1 Laboratory for Remediation Research, Plant Science Center, RIKEN, Wako, Saitama 351-0198, and Yokohama, Kanagawa 230-0045, Japan
2 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, RIKEN, Wako, Saitama 351-0198, Japan
6 Genetic 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
Correspondence
Makoto Kimura
mkimura{at}postman.riken.go.jp
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The GenBank/EMBL/DDBJ accession numbers for the sequences determined in this work are AB181459AB181481, 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 AO 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.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 UTP-ammonia 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, 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 pho5ura7 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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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
1.3clone-F and
1.3clone-R for a putative
-1,3-glucosidase gene (gene Q, orthologue of gene 1), and primers NADH-F and NADH-R for a putative NADH-cytochrome b5 reductase gene (gene J, orthologue of gene 6).
|
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 µg T-2 toxin ml1. 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 µg ampicillin ml1 and 34 µg chloramphenicol ml1. The bacterial cultures were incubated at 20 °C 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 µg ml1) for 3 days at 28 °C. Yeast cells were inoculated on YPD medium (1 % yeast extract, 2 % peptone and 2 % glucose) with 20 µg T-2 toxin ml1 and cultured for 2 days at 28 °C. 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 neighbour-joining (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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
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
-1,3-glucosidase gene (gene 1), an esterase gene (gene 2), a 3-hydroxyacyl-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 pCosFo1-12-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 non-trichothecene 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 trichothecene-producing (e.g. F. asiaticum, F. graminearum) and non-producing (e.g. F. oxysporum, F. fujikuroi) Gibberella species.
|
|
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 (3-ADON), 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-O-acetyltransferase 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
).
|
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).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 predating the evolutionary radiation of the trichothecene-producing 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 co-workers 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-non-producer 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 biosynthesis-related genes. In fact, O'Donnell and co-workers previously reported that the evolution of Tri101 tracks with the species 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 the genome of Coccidioides posadasii (minimum E value of e134; Query=FdTAT; score=476) and Coprinopsis cinerea (minimum E value of e121; 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 1278b (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 non-essential genes in yeasts (Thatcher et al., 1998
).
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Alexander, N. J., McCormick, S. P. & Hohn, T. M. (2002). The identification of the Saccharomyces cerevisiae gene AYT1(ORF-YLL063c) encoding an acetyltransferase. Yeast 19, 14251430.[CrossRef][Medline]
Ballance, D. J. (1986). Sequences important for gene expression in filamentous fungi. Yeast 2, 229236.[Medline]
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, 221227.[Medline]
Beremand, M. N. (1987). Isolation and characterization of mutants blocked in T-2 toxin biosynthesis. Appl Environ Microbiol 53, 18551859.[Medline]
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, 371374.[Medline]
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, 121133.[CrossRef][Medline]
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, 224233.[CrossRef][Medline]
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, 79367944.[CrossRef][Medline]
Clemens, S., Schroeder, J. I. & Degenkolb, T. (2001). Caenorhabditis elegans expresses a functional phytochelatin synthase. Eur J Biochem 268, 36403643.
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, 397406.[CrossRef][Medline]
Cundliffe, E. (1989). How antibiotic-producing organisms avoid suicide. Annu Rev Microbiol 43, 207233.[CrossRef][Medline]
Desjardins, A. E., Hohn, T. M. & McCormick, S. P. (1993). Trichothecene biosynthesis in Fusarium species: chemistry, genetics, and significance. Microbiol Rev 57, 595604.[Medline]
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 PlantMicrobe Interact 15, 91101.[Medline]
Hohn, T. M. & Beremand, P. D. (1989). Isolation and nucleotide sequence of a sesquiterpene cyclase gene from the trichothecene-producing fungus Fusarium sporotrichioides. Gene 79, 131138.[CrossRef][Medline]
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, 291295.[CrossRef][Medline]
Keller, N. P. & Hohn, T. M. (1997). Metabolic pathway gene clusters in filamentous fungi. Fungal Genet Biol 21, 1729.[CrossRef][Medline]
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, 121129.[Medline]
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, 16541661.
Kimura, M., Matsumoto, G., Shingu, Y., Yoneyama, K. & Yamaguchi, I. (1998b). The mystery of the trichothecene 3-O-acetyltransferase gene. Analysis of the region around Tri101 and characterization of its homologue from Fusarium sporotrichioides. FEBS Lett 435, 163168.[CrossRef][Medline]
Kimura, M., Shingu, Y., Yoneyama, K. & Yamaguchi, I. (1998c). Features of Tri101, the trichothecene 3-O-acetyltransferase gene, related to the self-defense mechanism in Fusarium graminearum. Biosci Biotechnol Biochem 62, 10331036.[Medline]
Kimura, M., Anzai, H. & Yamaguchi, I. (2001). Microbial toxins in plant-pathogen interactions: biosynthesis, resistance mechanisms, and significance. J Gen Appl Microbiol 47, 149160.[Medline]
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, 14371445.[CrossRef][Medline]
Kimura, M., Tokai, T., Matsumoto, G., Fujimura, M., Hamamoto, H., Yoneyama, K., Shibata, T. & Yamaguchi, I. (2003a). Trichothecene nonproducer Gibberella species have both functional and nonfunctional 3-O-acetyltransferase genes. Genetics 163, 677684.
Kimura, M., Tokai, T., O'Donnell, K., Ward, T. J., Fujimura, M., Hamamoto, H., Shibata, T. & Yamaguchi, I. (2003b). The trichothecene biosynthesis gene cluster of Fusarium graminearum F15 contains a limited number of essential pathway genes and expressed non-essential genes. FEBS Lett 539, 105110.[CrossRef][Medline]
Klich, M. A., Yu, J., Chang, P. K., Mullaney, E. J., Bhatnagar, D. & Cleveland, T. E. (1995). Hybridization of genes involved in aflatoxin biosynthesis to DNA of aflatoxigenic and non-aflatoxigenic aspergilli. Appl Microbiol Biotechnol 44, 439443.[CrossRef][Medline]
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, 98104.[CrossRef][Medline]
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, 29662972.
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, 21482154.
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, 19441954.
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, 52525256.
McCormick, S. P., Harris, L. J., Alexander, N. J., Ouellet, T., Saparno, A., Allard, S. & Desjardins, A. E. (2004). Tri1 in Fusarium graminearum encodes a P450 oxygenase. Appl Environ Microbiol 70, 20442051.
Meek, I. B., Peplow, A. W., Ake, C., Jr, Phillips, T. D. & Beremand, M. N. (2003). Tri1 encodes the cytochrome P450 monooxygenase for C-8 hydroxylation during trichothecene biosynthesis in Fusarium sporotrichioides and resides upstream of another new Tri gene. Appl Environ Microbiol 69, 16071613.
O'Donnell, K., Kistler, H. C., Tacke, B. K. & Casper, H. H. (2000). Gene genealogies reveal global phylogeographic structure and reproductive isolation among lineages of Fusarium graminearum, the fungus causing wheat scab. Proc Natl Acad Sci U S A 97, 79057910.
O'Donnell, K., Ward, T. J., Geiser, D. M., Corby Kistler, H. & Aoki, T. (2004). Genealogical concordance between the mating type locus and seven other nuclear genes supports formal recognition of nine phylogenetically distinct species within the Fusarium graminearum clade. Fungal Genet Biol 41, 600623.[CrossRef][Medline]
Page, R. D. M. (1996). TREEVIEW: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 12, 357358.[Medline]
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 during trichothecene mycotoxin production by Fusarium sporotrichioides. Appl Environ Microbiol 69, 59355940.
Sandrock, R. W., DellaPenna, D. & VanEtten, H. D. (1995). Purification and characterization of 2-tomatinase, an enzyme involved in the degradation of a-tomatine and isolation of the gene encoding
2-tomatinase from Septoria lycopersici. Mol PlantMicrobe Interact 8, 960970.[Medline]
Strimmer, K. & von Haeseler, A. (1996). Quartet puzzling: a quartet maximum-likelihood method for reconstructing tree topologies. Mol Biol Evol 13, 964969.
Takitani, S., Asabe, Y., Kato, T., Suzuki, M. & Ueno, Y. (1979). Spectrodensitometric determination of trichothecene mycotoxins with 4-(p-nitrobenzyl)pyridine on silica gel thin-layer chromatograms. J Chromatogr 172, 335342.[CrossRef][Medline]
Tanaka, A., Shiotani, H., Yamamoto, M. & Tsuge, T. (1999). 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 PlantMicrobe Interact 12, 691702.[Medline]
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, 253257.
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, 46734680.[Abstract]
Trail, F. & Common, R. (2000). Perithecial development by Gibberella zeae: a light microscopy study. Mycologia 92, 130138.
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, 2081720820.
Walton, J. D. (2000). Horizontal gene transfer and the evolution of secondary metabolite gene clusters in fungi: an hypothesis. Fungal Genet Biol 30, 167171.[CrossRef][Medline]
Ward, T. J., Bielawski, J. P., Kistler, H. C., Sullivan, E. & O'Donnell, K. (2002). Ancestral polymorphism and adaptive evolution in the trichothecene mycotoxin gene cluster of phytopathogenic Fusarium. Proc Natl Acad Sci U S A 99, 92789283.
Weltring, K. M., Turgeon, B. G., Yoder, O. C. & VanEtten, H. D. (1988). Isolation of a phytoalexin-detoxification gene from the plant pathogenic fungus Nectria haematococca by detecting its expression in Aspergillus nidulans. Gene 68, 335344.[CrossRef][Medline]
Wuchiyama, J., Kimura, M. & Yamaguchi, I. (2000). A trichothecene efflux pump encoded by Tri102 in the biosynthesis gene cluster of Fusarium graminearum. J Antibiot 53, 196200.[Medline]
Yang, G., Rose, M. S., Turgeon, B. G. & Yoder, O. C. (1996). A polyketide synthase is required for fungal virulence and production of the polyketide T-toxin. Plant Cell 8, 21392150.
Received 2 June 2004;
revised 18 October 2004;
accepted 4 November 2004.
HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
J MED MICROBIOL | ALL SGM JOURNALS |