a Dayoff amino acid substitutionmodel, a pairwise deletion for missing residues or gaps, and 1,000 boot-strap replicates. Prediction of mrGAT subcellular localization was per-formed with the program ProtComp (ver. 9.0, Softberry) and TargetP(ver. 1.1) (22).

Gene deletion and complementation. For functional studies, mrGATgene was deleted using an Agrobacterium-mediated transformationmethod as described in our previous study (23). In brief, the 5=- and3=-flanking regions of mrGAT were amplified by PCR using the genomicDNA as the template with the primer pairs mrGATUF/mrGATUR andmrGATDF/mrGATDR (see Table S1 in the supplemental material). Theamplified products were subsequently cloned into the PstI and SpeI re-striction sites of the binary vector pDHt-SK-ben (conferring resistanceagainst benomyl) for fungal transformation (23) to create the �mrGATdeletion mutant. To complement gene deletion, the mrGAT gene wasamplified together with its promoter and the 3=-untranslated region (3=-UTR) with the primers mrGATCompF and mrGATCompR, and theproduct was subcloned into the SpeI site of the binary vector pDHt-SK-Bar (conferring resistance against ammonium glufosinate) before fungaltransformation to obtain the complemented mutant (Comp). Transfor-mants were verified by PCR and reverse transcription (RT)-PCR analysesusing primers mrGATF and mrGATR (see Table S1 in the supplementalmaterial). The �-tubulin gene (MAA_02081) was used as the control andamplified using primers TubF and TubR. Deletion of mrDHAPAT was sim-ilarly performed using the primer pairs mrDHAPATUF/mrDHAPATUR,and mrDHAPATDUF/mrDHAPATDR, respectively (see Table S1 in thesupplemental material).

Examination of protein localization. To confirm ER localization ofmrGAT, the open reading frame (ORF) of mrGAT gene was amplified byPCR with the primer pairs mrGAT-GFP1F and mrGAT-GFP1R to deletethe stop codon and include the promoter region of the gene (1,970 bpupstream from the start codon). The Egfp gene was amplified from theplasmid pEGFP (14) with the primers mrGAT-GFP2F and mrGAT-GFP2R (see Table S1 in the supplemental material). The acquired frag-ments were purified and jointed together by a fusion PCR (23). The prod-uct was digested with the restriction enzymes SpeI and EcoRI and clonedinto the same enzyme-treated plasmid pDHt-SK-Bar (conferring resis-tance against glufosinate) for Agrobacterium-mediated transformation(23). The acquired mrGAT-GFP (green fluorescent protein) strain wascultured in SDB for 3 days, and the mycelia were washed twice withHanks’ balanced salt solution (Gibco) before being stained with the fluo-

rescent dye ER-Tracker Blue-White DPX (catalog number E-12353; In-vitrogen) for 30 min. The images were taken with a fluorescence micro-scope, BX51-33P (Olympus).

Appressorium induction and lipid droplet visualization. Conidiafrom the WT, �mrGAT, and Comp strains were inoculated into individ-ual polystyrene petri dishes (5.5 cm in diameter) containing 2 ml MMGlyat a final concentration of 2 � 105 spores ml�1. After incubation for 18 h,spore germination and appressorium differentiation rates were recordedfor �100 spores under a microscope. Appressoria were also induced onlocust (S. gregaria) hind wings as described previously (24). To visualizeand compare the formation of LDs, conidia, germlings, and appressoriafrom the WT, �mrGAT, and Comp strains were washed twice with phos-phate-buffered saline (PBS) and then stained with a fluorescent dye, Bo-dipy (catalog number D-3922; Invitrogen) for 30 min (17). The accumu-lation of intracellular LDs was observed under a transmission electronmicroscope (TEM) as described previously (23). Fungal samples werefixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) at 4°C for12 h, rinsed three times in phosphate buffer, and fixed overnight in 1%osmium tetroxide in 0.1 M cacodylate buffer (pH 7.0) at 4°C. After rinsingthree times in phosphate buffer, samples were dehydrated in an ethanolgradient series, infiltrated with a graded series of epoxy resin in epoxypropane, and then embedded in Epon resin and sectioned. The ultrathinsections were stained in 2% uranium acetate followed by lead citrate andvisualized under a transmission electron microscope (H-7650; Hitachi,Tokyo, Japan) operating at 80 kV.

Free-glycerol and -triacylglycerol assays. Conidia were collectedfrom 2-week-old cultures on PDA plates, while mycelia were collectedfrom 3-day-old cultures in SDB. Glycerol content in the samples wasassayed using the free-glycerol assay kit E1012 (Applygen TechnologiesInc., Beijing, China) at 550 nm, while triglycerides were assayed using thetriglyceride assay kit E1014 (Applygen Technologies Inc., Beijing, China)at 550 nm. First, all samples were washed twice with phosphate buffer,homogenized in extraction buffer, and centrifuged at 5,000 � g for 5 min.Supernatants were aliquoted and incubated with the reaction buffer for 30min before determining the optical density at 550 nm (OD550) using amicroplate reader (Varioskan Flash Multimode Reader; Thermo Scien-tific). Total protein concentration in supernatants was estimated using theBradford method. All experiments were repeated twice, and three repli-cates were maintained for each sample. Glycerol and triglyceride concen-trations in different samples were expressed as micromoles of glycerol ortriglyceride per milligram of total proteins, respectively.

Total lipid quantification. To determine the effect of mrGAT on lipidbiosynthesis, total lipid was quantified by a phosphoric acid-vanillinmethod (25). Conidia from WT, �mrGAT, and Comp strains were har-vested from the PDA plates incubated at 25°C for 20 days, and myceliawere collected from the SDB after incubation at 220 rpm and 25°C for 3days. For assays, spore suspensions (0.5 ml, 1.0 � 108 conidia ml�1) wereadded to glass tubes and 1 mg mycelium homogenates (dried overnight at150°C) was added to glass tubes with 0.5 ml water. Then, 2 ml of 18 MH2SO4 was added to each tube, boiled in a water bath for 10 min, andcooled for 5 min at room temperature before adding 5 ml phosphoricacid-vanillin reagent (1.2 g liter�1 vanillin and 200 ml of water, adjustedto 1 liter with 85% H3PO4). The tubes were then incubated at 37°C foranother 15 min and centrifuged to determine absorbance at 530 nm (17).All experiments were repeated twice. A standard curve was generatedusing triolein (Sigma) for quantification.

Western blot analysis. Proteins were extracted from fungal conidiaand mycelia with the RIPA lysis buffer (Thermo Scientific) containing 1mM protease inhibitor phenylmethylsulfonyl fluoride (PMSF). Proteinswere separated on 12% sodium dodecyl sulfate containing polyacryl-amide gels and transferred onto polyvinylidene difluoride membranes.The membranes were probed with the antibodies against the LD surfaceperilipin protein, MPL1, and �-tubulin separately (23).

Insect bioassays. To investigate the effect of mrGAT on fungal viru-lence, insect bioassays were conducted on second-day, fifth-instar Bom-

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byx mori larvae. Conidia from WT, �mrGAT, Comp, and �mrDHAPATstrains were applied topically by immersing the larvae in an aqueous sus-pension containing 5 � 106 conidia ml�1 for 1 min or by injection of a10-�l suspension containing 1 � 106 spores ml�1 into the second proleg.Each treatment had three replicates with 15 insects each, and all experi-ments were repeated three times. Larval mortality was recorded every 12h, and the median lethal time (LT50) was estimated by Kaplan-Meieranalysis using SPSS (ver. 13.0).

RESULTSCharacteristics of the mrGAT protein. The complete ORF ofmrGAT encodes a protein with 766 amino acids and a predictedmolecular mass of 84.9 kDa and pI of 9.63. MrGAT is a typicalmember of the Pfam 01553 family of acyltransferases and is ho-mologous to yeast GAT1 (GPT2p, 34% identity) and GAT2(SCT1p, 37% identity). Similar to other organisms, it contains thefour highly conserved AGPAT (1-acyl-sn-glycerol-3-phosphateacyltransferase) motifs for catalysis (motifs I and IV) and G3Pbinding (motifs II and III) (see Table S2 in the supplemental ma-terial). In silico analysis with the programs TargetP and ProtCompindicated with a high confidence score that mrGAT is localized tothe endoplasmic reticulum. Survey of the M. robertsii genomerevealed the presence of only one copy of the gene, which is similarto what is observed in other fungi from the subphylum Pezizomy-cotina (phylum Ascomycota) (Fig. 1). A single copy of GPAT wasalso found in the fission yeast (Schizosaccharomyces pombe) and ina chytrid (Batrachochytrium dendrobatidis). In contrast, two cop-

ies were found in the subphylum Saccharomycotina (Ascomy-cota), which includes the budding yeast, and four copies werefound in the zygomycete fungus Rhizopus delemar (Fig. 1). In thebasidiomycete fungal species, either one or two copies of theGPAT gene were found. Interestingly, a GPAT gene homolog wasabsent in the genomes of the microsporidian fungi. Relative to thewell-established fungal tree of life (26), the GPAT gene evolution-ary tree is not congruent with fungal speciation phylogeny acrossthe phyla. For example, chytrids are in general considered to havediverged early to form a basal clade within the fungal kingdom,while the GPAT tree is rooted by yeast GPAT and the chytridGPAT is more closely related to that of ascomycetes than zygomy-cetes (Fig. 1). A genome survey of mammalian DHAPAT protein-like genes showed that a single copy of the gene is present in thegenomes of M. robertsii and other ascomycete species of subphy-lum Pezizomycotina but not in the yeast species of Saccharomy-cotina (see Fig. S1 in the supplemental material).

Characterization of the mutants. In S. cerevisiae, GAT1 andGAT2 are functionally redundant because viability is not affectedby deletion of either gene; however, double deletions are lethal (8).To determine the effect of mrGAT on fungal viability, gene dele-tion and complementation were performed via Agrobacterium-mediated transformation, and the resultant strains were analyzedby PCR and RT-PCR (Fig. 2A and B). We found that the �mrGATstrain of M. robertsii was viable, although the ability to sporulateand form pigments was impaired compared to the WT and com-

FIG 1 Phylogenetic analysis of fungal glycerol-3-phosphate O-acyltransferases. Protein sequences were retrieved and aligned to generate a neighbor-joining treewith a Dayoff amino acid substitution model. Values above the branches were estimated based on 1,000 bootstrap replicates.

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plemented (Comp) strains (Fig. 2C and D). For example, aftergrowth on PDA for 14 days, the sporulation of the �mrGAT mu-tant (1.47 � 106 0.25 � 106 conidia cm�2) decreased drastically(P � 0.01) compared to the WT (22 � 106 5.046 � 106 conidiacm�2) and Comp (25.64 � 106 1.45 � 106 conidia cm�2)strains. This effect also persisted in the mutant after growth for upto 20 days (Fig. 2D). Consistent with the above in silico analysis,mrGAT was confirmed to target to the ER by green fluorescentprotein (GFP) fusion and ER-specific staining (see Fig. S2 in thesupplemental material).

Deletion of mrDHAPAT gene in M. robertsii did not result inobvious phenotype changes, including nonsignificant differencesof conidiation and insect-killing ability against the silkworm lar-vae between the WT and mutant (see Fig. S3 in the supplementalmaterial). In addition, we did not find obvious alternation in LDformation in the mutant cells compared with the WT (see Fig. S4in the supplemental material). For total lipid content, a differencewas found between the WT and �mrDHAPAT conidial samples(P 0.0127) but not between their mycelial samples (P 0.0948)(see Fig. S5 in the supplemental material). It is noteworthy thatrepeated trials failed to obtain the mrGAT and mrDHAPAT dou-ble deletion mutants in M. robertsii, implying a lethal effect.

Effects of mrGAT on the biosynthesis of triacylglycerol, glyc-erol, and total lipid content. The first step in the TAG biosynthe-sis pathway is the catalysis of G3P to LPA by GPAT (1, 4). Notsurprisingly, cellular accumulation of TAG was significantly (P �0.001) reduced in the conidia and mycelia of the null mutantcompared to WT and Comp (Fig. 3A). In addition, cellular glyc-erol in the �mrGAT mutant was also significantly (P � 0.01) lowerthan in WT and Comp (Fig. 3B). Quantification of total lipids inthe conidial samples showed that the �mrGAT mutant had only52% (19.51 0.22 �g 10�7 spores) of the total lipid content in theWT (37.25 0.18 �g 10�7 spores) (P 7.71�6e) and 67% of thatin Comp (28.87 0.38 �g10�7 spores) (P 2.64�5e), respec-tively (Fig. 3C). In mycelia, the total lipid content of the �mrGATmutant (40.00 0.85 �g mg�1, dry weight) was also significantlylower than that of WT (59.38 1.01 �g mg�1, dry weight; P 0.0011) and that of Comp (53.02 0.91 �g mg�1, dry weight; P 0.0052) (Fig. 3D).

Effect of mrGAT on lipid droplets formation. In eukaryoticcells, lipids such as neutral triglycerides are stored in LDs (27). Toexamine and compare the formation of LD in the WT and mutantstrains, TEM and fluorescent staining assays were conducted. Theresults indicated that, compared to WT and Comp, the �mrGATmutant stored far fewer LDs in the conidia (Fig. 4A to C; Fig. 5A)and no visible LDs were observed in �mrGAT mycelia (Fig. 4D toF; Fig. 5C). Relative to WT, fewer LDs were also observed in themutant germlings (Fig. 5B) and appressoria (Fig. 5D and E). For-mation and stabilization of LDs are essentially controlled by LDsurface perilipin proteins, such as the Mpl1 protein in M. robertsii(17). Since the LD formation was impaired in �mrGAT, we com-pared the accumulation pattern of Mpl1 between the WT and null

FIG 2 Gene disruption, complementation, and phenotyping in Metarhiziumrobertsii. (A) PCR confirmation. Genomic DNAs extracted from the wild-type(WT), �mrGAT, and gene complemented mutant (Comp) strains were used asthe templates for PCR. CK, negative control using water as the template. (B)RT-PCR verification of mrGAT gene in WT, �mrGAT, and Comp strains. Tub,�-tubulin gene. (C) Phenotypic characterization. In contrast to WT andComp, the �mrGAT mutant had impaired conidia (upper panels) and pig-ment production (lower panels show the reverse sides of the plates) aftergrowth on PDA for 2 weeks. (D) Quantification of conidial production by WT,�mrGAT, and Comp strains after growth on PDA for 14 or 20 days.

FIG 3 Quantification of triacylglycerol (TAG), glycerol, and total lipids. (A)Conidia harvested from PDA after 20 days of culture and mycelia collectedfrom SDB after 3 days of culture were used for TAG analysis to demonstrate thedifferences among the WT, �mrGAT, and Comp strains. (B) Differences inglycerol content among the WT, �mrGAT, and Comp strains. (C) Differencesin total lipid content in conidia among the WT, �mrGAT, and Comp strains.(D) Total lipid content variations in mycelia among the WT, �mrGAT, andComp strains. MDW, mycelium dry weight.

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mutant. Western blot analysis demonstrated the highly reducedaccumulation of Mpl1 in the conidia and mycelia of the �mrGATstrain compared to the WT and Comp (Fig. 4G), which is consis-tent with the failure in LD formation. This indicated an associa-tion between mrGAT and the perilipin protein MPL1.

mrGAT is required for full virulence in M. robertsii. We ob-served that deletion of mrGAT did not impair the formation ofappressoria on the surfaces of either hydrophobic plastic plates(Fig. 5D and E) or locust hind wings (see Fig. S6 in the supple-mental material). To determine the effect of mrGAT on fungalvirulence, we performed injection and immersion bioassays insilkworm larvae. The results demonstrated that the median le-thal time (LT50) for the topical infection of the �mrGAT mu-tant (LT50 4.30 0.15 days) was significantly longer than forthe WT (LT50 3.40 0.13 days, �2 20.23 and P � 0.0001)and Comp (LT50 3.36 0.12 days, �2 21.71 and P �0.0001) (Fig. 6A), which indicated impaired fungal virulenceafter deletion of mrGAT. However, when the fungal sporeswere injected directly into the insect hemocoels (body cavities),which bypasses insect cuticles, no significant differences wereobserved between the �mrGAT (LT50 1.97 0.04 days) andWT (LT50 1.95 0.06 days, �2 0.05 and P 0.8173)strains or between the �mrGAT and Comp (LT50 1.95 0.04days, �2 0.12 and P 0.7323) strains (Fig. 6B). These resultsindicated that deletion of mrGAT reduced virulence by impair-ing the ability of the fungus to penetrate host cuticle.

DISCUSSION

In this study, we present the characterization of mrGAT, which isa GPAT family protein, in a filamentous fungus. We found that incontrast to what occurs in the budding yeast, plant, nematode, andanimals, only a single copy of the GPAT gene is present in thegenomes of M. robertsii and other insect- and plant-pathogenicfungi belonging to the subphylum Pezizomycotina, Ascomycota.Unlike the lethal effect of GPAT isoform gene deletion in yeastsand nematodes (8, 10), the �mrGAT mutant was viable but hadreduced abilities to sporulate, accumulate TAG and glycerol, andform LD. The mrGAT null mutant could successfully form ap-pressoria, similar to WT; however, the mutant took a substantiallylonger time to kill insects than the WT and gene complementationmutant during topical infection but not during injection assays,indicating that the lack of mrGAT diminished fungal capacity topenetrate insect cuticles, which in turn reduced virulence. Dele-tion of a mammalian DHAPAT-like gene, mrDHAPAT, which isputatively involved in an alternate pathway for LPA production,in M. robertsii did not result in significant changes, if any at all, inmutant physiologies.

The G3P pathway is a crucial physiological process in TAG andphospholipid metabolisms and energy balance (1, 28). In differentorganisms, various numbers of GPAT isoforms have been re-ported, and the functions of each isoform in the G3P pathwayhave been presumed by incorporation of different fatty acid moi-eties into TAG (3). Different numbers of GPAT are also found inthe fungal lineage, with multiple copies observed in basal zygomy-cete species but only a single copy in most ascomycete species (Fig.1). This discordance is most likely the result of gene loss that issupported by the absence of GPAT in the lineage of obligate mi-crosporidia, which suffer extensive gene losses during host adap-tation (29). However, gene duplication events could not be pre-cluded due to the presence of multiple copies of GPAT in differentyeast, mushroom, and zygomycete species (Fig. 1). A single copyof GPAT found in M. robertsii and other fungal species in subphy-lum Pezizomycotina suggests that in these species the proteinfunctions solely in the G3P pathway. In contrast to the lethal effectof �GAT1 �GAT2 in yeast mutants (8), the �mrGAT mutant isviable but has a reduced yet detectable level of TAG in the mutantcells relative to the control (Fig. 3A). This finding suggests thatfilamentous fungi could have an alternate pathway for LPA and inturn TAG biosynthesis. Indeed, the presence of the mammalian-protein-like mrDHAPAT in M. robertsii but not in yeast couldexplain, at least in part, why the �mrGAT strain of M. robertsii isviable while �GAT1 �GAT2 strains of S. cerevisiae cease to grow.The fact that double deletion mutants of mrGAT and mrDHAPATcould not be acquired suggests a similar lethal effect by a completeabolishment of LPA production in the fungus. It remains to bedetermined whether like yeast GAT1 (7, 8), mrGAT could alsoconvert DHAP into 1-acyl-DHAP or not.

Besides the critical role in initiating TAG biosynthesis, individ-ual GPAT isoforms also contribute to cell polarized growth inyeast (12), LD size increase in fruit fly and mammalian cells (4),and mitochondrial fragmentation in nematode (10). In this study,we found that together with a reduction in cellular TAG level,�mrGAT mutants also had impaired sporulation (�90% reduc-tion compared to WT) and LD formation. Fungal conidiation/fertility has been known to be associated with cellular lipid com-position and dynamics in M. robertsii and Neurospora crassa (23,

FIG 4 Visualization of cellular lipid droplets (LDs) and Western blot analysisof LD surface protein. Conidia from the WT, �mrGAT, and Comp strainsharvested from PDA plates after growth for 20 days were used for TEM anal-ysis. In contrast to what was observed with the WT (A) and Comp (C) strains,accumulation of LDs (black arrows) was significantly reduced in the �mrGATmutant (B). Mycelia cultured in SDB for 3 days were also examined, and theresults showed that in comparison to the WT (D) and Comp (F) strains, novisible LDs were found in the �mrGAT mutant (E) (white arrows point tomitochondria). Bar, 2 �m. (G) Western blot analysis indicated that, in contrastto WT and Comp, cellular accumulation of the LD surface perilipin proteinMPL1 was significantly reduced in �mrGAT mycelia and conidia.

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30). Therefore, deletion of mrGAT leading to the failure of fungalsporulation could be due to TAG reduction, which in turn couldalter glycerolipid composition. Similarly, deletion of Acl-6 in C.elegans resulted in 70% of the mutants being sterile (10). In D.melanogaster, a DGAT protein (CG8112, isoform A) was also re-quired for oogenesis (31), and functional studies of membrane-bound O-acyltransferases (MBOATs) that contribute to the G3Ppathway showed that germ cell development, which is guided bylipid signals in fruit fly, requires redundant protein function (32).

In eukaryotes, TAG is stored as LD in every cell type. Except forthe observation that the GPAT4 is associated with LD size growthin flies and mammals (4), deletion of other GPAT isoforms doesnot directly abolish cellular LD formation in different organisms(1, 3). In this study, we provide the evidence to link GPAT with LDbiogenesis in a filamentous fungus in which the number of LDswas significantly reduced in null mutant conidia and completelydisappeared in mutant hyphae. LDs are independent organellesthat are composed of a neutral lipid core and a phospholipidmonolayer anchored by different LD-specific proteins (27). De-pending on the cell types, components of the neutral lipid corecontain TAG mainly (ca. 44%), DAG (1.6%), cholesteryl esters(ca. 34%), and unknown neutral lipids (ca. 20%) (18). Therefore,the significantly reduced TAG levels in the �mrGAT mutant couldhave contributed to the failure to form the neutral lipid core. In

addition, we found that the disruption of mrGAT impaired theaccumulation of MPL1 (Fig. 4G), the essential LD surface perili-pin protein localized on the phospholipid monolayer to maintainLD structure (17). Taken together, it is not surprising that LDformation is severely impaired or fails in the �mrGAT strain.

In both plant- and insect-pathogenic fungi such as M. oryzaeand M. robertsii, accumulation of a high concentration of glycerolfor building up turgor pressure within the appressorium is a pre-requisite for successful penetration of host cuticles (16, 17). In thisstudy, we found that mrGAT but not mrDHAPAT is required forthe full virulence of M. robertsii by contributing to fungal pene-tration of insect cuticles. Along with TAG reduction, glycerol con-centration was also reduced in �mrGAT cells (Fig. 3B), explainingthe loss/reduction of turgor pressure in mutant appressoria. Ourdata also suggest that similar to what is seen in M. oryzae (19),glycerol production in M. robertsii could be mainly from the lipol-ysis of TAG rather than from carbohydrate sources. In this respect,G3P production in filamentous fungi could be from the conver-sion of the glycolytic intermediate dihydroxyacetone phosphateby G3P dehydrogenase (GPD) rather than the phosphorylation ofglycerol by glycerol kinase (33). A genome survey found a putativeGPD (MAA_06993) in M. robertsii that is similar to the yeast iso-forms GPD1 (46% identity) and GPD2 (44%), which control G3Pproduction in S. cerevisiae (34).

FIG 5 Staining with fluorescent dye shows the accumulation of LDs in different cell types of M. robertsii. (A) LD accumulation in the conidia of wild-type (WT),�mrGAT, and Comp strains harvested from PDA plates after 20 days. (B) LD distribution in the germlings of WT, �mrGAT, and Comp strains grown in aminimum medium with 1% glycerol for 10 h. (C) LD accumulation in the mycelia of WT, �mrGAT, and Comp strains grown in SDB for 3 days. (D) LDdistribution in appressoria of WT, �mrGAT, and Comp strains induced on a hydrophobic surface for 24 h. (E) LD distribution in appressoria of WT, �mrGAT,and Comp strains induced on a hydrophobic surface for 48 h. Bar, 5 �m.

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In conclusion, our results reveal both the conservative and thedivergent roles of GPAT in the G3P pathway in a filamentousfungus model. Unlike what is seen in yeasts, plants, and animals,the single copy of mrGAT and the nonlethal effect of gene deletionin M. robertsii suggest that this fungus can serve as a better modelfor future studies to elucidate the G3P pathway in fungi, particu-larly to determine how the G3P pathway is mechanistically con-nected with LD biogenesis and contributes to fungal pathogenicprocesses.

ACKNOWLEDGMENTS

This study is supported by the National Natural Science Foundation ofChina (grant no. 31225023) and the National Hi-Tech Research and De-velopment Program of China (grant no. 2011AA10A204).

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