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High Trap Formation and Low Metabolite Production by Disruption of the PKS Gene Involved in the Biosynthesis of Arthrosporols from Nematode-Trapping Fungus Arthrobotrys oligospora Zifei Xu, Baile Wang, Hongkai Sun, Ni Yan, Zhijun Zeng, Keqin Zhang, and Xuemei Niu J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 30 Sep 2015 Downloaded from http://pubs.acs.org on October 8, 2015

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Journal of Agricultural and Food Chemistry

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High Trap Formation and Low Metabolite Production

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by Disruption of the PKS Gene Involved in the

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Biosynthesis of Arthrosporols from Nematode-Trapping

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Fungus Arthrobotrys oligospora

5 Zi-Fei Xu†, Bai-Le Wang†, Hong-Kai Sun†, Ni Yan†, Zhi-Jun Zeng†, Ke-Qin Zhang†, Xue-Mei Niu*,†

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†

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Resources of the Ministry of Education, Yunnan University, Kunming, 650091, People’s Republic of

Laboratory for Conservation and Utilization of Bio-Resources & Key Laboratory for Microbial

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China

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*Corresponding author (Tel: 86-871-65032538; Fax: 86-871-65034838: E-mail: [email protected])

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ACS Paragon Plus Environment

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ABSTRACT

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A group of morphology regulatory arthrosporol metabolites have been recently characterized from

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carnivorous fungus Arthrobotrys oligospora that can develop trapping networks to capture their prey. A

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combination of genetic manipulation and chemical analyses was applied to characterize the function of

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one polyketide synthase (PKS) gene AOL_s00215g283 in A. oligospora, which was putatively involved

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in the production of 6-methylsalicylic acid. HPLC analysis showed that the disruption of the PKS gene

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not only led to the total loss of the arthrosporol A, but also resulted in significant reduction in

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production of secondary metabolites in the cultural broth of the mutant ∆AOL_s00215g283 strain.

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Interestingly, the mutant strain displayed significant increases in the trap formation and the nematicidal

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activity by 10 times and twice, respectively, higher than the wildtype strain. These findings revealed a

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pathogenicity-related biosynthetic gene of this agriculturally important biological agent and have

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implications for establishment of efficient fungal biocontrol agents.

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KEYWORDS: Nematode-trapping fungus; Arthrobotrys oligospora; 6-methylsalicylic acid; biosynthesis; arthrosporol

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INTRODUCTION

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Plant parasitic nematodes are one of the most devastating pest groups responsible for insidious disease

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symptoms in different crops and cause significant global crop loss.1,2 Because of the banning of the most

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important chemical nematicides, the need for alternative management systems for the control of plant-

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parasitic nematodes has increased dramatically over the last decade. Therefore, biological control of

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phytonematodes has received an enhanced impetus.3

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Nematode-trapping fungi show a distinguishing feature, predatism, which is the ability to capture

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nematodes with developing specific trapping devices such as adhesive networks, adhesive knobs, and

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constricting rings to capture nematodes and then extract nutrients from their nematode prey.4 The

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formation of trapping devices by nematode-trapping fungi is an important indicator of their switch from

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the saprophytic to the predacious lifestyles. They play important roles in controlling nematode

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population density in diverse natural environments and are of great importance for improving

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agricultural production. The behavior and ecology of nematode-trapping fungi have been studied in the

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past in attempts to make them better biological control agents.5

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Arthrobotrys oligospora Fres., the first recognized nematode-trapping fungus,6 is the most commonly

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isolated and by far the most abundant nematode-trapping fungus in the environment.7 Strains of A.

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oligospora have been found in diverse soil environments including heavy metal-polluted soils and

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decaying wood where they live mainly as saprophytes.7.8 In the presence of nematodes, A. oligospora

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enters the predatory stage by forming complex three-dimensional networks to trap nematodes.9 It has

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been assumed to be among the biggest contributors in controlling the population of nematodes, and

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extensively studied as a model system for identifying and characterizing the ecology and biology of

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nematode-trapping fungi.3-10

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A unique class of the oligosporon metabolites have been reported from the strains of A. oligospora

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from The Netherlands,11 Australia12 and China,13 respectively. The skeletal feature of these secondary

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metabolites is a polyketide-derived epoxy nucleus combined with a terpenoid-derived linear farnesyl

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unit. These compounds exhibited interesting biological activities, including nematicidal and ACS Paragon Plus Environment

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antibacterial properties. Recently, we characterized a new group of oligosporon metabolites,

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arthrosporols (Figure 1), with a novel carbon scaffold from A. oligospora.14 The arthrosporol

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metabolites possessed the similar polyketide-derived epoxy nucleus combined with a six-membered

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monocyclic farnesyl unit and displayed significant autoregulatory effects on the formation of

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conidiophores and the transition of hypha to two dimensional network in A. oligospora, which correlate

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with fungal reproductive and defensive capabilities, respectively.14

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Up to now, the arthrosporols metabolites represented the most complex structural types of bioactive

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metabolites to be isolated and characterized from nematophagous fungi. Hybrid natural metabolites

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derived through mixed biosynthetic pathways, coupled with their interesting bioactivity profile, have

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been evoking our great interest in the biosynthesis of this class of naturally occurring metabolites from A.

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oligospora.

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We now report the identification of the PKS gene encoding 6-methylsalicylic acid synthase involved

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in the biosynthesis of the arthrosporol metabolites in A. oligospora. The effect of disruption of the PKS

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gene on production of arthrosporol metabolites was investigated. The formation of adhesive trapping

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networks and capture of nematodes by the mutant strain was also studied. Our results suggested that the

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PKS gene might play an important role in regulating the trap formation and the life-style switching of

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this fungus.

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MATERIALS AND METHODS

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Sequence and phylogenetic analysis of AOL_s00215g283 in A. oligospora

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Predicted AOL_s00215g283 in A. oligospora was annotated by BLAST searches against protein

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databases, and by Inter-ProScan searches against protein domain databases. The amino acid sequence of

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AOL_s00215g283 in A. oligospora was downloaded from the GenBank, and the sequences from

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different fungi were downloaded from GenBank. The amino acid sequences from different fungi were

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analyzed using the DNAman software package, version 5.2.2 (Lynnon Biosoft, St. Louis, Canada). A

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neighbor-joining tree was constructed using the Mega 5.1 software package. A web-based analysis ACS Paragon Plus Environment

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platform antiSMASH 2.0,15 was applied to perform genome mining of the biosynthetic gene clusters.

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The conserved functional domains were analyzed using InterProScan 4.8 with default parameter

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settings.

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Fungal material

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The strain A. oligospora YMF 1.3170 was isolated from Jiuquan, Gansu, People’s Republic of

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China,14 and identified as A. oligospora by morphological features. The isolate was deposited in the

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strain collection of Laboratory for Conservation and Utilization of Bio-Resources & Key Laboratory for

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Microbial Resources of the Ministry of Education, Yunnan University. After the conidia of A.

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oligospora YMF 1.3170 had developed on potato dextrose agar (PDA) slants in test tubes at 25 °C, the

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strain was kept at −30 °C as stock cultures. DNA extraction of A. oligospora YMF 1.3170 and PCR

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amplification of the ITS rRNA gene were carried out according to the literature. A characteristic

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fragment (GenBank, accession no. JX244893) was amplified by PCR using the species-specific primers

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ITS4 and ITS5, and the genome DNA of A. oligospora YMF 1.3170 as template. The BLAST searches

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in the GenBank showed that the DNA fragment was most similar to the published ITS sequences of A.

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oligospora, consistent with morphological identifications.

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Disruption vector construction

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The plasmid pAg1-H3,16 which was used for gene disruption vector construction and transformation,

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was maintained in Escherichia coli strain DH5а (Takara, Shiga, Japan). The strain A. oligospora YMF

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1.3170 used in this study was maintained on PDA at 28 °C. Genomic DNA of A. oligospora YMF

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1.3170 was extracted as previously described.17 Restriction endonucleases and DNA modifying enzymes

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were from New England Biolabs (Beverly, Massachusetts, MA). In-Fusion HD Cloning Kits were from

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Clontech Laboratories (Mountain View, California, CA). The left and right DNA fragments flanking the

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hygromycin resistant gene (hygR) in pAg1-H3 vector were PCR amplified from the genomic DNA of A.

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oligospora YMF 1.3170 by GXL high-fidelity DNA Polymerase (Takara, Shiga, Japan), using primer ACS Paragon Plus Environment

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283-5f:

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sets

GAGCTCGGTACCAAGGCCCGGGGGAGCCGCAGAGGACAACA;

283-5r:

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AGGCCTGATCATCGATGGGCCCCGACATGCCATGCCGATT;

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TCTAGAGGATCCCCCGACTAGTATTATCGGTAGCCTTTGTC;

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CACGAAGCTTGCATGCCTGCAGGAGGATTCGGTATTGTTGTT

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fragments (5' flank and 3' flank) were purified using NucleoSpin Gel and PCR Clean-up Kit

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(MACHEREY-NAGEL, Düren, Germany). The purified 5' flank DNA fragment was inserted into the

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Sma I and Apa I-digested pAg1-H3 vector by the in-fusion method to produce pAg1-H3-283-5′.

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Similarly, the 3' flank DNA fragment was inserted into the Spe I and Sbf I sites of pAg1-H3-283-5′ to

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generate the completed disruption vector pAg1-H3-283-5′-3′.

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The homologous fragment amplifications were carried out as follows. One microliter of the prepared

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genomic DNA template from A. oligospora YMF 1.3170 was added to a 50 µL PCR amplification

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system using GXL high-fidelity DNA polymerase following the manufacturer’s instructions (Takara).

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All PCRs were carried out in a Veriti 96-well thermal cycler (PE Applied Biosystems). The

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amplification program consisted of predenaturation at 98 °C for 3 min followed by 30 cycles of

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denaturation at 98 °C for 10 s, annealing at 57 °C for 15 s, and elongation at 68 °C for 2 min, with a

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final extension step at 68 °C for 10 min.

283-3f: and, respectively.

283-3r: The

DNA

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Preparation of fungal protoplasts

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Regeneration medium PDASS (PDA supplied with 0.6 M sucrose, 0.3 g/L yeast extract, 0.3 g/L tryptone,

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0.3 g/L peptone) was used for protoplast regeneration,18 and PDASS supplemented with 200 µg/mL

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hygromycin B (Amresco, Solon, OH) was used for selecting transformants. In the initial transformation

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for the disruption of the PKS gene, four 1cm diameter mycelia plugs from YMA medium (2 g/L yeast

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extract, 10 g/L malt extract, 18 g/L agar) for 7 days at 28 °C were inoculated into 100 mL of TG medium

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consisting of 1% tryptone (Oxoid, Basingstoke, UK) and 1% glucose and cultures at 30 °C at 180 rpm

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for 36 h. The mycelia were harvested and resuspended in 20 mL of a filter-sterilized enzyme solution

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that contained 120 mg of lysing enzymes (Sigma, St. Louis, MO), 0.4 mL of cellulase (Sigma, St. Louis, ACS Paragon Plus Environment

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MO), and 100 mg of snailase (Solarbio, Beijing, China) in 0.6 M MgSO4 at pH 6.0. Snailase, extracted

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from crop and digestive tract of snails, consists of more than 30 enzymes, including cellulase,

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hemicellulase, β-glucuronidase, etc. The suspension was incubated for 4 h at 28 °C on a rotary shaker at

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180 rpm. Protoplasts were collected by filtering through six layers of sterile lens-cleaning tissue and

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centrifuged at 1000 g. The protoplasts were washed twice with KTC (1.2 M KCl, 10 mM Tris-HCl, 50

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mM CaCl2) solution and finally resuspended in the same solution.

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Transformation of fungus A. oligospora

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The protoplast-based protocol for the gene disruption in A. oligospora YMF 1.3170 was performed as

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described as previous report,18 with modifications. 100 µL protoplasts (circa 8.0×107 mL-l) were mixed

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with 10 µg linear DNA in a 1.5 mL centrifuge tube. After 30 min of incubation on ice, 600 µL of PTC

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(50% polyethylene glycol 6,000, 20 mM Tris-HCl, pH 7.5, 50 mM CaCl2) was added into the mixture

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and mixed gently. After incubation at 28 °C for 1 h, regeneration for 12 h, the putatively transformed

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protoplasts were plated onto PDAS medium (PDA supplemented with 5 g/L molasses, 0.6 M saccharose,

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0.3 g/L yeast extract, 0.3 g/L tryptone, and 0.3 g/L casein peptone) containing 200 µg/mL of hygromycin

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B (Roche Corporation, Germany). The transformation colonies were selected after incubation at 28 °C

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for 6-7 days. After single spore isolation, the putative transformants were transferred onto TYGA

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medium alone (10 g/L tryptone, 10 g/L glucose, 5 g/L yeast extract, 5 g/L molasses, 18 g/L agar)

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containing 200 µg/mL of hygromycin B and incubated for 5 days at 28 °C. Genomic DNA of putative

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transformants were extracted and were verified by PCR to check for the integration of genes in the

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genome. The positive transformants were further confirmed by Southern blot analyses. Southern

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analysis was carried out according to the instructions provided by the North2South chemiluminescent

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hybridization

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(TTGGAGAAACCGAACAACCT) and 283-S2 (TTGGTGAGGAGTGAATAGCG) were used as

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Southern hybridization probes, and restriction enzyme SspI was used to digest the genomic DNA of the

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wildtype A. oligospora and the mutant strains for Southern analysis.

and

detection

kit

(Pierce,

Rockford,

IL).


The

primer

pair

283-S1

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Vegetative growth and conidia yield comparisons

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Nine mm diameter mycelia plugs from 7 day old fungal colonies of the wildtype and the mutant strains

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were inoculated onto 9 cm plates with different media (6 replicates/strain), respectively. The mycelia

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plugs of the same size and age were incubated at 28 °C at both light and dark conditions for 6–10 days,

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respectively. Radial colony growth rates and colony morphology were recorded at 24 h interval for 1

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week and growth rate calculated as mm/h each day. The growth rates and the colony morphology of the

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wildtype and the mutant strains were compared in PDA, YMA (2 g/L yeast extract, 10 g/L malt extract,

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18 g/L agar) and TYGA (10 g/L tryptone, 10 g/L glucose, 5 g/L yeast extract, 5 g/L molasses, 18 g/L

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agar) media, respectively. Similarly, to test the adaptive abilities, the growth rates were compared in

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TGA medium supplemented with stress-related compounds, including 0.1-0.3 mol/L NaCl, 5-15

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mmol/L H2O2, and 0.01%-0.03% sodium dodecyl sulfate (SDS), respectively.

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To assess sporulation capacities of the wildtype and the mutant strains, 100 µL aliquots of conidial

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suspension were spread on 9 cm plates with YMA medium and incubated for 10 days at 28 °C, the

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conidia were washed into 10 mL sterile water, followed by filtration through four layers of lens tissues

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to remove mycelial debris. The concentrations of the conidial suspension were determined with

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microscopic counts on a haemocytometer.18

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Induction of trap formation and virulence assays

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Caenorhabditis elegans (strain N2) was cultured in oatmeal medium at 22 °C for 6–7 days. The

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wildtype and the mutant strains were cultivated on agar plates by spreading approximately 106 conidia

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per plate. After 3 days of incubation at 28 °C, 300 adult nematodes were added into the middle of the

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plate. Fungal mycelia and traps were observed and the numbers of captured nematodes were recorded

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after 12 and 24 h, respectively, under light microscopy (Olympus, Tokyo, Japan). The experiments were

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performed in triplicate.

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Fermentation and extraction procedures.

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The wildtype A. oligospora and the ∆AOL_s00215g283 mutant strains cultured on PDA medium at 28

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°C for 7 days were inoculated into 500 mL ﬂasks containing 250 mL of production PD medium (potato

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200 g/L, dextrose 20 g/L). The culture broths of the 14-day-old liquid fermentations (1.5 L each strain)

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were filtered to separate the mycelia from the cultures. The culture filtrates were concentrated in vacuo

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to 50 mL and were exhaustively extracted overnight with ethyl acetate (1:1, v/v). The organic fractions

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were evaporated to dryness to give residues. The dried organic residues were then dissolved in 500 µL

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of methanol, filtered through 0.22 µm membranes, and further analyzed using HPLC-DAD and GC-MS.

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HPLC analysis

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HPLC-analysis was carried out using a HPLC-1200 Series (Agilent, Waldbronn, Germany) system

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equipped with a G1311A binary pump and G1315A photodiode array UV/Vis detector, employing the

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following instrumental conditions: column, CAPCELL PAK C18, 5 µm; 4.6 × 250 mm (Shiseido); The

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total flow rate was 1 mL/min; mobile phase A was 0.1% formic acid in water and mobile phase B was

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0.1% formic acid in acetonitrile. Column temperature was maintained at 40 °C. Injection volume for the

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extracts was 25 µL. The LC conditions were manually optimized based on separation patterns with the

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following gradient: 0-2 min, 10% B; 10 min, 25% B; 30 min, 35% B; 35 min, 50% B; 45 min, 90% B;

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47 min, 10% B; 49 min, 10% B. UV spectra were recorded at 220-400 nm. The standard sample

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arthrosporol A was isolated from wildtype A. oligospora according to the modified method described in

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the literature.14 A 14-day-old fermentation broth of strain A. oligospora YMF 1.3170 cultured on PDA

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medium was filtered to separate the mycelia from the culture. The culture filtrate was concentrated in

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vacuo and partitioned with acetyl acetate, and the organic part was evaporated to dryness to give an oily

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residue. The residue was loaded onto a macroporous resin column (7 x 80 cm) and eluted with

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H2O−MeOH (0%, 25%, 50%, 90%, each in 3 L) to yield four fractions (A−D) based on TLC behavior.

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Fraction C obtained on elution with 50−90% MeOH−H2O was further subjected to a Sephadex LH-20

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gel column (4 x 150 cm) eluting with MeOH (2.5 L) to yield six subfractions. Subfraction 3 was ACS Paragon Plus Environment

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repeatedly subjected to a Sephadex LH-20 gel column (3 x 100 cm) eluting with acetone (2.3 L), a C18

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column (3 x 50 cm) eluting with H2O−MeOH (35%, 37%, 39%, each in 1.8 L ), and a silica gel column

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(2 x 40 cm) eluting with CHCl3−MeOH (35:1, 20:1, each in 1.5 L) to obtain arthrosporol A. The

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purified sample arthrosporol A was elucidated and confirmed with NMR data.14

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GC-MS analysis, metabolite identification and semi-quantitation

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The GC-MS analysis was performed with a 5890 series II Plus gas chromatograph interfaced with a

217

model 5972 mass spectrometric detector (Hewlett-Packard, Avondale, PA) equipped with a 30 m long,

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0.25 mm i.d., and 0.5 µm film thickness HP5-MS capillary column. The temperature was programmed

219

from 100-300 °C at a rate of 5 °C/min. Helium was used as a carrier gas at a flow rate of 0.7 mL/min.

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The split ratio was 1:20, the injector temperature 280 °C, the interface temperature 300 °C, and the

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ionization voltage 70 eV.

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Identification of these peaks was performed through retention time index and mass spectrum.19

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Compounds were designated as metabolites if they were identified with a match 900 on a scale of 0 to

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1,000 and RI deviation, 3.0. The semi-quantitative analysis of the main compounds was carried out by

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internal normalization with the area of each compound. The addition of each area of the compounds

226

corresponds to 100% area.20

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Statistical analysis

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Statistical analysis were performed using the SPSS package, version 17.0 for Windows (Chicago, IL).

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Differences with P

High Trap Formation and Low Metabolite Production by Disruption of

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