Microbiology (2005), 151, 509–519
DOI 10.1099/mic.0.27435-0
Concordant evolution of trichothecene
3-O-acetyltransferase and an rDNA species
phylogeny of trichothecene-producing and nonproducing fusaria and other ascomycetous fungi
Takeshi Tokai,1,2,3 Makoto Fujimura,2 Hirokazu Inoue,3
Takayuki Aoki,4 Kunihiro Ohta,5,6 Takehiko Shibata,5
Isamu Yamaguchi1,7 and Makoto Kimura1,5,6
1
Laboratory for Remediation Research, Plant Science Center, RIKEN, Wako, Saitama
351-0198, and Yokohama, Kanagawa 230-0045, Japan
Correspondence
Makoto Kimura
2
[email protected]
Faculty of Life Science, Toyo University, Itakura, Gunma 374-0193, Japan
3
Laboratory of Genetics, Department of Regulation Biology, Faculty of Science, Saitama
University, Saitama City, Saitama 338-8570, Japan
4
Genetic Diversity Department, National Institute of Agrobiological Sciences (NIAS), Tsukuba,
Ibaraki 305-8602, Japan
5
Cellular and Molecular Biology Laboratory and 6Genetic Dynamics Research Unit Laboratory,
RIKEN, Wako, Saitama 351-0198, Japan
7
Laboratory for Adaptation and Resistance, Plant Science Center, RIKEN, Yokohama,
Kanagawa 230-0045, Japan
Received 2 June 2004
Revised
18 October 2004
Accepted 4 November 2004
The cereal pathogen Fusarium graminearum species complex (e.g. Fusarium asiaticum,
previously referred to as F. graminearum lineage 6) produces the mycotoxin trichothecene in
infected grains. The fungus has a gene for self-defence, Tri101, which is responsible for
3-O-acetylation of the trichothecene skeleton in the biosynthetic pathway. Recently,
trichothecene non-producers Fusarium oxysporum and Fusarium fujikuroi (teleomorph
Gibberella fujikuroi) were shown to have both functional (Tri201) and non-functional
(pseudo-Tri101) trichothecene 3-O-acetyltransferase genes in their genome. To gain
insight into the evolution of the trichothecene genes in Gibberella species, the authors
examined whether or not other (pseudo-)biosynthesis-related genes are found near Tri201.
However, sequence analysis of a 12 kb region containing Tri201 did not result in identification
of additional trichothecene (pseudo-)genes in F. oxysporum. In a further attempt to find other
trichothecene (pseudo-)genes from the non-producer, the authors examined whether or not the
non-trichothecene genes flanking the ends of the core trichothecene gene cluster (i.e. the Tri5
cluster) comprise a region of synteny in Gibberella species. However, it was not possible to
isolate trichothecene (pseudo-)genes from F. oxysporum (in addition to the previously identified
pseudo-Tri101), because synteny was not observed for this region in F. asiaticum and
F. oxysporum. In contrast to this unsuccessful identification of additional trichothecene
(pseudo-)genes in the non-producer, a functional trichothecene 3-O-acetyltransferase gene
could be identified in fusaria other than Gibberella: Fusarium decemcellulare and Fusarium
solani; and in an ascomycete from a different fungal genus, Magnaporthe grisea. Together with
the recent functional identification of Saccharomyces cerevisiae ScAYT1, these results are
Abbreviations: 3-ADON, 3-acetyldeoxynivalenol; DON, deoxynivalenol; NJ, neighbour-joining.
The GenBank/EMBL/DDBJ accession numbers for the sequences determined in this work are AB181459–AB181481, AB193099 and AB193100.
A list of hypothetical genes identified on Fusarium oxysporum cosmids is shown in Supplementary Table S1, an analysis of the region containing genes
A–O in F. oxysporum and Fusarium fujikuroi in Supplementary Fig. S1 and an analysis of the trichothecene 3-O-acetyltransferase of Magnaporthe grisea
in Supplementary Figure S2 with the online version of this paper at http://mic.sgmjournals.org.
0002-7435 G 2005 SGM
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 19 Jun 2017 00:58:48
Printed in Great Britain
509
T. Tokai and others
suggestive of a different evolutionary origin for the trichothecene 3-O-acetyltransferase gene
from other biosynthesis pathway genes. The phylogeny of the 3-O-acetyltransferase was mostly
concordant with the rDNA species phylogeny of these ascomycetous fungi.
INTRODUCTION
Trichothecenes are a large group of protein synthesis
inhibitors in eukaryotes. These secondary metabolites are
also known as mycotoxins and cause contamination of
agricultural and food products. Fungi in several different
genera, including Fusarium, Trichothecium and Myrothecium,
produce various types of trichothecenes (Desjardins et al.,
1993). The biosynthesis of trichothecene has perhaps been
most extensively studied in Fusarium species, such as the
T-2 toxin (a highly toxic trichothecene) producer Fusarium
sporotrichioides (Beremand, 1987; Brown et al., 2001; Keller
& Hohn, 1997), and in wheat head blight fungi such as
Fusarium asiaticum (previously referred to as Fusarium
graminearum lineage 6 (O’Donnell et al., 2004; Kimura et al.,
2003b; Lee et al., 2001, 2002; Wuchiyama et al., 2000) and
F. graminearum lineage 7 (Brown et al., 2001, 2002).
Following the isolation of Tri5 (Hohn & Beremand, 1989),
some but not all trichothecene genes were found to cluster
around this first gene in the biosynthetic pathway (Hohn
et al., 1993). Recently, at least two new genes in the pathway,
Tri1 (McCormick et al., 2004; Meek et al., 2003) and Tri16
(Peplow et al., 2003), were identified at another single
locus in the genome of F. sporotrichioides and F. graminearum (Brown et al., 2003). However, most of the other
trichothecene genes (either dispersed or clustered) remain
to be identified (Kimura et al., 2003b).
The resistance genes of antibiotic producers are often
linked to the biosynthesis gene clusters in the genome of
the producers (Cundliffe, 1989). We previously found
that 3-O-acetylation of the trichothecene skeleton is a
self-protection mechanism for F. asiaticum. Based on this
finding, the gene responsible, Tri101, was cloned by expression cloning using fission yeasts (Kimura et al., 1998a).
However, this gene, involved in resistance and biosynthesis (McCormick et al., 1999), was located between a UTPammonia ligase gene (ura7) and a phosphate permease
gene (pho5); that is, it was not clustered with other
trichothecene genes (Kimura et al., 1998b). Furthermore,
the trichothecene non-producers Fusarium oxysporum and
Fusarium fujikuroi [incorrectly referred to as Fusarium
moniliforme in our previous study (Kimura et al., 2003a);
teleomorph Gibberella fujikuroi] were unexpectedly found
to carry both functional and non-functional trichothecene
3-O-acetyltransferase genes (Tri201 and Tri101, respectively). These trichothecene non-producers possess a
pseudo-Tri101 between pho5 and ura7, which comprises
a region of microsynteny in Gibberella species. These
findings raise the possibility that the ancestor of F.
oxysporum was a trichothecene producer and that Tri201
is an original copy of a duplicated 3-O-acetyltransferase
gene involved in biosynthesis (Kimura et al., 2003a). In fact,
510
non-aflatoxigenic Aspergillus species are known to have a
dysfunctional aflatoxin gene cluster in their genome (Klich
et al., 1995; Kusumoto et al., 1998) and it is a question of
whether or not a similar case holds true for the trichothecene
gene cluster in Gibberella species.
In non-aflatoxigenic Aspergillus species that are phylogenetically close to aflatoxin-producing species (i.e. where
both belong to Aspergillus section Flavi), the genes for
aflatoxin biosynthesis have been identified by Southern
blot analysis (Klich et al., 1995). In contrast to these
Aspergillus species, the divergence of trichothecene producer and non-producer Gibberella species occurred long
ago, and functional 3-O-acetyltransferase genes have not
been detected on a Southern blot (Kimura et al., 1998b).
There are no extant strains with a near-isogenic background which may be used for a comparative analysis.
Chromosome numbers and gene orders could differ
between the producer and non-producer strains, and it is
difficult to trace the evolutionary events that have happened
to the ancestral Fusarium species (Kimura et al., 2001).
However, if the non-biosynthesis genes surrounding the
trichothecene genes are found in syntenic regions of the
F. graminearum species complex and of the F. oxysporum
genomes (e.g. the pho5–ura7 region containing Tri101), it
might be possible to gain insight into the evolution of the
gene cluster in the absence of the entire genome sequence.
We therefore constructed a cosmid library of F. oxysporum
to (1) identify genes on both sides of Tri201 and (2) isolate
orthologues of non-trichothecene genes demarcating the
core trichothecene gene cluster (i.e. the Tri5 cluster) (Kimura
et al., 2003b). These non-trichothecene genes were used
for a comparative analysis of F. asiaticum and F. oxysporum.
We also describe the identification and characterization of
functional trichothecene 3-O-acetyltransferase genes from
diverse fungal genera. These results are discussed in relation
to the evolutionary history of the trichothecene genes in
Gibberella species.
METHODS
Strains and growth conditions. F. asiaticum F15, F. oxysporum
IFO 31983, F. fujikuroi IFO 31251, Fusarium acuminatum IFO 7772
[referred to as Fusarium species IFO 7772 in our previous study
(Kimura et al., 2003a)], Fusarium decemcellulare (teleomorph;
Albonectria rigidiuscula) IFO 30918, Fusarium solani IFO 31094 and
Magnaporthe grisea (anamorph; Pyricularia oryzae) P2 have been
described previously (Banno et al., 2003; Kimura et al., 1998a,
2003a). Fusarium nygamai MAFF 239069, Fusarium nisikadoi MAFF
237507 and Fusarium avenaceum TNPG-3 are in Dr Aoki’s fungal
collection at the Ministry of Agriculture, Fishery, and Forestry of
Japan (MAFF) Genebank. Aspergillus nidulans (teleomorph, Emericella
nidulans) IFO 33017 was purchased from NITE Biological Resource
Center (NBRC), Kisarazu, Japan. Saccharomyces cerevisiae INVSc1 was
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 19 Jun 2017 00:58:48
Microbiology 151
Evolutionary history of trichothecene genes
obtained from Invitrogen and used throughout this study. Fungal strains
were maintained on Difco potato glucose agar (Becton Dickinson)
plates at 20 uC. Mycelia were grown on YG medium (0?5 % yeast
extract, 2 % glucose) and used for extraction of DNA and RNA.
Construction of a F. oxysporum cosmid library and PCR
screening of positive clones. A cosmid library of F. oxysporum
was constructed using the SuperCos1 cosmid vector (Stratagene),
following the manufacturer’s instructions. Individual cosmid clones
were distributed in 96-well plates (52 plates containing 4992 clones)
and aliquots (48 cosmid clones each) were used as templates for the
screening of positive clones by PCR. The following primers were
used for the isolation of F. oxysporum genes from the cosmid library
(see Table 1 for primer sequences): primers Tri201-F and Tri201-D3
for Tri201, primers tyrosinase-F and tyrosinase-R for a putative
tyrosinase gene (gene B, orthologue of gene 3), primers actylclone-F
and actylclone-R for a putative polysaccharide deacetylase gene
(gene S, orthologue of gene 4), primers b1.3clone-F and b1.3clone-R
for a putative b-1,3-glucosidase gene (gene Q, orthologue of gene 1),
and primers NADH-F and NADH-R for a putative NADHcytochrome b5 reductase gene (gene J, orthologue of gene 6).
Sequence analysis of cosmids containing non-trichothecene
genes. The internal sequence of pCosFo17-12-G was partially deter-
mined and mapped using PCR techniques. Briefly, the insert DNA
was divided into three regions, which are demarcated by gene B
(orthologue of gene 3) and/or gene J (orthologue of gene 6), and
these regions were amplified by long PCR with the following primer
pairs: Fotyrosinase-U32 and SuperT7, Fotyrosinase-D32 and FoNADHD32, and FoNADH-U32 and SuperT3. The amplified PCR products
were partially digested with Sau3AI and cloned in pUC18. Randomly
selected clones were sequenced by a single pass analysis and putative
gene-coding regions were predicted using the BLASTX analysis. To
determine the partial nucleotide sequences of these DNA fragments
(fragment I–fragment XV), primers were synthesized on the basis of
their provisional (i.e. with possible PCR errors) sequences and used
for direct DNA sequencing. The F. oxysporum genes identified on
pCosFo17-12-G (gene A–gene O) were mapped to the cosmid by
sequentially determining their relative positions by PCR.
Cloning of trichothecene 3-O-acetyltransferase genes (TAT)
from F. decemcellulare and F. solani. Twenty-one primers
were designed based on the nucleotide sequences of known trichothecene 3-O-acetyltransferase genes (i.e. Tri101, Tri201 and FdTAT
for cloning of FsoTAT) and used for RT-PCR of TAT. To concentrate the TAT mRNA species, RNA was prepared from mycelia
treated with 100 mg T-2 toxin ml21. Portions of FdTAT and FsoTAT
were amplified after extensive RT-PCR trials with possible pairs of
primers (see Table 1 for successful primer pairs). Based on the
internal sequences of the RT-PCR products, we designed primers
for vectorette PCR (TaKaRa LA PCR in vitro cloning kit; TAKARA
BIO) to clone the upstream and downstream regions of TAT.
Primers and cassette libraries listed in Table 1 were used for genomic walking by vectorette PCR.
In vitro acetyltransferase assays. The candidate 3-O-acetyltransferase genes, with the exception of FdTAT, were amplified by PCR
using KOD-plus DNA polymerase (TOYOBO) and the amplified
products (without non-synonymous substitutions) were cloned into
an expression vector, pET-12a (Novagen), between the NdeI and
BamHI sites. For construction of an FdTAT expression vector, the
PCR product (without non-synonymous substitutions) amplified by
LA-Taq (TAKARA BIO) was directly cloned in pCRT7/NT-TOPO
(Invitrogen). Primers listed in Table 1 were used for PCR.
The expression vectors were transformed to Escherichia coli Rosetta
(DE3) (Novagen) and grown on CircleGrow medium (Bio 101)
containing 100 mg ampicillin ml21 and 34 mg chloramphenicol ml21.
http://mic.sgmjournals.org
The bacterial cultures were incubated at 20 uC overnight with 1 mM
IPTG to induce expression of the acetyltransferase gene. Crude
recombinant enzymes were prepared from the bacteria and the
trichothecene 3-O-acetyltransferase activities were assayed as described previously (Kimura et al., 1998a). Ethylacetate-extracted
reaction mixtures were developed on TLC plates (Kieselgel; Merck)
with ethylacetate/toluene (3 : 1 v/v) as the solvent. Trichothecenes were
visualized with the chromogenic reagent 4-(p-nitrobenzyl)pyridine
following the standard detection method (Takitani et al., 1979).
Northern analyses of the 3-O-acetyltransferase genes. Fungal
spores (F. solani) or young mycelial plugs (F. decemcellulare and M.
grisea) were grown in YG medium containing T-2 toxin (20 or
100 mg ml21) for 3 days at 28 uC. Yeast cells were inoculated on
YPD medium (1 % yeast extract, 2 % peptone and 2 % glucose) with
20 mg T-2 toxin ml21 and cultured for 2 days at 28 uC. Fungal cells
were disrupted by a bead-beader (Micro Smash MS100-R; Tomy)
and total RNA was extracted using TRIzol (Invitrogen). Twenty
micrograms of RNA was used for Northern blot analysis, as described previously (Kimura et al., 1998b). Digoxigenin-labelled RNA
probes (DIG RNA Labelling kit SP6/T7; Roche Diagnostics) were
prepared by in vitro transcription of the entire coding region of the
TAT and ScAYT1 genes (PCR products used for construction of
E. coli expression vectors in the previous section) cloned in pGEM-T
Easy (Promega).
Sequence alignment and construction of phylogenetic trees.
Multiple alignment of the deduced trichothecene 3-O-acetyltransferase
sequences was done using the CLUSTALW program (Thompson et al.,
1994) in the GENETYX-MAC (version 12.0) software package (Software
Development, Tokyo). For construction of unrooted neighbourjoining (NJ) trees, we used sequence analysis tools available
at DDBJ (http://spiral.genes.nig.ac.jp/homology/welcome-e.shtml).
Alignment was made with the program CLUSTALW (DDBJ version)
with the BLOSUM scoring matrix (GAPOPEN, 15 for DNA and 10
for protein; GAPEXT, 6.66 for DNA and 0.2 for protein; GAPDIST,
8; MAXDIV, 40; ENDGAPS, off; NOPGAPS, off; NOHGAPS, off)
and then automatically converted to tree files using the tree-making
program TREE (DDBJ version). The trees were calculated with 1000
bootstrap replications (KIMURA, on; TOSSGAPS, on; SEED, 111),
and the results (downloaded as MIME type files) were visualized
using the Macintosh program TREEVIEW (Page, 1996). The following
28S/26S rDNA sequences were used for construction of species
phylogeny: AB084297 for F. asiaticum; AB084298 for F. sporotrichioides; AB084299 for F. oxysporum; AB084300 for F. fujikuroi;
AB084301 for F. acuminatum; AB084302 for F. decemcellulare;
AB084303 for F. solani; AF362554 for M. grisea; AY130346 for S.
cerevisiae. The phylogenetic analysis of the acetyltransferases was
also done using a maximum-likelihood method with the program
PUZZLE (Strimmer & von Haeseler, 1996) at the Institute Pasteur
(http://www.pasteur.fr/english.html).
RESULTS
Tri201 is absent from a microsynteny region of
the F. graminearum species complex and some
trichothecene non-producer Gibberella species
We isolated two cosmid clones containing Tri201 from a
F. oxysporum genome library distributed in 96-well plates.
The nucleotide sequence of the region around Tri201
(12 396 bp; AB181461) was determined by sequencing
these cosmid clones. By using RNA isolated from mycelia
grown on YG medium, the transcription of candidate
genes was examined by RT-PCR. A BLASTX search revealed a
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 19 Jun 2017 00:58:48
511
T. Tokai and others
Table 1. Primers used for cloning and characterization of the 3-O-acetyltransferase genes
Primer names
Primer sequences
Tri201-F
Tri201-D3
Gibbe-201checkU
59-CACTAAACGTTACAAACATGGTCGAC-39
59-AGCTCGGATGATGTCGAATTATGTCA-39
59-GATTCACAACGCACGGAAGAAACGCGAG-39
Gibbe-201checkD3
59-TGTCRTAGTGGAATTTCCATGTTCCG-39
tyrosinase-F
59-CAAAGATCCCGTCGTCCGCAAAGAATGGCG-39
tyrosinase-R
59-ATTGGCATGGTGGAGGAAGAAGAGAGGCTC-39
actylclone-F
59-ACCGTTGCGCTCACTTTCGATGACGGTC-39
acetylclone-R
59-GGGGTAGTATGTTCTGCACGGTGTTTTG-39
b1.3clone-F
59-TCCACCGACGATGCTCACTTTGGCCGTG-39
b1.3clone-R
59-CTTGGTAGGCCAGCCGGTCTCACCAACC-39
NADH-F
59-GCTTGGATTGCCTATTGGACAGCATGTC-39
NADH-R
59-CATCCCTGGCGGACCACATAAGAATACC-39
SuperT7
SuperT3
Fotyrosinase-U32
Fotyrosinase-D32
FoNADH-U32
FoNADH-D32
Tri201-DwU1
Tri201-D39
Fd 201-Uw07
Fd201-Dw01
decem-L-U-CAS
59-GCGGCCGCATAATACGACTCACTATTG-39
59-GGCCGCAATTAACCCTCACTAAAG-39
59-TCTGTCTTCGCAACCTCCCCAAAAAGGCATAC-39
59-AGATTCGTGTTGGCAAAAGGTCCATCGACGAC-39
59-TTACTCGCCACTTCCTCTCAAGCAAAAGACCC-39
59-GTCACATACCCAGACCCAAACTTCCAATTCGC-39
59-AGTTCCCCATGAGCATGTTTGACGAG-39
59-AGCTCGGATGATGTCGAATTATG-39
59-TGCCCATGCTGAGGATCTAC-39
59-GCTTTTGATAGGAGGCGGATGATCTC-39
59-CACAGCATTCATCTGGCAGTGCGTATGCCGTG-39
decem-L-U-nest-CAS
59-CCAGAACATGACCTACCACAACGCAACAGTGC-39
decem-L-D-CAS
59-AATGGCAGTCTTCCGTTCTGTGGTCATTGCGG-39
decem-L-D-nest-CAS
59-CGGCAGGCTTTTGATAGGAGGCGGATGATCTC-39
solani-L-U-CAS
59-ACCCTGAGCCTGTGCTGATCTTTCAACTCAAC-39
solani-L-U-nest-CAS
59-ATTAAGGGTGGCCTGATATTGACCGTCAACGG-39
solani-L-D-CAS
59-TTTCCCTCGCTTTCATCGAAGCACTTGACCTG-39
solani-L-D-nest-CAS
59-CTTGTTGAAGGTATCGACGATGGTAGGGTGAG-39
Nde-Fo201
Bam-Fo201
Nde-Gf201
Bam-Gf201
Fd201-TOPO/ATG
59-GTTACACATATGGTCGACCTAGACATAG-39
59-CATTGGATCCCCGCGAGCAACCGGAAAC-39
59-CACACCATATGACAGCACTAAACACCAC-39
59-TAGTCCGGGATCCGCATATTGTCTCCG-39
59-ATGGACTCTTTAAACACACAAGCCCTAG-39
Fd201-TOPO/TAG
59-ACGCTATTTTGACTGCTGTCATGGTGTC-39
512
Comments
Primer for isolation of cosmids containing FoTri201
Primer for isolation of cosmids containing FoTri201
Primer for amplification of the microsynteny region of
Gibberella species
Primer for amplification of the microsynteny region of
Gibberella species
Primer for isolation of cosmids containing orthologue
of gene 3
Primer for isolation of cosmids containing orthologue
of gene 3
Primer for isolation of cosmids containing orthologue
of gene 4
Primer for isolation of cosmids containing orthologue
of gene 4
Primer for isolation of cosmids containing orthologue
of gene 1
Primer for isolation of cosmids containing orthologue
of gene 1
Primer for isolation of cosmids containing orthologue
of gene 6
Primer for isolation of cosmids containing orthologue
of gene 6
T7 primer on SuperCos1
T3 primer on SuperCos1
Primer for long PCR of pCosFo17-12-G
Primer for long PCR of pCosFo17-12-G
Primer for long PCR of pCosFo17-12-G
Primer for long PCR of pCosFo17-12-G
Forward primer for amplification of partial FdTAT region
Reverse primer for amplification of partial FdTAT region
Forward primer for amplification of partial FsoTAT region
Reverse primer for amplification of partial FsoTAT region
Primer for vectorette PCR of FdTAT downstream region
(FbaI cassette)
Nested primer for vectorette PCR of FdTAT downstream
region (FbaI cassette)
Primer for vectorette PCR of FdTAT upstream region
(XbaI cassette)
Nested primer for vectorette PCR of FdTAT upstream
region (XbaI cassette)
Primer for vectorette PCR of FsoTAT downstream region
(HindIII cassette)
Nested primer for vectorette PCR of FsoTAT downstream
region (HindIII cassette)
Primer for vectorette PCR of FsoTAT upstream region
(XbaI cassette)
Nested primer for vectorette PCR of FsoTAT upstream
region (XbaI cassette)
Forward primer for cloning of FoTri201 into pET-12a
Reverse primer for cloning of FoTri201 into pET-12a
Forward primer for cloning of GfTri201 into pET-12a
Reverse primer for cloning of GfTri201 into pET-12a
Forward primer for cloning of FdTAT into pCRT7/NTTOPO
Reverse primer for cloning of FdTAT into pCRT7/NTTOPO
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 19 Jun 2017 00:58:48
Microbiology 151
Evolutionary history of trichothecene genes
Table 1. cont.
Primer names
Primer sequences
Comments
Nde-Fso201
Bam-Fso201
ScAYT1-Nde
ScAYT1-Bam
Mg9777.1-Nde
Mg9777.1-Bam
59-CTTCCATATGGATACCCCAGACACTAGC-39
59-AGCTTAGATCTTCTACCCGATAAATCGG-39
59-TAGACATATGTTTAGAGTCAAGATCATC-39
59-ACAAGGATCCCTATTGGTAATATATTAC-39
59-TACCCCATATGGACGGTCTACACACC-39
59-TTGGGATCCGATCTAACCCAAATATG-39
Forward primer for cloning of FsoTAT into pET-12a
Reverse primer for cloning of FsoTAT into pET-12a
Forward primer for cloning of ScAYT I into pET-12a
Reverse primer for cloning of ScAYT I into pET-12a
Forward primer for cloning of MgTAT into pET-12a
Reverse primer for cloning of MgTAT into pET-12a
transcribed hypothetical gene similar to Drosophila melanogaster CG15021 (encoding a protein similar to RE17165p;
accession no. AAL48746) and a putative b-D-galactosidase
gene (non-transcribed under the above experimental conditions) around Tri201, but no trichothecene (pseudo-)
genes were found nearby (Fig. 1). The hypothetical gene,
Tri201, and a putative b-D-galactosidase gene were also
arranged in the same manner in F. fujikuroi with respect
to gene order and orientation. In F. asiaticum F15 and F.
acuminatum IFO 7772 (which have a functional Tri101),
however, these two non-trichothecene genes were adjacent
to each other, and there was no (pseudo-)Tri201 between
these genes.
We further investigated the arrangement of genes at this
locus using other trichothecene non-producing Gibberella
species. For this purpose, the regions between the hypothetical gene and a putative b-D-galactosidase gene were
amplified by long PCR with LA-Taq using primers
Gibbe-201checkU and Gibbe-201checkD3 (shown as
arrowheads in Fig. 1). Sequence analysis of the amplified
products revealed that F. nygamai (AB193099) and F.
nisikadoi (AB193100) carry Tri201 between these two
genes. In addition to F. acuminatum, F. avenaceum did
not have this (pseudo-)gene. These results suggest that
some trichothecene-non-producing Gibberella species lack
Tri201 in the microsynteny region.
Fig. 1. Comparison of the microsynteny regions of Gibberella species, which contain a transcribed hypothetical gene (similar
to D. melanogaster CG15021) and a putative b-D-galactosidase gene. Experimentally determined exons are represented as
filled boxes (black for Tri201 and grey for other genes); predicted exons of non-transcribed (i.e. vegetatively growing mycelia)
genes are shown as open boxes. While the trichothecene non-producers F. oxysporum and F. fujikuroi had a functional
Tri201 between a hypothetical gene and a putative b-D-galactosidase gene (accession nos AB181461 and AB181462,
respectively), Tri201 was missing from this region in F. asiaticum and F. acuminatum (AB181459 and AB181460,
respectively); the latter two species carry a functional Tri101 in the pho5–ura7 syntenic region. Filled and empty arrowheads
denote primers Gibbe-201checkU and Gibbe-201checkD3, respectively. Three additional Fusarium strains, F. nygamai,
F. nisikadoi and F. avenaceum, were used for the PCR analysis. The PCR products amplified by these primers are shown
below the map.
http://mic.sgmjournals.org
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 19 Jun 2017 00:58:48
513
T. Tokai and others
Non-trichothecene genes flanking the Tri5
cluster do not comprise a region of synteny in
Gibberella species
The 25 kb Tri5 cluster is surrounded by putative tyrosinase
(gene 3) and polysaccharide deacetylase (gene 4) genes in
F. asiaticum F15 (Kimura et al., 2003b). On both sides of
these non-trichothecene genes, there are a putative b-1,3glucosidase gene (gene 1), an esterase gene (gene 2), a 3hydroxyacyl-CoA dehydrogenase gene (gene 5), and a
NADH-cytochrome b5 reductase gene (gene 6). Among
these genes, we focused on the cluster-flanking genes,
gene 3 and gene 4, and the most distal genes, gene 1 and
gene 6 (see Fig. 2). Primers were synthesized on the basis
of the sequences of these non-trichothecene genes (see
Table 1) and used to amplify portions of their F. oxysporum orthologues. By sib selection, the non-trichothecene
genes were identified in a pool of cosmids distributed in
96-well plates, and a single cosmid was subsequently
isolated. Although orthologues of gene 3 and gene 6 were
located on a single cosmid, pCosFo17-12-G (gene B and
gene J, respectively), gene 1 and gene 4 were identified on
different cosmids, pCosFo15-3-H (gene Q) and pCosFo112-D (gene S), respectively (Fig. 2). These three cosmids
did not overlap each other, as assessed by PCR with primers
on the basis of the insert T3 and T7 end-sequences (data
not shown). Shotgun sequencing of pCosFo17-12-G, combined with PCR mapping and BLASTX analysis, resulted in
the identification of 15 putative non-trichothecene genes
(genes A to O, with identification of gene L as an orthologue of gene 5; see also Supplementary Table S1 with the
online version of this paper at http://mic.sgmjournals.org
for BLASTX results of these F. oxysporum genes) in the
neighbourhood of gene B and gene J, but no candidate
trichothecene (pseudo-)genes were found nearby. As shown
in Fig. 2, most orthologues of these genes were identified on
different chromosomes (see also Supplementary Table S1;
genes A, C, D, E, F, G, K, M, N and O are on different
contigs) of F. graminearum PH-1 (NRRL 31084) (Trail &
Common, 2000), for which the complete genome sequence
is available (Broad Institute; http://www.broad.mit.edu/
annotation/fungi/fusarium/). Orthologues of the five nontrichothecene genes on pCosFo15-3-H and pCosFo1-12-D
(i.e. genes P to T in Fig. 2) were not mapped to a single
contig of F. graminearum PH-1 (Supplementary Table S1).
In F. oxysporum and F. fujikuroi, most genes identified on
pCosFo17-12-G comprised a region of synteny, as assessed
by PCR mapping (Supplementary Fig. S1 with the online
version of this paper at http://mic.sgmjournals.org). These
results indicate that non-trichothecene genes demarcating
the Tri5 cluster in the F. graminearum species complex are
not organized in a region of synteny in trichotheceneproducing (e.g. F. asiaticum, F. graminearum) and nonproducing (e.g. F. oxysporum, F. fujikuroi) Gibberella species.
Trichothecene 3-O-acetyltransferase genes in
Fusarium species that belong to teleomorph
genera other than Gibberella
Although our previous analysis focusing on pho5 and
ura7 did not result in the identification of trichothecene
3-O-acetyltransferase genes (TAT) from Fusarium strains
Fig. 2. Comparative analysis of F. asiaticum and F. oxysporum focusing on the non-trichothecene genes at either end of the
Tri5 cluster. Solid and dotted lines represent sequenced and non-sequenced (but mapped) regions, respectively. F.
oxysporum genomic DNA regions are enclosed by dotted ovals. The scale bar (5 kb) applies to both F. asiaticum and F.
oxysporum genomic DNA regions. Although orthologues of F. asiaticum non-trichothecene genes (gene 3, gene 5 and gene
6) were found on a single cosmid pCosFo17-12-G (gene B, gene L and gene J, respectively), further sequence analyses
(i.e. BLASTX analysis and PCR mapping of randomly sequenced regions) of this cosmid did not support the presence of
(pseudo-)trichothecene genes or microsyntenic regions in F. oxysporum. Chromosome numbers (e.g. Fg: chr-1) of F.
graminearum PH-1 orthologues are shown in parentheses.
514
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 19 Jun 2017 00:58:48
Microbiology 151
Evolutionary history of trichothecene genes
belonging to teleomorph genera other than Gibberella
(Kimura et al., 2003a), we found that F. decemcellulare
and F. solani were able to acetylate C-3 of trichothecenes
(data not shown). Portions of the 3-O-acetyltransferase
genes (referred to as FdTAT and FsoTAT from F. decemcellulare and F. solani, respectively) were amplified by RTPCR using RNA isolated from T-2 toxin-treated mycelia.
Concentration of the TAT mRNA species by treatment
with T-2 toxin was necessary to achieve a successful PCR
with primers (designed on the basis of the sequences of
other Fusarium strains) that do not perfectly match the
TAT genes. Based on the internal sequences of the amplified cDNA fragment, upstream and downstream coding
regions of TAT were obtained from genomic DNA by
vectorette PCR with the primers listed in Table 1. We
further obtained the regions upstream and downstream
of TAT by conducting extensive rounds of PCR walking
using vectorette PCR (primers not shown). The combined
nucleotide sequences containing FdTAT and FsoTAT are
available from GenBank under the accession numbers
AB181463 (5692 bp) and AB181464 (5392 bp), respectively.
At position 23 relative to the coding regions of FdTAT
and FsoTAT (1374 bp and 1380 bp, respectively), we found
‘A’, which is highly conserved in translational initiation
sites of fungal genes (Ballance, 1986). Also, the coding
regions of the genes were not interrupted by introns. These
features are similar to those of other trichothecene 3-Oacetyltransferase genes of Gibberella. At the amino acid
sequence level, FdTAT and FsoTAT were both 61?9 %
identical to FgTRI101 (BAA24430), while FdTAT and
FsoTAT were 73?5 % identical to each other. They contained
the sequence motifs HXXMDMXG and DFGXGLGXP (see
Fig. 3, bold), which are well conserved among trichothecene 3-O-acetyltransferase genes of Gibberella species.
Fig. 3. Alignment of conserved amino acid sequences of functional trichothecene 3-O-acetyltransferases from ascomycetous
fungi. The sequence alignment was made with the deduced
amino acid sequences of the acetyltransferase gene from F.
asiaticum F15 (FasTRI101), F. sporotrichioides IFO 9955
(FsTRI101), F. acuminatum IFO 7772 [FacTRI101; referred to
as FspTRI101 in our previous study (Kimura et al., 2003a)], F.
oxysporum IFO 31983 (FoTRI201), F. fujikuroi IFO 31251
(FfTRI201), F. decemcellulare IFO 30918 (FdTAT), F. solani
IFO 31094 (FsoTAT), M. grisea P2 (MgTAT) and S. cerevisiae
INVSc1 (ScAYT1). The sequence motifs conserved among
these functional 3-O-acetyltransferases are shown in bold (conserved among Fusarium species) and boxed (conserved among
ascomycota).
http://mic.sgmjournals.org
Characterization of the trichothecene
3-O-acetyltransferase genes from ascomycetous
fungi
The recent completion of genome sequencing projects for
several fungi (Broad Institute/MIT; as of June, 2004) has
revealed hypothetical genes homologous to Tri101 from
M. grisea (hypothetical genes; locus ID MG09777.4 encoding a hypothetical protein XP_364932, designated MgTAT
in this study, and locus ID MG08440.4 encoding a hypothetical protein XP_362997), A. nidulans (hypothetical
gene; locus ID AN3384.2 encoding a hypothetical protein
EAA63352) and S. cerevisiae (gene name, ScAYT1), but not
from Neurospora crassa. In addition, there is a trichothecene 3-O-acetyltransferase pseudogene in the genome of
Schizosaccharomyces pombe (SPCC338.19; see annotation
of AL023781). None of these ascomycetous fungi produce
trichothecenes. Among these genes, ScAYT1 is known as a
functional acetyltransferase gene (Alexander et al., 2002).
The function of these hypothetical genes was examined
with in vitro acetyltransferase assays using trichothecenes
as a substrate. FdTAT, FsoTAT, MgTAT, ScAYT1 and the
hypothetical genes MG08440.4 and An3384.2 were
expressed in E. coli using the T7 expression system. Almost
equal amounts of recombinant protein in soluble fraction
were used for the assay (Fig. 4a). As shown in Fig. 4(b),
deoxynivalenol (DON) was specifically acetylated at C-3
of the trichothecene skeleton by the crude recombinant
enzyme fractions, except those of MG08440.4 and An3384.2
(data not shown). While TAT from fusaria showed equally
strong 3-O-acetyltransferase activities with DON, MgTAT
was less active (see TLC at 1 min incubation). ScAYT1
showed much less DON acetylase activity than MgTAT,
but an expected product, 3-acetyldeoxynivalenol (3ADON), became detectable after 3 h incubation. In addition to DON, T-2 toxin also served as a substrate for
these recombinant proteins (data not shown). These results
demonstrate that several ascomycetous fungi distantly
related to the trichothecene producers (i.e. M. grisea and
S. cerevisiae) have genes that show trichothecene 3-Oacetyltransferase activity. In support of the results, the
deduced amino acid sequences of these functional homologues showed the conserved sequence motifs HXXMDXXG
and DFXXGXGXP (Fig. 3, bold and boxed), and were
43?9 % (MgTAT) and 43?5 % (ScAYT1) identical to that of
FasTRI101 from F. asiaticum F15 (Kimura et al., 1998a).
Expression of the gene in these fungi was also investigated
by Northern blot analysis. Fungal cells were treated with T-2
toxin (100 mg ml21 for F. decemcellulare and F. solani and
20 mg ml21 for M. grisea and S. cerevisiae, respectively) and
total RNA was isolated before and after the toxin treatment to compare the transcript level of the resistance
gene. Acetyltransferase gene expression was significantly
induced by the toxin treatment in F. solani and to a lesser
extent in F. decemcellulare, but was unchanged in M. grisea
and S. cerevisiae (Fig. 4c). While S. cerevisiae constitutively
expressed the ScAYT1 gene irrespective of the culture
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 19 Jun 2017 00:58:48
515
T. Tokai and others
conditions and specifically acetylated C-3 of T-2 toxin
(20 mg ml21) in the medium, expression of MgTAT was
not detected in M. grisea. However, DON (20 mg ml21) was
acetylated at C-3 by the M. grisea culture and the transcript
of MgTAT was detected in the toxin-treated mycelia by
RT-PCR (Supplementary Fig. S2 with the online version of
this paper at http://mic.sgmjournals.org), which was used
to detect small amounts of RNA below the detection limit
of Northern blot analysis (Kimura et al., 1998c).
(a)
(b)
Phylogenetic relationships
Although there is no evidence of orthologous relationships
between these trichothecene 3-O-acetyltransferase genes,
they all have similar functions in that the gene products
confer trichothecene resistance to the fungal strains in which
they exist. We therefore constructed an unrooted phylogenetic tree based on the amino acid sequences of these
trichothecene 3-O-acetyltransferases to establish the evolutionary links among this enzyme family. As shown in Fig. 5,
the NJ tree was basically concordant with the species
phylogeny (see 28S/26S rDNA tree of the fungi in Fig. 5),
except for the phylogenetic positions of TAT from F.
decemcellulare and F. solani. An essentially identical tree
was obtained by the maximum-likelihood method with the
program PUZZLE (data not shown).
(c)
DISCUSSION
Fig. 4. Characterization of the trichothecene 3-O-acetyltransferase genes from ascomycetous fungi. (a) Recombinant proteins
[including FasTRI101, most of which was recovered as an
inclusion body in E. coli HMS174 (DE3) in our previous study
(Kimura et al., 1998a)] were recovered in soluble fractions
when overexpressed in E. coli Rosetta (DE3) grown at 20 6C.
Total proteins were separated by 12?5 % SDS-PAGE and the
gel was stained with Coomassie brilliant blue. Soluble enzyme
fractions (equal amounts included in the reaction mixture) used
in the acetyltransferase assay were loaded on each lane. M,
protein marker; C, crude cell extract of control E. coli; Fas,
crude cell extract of E. coli overexpressing FasTRI101; Fd,
crude cell extract of E. coli overexpressing FdTAT; Fso, crude
cell extract of E. coli overexpressing FsoTAT; Mg, crude cell
extract of E. coli overexpressing MgTAT; Sc, crude cell extract
of E. coli overexpressing ScAYT1. (b) TLC of in vitro acetylation assay using DON as a substrate. Forty micrograms each
of DON (lane 1) and 3-ADON (lane 2) were loaded as
standard samples. DON was incubated with acetyl-CoA and
soluble E. coli cell extract of control (lane 3), recombinant
FasTRI101 (lane 4) (Kimura et al., 1998a), recombinant FdTAT
(lane 5), recombinant FsoTAT (lane 6), recombinant MgTAT
(lane 7) and recombinant ScAYT1 (lane 8) for 1 min (left
panel) or 3 h (right panel). (c) Northern blot analysis of the
acetyltransferase genes. The complementary strand RNA
probes were hybridized to the total RNA of the fungi grown in
the presence (+) or absence (”) of T-2 toxin.
516
The discovery of Tri201 in trichothecene-non-producer
Gibberella species raised the possibility that they were once
trichothecene producers and that extant non-producers
(e.g. F. oxysporum) arose subsequently by accumulating
mutations in the trichothecene genes (Kimura et al., 2003a).
In this study, we examined restricted genomic regions of
F. oxysporum (i.e. the Tri201 locus and hypothetical Tri5
cluster locus) on the basis of the assumption that trichothecene (pseudo-)genes, if they exist, may be located in
microsynteny regions of F. oxysporum and F. asiaticum,
as are the (pseudo-)Tri101 genes (Kimura et al., 2003a).
However, we were not able to obtain evidence either for or
against the presence of putative trichothecene (pseudo-)
genes in F. oxysporum (see Figs 1 and 2), other than the
previously identified Tri201.
Horizontal gene transfer is one possible explanation for
the acquisition of a secondary metabolism gene cluster
(Walton, 2000). However, only a few cases are supported
by experimental data, such as the T-toxin biosynthesis
gene cluster of Cochliobolus heterostrophus (Yang et al.,
1996), the HC-toxin biosynthesis gene cluster of Cochliobolus carbonum (Ahn & Walton, 1996) and the AK-toxin
biosynthesis gene cluster of Alternaria alternata (Tanaka
et al., 1999). In these cases, the toxin biosynthesis genes
are present only in some isolates of a species and are
completely absent from others in the near-isogenic background. Unlike these cases, the evolutionary history of
the trichothecene genes (e.g. whether or not the biosynthesis genes were acquired by horizontal gene transfer
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 19 Jun 2017 00:58:48
Microbiology 151
Evolutionary history of trichothecene genes
Fig. 5. Evolutionary relatedness of ascomycetous fungi used in this study (left) and
their trichothecene 3-O-acetyltransferase
protein family (right). Unrooted phylogenetic
trees were constructed using the NJ method
and downloaded from the DDBJ Web
server. The scale bars indicate the branch
length corresponding to the mean number of
differences (0.1) per residue along each
branch. Bootstrap values supporting the
branches connecting the subgroups are indicated. A 3-O-acetyltransferase tree with the
same topology was constructed using the
maximum-likelihood method accomplished
with PUZZLE. An enhanced view of the
Fusarium species branch is shown in the
species phylogeny (28S/26S rDNA tree) of
the fungi used in this study. Fas, F. asiaticum; Fs, F. sporotrichioides; Fo, F. oxysporum; Ff, F. fujikuroi; Fac, F. acuminatum;
Fd, F. decemcellulare; Fso, F. solani; Mg, M.
grisea; Sc, S. cerevisiae.
predating the evolutionary radiation of the trichotheceneproducing Fusarium species) currently remains unclear due
to limited sequence information on phylogenetically close
trichothecene-non-producer Gibberella species. The entire
genome sequence of F. oxysporum and other related species
may be necessary for comparative intraspecific sequence
analysis of trichothecene-producer and non-producer
fusaria. It should be noted, however, that Ward and coworkers specifically ruled out horizontal gene transfer in
the case of the type B trichothecene-producing Fusarium
species; the toxin gene cluster evolution was discordant
with the species phylogeny inferred from genes outside the
trichothecene core cluster, and a novel form of balancing
selection was suggested to explain this discordance (Ward
et al., 2002).
The occurrence of Tri201 only in the trichothecene-nonproducer Gibberella species cannot be explained by recent
horizontal gene acquisition, since the phylogeny of these
trichothecene 3-O-aceyltransferases is concordant with the
species phylogeny. This indicates that the acetyltransferase
gene experienced duplication (i.e. Tri201 generated) and
inactivation (i.e. Tri101 inactivated) in the evolutionary
history of F. oxysporum and F. fujikuroi. In addition to the
teleomorph genus Gibberella, we also extended our investigations of trichothecene 3-O-acetyltransferase genes to F.
decemcellulare and F. solani, which belong to the teleomorph genera Albonectria and Neocosmospora, respectively.
The unexpected discovery of a functional TAT in these phylogenetically distant Fusarium species raised an alternative
possibility: that the trichothecene 3-O-acetyltransferase
gene is just an antibiotic resistance gene and has a different
evolutionary history from other trichothecene biosynthesisrelated genes. In fact, O’Donnell and co-workers previously
reported that the evolution of Tri101 tracks with the species
http://mic.sgmjournals.org
phylogeny within the F. graminearum species complex
(O’Donnell et al., 2000) and, in this context, our present
results extend the applicability of their findings to other
fusaria.
As more genomes are being sequenced and made publicly
available, homology-based searches have revealed the existence of unexpected genes. For example, the occurrence
of phytochelatin (formerly believed to be a plant-specific
peptide) in animals was not documented until a homologue of the phytochelatin synthase gene was found in the
genome of Caenorhabditis elegans (Clemens et al., 2001;
Vatamaniuk et al., 2001). Recently, sequencing efforts
have also been made for several filamentous ascomycetes
(A. nidulans, M. grisea, F. graminearum, Sch. pombe and
N. crassa) in addition to S. cerevisiae (Mannhaupt et al.,
2003). The genome information has revealed the presence
of putative trichothecene 3-O-acetyltransferase gene homologues in most of these fungi. Functional identification of
the trichothecene 3-O-acetyltransferase genes of M. grisea
(i.e. MgTAT) and S. cerevisiae (i.e. ScAYT1) appears to be in
support of the alternative idea that this resistance gene is
widely distributed among various fungal species and has
a different evolutionary history from other trichothecene
genes of the F. graminearum species complex. If this is the
case, the ancestors of N. crassa and Sch. pombe may once
have possessed the 3-O-acetyltransferase gene, which was
subsequently lost or inactivated due to a lack of selective
constraints.
After we finished the analysis of these functional trichothecene 3-O-acetyltransferase genes, more fungal genomes
were sequenced and released into the public domain in
GenBank. A TBLASTN search of these acetyltransferase
genes further revealed highly homologous sequences in
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 19 Jun 2017 00:58:48
517
T. Tokai and others
the genome of Coccidioides posadasii (minimum E value
of e2134; Query=FdTAT; score=476) and Coprinopsis
cinerea (minimum E value of e2121; Query=FdTAT;
score=435). Although the functions of these genes are
not known, the conserved sequence motifs HXXMDXXG
and DFXXGXGXP were conserved in these homologues,
suggesting that they may also code for functional trichothecene 3-O-acetyltransferases.
It is rather a surprise that several fungi [at least seven distinct species functionally identified in this study and
previous studies (Alexander et al., 2002; Kimura et al.,
2003a)] that do not produce trichothecenes carried functional trichothecene 3-O-acetyltransferase genes without
accumulating mutations during their evolution. Unlike
prokaryotic antibiotic resistance genes, which are often
found on plasmids or transposons, there are no structural
features that greatly accelerate horizontal transmission of
the trichothecene resistance genes. In such cases, the persistence of this non-essential gene depends on the selective
advantage that it confers to the organism in which it exists
(Walton, 2000). For example, certain phytopathogenic
fungi possess inactivating genes for the plant antibiotics
phytoalexins (Covert et al., 1996; Weltring et al., 1988) and
‘phytoanticipins’ (Bowyer et al., 1995; Glenn et al., 2002;
Sandrock et al., 1995), which is reasonably explained by
the functional advantages for the fungi. However, unlike
these cases, the presence of 3-O-acetyltransferase genes
in diverse groups of trichothecene-non-producer fungi
is unusual in that these fungi do not necessarily inhabit
an environment frequently exposed to this group of
antibiotics.
To our knowledge, there are only two other examples of
fungal antibiotic resistance genes (by means of inactivation) that are not subject to selective constraints: MPR1 of
S. cerevisiae strain S1278b (Kimura et al., 2002) and BSD
of Aspergillus terreus (Kimura et al., 1994). MPR1 and
BSD code for acetyltransferase and deaminase, respectively,
and they confer resistance to the toxic proline analogue
L-azetidine-2-carboxylic acid and the aminoacylnucleoside
antibiotic blasticidin S, respectively. Both genes have
highly conserved homologues in other fungi (e.g. the
MPR1 homologue in N. crassa and the BSD homologue in
C. posadasii), suggesting that these resistance genes are
widely distributed across genera in fungi. Although the
eukaryotic antibiotic resistance genes apparently have no
physiological functions, they may actually contribute to
‘marginal fitness’, as demonstrated for seemingly nonessential genes in yeasts (Thatcher et al., 1998).
Ahn, J. H. & Walton, J. D. (1996). Chromosomal organization of
TOX2, a complex locus controlling host-selective toxin biosynthesis
in Cochliobolus carbonum. Plant Cell 8, 887–897.
Alexander, N. J., McCormick, S. P. & Hohn, T. M. (2002). The
identification of the Saccharomyces cerevisiae gene AYT1(ORFYLL063c) encoding an acetyltransferase. Yeast 19, 1425–1430.
Ballance, D. J. (1986). Sequences important for gene expression in
filamentous fungi. Yeast 2, 229–236.
Banno, S., Kimura, M., Tokai, T. & 7 other authors (2003). Cloning
and characterization of genes specifically expressed during infection
stages in the rice blast fungus. FEMS Microbiol Lett 222, 221–227.
Beremand, M. N. (1987). Isolation and characterization of mutants
blocked in T-2 toxin biosynthesis. Appl Environ Microbiol 53, 1855–1859.
Bowyer, P., Clarke, B. R., Lunness, P., Daniels, M. J. & Osbourn,
A. E. (1995). Host range of a plant pathogenic fungus determined by
a saponin detoxifying enzyme. Science 267, 371–374.
Brown, D. W., McCormick, S. P., Alexander, N. J., Proctor, R. H. &
Desjardins, A. E. (2001). A genetic and biochemical approach to
study trichothecene diversity in Fusarium sporotrichioides and
Fusarium graminearum. Fungal Genet Biol 32, 121–133.
Brown, D. W., McCormick, S. P., Alexander, N. J., Proctor, R. H. &
Desjardins, A. E. (2002). Inactivation of a cytochrome P-450 is a
determinant of trichothecene diversity in Fusarium species. Fungal
Genet Biol 36, 224–233.
Brown, D. W., Proctor, R. H., Dyer, R. B. & Plattner, R. D. (2003).
Characterization of a Fusarium 2-gene cluster involved in trichothecene C-8 modification. J Agric Food Chem 51, 7936–7944.
Clemens, S., Schroeder, J. I. & Degenkolb, T. (2001). Caenorhabditis
elegans expresses a functional phytochelatin synthase. Eur J Biochem
268, 3640–3643.
Covert, S. F., Enkerli, J., Miao, V. P. & VanEtten, H. D. (1996). A
gene for maackiain detoxification from a dispensable chromosome
of Nectria haematococca. Mol Gen Genet 251, 397–406.
Cundliffe, E. (1989). How antibiotic-producing organisms avoid
suicide. Annu Rev Microbiol 43, 207–233.
Desjardins, A. E., Hohn, T. M. & McCormick, S. P. (1993).
Trichothecene biosynthesis in Fusarium species: chemistry, genetics,
and significance. Microbiol Rev 57, 595–604.
Glenn, A. E., Gold, S. E. & Bacon, C. W. (2002). Fdb1 and Fdb2,
Fusarium verticillioides loci necessary for detoxification of preformed
antimicrobials from corn. Mol Plant–Microbe Interact 15, 91–101.
Hohn, T. M. & Beremand, P. D. (1989). Isolation and nucleotide
sequence of a sesquiterpene cyclase gene from the trichotheceneproducing fungus Fusarium sporotrichioides. Gene 79, 131–138.
Hohn, T. M., McCormick, S. P. & Desjardins, A. E. (1993). Evidence
for a gene cluster involving trichothecene-pathway biosynthetic genes
in Fusarium sporotrichioides. Curr Genet 24, 291–295.
Keller, N. P. & Hohn, T. M. (1997). Metabolic pathway gene clusters
in filamentous fungi. Fungal Genet Biol 21, 17–29.
Kimura, M., Kamakura, T., Tao, Q. Z., Kaneko, I. & Yamaguchi, I.
(1994). Cloning of the blasticidin S deaminase gene (BSD) from
Aspergillus terreus and its use as a selectable marker for Schizosaccharomyces pombe and Pyricularia oryzae. Mol Gen Genet 242,
121–129.
ACKNOWLEDGEMENTS
This research was supported in part by a grant for ‘Integrated Research
on the Safety and Physiological Function of Food’ to M. K. from the
Ministry of Agriculture, Fishery, and Forestry of Japan (MAFF). T. T.
was financially supported from RIKEN by a Junior Research Associate
(JRA) programme for graduate students.
518
REFERENCES
Kimura, M., Kaneko, I., Komiyama, M., Takatsuki, A., Koshino, H.,
Yoneyama, K. & Yamaguchi, I. (1998a). Trichothecene 3-O-
acetyltransferase protects both the producing organism and transformed yeast from related mycotoxins. Cloning and characterization
of Tri101. J Biol Chem 273, 1654–1661.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 19 Jun 2017 00:58:48
Microbiology 151
Evolutionary history of trichothecene genes
Kimura, M., Matsumoto, G., Shingu, Y., Yoneyama, K. &
Yamaguchi, I. (1998b). The mystery of the trichothecene 3-O-
O’Donnell, K., Ward, T. J., Geiser, D. M., Corby Kistler, H. & Aoki, T.
(2004). Genealogical concordance between the mating type locus and
acetyltransferase gene. Analysis of the region around Tri101 and
characterization of its homologue from Fusarium sporotrichioides.
FEBS Lett 435, 163–168.
seven other nuclear genes supports formal recognition of nine
phylogenetically distinct species within the Fusarium graminearum
clade. Fungal Genet Biol 41, 600–623.
Kimura, M., Shingu, Y., Yoneyama, K. & Yamaguchi, I. (1998c).
Page, R. D. M. (1996). TREEVIEW: an application to display
Features of Tri101, the trichothecene 3-O-acetyltransferase gene,
related to the self-defense mechanism in Fusarium graminearum.
Biosci Biotechnol Biochem 62, 1033–1036.
phylogenetic trees on personal computers. Comput Appl Biosci 12,
357–358.
Kimura, M., Anzai, H. & Yamaguchi, I. (2001). Microbial toxins in
Peplow, A. W., Meek, I. B., Wiles, M. C., Phillips, T. D. & Beremand,
M. N. (2003). Tri16 is required for esterification of position C-8
plant-pathogen interactions: biosynthesis, resistance mechanisms,
and significance. J Gen Appl Microbiol 47, 149–160.
during trichothecene mycotoxin production by Fusarium sporotrichioides. Appl Environ Microbiol 69, 5935–5940.
Kimura, Y., Nakamori, S. & Takagi, H. (2002). Polymorphism of the
MPR1 gene required for toxic proline analogue resistance in the
Saccharomyces cerevisiae complex species. Yeast 19, 1437–1445.
Sandrock, R. W., DellaPenna, D. & VanEtten, H. D. (1995). Purification and characterization of b 2-tomatinase, an enzyme involved in
the degradation of a-tomatine and isolation of the gene encoding b
Kimura, M., Tokai, T., Matsumoto, G., Fujimura, M., Hamamoto, H.,
Yoneyama, K., Shibata, T. & Yamaguchi, I. (2003a). Trichothecene
2-tomatinase from Septoria lycopersici. Mol Plant–Microbe Interact 8,
960–970.
nonproducer Gibberella species have both functional and nonfunctional 3-O-acetyltransferase genes. Genetics 163, 677–684.
Strimmer, K. & von Haeseler, A. (1996). Quartet puzzling: a quartet
maximum-likelihood method for reconstructing tree topologies. Mol
Biol Evol 13, 964–969.
Kimura, M., Tokai, T., O’Donnell, K., Ward, T. J., Fujimura, M.,
Hamamoto, H., Shibata, T. & Yamaguchi, I. (2003b). The
Takitani, S., Asabe, Y., Kato, T., Suzuki, M. & Ueno, Y. (1979).
trichothecene biosynthesis gene cluster of Fusarium graminearum
F15 contains a limited number of essential pathway genes and
expressed non-essential genes. FEBS Lett 539, 105–110.
Spectrodensitometric determination of trichothecene mycotoxins
with 4-(p-nitrobenzyl)pyridine on silica gel thin-layer chromatograms. J Chromatogr 172, 335–342.
Klich, M. A., Yu, J., Chang, P. K., Mullaney, E. J., Bhatnagar, D. &
Cleveland, T. E. (1995). Hybridization of genes involved in aflatoxin
Tanaka, A., Shiotani, H., Yamamoto, M. & Tsuge, T. (1999).
biosynthesis to DNA of aflatoxigenic and non-aflatoxigenic aspergilli.
Appl Microbiol Biotechnol 44, 439–443.
Kusumoto, K. I., Yabe, K., Nogata, Y. & Ohta, H. (1998). Aspergillus
oryzae with and without a homolog of aflatoxin biosynthetic gene
ver-1. Appl Microbiol Biotechnol 50, 98–104.
Lee, T., Oh, D. W., Kim, H. S., Lee, J., Kim, Y. H., Yun, S. H. & Lee,
Y. W. (2001). Identification of deoxynivalenol- and nivalenol-
producing chemotypes of Gibberella zeae by using PCR. Appl
Environ Microbiol 67, 2966–2972.
Lee, T., Han, Y. K., Kim, K. H., Yun, S. H. & Lee, Y. W.
(2002). Tri13 and Tri7 determine deoxynivalenol- and nivalenol-
producing chemotypes of Gibberella zeae. Appl Environ Microbiol 68,
2148–2154.
Mannhaupt, G., Montrone, C., Haase, D. & 8 other authors (2003).
What’s in the genome of a filamentous fungus? Analysis of the
Neurospora genome sequence. Nucleic Acids Res 31, 1944–1954.
McCormick, S. P., Alexander, N. J., Trapp, S. E. & Hohn, T. M.
(1999). Disruption of TRI101, the gene encoding trichothecene
3-O-acetyltransferase, from Fusarium sporotrichioides. Appl Environ
Microbiol 65, 5252–5256.
McCormick, S. P., Harris, L. J., Alexander, N. J., Ouellet, T.,
Saparno, A., Allard, S. & Desjardins, A. E. (2004). Tri1 in Fusarium
Insertional mutagenesis and cloning of the genes required for
biosynthesis of the host-specific AK-toxin in the Japanese pear
pathotype of Alternaria alternata. Mol Plant–Microbe Interact 12,
691–702.
Thatcher, J. W., Shaw, J. M. & Dickinson, W. J. (1998). Marginal
fitness contributions of nonessential genes in yeast. Proc Natl Acad
Sci U S A 95, 253–257.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W:
improving the sensitivity of progressive multiple sequence alignment
through sequence weighting, position-specific gap penalties and
weight matrix choice. Nucleic Acids Res 22, 4673–4680.
Trail, F. & Common, R. (2000). Perithecial development by Gibberella
zeae: a light microscopy study. Mycologia 92, 130–138.
Vatamaniuk, O. K., Bucher, E. A., Ward, J. T. & Rea, P. A. (2001).
A new pathway for heavy metal detoxification in animals. Phytochelatin synthase is required for cadmium tolerance in Caenorhabditis elegans. J Biol Chem 276, 20817–20820.
Walton, J. D. (2000). Horizontal gene transfer and the evolution of
secondary metabolite gene clusters in fungi: an hypothesis. Fungal
Genet Biol 30, 167–171.
Ward, T. J., Bielawski, J. P., Kistler, H. C., Sullivan, E. & O’Donnell, K.
(2002). Ancestral polymorphism and adaptive evolution in the
graminearum encodes a P450 oxygenase. Appl Environ Microbiol 70,
2044–2051.
trichothecene mycotoxin gene cluster of phytopathogenic Fusarium.
Proc Natl Acad Sci U S A 99, 9278–9283.
Meek, I. B., Peplow, A. W., Ake, C., Jr, Phillips, T. D. & Beremand,
M. N. (2003). Tri1 encodes the cytochrome P450 monooxygenase for
Weltring, K. M., Turgeon, B. G., Yoder, O. C. & VanEtten, H. D.
(1988). Isolation of a phytoalexin-detoxification gene from the plant
C-8 hydroxylation during trichothecene biosynthesis in Fusarium
sporotrichioides and resides upstream of another new Tri gene. Appl
Environ Microbiol 69, 1607–1613.
pathogenic fungus Nectria haematococca by detecting its expression
in Aspergillus nidulans. Gene 68, 335–344.
O’Donnell, K., Kistler, H. C., Tacke, B. K. & Casper, H. H.
(2000). Gene genealogies reveal global phylogeographic structure
efflux pump encoded by Tri102 in the biosynthesis gene cluster of
Fusarium graminearum. J Antibiot 53, 196–200.
and reproductive isolation among lineages of Fusarium graminearum, the fungus causing wheat scab. Proc Natl Acad Sci U S A
97, 7905–7910.
Yang, G., Rose, M. S., Turgeon, B. G. & Yoder, O. C. (1996). A
http://mic.sgmjournals.org
Wuchiyama, J., Kimura, M. & Yamaguchi, I. (2000). A trichothecene
polyketide synthase is required for fungal virulence and production
of the polyketide T-toxin. Plant Cell 8, 2139–2150.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Mon, 19 Jun 2017 00:58:48
519