Fungal Genetics and Biology 41 (2004) 189–198

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Fungal Genetics and Biology 41 (2004) 189–198Fungal Genetics and Biology 41 (2004) 189–198

The determinant step in ergot alkaloid biosynthesisby an endophyte of perennial ryegrass

Jinghong Wang,a Caroline Machado,a Daniel G. Panaccione,b Huei-Fung Tsai,a,1

and Christopher L. Schardla,*

a Department of Plant Pathology, University of Kentucky, Lexington, KY 40546-0312, USAb Division of Plant and Soil Sciences, West Virginia University, Morgantown, WV 26506-6058, USA

Received 29 March 2003; accepted 6 October 2003

Abstract

Many cool-season grasses harbor fungal endophytes in the genus Neotyphodium, which enhance host fitness, but some also

produce metabolites—such as ergovaline—believed to cause livestock toxicoses. In Claviceps species the first step in ergot alkaloid

biosynthesis is thought to be dimethylallyltryptophan (DMAT) synthase, encoded by dmaW, previously cloned from Claviceps

fusiformis. Here we report the cloning and characterization of dmaW from Neotyphodium sp. isolate Lp1, an endophyte of perennial

ryegrass (Lolium perenne). The gene was then disrupted, and the mutant failed to produce any detectable ergovaline or simpler ergot

and clavine alkaloids. The disruption was complemented with the C. fusiformis gene, which restored ergovaline production. Thus,

the biosynthetic role of DMAT synthase was confirmed, and a mutant was generated for future studies of the ecological and ag-

ricultural importance of ergot alkaloids in endophytes of grasses.

� 2003 Elsevier Inc. All rights reserved.

1. Introduction

Fungal endophytes in the genus Neotyphodium (sex-

ual state¼Epichlo€ee) form symbiotic and often mutual-

istic associations with cool-season grasses (subfamily

Po€ooideae). Their hosts include several important forage

and turf grasses, to which the endophytes confer en-

hanced resistance to insect and mammalian herbivores,

as well as enhanced drought tolerance, nematode resis-tance, mineral uptake, competitiveness, growth, and

fecundity (Clay and Schardl, 2002; Malinowski and

Belesky, 2000). Endophytic Neotyphodium spp. inhabit

apoplasts of their hosts, colonize all aerial tissues in-

cluding shoot and floral meristems, leaves, culms, sto-

lons, and seeds, and in the case of the asexual

endophytes, can only be transmitted vertically by sys-

* Corresponding author. Fax: 1-859-323-1961.

E-mail addresses: schardl@uky.edu, schardl@pop.uky.edu (C.L.

Schardl).1 Present address: NIAID, National Institutes of Health, Bldg. 10,

Rm. 11C304, 10 Center Drive, MSC 1882, Bethesda, MD 20892-1882,

USA.

1087-1845/$ - see front matter � 2003 Elsevier Inc. All rights reserved.

doi:10.1016/j.fgb.2003.10.002

temic infection of embryos and seedlings (Freeman,1904; Sampson, 1937). Benefits conferred to plants are

partially due to production by endophytes of biologi-

cally active alkaloids (Bush et al., 1997). Profiles of al-

kaloids vary among endophyte species and genotypes,

and altogether four alkaloid classes are known from

Neotyphodium spp. Of these, the ergot alkaloids and

lolitrems (indolediterpenes) cause neurotoxic effects on

grazing or granivorous vertebrates, peramine (a pyrrol-opyrazine) is an insect feeding deterrent, and lolines

(saturated 1-aminopyrrolizidines) are insecticidal (Bush

et al., 1997; Wilkinson et al., 2000).

Endophytes in forage grasses have elicited consider-

able ambivalence because, on the one hand they can

greatly enhance long-term persistence of pasture stands

subject to biotic or abiotic stress factors, whereas on the

other hand they can cause episodes of toxicosis to live-stock andwildlife (Thompson and Stuedemann, 1993). In

the United States, cattle and horses grazed on tall fescue

with the endophyte Neotyphodium coenophialum exhibit

symptoms of toxicosis including poor weight gain, low

fertility, agalactia, fat necrosis, and, in extreme cases,

gangrene and death (Thompson and Stuedemann, 1993).

190 J. Wang et al. / Fungal Genetics and Biology 41 (2004) 189–198

Consequently, annual economic losses to the US beefindustry were estimated in 1990 at $600 million (Hove-

land, 1993), with undetermined but substantial losses in

dairy, horses, and sheep as well. The symptoms are

reminiscent of poisoning by the infamous ergot fungus,

Claviceps purpurea (Raisbeck et al., 1991), which is a

relative of the Neotyphodium spp. (Kuldau et al., 1997).

Much of the biological activity of ergot is associated with

the ergopeptines, abundant and particularly potentpeptide derivatives of lysergic acid. For this reason, er-

govaline, the ergopeptine produced by Neotyphodium

spp., has been the subject of considerable research and is

thought to be a significant factor in toxicoses associated

with endophyte-infected ryegrasses and fescues (Bacon et

al., 1986; Cross, 2003). However, the roles of the simpler

lysergic acid derivatives and the clavines (which include

lysergic acid precursors and other derivatives) have notbeen assessed due to the technical challenges of alkaloid

purification or other means of conducting controlled

experiments. Endophytes mutated in specific biosyn-

thetic genes would provide raw material for controlled

tests to determine the roles of ergot alkaloids in livestock

toxicosis and other ecological factors.

Ergot alkaloid biosynthesis in Claviceps species (ergot

fungi) involves multiple steps, most of which have beenelucidated (Tudzynski et al., 2001). Mothes et al. (1958)

proposed that the ergoline ring system was derived from

prenylated tryptophan. Gr€ooger et al. (1960, 1959), and

Birch et al. (1960) demonstrated incorporation of radio-

labeled tryptophan and mevalonate into the ergoline ring

system. Heinstein et al. (1971) partially purified a dim-

ethylallyl-pyrophosphate: LL-tryptophan dimethylallyl

transferase (DMAT synthase) from a Claviceps sp. Thisactivity, which yields dimethylallyltryptophan (DMAT;

Fig. 1), was proposed to be the first pathway specific step

in alkaloid biosynthesis. DMAT synthase also has been

implicated as a key regulatory step for the pathway (Floss

et al., 1974; Krupinski et al., 1976).

Gebler and Poulter (1992) purified DMAT synthase

from Claviceps fusiformis and characterized it as a ho-

modimer of 52-kDa subunits. Tsai et al. (1995) thencloned the dmaW gene encoding this enzyme, and

expressed it in yeast to confirm the biochemical activity

Fig. 1. Activity of DMAT synthase. The 4-position of the tryptophan

aromatic ring system is prenylated by electrophilic substitution (Gebler

et al., 1992).

of the gene product. Tudzynski et al. (1999) identified arelated gene in a likely ergot alkaloid biosynthesis gene

cluster in C. purpurea strain P1.

Here we employed a Neotyphodium sp. endophyte of

perennial ryegrass (Lolium perenne) to conduct a genetic

test of the hypothesis that DMAT synthase is required

for ergot alkaloid production. We cloned and disrupted

dmaW in Neotyphodium sp. isolate Lp1, complemented

the mutant with the C. fusiformis gene, and introducedthe wild type, dmaW disruptant and complemented

strains into ryegrass plants to test for expression of

clavine and ergot alkaloids.

2. Materials and methods

2.1. Biological materials

Neotyphodium sp. Lp1, a natural hybrid of Neoty-

phodium lolii and Epichlo€ee typhina (Schardl et al., 1994),

was isolated by the method of Latch et al. (1984), as

modified by Schardl and An (1993), from surface-ster-

ilized leaf sheaths of infected L. perenne (perennial rye-

grass). Claviceps purpurea ATCC 20102 was obtained

from the American Type Culture Collection (Manassas,VA, USA; note that Accession No. 20102 has since been

reassigned by ATCC). The fungal cultures were rou-

tinely maintained on potato dextrose agar (PDA; Difco,

Detroit, MI, USA) plates at 21 �C in the dark.

Endophyte-minus seed of the tetraploid perennial

ryegrass (L. perenne) cultivar, Rosalin, was generously

provided by Dr. Kenneth Hignight (Advanta Seeds

Pacific, Albany, OR, USA). Symbiota of perennialryegrass with the wild type and other fungal strains in

this study were generated and maintained as previously

described (Panaccione et al., 2001).

Fungal DNA was isolated as previously described

(Byrd et al., 1990).

2.2. Southern-blot analysis

Hybridization probes were prepared by PCR with

dTTP partly substituted with hapten-labeled dUTP

(135 lM dTTP and 15 lM digoxigenin-dUTP; Roche–

Boehringer, Indianapolis, IN, USA) (Gebeyehu et al.,

1987). Genomic DNA was digested with restriction en-

donucleases as indicated, separated by agarose gel

electrophoresis, and transferred onto nylon membrane

(Hybond-N+; Amersham–Pharmacia Biotech, Piscata-way, NJ, USA) by the alkaline-blot method (Ausubel et

al., 2001). Blots were prehybridized, then hybridized at

42 �C with denatured probe, in 50% formamide, 6�sodium chloride/sodium citrate solution (SSC), 5�Denhardt�s solution (Ausubel et al., 2001), 0.1% sodium

dodecylsulfate (SDS), 0.1% sodium diphosphate, 50mM

Tris(hydroxymethyl)aminomethane (Tris)–HCl, pH 7.5,

J. Wang et al. / Fungal Genetics and Biology 41 (2004) 189–198 191

and 100 lg/ml denatured herring sperm DNA. Themembranes were then washed in 300ml of 2� SSC, 0.1%

SDS at room temperature for 5min, at 55 �C for 20min,

and at 55 �C for 30min. Immunological detection em-

ployed anti-digoxigenin antibody conjugated to alkaline

phosphatase and CSPD substrate (Roche–Boehringer).

The filters were exposed to double emulsion film (Bio-

Max Light-2, Sigma Chemical, St. Louis, MO, USA) to

record the chemiluminescent signal.

2.3. Sequence determinations and alignments

Cosmid and plasmid DNA templates were isolated by

using the Wizard Plus Maxipreps DNA Purification

System (Promega, Madison, WI, USA). Approximately

1 lg cosmid DNA or 200 ng plasmid DNA was used as

template in each sequencing reaction. PCR fragmentswere cleaned using Qiagen PCR purification kit (Qiagen,

Valencia, CA, USA), then 200–400 ng was used as se-

quencing templates. Sequencing reactions were carried

out by the Sanger random chain termination method

with BigDye Terminator Cycle Sequence Kit (Applied

Biosystems, Foster City, CA, USA), according to

manufacturer�s suggestions.Deduced amino acid sequences were aligned by using

Pileup, implemented in Seqweb Version 1.1 [Genetics

Computer Group (GCG), Wisconsin Package]. The

parameter settings of gap creation penalty and gap ex-

tension penalty were 8 and 2, respectively.

2.4. Cosmid library construction and screening

DNA was prepared from C. purpurea ATCC 20102,partially digested with MboI, and fragments of ca. 30–

50 kb isolated as described previously (Byrd et al., 1990).

The fragments were half-filled with dATP and dGTP.

Cosmid vector pMOcosX (Orbach, 1994) was digested by

XhoI, and then half-filled with dCTP and dTTP. Vector

and inserts were mixed at approx. 1:1 molar ratio, and

ligated with T4 DNA ligase. In vitro packaging of the li-

gation mix and transfection of Escherichia coli were car-ried out using Gigapack III XL packaging extract

(Stratagene Cloning Systems, La Jolla, CA, USA). Each

ampicillin-resistant colony was used to inoculate 200 lllibrary broth [a 9:1 mix of LB broth and 10� freezer

medium consisting of 360mM K2HPO4, 132mM

KH2PO4, 17mM Na-citrate, 4mM MgSO4, 68mM

(NH4)2SO4, and 44% glycerol] in a well of a 96-well mi-

crotitre plate, and grownovernightwith gentle shaking.Atotal of 3840 clones were arrayed and stored at )80 �C.

The C. purpurea cosmid library was screened as fol-

lows: a pool of all clones from each 96-well plate was

inoculated into LB with 100 lgml�1 ampicillin, and

grown overnight at 30 �C with vigorous shaking. Cells

were collected and cosmid DNAwas prepared byWizard

Plus Minipreps DNA Purification System (Promega).

The pooled cosmid DNAs were slot-blotted (Ausubel etal., 2001) onto a Hybond-N+ nylon membrane. The

probe was a digoxigenin-labeled full-length copy of

dmaW from C. fusiformis, generated by PCR using, as

template, pKAES132 (Tsai et al., 1995), and as primers,

50-GCGTCGACACGATGATGACAAAA GC-30 and

50-GCGTCGACGCAAAGACCCTTGACAT-30. Probepreparation and hybridization protocols were as de-

scribed above. Positive plates, then positive rows of thoseplates, then the positive clones were identified.

2.5. Amplification and cloning dmaW and flanking DNA

Sequence comparison of dmaW from C. fusiformis

ATCC 26245 (Tsai et al., 1995) and C. purpurea ATCC

20102 (this work) revealed potentially conserved amino

acid sequences, and degenerate oligonucleotide primersdeg1, deg2, deg3, and deg4 were designed based on those

regions (Fig. 2, Table 1). The primers were used in PCR

with genomic DNA of Neotyphodium sp. Lp1 as tem-

plate. Each 50 ll reaction mixture contained 200 ng

fungal genomic DNA template, 200 lM each dNTP,

2 lM each of two degenerate primers, 1�PCR buffer

(Applied Biosystems), and 2.5U TaqGold DNA poly-

merase (Applied Biosystems). Reaction mixtures wereheld at 93 �C for 9min, followed by 95 �C for 3min, then

subjected to 35 cycles of 94 �C for 45 s, 53 �C for 45 s,

72 �C for 80 s; then a final 5min incubation at 72 �C. ThePCR products were electrophoresed in low-melting-

temperature agarose (BioWhittaker Molecular Appli-

cations, Rockland, ME, USA), and fragments of the

expected sizes were excised. The gel slices were melted,

and 2 ll of each was used as template in a second roundof PCR with similar temperature cycling and variants of

the degenerate primers incorporating restriction endo-

nuclease cleavage sites as follows: deg1-EcoRI and deg2-

EcoRI had 50-CGGAATTC extensions; deg3-XbaI and

deg4-XbaI had 50-CGTCTAGA extensions. The ampli-

fication products were digested with EcoRI and XbaI,

gel purified, cloned into pBluescriptKS(+) (Stratagene

Cloning Systems, La Jolla, CA, USA), and sequenced.The 50- and 30-portions and flanking regions of dmaW

from Neotyphodium sp. Lp1 were amplified by cassette-

mediated PCR, with the Panvera (Madison, WI, USA)

LA-PCR kit, according to manufacturer�s suggestions.

Sequences of the dmaW-specific primers are available

from the authors on request.

2.6. Gene expression in yeast and enzyme activity assay

Coding sequences of dmaW from C. purpurea and

Neotyphodium sp. Lp1 were cloned into yeast expression

vector pMDM281, then yeast was transformed, grown,

induced and extracted, and DMAT Synthase activity

assays were conducted as previously described (Tsai

et al., 1995).

Fig. 2. Pileup alignment of amino acid sequences deduced from dmaW

of C. fusiformis (Cf), C. purpurea (Cp), and Neotyphodium sp. (Lp1).

Dots in sequences indicate alignment gaps, implying deletion or in-

sertion events in evolution of these genes. The consensus sequence

(Con) is in bold, and identifies positions identical in all confirmed

active dmaW genes; dashes indicate nonidentical positions. Conserved

intron positions (^) are: intron 1 after the second position of codon

397 in the alignment; intron 2 after the first position of codon 438.

Underlined sequences indicate regions on which degenerate primers

were designed, based on identity or near identity between Cf and Cp.

GenBank Accession Nos. for the nucleotide sequences are AY259837–

AY259840, AY262013, and AY262014.

192 J. Wang et al. / Fungal Genetics and Biology 41 (2004) 189–198

2.7. Generation and complementation of the dmaW

knockout

A targeting vector for gene disruption was con-

structed as follows: a 3.0-kb fragment containing 50-flanking sequence up to, but not including the dmaW

start codon, was amplified by PCR using primer SacII-Ns-dmaW()3011/)2981)d and BamHI-Ns-dmaW()1/)30)u, then cloned into pBluescriptKS(+) that was cut

with SacII and BamHI. PCR amplification with primer

Ns-dmaW(84.3/94.2)d and SalI-Ns-dmaW(3415/3383)u,

followed by digestion with BamHI and SalI, yielded a

3.1-kb fragment containing the Lp1 dmaW starting from

codon 114 into the 30-flanking region. This fragment was

then cloned adjacent to the 50-flanking sequence betweenthe BamHI and SalI sites. A cassette containing a hy-

gromycin resistance gene (hph) and controlling se-

quences (Panaccione et al., 2001) was cloned into the

BamHI site between the two dmaW-flanking fragments.

The resulting construct, pKAES170, was linearized with

SalI prior to introduction into Lp1 by transformation

(cleavage of the other end with SacII was impractical

due to a SacII site in the hph).Protoplasts of Neotyphodium sp. Lp1 were prepared

as described by Murray et al. (1992), except that the

mycelium was treated with 1.3mgml�1 Novozyme and

20mgml�1 glucanase. Then protoplasts were trans-

formed with the linearized construct by electroporation

as described by Tsai et al. (1992). After electroporation,

aliquots of the mixture were each mixed with 5ml

molten regeneration medium (Panaccione et al., 2001)and poured onto a regeneration medium plate contain-

ing hygromycin B (CalBiochem, La Jolla, CA, USA) at

300 lgml�1. The plates were incubated at 21 �C for 3–4

weeks and viable fungal colonies were transferred to

PDA with hygromycin B (200 lgml�1) for sporulation

and single-spore isolation. Hygromycin-resistant trans-

formants were screened for homologous recombination

by PCR using primers Ns-dmaW(31/37.2)d and Ns-dmaW(278/273.2)u. Clones that did not yield amplifi-

cation products were further analyzed by Southern-blot

analysis using, as probe, a mixture of two fragments

from the Neotyphodium sp. Lp1 dmaW region. These

fragments were amplified and labeled with digoxigenin

by PCR with the following primers (Table 1):

Ns-dmaW(44.2/38)u and Ns-dmaW()476/)457)d for

the probe just upstream of the dmaW coding region; andNs-dmaW(146/152.2)d and Ns-dmaW(445.1/438.3)u for

the coding sequence.

In the complementation vector construct,

pKAES157, dmaW cDNA from C. fusiformis was placed

under control of the promoter of the E. typhina b-tu-bulin gene (tub2; Tsai et al., 1992) by a PCR procedure

similar to that used previously for a Protub2-fusion to

Fusarium solani khs (Li et al., 1995). The product (Ptub2–dmaW) included the 230 bp 50 to the translational start

of tub2, followed by the dmaW coding and 30-untrans-lated sequences from pKAES132 (Tsai et al., 1995). The

product was cloned into pCB1004 (Carroll et al., 1994)

to generate pKAES157. Protoplasts from the dmaW

knockout mutant were prepared as described by Murray

et al. (1992) except that mycelium was treated with

Table 1

Primers used in this study

Primer name Sequencea Targetb Positionc

deg1 GARCARMGNYTNTGGTGGCA Cp dmaW-1 cd 25.1/31.2

deg2 GGNATHTTYAARAARCAYAT Cp dmaW-1 cd 56.1/62.2

deg3 ARNGTCCANARRTCYTCCAT Cp dmaW-1 cd 283.1/277.2

deg4 TANACYTGNGGYTCNGGCAT Cp dmaW-1 cd 350.1/344.2

SacII-Ns-dmaW(-3011/-2981)d GCCCGCGGAGTATAGTATAATATATAACCCTTATTATAC Ns dmaW nt )3011/)2981BamHI-Ns-dmaW()1/)30)u GCGGATCCTGTGAAGAAGAGGACGAGCGTAATAGCCCT Ns dmaW nt )1/)30Ns-dmaW(84.3/94.2)d CGGAACCCCGTTTGAGCTAAGTCTTAATTG Ns dmaW cd 84.3/94.2

SalI-Ns-dmaW(3415/3383)u GCGTCGACGCTAACTACTAGTTATCTTATTATAAAGATACC Ns dmaW nt +3415/+3383

Ns-dmaW(31/37.2)d CACAGCACGGCGCCAATGTT Ns dmaW cd 31.1/37.2

Ns-dmaW(278/273.2)u CGCTGGCAACGAGACCATTC Ns dmaW cd 278.3/273.2

Ns-dmaW(44.2/38)u GCAGTTTGGAGTATCTTTTG Ns dmaW cd 44.2/38.1

Ns-dmaW()476/)457)d TCTATGTCAGAATCGTACCG Ns dmaW nt )476/)457Ns-dmaW(146/152.2)d CAAGAGCTTACACTTGACGC Ns dmaW cd 146.1/152.2

Ns-dmaW(445.1/438.3)u CTCGCCGGCATGCGTCAAAA Ns dmaW cd 445.1/438.3

a Sequences written 50–30. R¼A or G; M¼A or C; Y¼T or C; H¼A, T, or C; N¼A, G, C, or T.bAbbreviations used: Cp, Claviceps purpurea ATCC 20102, Ns, Neotyphodium sp. Lp1.cAll codon (cd) or nucleotide (nt) positions relative to the likely start codon, indicated as 50-end/30-end of the oligonucleotide.

J. Wang et al. / Fungal Genetics and Biology 41 (2004) 189–198 193

10mgml�1 Novozyme. The protoplasts were cotrans-

formed, by electroporation (Tsai et al., 1992), with

pKAES157 (linearized) in a 1:1 molar ratio with pAN8-

1 (Mattern et al., 1988), and transformants were selected

with 74 lgml�1 phleomycin (Sigma). The transformantswere screened by PCR with primers targeted to the ends

of the Ptub2–dmaW construct.

2.8. Alkaloid analyses

All analyses were conducted on pseudostems of

symbiota or uninfected plants of cv. Rosalin. Ergova-

line, and ergine (the simple amide of lysergic acid) wereanalyzed by high-pressure liquid chromatography

(HPLC) with fluorescence detection, employing a pro-

tocol adapted from Spiering et al. (2002). Briefly, 50mg

of freeze-dried and milled pseudostems were extracted in

0.5ml of 2-propanol–water–lactic acid (50:50:1) con-

taining ergotamine tartrate as internal standard (at

1.111 lgml�1). Samples were agitated on a vortex mixer

for 1min, then left to stand in the dark at room tem-perature for 2 h before solid material was pelleted by

centrifugation at 6000g for 10min. Extract (20 ll) wasanalyzed on a Jasco HPLC (model PU-980 program-

mable pump with gradient maker LG-980-02) equipped

with a Phenomenex Prodigy reverse-phase C18 column

(150mm� 4.6mm, 5 lm i.d.) and Shimadzu model 551

fluorescence detector set with excitation and emission

wavelengths of 310 and 410 nm, respectively. Injectedsamples were subjected to a gradient in which mobile

phases A (15% acetonitrile + 85% aqueous 0.1M am-

monium acetate) and B (75% acetonitrile + 25% aqueous

0.1M ammonium acetate) were mixed as follows: 0min,

100% A+0% B; 25min, 85% A+15% B; 45min, 50%

A+50% B; 50min, 30% A+70% B; 55min, 0%

A+100% B; 60min, 0% A+100% B; and 65min, 100%

A+0% B. Flow rate was 1mlmin�1.

Chanoclavine was analyzed from crude extracts

(prepared as described above) by liquid chromatogra-

phy–electrospray ionization mass spectrometry (LC–

MS) as described by Panaccione et al. (2003). Ions with

a m/z of 257 ([chanoclavine I +H]þ and isomers) weredetected with the deflector voltage set at 45V for mini-

mal fragmentation, and monitored in selected ion mode.

Lolitrems were extracted and analyzed by a modifi-

cation of the isocratic HPLC method of Gallagher et al.

(1985). Lolitrems were separated on an Alltech (Deer-

field, IL, USA) Alltima silica column (150mm� 4.6mm,

5 lm particle size) with a mobile phase of dichlorome-

thane–acetonitrile–water (86:14:0.1) applied at1mlmin�1, and detected by fluorescence (excitation at

265 nm and emission at 440 nm).

3. Results

3.1. The dmaW gene of Claviceps purpurea

A cosmid library of genomic DNA from C. purpurea

ATCC 20102 was constructed and screened with a C.

fusiformis dmaW probe. Three clones (26A07, 27A01,

and 29A05) were identified that had overlapping geno-

mic segments. The dmaW-related sequence in those

cosmids, designated C. purpurea dmaW-1, was deter-

mined to be nearly identical to that previously reported

for C. purpurea P1 (Tudzynski et al., 1999).Previously, the identity of the C. fusiformis dmaW

gene product had been established by expression in

yeast, whereby the enzyme was assayed by measuring

tryptophan-dependent generation of nonvolatile label

from [3H]DMAPP (Tsai et al., 1995). The same ap-

proach was applied to C. purpurea dmaW-1. The coding

sequence, with introns removed, was cloned into a yeast

expression system. Extracts from transformed, induced

Table 2

DMAT synthase activities from Claviceps spp. dmaW coding regions

expressed in yeast

Gene origin Construct Orientation Sp. act.a

C. fusiformis pKAES135 + 170� 13

N/A Vector N/A )13� 3

C. purpurea pKAES155 ) 13� 4

C. purpurea pKAES153 + 145� 10

C. purpurea pKAES154 + 262� 6

a pmolmin�1 mg�1.

Fig. 3. (A) dmaW segments from Neotyphodium sp. Lp1, PCR-am-

plified using degenerate primer deg1 with deg4 (lane 1), and deg1 with

deg3 (lane 2). (B) Southern-blot analysis demonstrating that ergova-

line-producing endophytes possessed sequences that cross-hybridized

with Neotyphodium sp. Lp1 dmaW. Lanes contained genomic DNA

from Neotyphodium lolii e45 (lane 1), Epichlo€ee typhina ATCC 200736

(lane 2), Neotyphodium sp. Lp1 (lane 3), Neotyphodium sp. e41 (lane 4;

from a Mediterranean tall fescue), N. coenophialum ATCC 90664 (lane

5), Neotyphodium uncinatum CBS 102646 (lane 6), Epichlo€ee baconii

ATCC 200745 (lane 7), E. baconii ATCC 90167 (lane 8), and Epichlo€eefestucae ATCC 90660 (lane 9). Detection (+) or lack of detection ()) ofergovaline in symbiota with the each endophyte is indicated, and is

based on this or other studies (see text). The two bands in lane 5 arise

from the two alleles in N. coenophialum.

194 J. Wang et al. / Fungal Genetics and Biology 41 (2004) 189–198

yeast cells had substantial DMAT synthase activities,

not significantly different from those directed by the C.

fusiformis dmaW sequence in the same system (Table 2),

demonstrating that C. purpurea dmaW-1 encoded an

active enzyme.

3.2. dmaW from Neotyphodium sp. Lp1

An alignment of the deduced products of C. purpurea

dmaW-1 and C. fusiformis dmaW showed several puta-tively conserved blocks of amino acids (Fig. 2). Four of

these blocks—EQRLWWH (positions 25–31), GI-

FKKHI (positions 56–62), MEDLWTL (positions 285–

291), and MPEPQVY (positions 352–358)—were chosen

as the basis to design degenerate primers deg1, deg2,

deg3, and deg4, respectively (Table 1). These primers

were then used to PCR-amplify putative dmaW seg-

ments from Neotyphodium sp. Lp1, yielding fragmentswith the expected sizes (Fig. 3A). A 783-bp fragment

was amplified by PCR with primers deg1 and deg3, and

a 984-bp fragment was obtained with primers deg1 and

deg4. Neither PCR involving deg2 gave the expected

product. The 783-bp fragment was cloned and se-

quenced, revealing one continuous open reading frame.

The gene was then completely sequenced by a series of

cassette-mediated PCR walks into the genome 50 and 30

of the known 783-bp sequence, then the amplified

fragments were sequenced directly. The deduced amino

acid sequence of dmaW from Neotyphodium sp. Lp1

(Fig. 2) had 65% identity to that of C. purpurea and 60%

identity to that of C. fusiformis. The positions of two

putative introns (Fig. 2) were similar to those in the

homologs from Claviceps spp.

The number of dmaW alleles in each of eight Neoty-

phodium spp. and 10 Epichlo€ee spp. isolates was investi-

gated by Southern analysis of genomic DNAs digested

with restriction endonucleases EcoRI (Fig. 3B), PstI,

HindIII, SalI, XhoI, and XbaI (data not shown). The

results indicated only one dmaW allele in Neotyphodium

sp. Lp1. Other endophytes had zero, one or two alleles

in evidence (Fig. 3B). Those with no dmaW alleles had

previously tested negative for ergovaline, whereas thoseshowing strong hybridization to dmaW were known to

produce ergovaline in symbio (Bush et al., 1997; Leu-

chtmann et al., 2000). Only one isolate (E. baconii

ATCC 200745) showed faint hybridization, and its al-

kaloid profile was unknown.

A functional test of the Neotyphodium sp. Lp1 dmaW

coding sequence was conducted by expression in yeast,

similar to the tests of C. fusiformis dmaW (Tsai et al.,

1995) and C. purpurea dmaW (described above). How-

ever, no significant activity was detected in experiments

with the endophyte dmaW sequence.

3.3. dmaW knockout and complementation

A construct (pKAES170) for disrupting dmaW in

Neotyphodium sp. Lp1 was generated by substituting a

hygromycin phosphotransferase (hph) gene cassette for

341 bp of the gene from the start codon to the first

BamHI site (Fig. 4A). The resulting plasmid was thenintroduced into the endophyte by electroporation, and

transformants were screened by a negative PCR test for

homologous recombination; the test employed two

dmaW-specific primers, one of which was complemen-

tary to the region deleted in the mutant construct. Out

of 125 transformants screened, one failed to yield a PCR

product and was, therefore, a candidate knockout mu-

tant. In a Southern analysis (Fig. 4) 1.8- and 4.7-kbEcoRI fragments from the mutant hybridized with the

dmaW probe, but only the expected 4.1-kb fragment was

Fig. 4. (A) Maps of the wild type dmaW locus, targeting vector

(knockout construct) and expected map of the locus following dis-

ruption. Vertical lines indicate EcoRI sites, and double-headed arrows

indicate expected EcoRI fragments with sizes given in kb. (B) South-

ern-blot analysis indicating successful knockout of dmaW in Neoty-

phodium sp Lp1. Lanes contain total DNA of the wild type endophyte

(wt; lane 1), two single-spore isolates of the disruptant (dmaW ko;

lanes 2 and 3), and an Lp1 transformant with ectopic integration of the

vector (ec; lane 4). DNAs were digested with EcoRI. (In wt lanes,

bands above the main band are from incomplete digests.) The blot was

probed with a mixture of two labeled fragments, one extending from

476 bp upstream of the start codon to codon 44, and the other from

codons 146 to 445. Replacement of the 4.1-kb EcoRI fragment with

1.8- and 4.7-kb fragments indicated disruption of the gene.

Fig. 5. HPLC determinations of ergovaline (42.3min) and its stereo-

isomer ergovalinine (51.4min) in perennial ryegrass without endophyte

(e-), or symbiotic with wild type Neotyphodium sp. Lp1 (wt), the dmaW

knockout mutant (ko) or the mutant complemented with a construct

encoding C. fusiformis DMAT synthase (ct). Detection was by fluo-

rescence with excitation at 310 nm and emission at 410 nm. Asterisks

mark the positions or expected positions of ergovaline and its stereo-

isomer ergovalinine.

J. Wang et al. / Fungal Genetics and Biology 41 (2004) 189–198 195

observed in the wild type. These results indicated that

dmaW was replaced with the mutant dmaW, and that

both the 30- and 50-flanking regions were intact in the

mutant, hereafter designated dmaW ko.To complement the dmaW ko mutant, the C. fusi-

formis gene was placed under the control of an E.

typhina b-tubulin gene (tub2) promoter in the plasmid

construct pKAES157, which was then introduced into

the dmaW ko. Out of 20 independent transformants

screened, six possessed pKAES157 integrated in the

genome. The pKAES157 transformants are collectively

designated dmaW ct hereafter.Grass-endophyte symbiota were established consist-

ing of Lp1 wild type (wt), dmaW ko or dmaW ct in

perennial ryegrass cv. Rosalin. The frequencies of en-

dophyte infection after inoculation of perennial ryegrass

were 16.6, 13.5, and 18% for wt, dmaW ko, and dmaW

ct, respectively, suggesting that compatibility with the

host was uncompromised by the prior manipulations. Ina previous study (Panaccione et al., 2001), �Rosalin�plants were generated with a knockout mutation of lpsA

(lpsA ko), believed to encode the penultimate step in

ergovaline biosynthesis. To obtain endophyte-minus

controls, uninfected �Rosalin� seeds were grown and

seedlings remained uninoculated. The resulting plants

were tested by tissue-print immunoblot (Gwinn et al.,

1991), confirming that they lacked any Neotyphodium

endophyte.

Plants infected with the wild type Neotyphodium sp.

Lp1 accumulated high concentrations of ergovaline

(range from 4.6 to 7.8 lg g�1 dry weight of infected tis-

sue) and lesser amounts of the simple amide of lysergic

acid, ergine (range from 0.27 to 0.63 lg g�1). Plants with

dmaW ko, as well as endophyte-minus plants, had no

detectable ergovaline (Fig. 5) or ergine (lysergic acidamide; data not shown). Chanoclavine, the first accu-

mulating intermediate of the pathway, was detected

qualitatively by LC–MS in symbiota containing Lp1 or

its lpsA knockout derivative (blocked at a step down-

stream of chanoclavine), but not in the dmaW ko-in-

fected or endophyte-minus perennial ryegrass (Fig. 6).

Chanoclavine I standard co-chromatographed with the

257m/z molecular ion detected in Lp1 and the lpsA

knockout (Fig. 6). The series of nearly co-migrating

species are likely to be one or more of the four described

stereoisomers of chanoclavine I. Additional alkaloid

analyses (not shown) demonstrated that the clavines,

agroclavine, and its oxidized derivative setoclavine, were

readily detectable in Lp1-containing symbiota but not in

symbiota containing the dmaW ko.

Five of the dmaW ct transformants were introducedinto perennial ryegrass, and the symbiota tested for

Fig. 6. LC–MS determination of chanoclavine accumulation in sym-

biota with wt or genetically modified Neotyphodium sp. Lp1. A broad

peak containing chanoclavine isomers was evident in symbiota with

wt, as well as with a mutant Lp1 in which the lysergyl peptide syn-

thetase gene lpsA was knocked out (lpsA ko), but was absent in sym-

biota with the dmaW knockout (dmaW ko). Electrospray ionization

was in the positive mode and detection was by selected ion monitoring

at m=z ¼ 257. Chanoclavine I was used as standard. The shoulders

preceding the chanoclavine I peaks in the wt and lpsA ko symbiota

were likely due to one or more of the four stereoisomers of chano-

clavine (Gr€ooger and Floss, 1998). Int., relative intensity.

196 J. Wang et al. / Fungal Genetics and Biology 41 (2004) 189–198

ergot alkaloids. Each symbiotum possessed a small but

clearly identifiable amount of ergovaline and its isomer

ergovalinine (mean of 0.15 lg g�1 dry wt infected plant

material measured as both isomers). Small peaks cor-

responding to ergine and chanoclavine I were detectable

in some but not all symbiota with dmaW ct, indicatingthat these alkaloids were present in quantities near the

limits of detection of the assays. This observation is not

surprising considering that ergine accumulation is vari-

able but commonly detected in Lp1-containing symbiota

at concentrations that are approx. 4% of the measured

concentration of ergovaline (Panaccione et al., 2003).

Therefore, concentrations of ergine in symbiota with the

complemented endophyte, which contained an averageof 0.15 lg g�1 ergovaline, frequently would be below the

0.01 lg g�1 limit of detection of this assay.

Isoprene and indole are components common to er-

got alkaloids and lolitrems, and lolitrem B has been

reported in very low amounts in ryegrass plants symbi-

otic with Lp1 (Bush et al., 1997). Therefore, we tested

whether elimination of the ergot alkaloid pathway

caused reallocation of precursors into lolitrem B. Inboth the wild type and dmaW ko symbiota lolitrem B

was undetectable (data not shown), indicating that such

reallocation had not occurred.

4. Discussion

This study constitutes the first genetic test of the roleof DMAT synthase in ergot alkaloid biosynthesis.

Feeding experiments in Claviceps spp. fermentation

cultures have demonstrated that mevalonate and tryp-

tophan are precursors to the ergot and clavine alkaloids,so DMAT synthase was predicted to play a role as the

determinant step in the pathway (Lee et al., 1976). Un-

der a variety of induction and suppression conditions

DMAT synthase activity correlates with production of

ergopeptine or clavine alkaloids in Claviceps spp. cul-

tures (Krupinski et al., 1976). Previously, the dmaW

gene of C. fusiformis was demonstrated to encode au-

thentic DMAT synthase (Tsai et al., 1995). Here weestablished that DMAT synthase is indeed required for

the biosynthesis of ergovaline (the main product of the

ergot alkaloid pathway in endophytes), ergine (an al-

ternate product of the pathway), and chanoclavine (a

critical early intermediate). We eliminated the dmaW

homolog identified in the endophyte strain Lp1, and

observed that the mutant (dmaW ko) produced none of

these metabolites under the conditions conducive forproduction by the wild type, i.e., when symbiotic with

perennial ryegrass. We then demonstrated that the C.

fusiformis dmaW complemented the dmaW ko and re-

stored production of ergovaline.

The C. purpurea gene cloned from isolate P1 (closely

related to that of ATCC 20102) had previously been

proposed as a functional DMAT synthase gene based on

its location near the apparent lysergyl peptide synthe-tase-encoding gene, cpps1 (Tudzynski et al., 1999).

However, Southern-blot analysis (not shown) indicated

that ATCC 20102 and other C. purpurea isolates had

two or more loci with dmaW-related sequences (and it is

also conceivable that divergent paralogs were unde-

tected), so it was essential to show the function of the

cloned gene. We demonstrated, by expression of C.

purpurea dmaW cDNA in yeast, that this gene encodedactive DMAT synthase. This information was critical

for our effort to obtain endophyte homologs of the

Claviceps spp. dmaW genes by a strategy based on

identification of putatively conserved regions in C. fu-

siformis and C. purpurea. Since DMAT synthase con-

tains no known domains or signatures reminiscent of

other prenyl transferases, only comparison of known

dmaW genes could suggest possible conserved regions;the comparison would have been invalid if one of the

genes were nonfunctional.

Once the putative endophyte homolog was identified,

its function was tested by gene knockout. This approach

had the following advantages: (1) the gene cloned from

Neotyphodium sp. Lp1 was demonstrated to encode

DMAT synthase, since the corresponding ko mutant

was complemented by a gene that was previously dem-onstrated to encode this enzyme and (2) the test pro-

vided a definitive confirmation that DMAT synthase is

required for ergot alkaloid production in the endophyte.

The complementation of dmaW ko with the C. fusi-

formis gene provided confirmatory evidence that the

mutation was indeed responsible for loss of ergovaline

production. HPLC analysis of symbiota with the

J. Wang et al. / Fungal Genetics and Biology 41 (2004) 189–198 197

complemented strains (dmaW ct) indicated definitivepeaks with the retention times of both ergovaline and its

isomer, ergovalinine, whereas these peaks were com-

pletely absent in the endophyte-minus plants and from

symbiota with dmaW ko mutants (see Fig. 5). An at-

tempt to complement with the native endophyte gene

was unsuccessful because no stable co-transformants

were obtained. No further attempts were made with the

endophyte gene in light of the successful complementa-tion by C. fusiformis dmaW. The latter was the most

thoroughly characterized of the dmaW genes with re-

spect to the enzyme activity of the gene product and

authentication of the enzyme product (Tsai et al., 1995).

Thus, the complementation specifically confirmed that

the enzyme, DMAT synthase, was necessary for ergot

alkaloid production.

Two results were unexpected. First, the level of ex-pression of ergovaline and other ergot alkaloids in the

complemented strains (dmaW ct) was very low. It is

possible that the low ergot alkaloid levels were due to

poor expression (or poor stability) of the C. fusiformis

protein in the dmaW ct transformants. The promoter

used to express the C. fusiformis gene was from the b-tubulin gene of E. typhina, one of the ancestors of

Neotyphodium sp. Lp1. Furthermore, this promoter hadpreviously been used to drive hph expression in vectors

used for N. coenophialum transformation (Tsai et al.,

1992). We chose to use this promoter rather than the

native promoter from C. fusiformis because we had no

prior information that the native promoter would be

appropriately regulated in the endophyte. We can only

speculate why there was low expression of DMAT

synthase in dmaW ct. A possibility is that there wasselection against constitutive expression in the trans-

formants. Such selection may be expected if constitutive

expression caused depletion of cellular pools of essential

metabolites (tryptophan and DMAPP), or if the enzyme

product, DMAT, was toxic to the fungus. It is possible

that for this reason the only dmaW ct transformants that

were obtained were those that, perhaps due to positional

effects, produced very low levels of the enzyme.The second unexpected result was that the coding

region of Neotyphodium sp. Lp1 dmaW, placed into the

yeast expression system, failed to give detectable DMAT

synthase activity. Again, we can only speculate on pos-

sible reasons. It is possible, for example, given the con-

siderable sequence differences from the Claviceps spp.

genes, that mRNA from the endophyte gene, or the

protein product was unstable in the yeast system. Al-ternatively, the assay conditions, which had been de-

signed for the C. fusiformis enzyme (Gebler and Poulter,

1992), might have been unsuitable for the endophyte

DMAT synthase. Also it should be noted that the entire

coding sequence and approximately 300 bp of 30-un-translated region was cloned into the vector in the

construct of C. purpurea dmaW, whereas the endophyte

gene construct did not include the 30-untranslated re-gion. So, it is conceivable that the native dmaW 30-un-translated region may be important for the stability of

the mRNA. Whatever the reason for this result, the

genetic analyses employing gene knockout and com-

plementation definitively demonstrated function of the

endophyte dmaW gene.

Eliminating from endophytes of forage grasses the

factors responsible for fescue toxicosis is a long-termresearch goal. Although Neotyphodium spp. endophytes

provide profound benefits and play important roles in

grass protection and fitness, ergot alkaloids are generally

anti-mammalian and are thought to be a major cause of

livestock toxicosis. Modified endophytes incapable

of producing ergot alkaloids will allow definitive tests of

ergot alkaloid effects on livestock, as well as the means

to determine whether ergot alkaloids play other roles ingrass fitness and protection. By eliminating the first

enzyme in the pathway, and thus all ergot alkaloids, the

dmaW knockout described here provides a particularly

valuable tool for assessing the contribution of these

metabolites to the various endophyte-associated traits.

Moreover, the dmaW ko strain may serve as a standard,

against which can be compared strains that contain later

blocks in the pathway (e.g., in the lysergyl peptide syn-thetase (Panaccione et al., 2001)) and, thus produce

some subset of the ergot alkaloids.

Acknowledgments

We thank Alfred D. Byrd and Walter Hollin for

technical assistance. Alkaloid analyses were conducted

during a visit by D.G.P. to AgResearch Grasslands,

Palmerston North, New Zealand, in collaboration with

Brian Tapper, Geoff Lane, Elizabeth Davies, and Karl

Fraser. Authentic standards of ergot alkaloids were

provided by Forrest Smith (Auburn University) and

Miroslav Flieger (Czech Academy of Science). Thiswork was supported by Grant 2001-35319-10930 from

the United States Department of Agriculture National

Research Initiative. This is publication No. 03-12-113 of

the Kentucky Agricultural Experiment Station, pub-

lished with approval of the director.

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