1

1

Glycerol-3-phosphate acyltransferase contributes to triacylglycerol biosynthesis, 2

lipid droplet formation and host invasion in Metarhizium robertsii 3

4

Qiang Gao, Yanfang Shang, Wei Huang, and Chengshu Wang* 5

6

Key Laboratory of Insect Developmental and Evolutionary Biology, Institute of Plant 7

Physiology and Ecology, Shanghai Institutes for Biological Sciences, Chinese 8

Academy of Sciences, Shanghai 200032, China 9

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11

*To whom correspondence may be addressed: 12

Chengshu Wang, 13

Tel.: (86) 21 5492 4157; 14

Fax: (86) 21 5492 4015; 15

Email: cswang@sibs.ac.cn 16

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AEM Accepts, published online ahead of print on 27 September 2013Appl. Environ. Microbiol. doi:10.1128/AEM.02905-13Copyright © 2013, American Society for Microbiology. All Rights Reserved.

2

Enzymes involved in the triacylglycerol (TAG) biosynthesis have been well 18

studied in the model organisms of yeasts and animals. Among these, the isoforms 19

of glycerol-3-phosphate acyltransferase (GPAT) redundantly catalyze the first 20

and rate-limiting step in glycerolipid synthesis. Here, we report the functions of 21

mrGAT, a GPAT ortholog, in an insect pathogenic fungus Metarhizium robertsii. 22

Unlike yeasts and animals, a single copy of the mrGAT gene is present in the 23

fungal genome and the gene deletion mutant is viable. Compared to the wild type 24

and gene-rescued mutant, ΔmrGAT demonstrated reduced abilities to produce 25

conidia and synthesize TAG, glycerol and total lipids. More importantly, we 26

found that mrGAT is localized to the endoplasmic reticulum and directly linked 27

to the formation of lipid droplets (LDs) in fungal cells. Insect bioassay results 28

showed that mrGAT is required for full fungal virulence by aiding fungal 29

penetration of host cuticles. Data from this study not only advance our 30

understanding of GPAT functions in fungi, but also suggest that filamentous 31

fungi such as M. robertsii can serve as a good model to elucidate the role of 32

glycerol phosphate pathway in fungal physiology, particularly to determine the 33

mechanistic connection of GPAT with LD formation. 34

35

36

3

The glycerol phosphate pathway is the major pathway for triglyceride biosynthesis in 37

various organisms ranging from bacteria to animals to provide the crucial energy 38

molecules as well as serve as a repository for biosynthesis of fatty acids and 39

phospholipids (1). Among the various enzymes involved in this pathway, 40

glycerol-3-phosphate acyltransferase (GPAT) is the first enzyme and catalyzes the 41

acylation of glycerol 3-phosphate (G3P) that results in lysophosphatidic acid (LPA), 42

which is the precursor for the biosynthesis of phosphatidic acid, diacylglycerol (DAG) 43

and triacylglycerol (TAG) (2, 3). Different isoforms of GPAT have been characterized 44

in yeast, plants and animals. For example, two redundant GPATs, i.e. GAT1 (GPT2p) 45

and GAT2 (SCT1p) have been identified in the budding yeast Saccharomyces 46

cerevisiae, three copies each in Drosophila melanogaster and C. elegans, four each in 47

human and mouse, and eight copies in the plant, Arabidopsis thaliana (3, 4). While 48

most GPATs acylate the sn-1 position of G3P to produce LPA, the GPAT4 and GPAT6 49

isoforms in A. thaliana are involved in cutin biosynthesis and predominantly esterify 50

acyl groups at the sn-2 position of G3P to produce sn-2 monoacylglycerol (5). In 51

animals, a peroxisomal dihydroxyacetone-phosphate acyltransferase (DHAPAT) can 52

provide an alternate route for LPA production by acylation of dihydroxyacetone 53

phosphate (DHAP) and the subsequent reduction of 1-acyl-DHAP to LPA (6). Yeast 54

GAT1 could also convert DHAP into 1-acyl DHAP (7,8) and the later could be further 55

reduced to LPA by 1-acyldihydroxyacetone-phosphate reductase (AYR1) in yeast (9). 56

Fungal-like DHAPAT is still unknown. 57

Mammalian GPAT1 and GPAT2 are localized on the outer membrane of 58

4

mitochondria (MT) while GPAT3 and GPAT4 are targeted to the endoplasmic 59

reticulum (ER) (3, 10). A recent study showed that D. melanogaster and mammalian 60

GPAT4 isoforms could be relocalized from ER to the surface of nascent lipid droplets 61

(LDs) where they mediate LD growth (4). Similarly, GPAT isoforms, ACL-4 and 62

ACL-5 from C. elegans are also localized on the ER membrane. However, ACL-6 is a 63

MT-type GPAT, which is required to control MT fusion and nematode oogenesis (10). 64

Yeast GAT1 and GAT2 also called as microsomal GPATs, are ER-type GPATs (11) 65

and contribute to polarized cell growth (12). Deletion of either gene does not affect 66

yeast cell growth, however, the double deletion mutants are not viable (13). The GPAT 67

homolog has not been characterized thus far in filamentous fungi. 68

The ubiquitous insect pathogenic fungus M. robertsii is a biocontrol agent used 69

worldwide to control different insect pests (14, 15). Similar to plant pathogens, insect 70

pathogens such as M. robertsii infect hosts by penetrating host cuticles (16), which is 71

mediated by high concentrations of glycerol within the infection structure, appressoria 72

(17, 18). It has been shown that high concentration of glycerol in the appressoria of 73

the plant pathogen, Magnaporthe oryzae, are largely lipolytic products that originated 74

from stored TAG [ca. 44% of LD components, (19)] rather than from carbohydrate 75

sources by on-site biosynthesis (20). It is possible that the GPAT(s) in fungal 76

pathogens also contribute to fungal virulence. To investigate this, we studied the 77

functional characteristics of a GPAT from the entomopathogenic fungus, M. robertsii, 78

designated as mrGAT (MAA_02162) (21). Our data indicate the presence of a single 79

copy of mrGAT in the genome of M. robertsii and the gene deletion mutant although 80

5

viable has significantly impaired TAG accumulation, LD formation and virulence 81

against insect hosts. We also found a single copy of human DHAPAT (NP_055051) 82

like protein gene present in the genome of M. robertsii (MAA_02767, 25% identity 83

with human DHAPAT; designated as mrDHAPAT). Deletion of this gene did not 84

result in any obvious phenotypic and physiological changes in the mutants when 85

compared with the wild-type strain. 86

87

MATERIALS AND METHODS 88

Strains and culture conditions. To collect conidia for the experiments, the wild-type 89

(WT) strain ARSEF2575 of M. robertsii was grown on Potato dextrose agar (PDA, 90

Difco) at 25°C for 20 days. Spore germination and appressorium induction assays 91

were conducted using locust (Schistocerca gregaria) hind wings or the minimal 92

medium (MM: NaNO3, 6 g L-1, KCl, 0.52 g L-1, MgSO4·7H2O, 0.52 g L-1, KH2PO4, 93

0.25 g L-1) amended with 1% glycerol as the sole carbon resource (MMGly) (15). For 94

genomic DNA and RNA extractions, fungal spores were cultured in Sabouraud 95

dextrose broth (SDB, Difco) at 25°C and 200 rpm for 3 days in a rotary shaker. 96

Phylogenetic analysis. To determine the phylogeny of mrGAT across fungal 97

lineages, the homologs of mrGAT were retrieved from selected fungal pathogens and 98

saprophytes of ascomycetes, basidiomycetes, microsporidia, chytrid and zygomycetes 99

with well-annotated genomes using Blastp searches with a cutoff E value of <1e-100. 100

Sequence alignment was conducted using CLUSTAL X 2.0, and a neighbor joining 101

tree was generated using MEGA 5.2 (22) with a Dayoff amino acid substitution model, 102

6

a pairwise deletion for missing residues or gaps and 1000 bootstrap replicates. 103

Prediction of mrGAT subcellular localization was performed with the program 104

ProtComp (ver. 9.0, Softberry) and TargetP (ver. 1.1) (23). 105

Gene deletion and complementation. For functional studies, mrGAT gene was 106

deleted using an Agrobacterium-mediated transformation method as described in our 107

previous study (24). In brief, the 5'- and 3'- flanking regions of mrGAT were amplified 108

by PCR using the genomic DNA as a template with the primer pairs 109

mrGATUF/mrGATUR, and mrGATDF/mrGATDR (Table S1). The amplified products 110

were subsequently cloned into the PstI and SpeI restriction sites of the binary vector 111

pDHt-SK-ben (conferring resistance against benomyl) for fungal transformation (24) 112

to create the deletion mutant (ΔmrGAT). To complement gene deletion, the mrGAT 113

gene was amplified together with its promoter and 3′- UTR region with the primers 114

mrGATCompF and mrGATCompR and the product was sub-cloned into the SpeI site 115

of the binary vector pDHt-SK-Bar (conferring resistance against ammonium 116

glufosinate) before fungal transformation to obtain the complemented mutants 117

(Comp). Transformants were verified by PCR and RT-PCR analyses using primers 118

mrGATF and mrGATR (Table S1). The β-tubulin gene (MAA_02081) was used as the 119

control and amplified using primers TubF and TubR. Deletion of mrDHAPAT was 120

similarly performed using the primer pairs mrDHAPATUF/mrDHAPATUR, 121

mrDHAPATDUF/mrDHAPATDR, respectively (Table S1). 122

Examination of protein localization. To confirm ER-localization of mrGAT, the 123

ORF of mrGAT gene was amplified by PCR with the primer pairs mrGAT-GFP1F and 124

7

mrGAT-GFP1R to delete the stop codon and include the promoter region of the gene 125

(1970 bp upstream the start codon). The Egfp gene was amplified from the plasmid 126

pEGFP (15) with the primers mrGAT-GFP2F and mrGAT-GFP2R (Table S1). The 127

acquired fragments were purified and jointed together by a fusion PCR reaction (24). 128

The product was digested with the restriction enzymes SpeI and EcoRI and cloned 129

into the same enzyme-treated plasmid pDHt-SK-Bar (conferring resistance against 130

glufosinate) for Agrobacterium-mediated transformation (24). The acquired 131

mrGAT-GFP strain was cultured in SDB for 3 days and the mycelia were washed 132

twice with the Hank’s balanced salt solution (GIBCO) before staining with the 133

fluorescent dye ER-TrackerTM Blue-White DPX (E12353, Invitrogen) for 30 min. The 134

images were taken with a fluorescence microscopy BX51-33P (Olympus). 135

Appressorium induction and lipid droplets visualization. Conidia from the WT, 136

ΔmrGAT and Comp were inoculated into individual polystyrene Petri dishes (5.5 cm 137

in diameter) containing 2 ml MMGly medium at a final concentration of 2×105 spores 138

ml-1. After incubation for 18 hrs, spore germination and appressorium differentiation 139

rates were recorded for more than one hundred spores under a microscope. 140

Appressoria were also induced on locust (S. gregaria) hind wings as described 141

previously (25). To visualize and compare the formation of LDs, conidia, germlings 142

and appressoria from the WT, ΔmrGAT and Comp were washed twice with PBS and 143

then stained with a fluorescent dye Bodipy (D3922, Invitrogen) for 30 min (18). The 144

accumulation of intracellular LDs was observed under a transmission electron 145

microscope (TEM) as described previously (24). Fungal samples were fixed in 2.5% 146

8

glutaraldehyde in 0.1 M phosphate buffer (pH 7.2) at 4°C for 12 hrs, rinsed three times 147

in phosphate buffer and fixed overnight in 1% osmium tetroxide in 0.1 M cacodylate 148

buffer (pH 7.0) at 4°C. After rinsing three times in phosphate buffer, samples were 149

dehydrated in a gradient ethanol series, infiltrated with a graded series of epoxy resin 150

in epoxy propane, and then embedded in Epon resin and sectioned. The ultrathin 151

sections were stained in 2% uranium acetate followed by lead citrate and visualized 152

under a transmission electron microscope (Hitachi, H-7650, Tokyo, Japan) operating 153

at 80 kV. 154

Free glycerol and triacylglycerol assays. Conidia were collected from two week 155

old cultures on PDA plates, while mycelia were collected from 3 days old cultures in 156

SDB. Glycerol content in the samples were assayed using a free glycerol assay kit 157

E1012 (Applygen Technologies Inc., Beijing, China) at 550nm, while triglycerides 158

were assayed using a triglyceride assay kit E1014 (Applygen Technologies Inc., 159

Beijing, China) at 550nm. First, all samples were washed twice with phosphate buffer, 160

homogenized in extraction buffer and centrifuged at 5,000 g for 5 min. Supernatants 161

were aliquoted and incubated with the reaction buffer for 30 min before determining 162

the OD at 550 nm using a microplate reader (Varioskan Flash Multimode Reader, 163

Thermo Scientific). Total protein concentration in supernatants was estimated using 164

the Bradford method. All experiments were repeated twice and three replicates were 165

maintained for each sample. Glycerol and triglyceride concentrations in different 166

samples were expressed as micromole glycerol or triglyceride per milligram of total 167

proteins, respectively. 168

9

Total lipid quantification. To determine the effect of mrGAT on lipid biosynthesis, 169

total lipid was quantified by a phosphoric acid-vanillin method (26). Conidia from 170

WT, ΔmrGAT and Comp were harvested from the PDA plates incubated at 25°C for 171

20 days, and mycelia were collected from the SDB broth after incubation at 220 rpm, 172

25°C for 3 days. For assays, spore suspensions (0.5 ml 1.0×108 conidia ml-1) were 173

added to glass tubes and 1 mg mycelia homogenates (dried overnight at 150°C) was 174

added to glass tubes with 0.5 ml water. Then, 2 ml of 18 M H2SO4 was added to each 175

tube and boiled in a water bath for 10 min, cooled for 5 min at room temperature 176

before adding 5 ml phosphoric acid-vanillin reagent (1.2 g L-1 vanillin, 200 ml of 177

water and adjusted to 1 L with 85% H3PO4). The tubes were then incubated at 37°C 178

for another 15 min and centrifuged to determine absorbance at 530 nm (18). All 179

experiments were repeated twice. A standard curve was generated using triolein 180

(Sigma) for quantification. 181

Western blot analysis. Proteins were extracted from fungal conidia and mycelia 182

with the RIPA lysis buffer (Thermo Scientific) containing 1mM of the protease 183

inhibitor, phenylmethylsulfonyl fluoride (PMSF). Proteins were separated on 12% 184

sodium dodecyl sulfate containing polyacrylamide gels and transferred onto 185

polyvinylidene difluoride membranes. The membranes were probed with the 186

antibodies against the LD surface perilipin protein, MPL1, and β-tubulin, respectively 187

(24). 188

Insect Bioassays. To investigate the effect of mrGAT on fungal virulence, insect 189

10

bioassays were conducted on second day, fifth-instar Bombyx mori larvae. Conidia 190

from WT, ΔmrGAT, Comp and ΔmrDHAPAT strains were applied topically by 191

immersing the larvae in an aqueous suspension containing 5×106 conidia ml-1 for 1 192

min or by injection to the second proleg a 10 μl suspension containing 1×106 spores 193

ml-1. Each treatment had three replicates with 15 insects each and all experiments 194

were repeated three times. Larval mortality was recorded every 12 hrs and the median 195

lethal time (LT50) was estimated by Kaplan-Meier analysis using SPSS (ver. 13.0). 196

197

RESULTS 198

Characteristics of the mrGAT protein. The complete open reading frame (ORF) of 199

mrGAT encodes a protein with 766 amino acids and a predicted molecular weight of 200

84.9 kDa and pI 9.63. MrGAT is a typical member of Pfam 01553 family of 201

acyltransferases and is homologous to yeast GAT1 (GPT2p, 34% identity) and GAT2 202

(SCT1p, 37% identity). Similar to other organisms it contains the four highly 203

conserved AGPAT (1-acyl-sn-glycerol-3-phosphate acyltransferase) motifs for 204

catalysis (motifs I and IV) and G3P binding (motifs II and III) (Table S2). In silico 205

analysis with the programs TargetP and ProtComp indicated with a high confidence 206

score that mrGAT is localized in the endoplasmic reticulum. Survey of the M. 207

robertsii genome revealed the presence of only one copy of the gene, which is similar 208

to other fungi from the subphylum Pezizomycotina (Phylum: Ascomycota) (Fig. 1). 209

Single copy of GPAT was also found in the fission yeast (Schizosaccharomyces pombe) 210

11

and chytrid (Batrachochytrium dendrobatidis). In contrast, two copies were found in 211

the subphylum Saccharomycotina (Ascomycota), which includes the budding yeast 212

and four copies in the zygomycete fungus, Rhizopus delemar (Fig. 1). In the 213

basidiomycete fungal species either one or two copies of the GPAT gene was found. 214

Interestingly, a GPAT homolog was absent in the genomes of the microsporidian fungi. 215

Relative to the well-established fungal tree-of-life (27), GPAT gene evolutionary tree 216

is not congruent with fungal speciation phylogeny across the phyla. For example, 217

chytrids are in general considered to have diverged early to form a basal-clade within 218

the fungal kingdom while the GPAT tree is rooted by yeast GPATs, and the chytrid 219

GPAT is more closely related to ascomycetes than zygomycetes (Fig. 1). Genome 220

survey of mammalian-like DHAPAT genes showed that a single copy of the gene is 221

present in the genomes of M. robertsii and other ascomycete species of 222

Pezizomycotina subphylum, but not in the yeast species of Saccharyomycotina (Fig. 223

S1). 224

Characterization of the mutants. In S. cerevisiae, GAT1 and GAT2 are 225

functionally redundant because viability is not affected by deletion of either gene, 226

however, double deletion mutants are lethal (13). To determine the effect of mrGAT 227

on fungal viability gene deletion and complementation were performed via 228

Agrobacterium -mediated transformation, and the resultant strains were analyzed by 229

PCR and RT-PCR (Fig. 2 A and B). We found that ΔmrGAT strain of M. robertsii was 230

viable although the ability to sporulate and form pigments were impaired when 231

compared to the WT and complemented (Comp) strains (Fig. 2 C and D). For example, 232

12

after growth on PDA for 14 days, the sporulation of ΔmrGAT [(1.47±0.25)×106 233

conidia cm-2] decreased drastically (P < 0.01) when compared to the WT 234

[(22±5.046)×106 conidia cm-2] and Comp [(25.64±1.45)×106 conidia cm-2] strains. 235

This effect also persisted in the mutant after growth for up to 20 days (Fig. 2D). 236

Consistent with above in silico analysis, mrGAT was confirmed to target to the ER by 237

GFP fusion and ER specific staining (Fig. S2). 238

Deletion of mrDHAPAT gene in M. robertsii did not result in obvious phenotype 239

changes including non-significant differences of conidiation and insect-killing ability 240

against the silkworm larvae between the WT and mutant (Fig. S3). In addition, we did 241

not find obvious alternation in LD formation in the mutant cells when compared with 242

the WT (Fig. S4). For total lipid content, a difference was found between the WT and 243

ΔmrDHAPAT conidial samples (P = 0.0127) but not between their mycelial samples 244

(P = 0.0948) (Fig. S5). It is noteworthy that the repeated trials failed to obtain the 245

mrGAT and mrDHAPAT double deletion mutants in M. robertsii, implying a lethal 246

effect. 247

Effects of mrGAT on the biosynthesis of triacylglycerol, glycerol and total lipid 248

content. The first step in the TAG biosynthesis pathway is the catalysis of G3P to 249

LPA by GPAT (1, 4). Not surprisingly, cellular accumulation of TAG was significantly 250

(P < 0.001) reduced in the conidia and mycelia of the null mutant when compared to 251

WT and Comp (Fig. 3A). In addition, cellular glycerol in ΔmrGAT was also 252

significantly (P < 0.01) lower when compared to WT and Comp (Fig. 3B). 253

Quantification of total lipids in the conidial samples showed that ΔmrGAT (19.51±254

13

0.22 μg 10-7 spores) had only 52% of the total lipid content in the WT (37.25±0.18 μg 255

10-7 spores) (P = 7.71-6e) and 67% in Comp (28.87±0.38 μg10-7 spores) (P = 2.64-5e), 256

respectively (Fig. 3C). In mycelia, total lipid content of ΔmrGAT (40.00±0.85 μg mg-1 257

dry weight) was also significantly lower than WT (59.38±1.01 μg mg-1 dry weight; P 258

= 0.0011) and Comp (53.02±0.91 μg mg-1 dry weight; P = 0.0052), respectively (Fig. 259

3D). 260

Effect of mrGAT on lipid droplets formation. In eukaryotic cells, lipids such as 261

neutral triglycerides are stored in lipid droplets (LDs) (28). To examine and compare 262

the formation of LD in the WT and mutant strains, TEM and fluorescent staining 263

assays were conducted. The results indicated that, in contrast to WT and Comp, 264

ΔmrGAT stored much fewer LDs in the conidia (Fig. 4A to C; Fig. 5A) and no visible 265

LDs were observed in ΔmrGAT mycelia (Fig. 4D to F; Fig. 5C). Relative to WT, 266

fewer LDs were also observed in the mutant germlings (Fig. 5B) and appressoria (Fig. 267

5D and E). Formation and stabilization of LDs are essentially controlled by LD 268

surface perilipin proteins, such as the Mpl1 protein in M. robertsii (18). Since the LD 269

formation was impaired in ΔmrGAT, we compared the accumulation pattern of Mpl1 270

between the WT and null mutant. Western blot analysis demonstrated the highly 271

reduced accumulation of Mpl1 in the conidia and mycelia of ΔmrGAT when compared 272

to the WT and Comp (Fig. 4 G), which is consistent with the failure in LD formation. 273

This indicated an association between mrGAT and the perilipin protein, MPL1. 274

mrGAT is required for full virulence in M. robertsii. We observed that deletion of 275

mrGAT did not impair formation of appressorium on the surfaces of either 276

14

hydrophobic plastic plates (Fig. 5D and E) or locust hind wings (Fig. S6). To 277

determine the effect of mrGAT on fungal virulence, we performed topical injection 278

and immersion bioassays in silkworm larvae. The results demonstrated that the 279

median lethal time (LT50) for the topical infection of ΔmrGAT (LT50=4.30 ± 0.15 days) 280

was significantly longer than the WT (LT50=3.40 ±0.13 days, χ2=20.23 and P<0.0001) 281

and Comp (LT50=3.36 ± 0.12 days, χ2=21.71 and P<0.0001) (Fig. 6A), which 282

indicated impaired fungal virulence after deletion of mrGAT. However, when the 283

fungal spores were injected directly into the insect hemocoels (body cavities), which 284

bypasses insect cuticles, no significant differences were observed between the 285

ΔmrGAT (LT50=1.97 ± 0.04 days) and WT (LT50=1.95 ± 0.06 days, χ2=0.05 and 286

P=0.8173) or between the ΔmrGAT and Comp (LT50=1.95 ± 0.04 days, χ2=0.12 and 287

P=0.7323) (Fig. 6B). These results indicated that deletion of mrGAT reduced 288

virulence by impairing the ability of the fungus to penetrate host cuticle. 289

290

DISCUSSION 291

In this study, we present the characterization of mrGAT, which is a GPAT family 292

protein, in a filamentous fungus. We found that in contrast to the budding yeast, plant, 293

nematode and animals, only a single copy of the GPAT gene is present in the genomes 294

of M. robertsii and other insect and plant pathogenic fungi belonging to the 295

subphylum Pezizomycotina, Ascomycota. Unlike the lethal effect of GPAT isoform 296

gene deletion in yeasts and nematodes (10, 13), ΔmrGAT was viable but had reduced 297

15

abilities to sporulate, accumulate TAG and glycerol, and form LD. The mrGAT null 298

mutant could successfully form appressoria similar to WT, however, the mutant took a 299

substantially longer time to kill insects than the WT and gene complementation 300

mutant during topical infection but not during injection assays, indicating that the lack 301

of mrGAT diminished fungal capacity to penetrate insect cuticles, which in turn 302

reduced virulence. Deletion of a mammalian DHAPAT-like gene mrDHAPAT, which 303

is putatively involved in an alternate pathway for LPA production, in M. robertsii did 304

not result in significant changes, if not all, in mutant physiologies. 305

The G3P pathway is a crucial physiological process in TAG and phospholipid 306

metabolisms and energy balance (1, 29). In different organisms various numbers of 307

GPAT isoforms have been reported and the functions of each isoform in the G3P 308

pathway have been presumed by incorporation of different fatty acid moieties into 309

TAG (3). Different numbers of GPAT are also found in the fungal lineage with 310

multiple copies observed in basal zygomycete species but a single copy in most 311

ascomycete species (Fig. 1). This discordance is most likely the result of gene loss 312

that is supported by the absence of GPAT in the lineage of obligate microsporidia, 313

which suffer extensive gene losses during host adaptation (30). However, gene 314

duplication events could be not precluded due to the presence of multiple copies of 315

GPAT in different yeast, mushroom and zygomycete species (Fig. 1). A single copy of 316

GPAT found in M. robertsii and other fungal species in the Pezizomycotina 317

subphylum suggests that in these species the protein functions solely in the G3P 318

pathway. In contrast to the lethal effect of ΔGAT1ΔGAT2 in yeast mutants (13), 319

16

ΔmrGAT is viable but has reduced yet detectable level of TAG in the mutant cells 320

relative to the control (Fig. 3A). This finding suggests that filamentous fungi could 321

have an alternate pathway for LPA and in turn TAG biosynthesis. Indeed the presence 322

of a mammalian-like mrDHAPAT in M. robertsii but not in yeast could explain, at 323

least in part, why ΔmrGAT strain of M. robertsii is viable while ΔGAT1ΔGAT2 strains 324

of S. cerevisiae cease to grow. The fact that double deletion mutants of mrGAT and 325

mrDHAPAT could not be acquired suggests a similar lethal effect by a complete 326

abolishment of LPA production in the fungus. It remains to be determined whether 327

like yeast GAT1 (7,8), mrGAT could also convert DHAP into 1-acyl-DHAP or not. 328

Besides the critical role in initiating TAG biosynthesis, individual GPAT isoforms 329

also contribute to cell polarized growth in yeast (12), LD size increase in fruit-fly and 330

mammalian cells (4) and mitochondrial fragmentation in nematode (10). In this study, 331

we found that together with a reduction in cellular TAG level, ΔmrGAT also had 332

impaired sporulation (>90% reduction when compared to WT) and LD formation. 333

Fungal conidiation/fertility has been known to be associated with cellular lipid 334

composition and dynamics in M. roberstii and N. crassa (24, 31). Therefore, deletion 335

of mrGAT leading to the failure of fungal sporulation could be due to TAG reduction, 336

which in turn could alter glycerolipid composition. Similarly, deletion of Acl-6 in C. 337

elegans resulted in 70% of the mutants being sterile (10). In D. melanogaster, a 338

DGAT protein (CG8112, isoform A) was also required for oogenesis (32) and 339

functional studies of membrane-bound O-acyltransferases (MBOATs) that contributes 340

to G3P pathway showed that germ cell development, which is guided by lipid signals 341

17

in fruit fly, requires redundant protein function (33). 342

In eukaryotes TAG is stored as LD in every cell type. Except for the observation 343

that the GPAT4 is associated with LD size growth in flies and mammals (4), deletion 344

of other GPAT isoforms does not directly abolish cellular LD formation in different 345

organisms (1, 3). In this study, we provide the evidence to link GPAT with LD 346

biogenesis in a filamentous fungus where the number of LDs was significantly 347

reduced in null mutant conidia and completely disappeared in mutant hyphae. LDs are 348

independent organelles that are composed of a neutral lipid core and a phospholipid 349

monolayer anchored by different LD-specific proteins (28). Depending on the cell 350

types, components of the neutral lipid core mainly contain TAG (ca. 44%), DAG 351

(1.6%), cholesteryl esters (ca. 34%) and unknown neutral lipids (ca. 20%) (19). 352

Therefore, the significantly reduced TAG levels in ΔmrGAT could have contributed to 353

the failure to form the neutral lipid core. In addition, we found that the disruption of 354

mrGAT impaired the accumulation of MPL1 (Fig. 4G), the essential LD surface 355

perilipin protein localized on the phospholipid monolayer to maintain LD structure 356

(18). Taken together, it is not surprising that LD formation is severely impaired or 357

failed in ΔmrGAT. 358

In both plant and insect pathogenic fungi such as M. oryzae and M. robertsii, 359

accumulation of a high concentration of glycerol for building up turgor pressure 360

within the appressorium is a prerequisite for successful penetration of host cuticles 361

(17, 18). In this study, we found that mrGAT but not mrDHAPAT is required for the 362

full virulence of M. robertsii by contributing to fungal penetration of insect cuticles. 363

18

Along with TAG reduction, glycerol concentration was also reduced in ΔmrGAT cells 364

(Fig. 3B) explaining the loss/reduction of turgor pressure in mutant appressoria. Our 365

data also suggest that similar to M. oryzae (20) glycerol production in M. robertsii 366

could be mainly from the lipolysis of TAG rather than from carbohydrate sources. In 367

this respect, G3P production in filamentous fungi could be from the conversion of the 368

glycolytic intermediate dihydroxyacetone phosphate by G3P dehydrogenase (GPD) 369

rather than the phosphorylation of glycerol by glycerol kinase (34). A genome survey 370

found a putative GPD (MAA_06993) in M. robertsii that is similar to yeast isoforms, 371

GPD1 (46% identity) and GPD2 (44%) that control G3P production in S. cerevisiae 372

(35). 373

In conclusion, our results reveal both the conservative and divergent roles of GPAT 374

in the G3P pathway in a filamentous fungus model. Unlike yeasts, plants and animals, 375

a single copy of mrGAT and the non-lethal effect of gene deletion in M. robertsii 376

suggests that this fungus can serve as a better model for future studies to elucidate the 377

G3P pathway in fungi, particularly to determine how the G3P pathway is 378

mechanistically connected with LD biogenesis and contribute to fungal pathogenic 379

processes. 380

381

ACKNOWLEDGMENT 382

This study is supported by the National Natural Science Foundation of China (Grant 383

No. 31225023) and the National Hi-Tech Research and Development Program of 384

China (Grant No. 2011AA10A204). 385

386

19

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496

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Figure legends: 497

FIG 1 Phylogenetic analysis of fungal glycerol-3-phosphate O-acyltransferases. 498

Protein sequences were retrieved and aligned to generate a neighbor joining tree with 499

a Dayoff amino acid substitution model. Values above the branches were estimated 500

based on 1000 bootstrap replicates. 501

502

FIG 2 Gene disruption, complementation and phenotyping in Metarhizium robertsii. 503

(A) PCR confirmation. Genomic DNA extracted from the wild type (WT), ΔmrGAT 504

and gene complemented mutant (Comp) were used as templates for PCR. CK, 505

negative control using water as template. (B) RT-PCR verification of mrGAT gene in 506

WT, ΔmrGAT and Comp. Tub, β-tubulin gene. (C) Phenotypic characterization. In 507

contrast to WT and Comp, ΔmrGAT had impaired conidia (upper panels) and pigment 508

production (lower panels show the reverse sides of the plates) after growth on PDA 509

for two weeks. (D) Quantification of conidial production by WT, ΔmrGAT and Comp 510

after growth on PDA for 14 or 20 days. 511

512

FIG 3 Quantification of triacylglycerol (TAG), glycerol and total lipids. (A) Conidia 513

harvested from the PDA after 20 days of culture and mycelia collected from SDB 514

after three days of culture were used for TAG analysis to demonstrate the differences 515

among the wild type (WT), ΔmrGAT and Comp. (B) Differences in glycerol content 516

among the WT, ΔmrGAT and Comp strains. (C) Differences in total lipid content in 517

conidia among the WT, ΔmrGAT and Comp strains. (D) Total lipid content variations 518

in mycelia among the WT, ΔmrGAT and Comp strains. MDW, mycelium dry weight. 519

23

520

FIG 4 Visualization of cellular lipid droplets (LDs) and western blot analysis of LD 521

surface protein. Conidia from the wild type (WT), ΔmrGAT and Comp mutant 522

harvested from PDA plates after growth for 20 days were used for TEM analysis. In 523

contrast to the WT (A) and Comp (C), accumulation of LDs (black arrows) was 524

significantly reduced in ΔmrGAT (B). Mycelia cultured in SDB for three days were 525

also examined and the results showed that in comparison to the WT (D) and Comp (F), 526

no visible LDs were found in ΔmrGAT (E, white arrows point to mitochondria). Bar, 2 527

μm. (G) Western blot analysis indicated that, in contrast to WT and Comp, cellular 528

accumulation of the LD surface perilipin protein, MPL1, was significantly reduced in 529

ΔmrGAT mycelia and conidia. 530

531

FIG 5 Staining with fluorescent dye shows the accumulation of LDs in different cell 532

types of M. robertsii. (A) LD accumulation in the conidia of wild type (WT), ΔmrGAT 533

and Comp harvested from PDA plates after 20 days. (B) LD distribution in the 534

germlings of WT, ΔmrGAT and Comp grown in a minimum medium with 1% glycerol 535

for 10 hrs. (C) LD accumulation in the mycelia of WT, ΔmrGAT and Comp grown in 536

SDB for three days. (D) LD distribution in appressoria of WT, ΔmrGAT and Comp 537

induced on a hydrophobic surface for 24 hrs. (E) LD distribution in appressoria of WT, 538

ΔmrGAT and Comp induced on a hydrophobic surface for 48 hrs. Bar, 5 μm. 539

540

FIG 6 Insect bioassays. (A) Survival of silkworm larvae after topical application of 541

24

the conidial suspension (1×107 conidia ml-1) from WT, ΔmrGAT and Comp strains. 542

Control insects were treated with 0.05% Tween-20 for 30 seconds. (B) Survival of 543

silkworm larvae following an injection of 10μl of 1×106 conidia ml-1 suspensions 544

from WT, ΔmrGAT and Comp strains into the second proleg of larvae. Control insects 545

were injected with 10μl 0.05% Tween-20. 546

547

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549