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Applied and Environmental Microbiology, December 2008, p. 7790-7801, Vol. 74, No. 24
0099-2240/08/$08.00+0     doi:10.1128/AEM.01819-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Loss of Gibberellin Production in Fusarium verticillioides (Gibberella fujikuroi MP-A) Is Due to a Deletion in the Gibberellic Acid Gene Cluster{triangledown}

Christiane Bömke,1 Maria C. Rojas,2 Peter Hedden,3 and Bettina Tudzynski1*

Westfälische Wilhelms-Universität Münster, Institut für Botanik, Schlossgarten 3, 48149 Münster, Germany,1 Laboratorio de Bioorgánica, Departamento de Química, Facultad de Ciencias, Universidad de Chile, Casilla 653, Santiago, Chile,2 Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, United Kingdom3

Received 6 August 2008/ Accepted 20 October 2008


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ABSTRACT
 
Fusarium verticillioides (Gibberella fujikuroi mating population A [MP-A]) is a widespread pathogen on maize and is well-known for producing fumonisins, mycotoxins that cause severe disease in animals and humans. The species is a member of the Gibberella fujikuroi species complex, which consists of at least 11 different biological species, termed MP-A to -K. All members of this species complex are known to produce a variety of secondary metabolites. The production of gibberellins (GAs), a group of diterpenoid plant hormones, is mainly restricted to Fusarium fujikuroi (G. fujikuroi MP-C) and Fusarium konzum (MP-I), although most members of the G. fujikuroi species complex contain the GA biosynthesis gene cluster or parts of it. In this work, we show that the inability to produce GAs in F. verticillioides (MP-A) is due to the loss of a majority of the GA gene cluster as found in F. fujikuroi. The remaining part of the cluster consists of the full-length F. verticillioides des gene (Fvdes), encoding the GA4 desaturase, and the coding region of FvP450-4, encoding the ent-kaurene oxidase. Both genes share a high degree of sequence identity with the corresponding genes of F. fujikuroi. The GA production capacity of F. verticillioides was restored by transforming a cosmid with the entire GA gene cluster from F. fujikuroi, indicating the existence of an active regulation system in F. verticillioides. Furthermore, the GA4 desaturase gene des from F. verticillioides encodes an active enzyme which was able to restore the GA production in a corresponding des deletion mutant of F. fujikuroi.


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INTRODUCTION
 
Fusarium verticillioides (Sacc.) Nirenberg (synonym Fusarium moniliforme; teleomorph Gibberella fujikuroi MP-A or Gibberella moniliformis) is a widespread pathogen of maize plants causing ear and stalk rot disease (4, 10) and belongs to the Gibberella fujikuroi (Sawada) species complex. This complex includes important fungal pathogens of a variety of crops, such as maize, rice, barley, sugarcane, pine, mango, pineapple, and sorghum (26). It comprises 11 sexually fertile biological species, also known as mating populations (MPs; MP-A to -K), and more than 30 anamorphic species (genus Fusarium, sections Liseola, Elegans, and Dlaminia) (21, 24, 44, 45, 65). Members of the species complex produce a broad variety of mycotoxins and other secondary metabolites, such as moniliformin (25), fusaric acid (1), beauvericin (30), gibberellins (GAs) (58), bikaverin (29), and fumonisins (3, 4, 39, 43).

It is noteworthy that some secondary metabolites are produced by more than one species in the complex, whereas other metabolites are produced only by one species. Thus, fumonisins are produced by F. proliferatum and F. fujikuroi, as well as by F. verticillioides (12, 37, 49, 51), moniliformin by F. verticillioides, F. fujikuroi and F. thapsinum (25, 38), and beauvericin by isolates of F. fujikuroi, F. proliferatum, and F. subglutinans (12, 55). In contrast, the production of GAs, a group of plant hormones, is so far mainly restricted to F. fujikuroi (MP-C) and F. konzum (MP-I), although several species of the G. fujikuroi species complex contain the entire GA-biosynthetic gene cluster (32, 33, 34).

The genetics and biochemistry of GA production in the rice pathogen F. fujikuroi have been well characterized in recent years (53, 61, 62, 63). The GA biosynthesis genes in F. fujikuroi are organized in a gene cluster (28, 58), as is often the case for secondary-metabolite biosynthesis pathways in fungi, e.g., the genes for the biosynthesis of fumonisins in F. verticillioides (48), aurofusarin in Fusarium graminearum (35), and aflatoxin and sterigmatocystin in Aspergillus species (20).

Beside genes encoding a pathway-specific geranylgeranyl diphosphate synthase (ggs2) and the bifunctional ent-copalyl diphosphate/ent-kaurene synthase (cps/ks), the GA gene cluster includes four cytochrome P450 monooxygenase genes (P450-1 to P450-4) and the GA4 desaturase gene (des) (53, 58, 61, 62, 63). Thirteen enzymatic steps are involved in the biosynthesis of gibberellic acid (GA3) from geranylgeranyl diphosphate, indicating that most of the cluster genes encode multifunctional enzymes (Fig. 1). Recently, we demonstrated that the expression of six of the seven genes is under positive control by the general transcription factor AreA (40, 60) and that high nitrogen levels, which suppress AreA activity, virtually eliminate the expression of these genes and GA production. We have also characterized the cytochrome P450 oxidoreductase gene, cpr, and showed that CPR is essential for the functionality of the GA-specific cytochrome P450 monooxygenases in this fungus (31).


Figure 1
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  		FIG. 1. GA-biosynthetic pathway in F. fujikuroi. The major pathway is indicated by bold arrows.

Here we show that the genome of F. verticillioides contains a rudimentary GA gene cluster due to the complete absence of five of the seven biosynthetic genes. We found that the only complete remaining gene, Fvdes (F. verticillioides des), encoding the GA4 desaturase, is active and is able to complement the block in GA3 biosynthesis in the F. fujikuroi des deletion (Ff{Delta}des) mutant. Furthermore, we show that F. verticillioides produces a full spectrum of GAs after transformation with the entire GA gene cluster from F. fujikuroi, indicating an active regulation system in F. verticillioides.


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MATERIALS AND METHODS
 
Fungal strains.
Fusarium fujikuroi (MP-C) strains IMI58289 (Commonwealth Mycological Institute, Kew, United Kingdom) and m567 (Fungal Culture Collection, Weimar, Germany) are GA-producing wild-type strains. F. verticillioides (MP-A) strains A00149 (FGSC7415), A00999 (FGSC7603), A02949, A03823 (FRC M1212), A03824 (JFK), A04516 (FGSC7606), A04362 (NRRL22052), A04367 (JFL), A04796 (JFL), and A04801 (MRC4315) are isolates from maize kernels at different geographic locations and were kindly provided by J. F. Leslie (Kansas State University, KA). Strain Ff-{Delta}orf3-T33 (Ff{Delta}des) is a des deletion mutant of F. fujikuroi strain IMI58289, lacking GA3 and GA7 production (63). The GA-deficient strain SG139 is a UV-induced F. fujikuroi mutant that lacks the whole GA gene cluster (14, 61). It was kindly provided by E. Cerdá-Olmedo and J. Avalos (University of Seville, Seville, Spain). Mutant B1-41a, obtained by UV irradiation of F. fujikuroi strain GF-1a (2), was donated by J. MacMillan (University of Bristol, Bristol, United Kingdom). This strain was shown to contain a point mutation in the P450-4 locus and is GA deficient.

Bacterial strains and plasmids.
Escherichia coli strain Top10F' (Invitrogen, Groningen, The Netherlands) was used for plasmid propagation. Vectors pUCBM20 (Boehringer, Mannheim, Germany) and pUC19 (Fermentas, St. Leon-Rot, Germany) were used to clone DNA fragments carrying the Fusarium cluster genes and gene fragments from F. fujikuroi and F. verticillioides. Cosmid pCos1, derived from a cosmid library based on strain m567, contains the entire GA gene cluster, including the noncoding 5' and 3' regions (about 40 kb) and, additionally, the hygromycin resistance cassette for selection (P. Linnemannstöns and B. Tudzynski, unpublished data). Plasmid pDesA was constructed by PCR using primer pair des-Prom and P450-4-GD1 and contains the full-length Fvdes gene from strain A00149, encoding the GA4 desaturase, and the rudimentary P450-4 gene, encoding ent-kaurene oxidase. For functional studies with FvP450-4, vectors pFv-P450-4 (genomic gene copy with natural promoter region) and pFfP4prom::FvP4 (genomic gene copy under the control of the F. fujikuroi P450-4 promoter) were generated. To obtain expression vector pFv-P450-4, the genomic copy of FvP450-4 with the putative promoter was amplified by using primer pair Fv-P450-4-Prom-R2 and Fv-P450-4-F1 and cloned into pCR2.1TOPO. For expression of the FvP450-4 gene under the control of the corresponding F. fujikuroi promoter, the gene was amplified with an introduced NcoI site at the translation start codon (primer combination Fv-P450-4-Prom-NcoI-R1 and Fv-P450-4-F1), cloned into pCR2.1TOPO, excised with EcoRI, and ligated into pUC19. The F. fujikuroi promoter was generated by PCR using primers Ff-P450-4-1-Prom-NcoI-F1 and Ff-P450-4-1-Prom-NcoI-R1, introducing NcoI sites at the 5' and 3' ends. It was ligated into the NcoI-digested pUC19 containing FvP450-4, generating plasmid pFfP4prom::FvP4. In cotransformation experiments, pNR1 (nourseothricin resistance) (23, 31) or pAN7-1 (hygromycin resistance) (50), respectively, were used for selection of transformants.

Media and culture conditions.
For DNA isolation, Fusarium strains were grown for 3 days at 28°C on cellophane sheets (Alba Gewürze, Bielefeld, Germany) placed on CM agar (46). The harvested mycelium was frozen in liquid nitrogen, lyophilized for 24 h, and ground to a fine powder with a mortar and pestle. F. verticillioides strains were cultivated on V8 juice agar (200 ml/liter V8 juice, 0.3% CaCO3, and 2% agar) for sporulation. For RNA isolation, fungal strains were grown in 100%, 20%, or 0% ICI medium (16), containing 8% glucose, 0.5% MgSO4, 0.1% KH2PO4, and 0.5, 0.1, or 0% NH4NO3, respectively.

For analysis of gene expression, Fusarium strains were cultivated for 7 to 10 days in 20% ICI medium on a rotary shaker at 28°C. To elucidate nitrogen regulation, strains were harvested after 4 days of cultivation in 10% ICI medium, the mycelium was washed, and 1.5-g (wet weight) amounts were transferred to 100 ml of 0% or 100% ICI medium for 3 h. For GA production, the strains were grown for 10 days on a rotary shaker (190 rpm) at 28°C in 300-ml Erlenmeyer flasks containing 100 ml of 20% ICI medium.

DNA and RNA isolation.
Genomic DNA was isolated from lyophilized mycelium as described by Doyle and Doyle (13). Plasmid DNA was extracted by using Qiagen columns following the manufacturer's protocol (Qiagen, Hilden, Germany). RNA was isolated by using an RNAgents total RNA isolation kit (Promega, Mannheim, Germany).

PCR.
The PCR mixture contained 25 ng DNA, 10 ng of each primer (Operon Biotechnologies GmbH, Köln, Germany), 0.2 mM deoxynucleoside triphosphates, and 1 U DNA polymerase (BioTherm; GeneCraft GmbH, Lüdinghausen, Germany) in a total volume of 50 µl. PCR was carried out at 94°C for 4 min, followed by 36 cycles of 94°C for 1 min, 50 to 60°C for 1 min, 70°C for 1 to 4.5 min, and a final 10 min at 70°C. Annealing temperature and elongation time were applied differently, depending on the annealing temperature of each primer and the length of the amplified fragment. For analysis of the GA gene cluster and cloning of the remaining GA genes in F. verticillioides (MP-A) strain A00149, the following primers (Table 1) were synthesized on the basis of sequence data from F. fujikuroi genes and the published F. verticillioides genome sequence (Fusarium verticillioides Sequencing Project, Broad Institute of Harvard and MIT [http://www.broad.mit.edu]).


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  		TABLE 1. Primers used in this study

For reverse transcription-PCR (RT-PCR), 1 µg of total RNA of nitrogen-starved wild-type mycelium served as template to create cDNA by using a One-Step quantitative RT-PCR kit (Invitrogen, Groningen). For RT-PCR analysis of the des and P450-4 genes, primer pairs des-1 (5'-GCC AGT GCG CAA GAG TGT CAC TGC-3') and des-2 (5'-TCT CAC TTC CTC CTT GTC AGT TCC-3') and Fv-P450-4-RT-1 (5'-GCA TCG CGT GAG AGG GGA CGG C-3') and Fv-P450-4-RT-2 (5'-GCA GAA CCG TGT GAT TCA GC-3'), respectively, were used.

Southern and Northern blot analysis.
After digestion with restriction endonucleases and electrophoresis, genomic DNA was transferred onto Hybond N+ filters (Amersham Pharmacia, Freiburg, Germany). 32P-labeled probes were prepared by using the random oligomer-primer method (36). Filters were hybridized at 65°C in 5x Denhardt's solution containing 5% dextran sulfate (36). Filters were washed at the same temperature used for hybridization in 1x SSPE (1x SSPE is 0.18 M NaCl, 10 mM NaH2PO4, and 1 mM EDTA [pH 7.7]), 0.1% sodium dodecyl sulfate.

Northern blot hybridizations were accomplished by the method of Church and Gilbert (8). The F. fujikuroi rRNA gene was used as a control hybridization probe to confirm RNA transfer.

Sequencing.
DNA sequencing of recombinant plasmid clones was accomplished with the automatic sequencer LI-COR 4200 (MWG, München, Germany). The two strands of overlapping subclones obtained from the genomic DNA clones were sequenced by using the universal and the reverse primers. DNA and protein sequences were aligned by using DNAStar (Madison, WI).

Transformation of Fusarium strains.
The preparation of protoplasts and the transformation procedure were carried out as previously described (59), with the following modifications for strain A00149: strain A00149 was precultivated for 7 to 10 days on V8 agar. About 5 x 108 spores were inoculated into 100 ml CM medium. For complementation experiments, 107 protoplasts (in 50 µl) of strain A00149, Ff-{Delta}orf3-T33 (Ff{Delta}des), SG139, or B1-41a were transformed with up to 15 µg of the cosmid pCos1, carrying the entire GA gene cluster from F. fujikuroi, or one of the circular complementation vectors pDesA, pFv-P450-4, and pFfP4prom::FvP4. Plasmids were cotransformed with pNR1 (nourseothricin resistance marker) (31) or pAN7-1 (hygromycin resistance) (50).

Transformed protoplasts were regenerated at 28°C on complete regeneration agar [0.7 M sucrose, 0.05% yeast extract, 0.1% (NH4)2SO4] containing 120 µg/ml hygromycin B (Calbiochem, Bad Soden, Germany) or 120 µg/ml nourseothricin (Werner BioAgents, Jena, Germany) for 6 to 7 days.

GA assays.
The complete GA spectrum produced by the different strains and transformants was determined by gas chromatography-mass spectrometry (GC-MS) in ethyl acetate extracts of culture filtrates as described previously (63). Gibberellic acid (GA3) was quantified in the culture fluid of B1-41a and B1-41a transformants by GC-MS after 5 days of incubation in 0% ICI medium (16). For incubation with [14C]GA4, cultures grown in 40% ICI were transferred to 0% ICI (10 ml) buffered at pH 3.0 and 300,000 dpm [14C]GA4 was added as a methanol solution. The mixture was further incubated for 3 days at 28°C, the culture fluid was separated by filtration, and GAs were extracted as described previously and analyzed by GC-MS (63).

Nucleotide sequence accession numbers.
The gene sequences for Fvdes and FvP450-4 of F. verticillioides A00149 have been deposited in the GenBank database under accession numbers AM946176 and AM946177, respectively.


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RESULTS
 
Presence of a remnant GA gene cluster in F. verticillioides (MP-A) strains.
Previously, we have shown that F. verticillioides strains A00999 and A00149 contain only two of the seven GA-biosynthetic genes in the genome; these encode the GA4 desaturase (DES) and the ent-kaurene oxidase (P450-4) (33). To confirm that this finding is a general feature in F. verticillioides, we analyzed eight additional F. verticillioides strains, all isolated from maize kernels in different regions of the United States (J. F. Leslie, personal communication), by Southern blot analysis using the seven F. fujikuroi GA-biosynthetic genes as probes. All F. verticillioides strains revealed hybridizing bands for des and P450-4 but no hybridization signals for the other GA cluster genes (Fig. 2, results shown for des, P450-4, and P450-1). Therefore, F. verticillioides contains only the left border of the GA gene cluster found in F. fujikuroi and in most of the other species in the G. fujikuroi species complex (33).


Figure 2
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  		FIG. 2. Southern blot analysis of F. fujikuroi IMI58289 and several F. verticillioides isolates. The genomic DNA of all strains was digested with HindIII and hybridized with a 1:2 mixture of the gene probes from F. fujikuroi and F. verticillioides (for des and P450-4) or with the F. fujikuroi gene (P450-1). Ff, F. fujikuroi; Fv, F. verticillioides.

BLAST comparison of the genes flanking the F. fujikuroi GA gene cluster to the corresponding region (Broad Institute; http://www.broad.mit.edu) flanking the remnant putative F. verticillioides GA genes identified some highly homologous regions, as well as some striking differences (Fig. 3). Thus, the composition of the genomic region upstream of des is almost identical to that in F. fujikuroi. Genes encoding a sugar membrane transporter (smt) (64) and an ankyrin domain-containing protein (ank), as well as an alcohol and an aldehyde dehydrogenase (alc-dh and ald-dh) (reviewed in reference 57), are located upstream of des in both species (Fig. 3). However, smt and ank, which are immediately adjacent to des and share a bidirectional promoter region, are inverted and orientated in the opposite direction in Fusarium verticillioides compared with their orientation in F. fujikuroi. Interestingly, about 50 kb downstream of FvP450-4 is a region that shares 90% identity to the genomic region downstream of the F. fujikuroi GA gene cluster (orf1, orf2, and mfs), though in the opposite direction, suggesting an inversion of this genomic fragment (Fig. 3).


Figure 3
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  		FIG. 3. Comparison of the GA gene clusters in F. fujikuroi and F. verticillioides. Crosses indicate gene inversions, dashed lines demonstrate putative inversions, and arrows show the orientation of transcription. GA genes are shown in gray, and genes not belonging to the cluster are colored.

Expression of the two putative GA biosynthesis genes.
To analyze the expression of the two remaining GA-biosynthetic genes, Northern blot analyses were carried out with RNA isolated from different strains of F. verticillioides and compared to that from F. fujikuroi by using des and P450-4 as probes (Fig. 4). In addition, the filter was probed with the P450-3 gene, which is located at the right border of the cluster in F. fujikuroi and is missing in the remnant F. verticillioides cluster (Fig. 3). In F. fujikuroi, all three genes were expressed under conditions of nitrogen starvation, but no signal was obtained for des and P450-4 in F. verticillioides. Although we expected FvP450-4 not to be expressed or to be expressed only at low levels due to the missing homologous promoter region, there is no obvious reason for the lack of des expression. To scrutinize the expression of these genes, we performed RT-PCR analyses with primers des-1 and des-2 (des) and Fv-P450-4-RT-1 and Fv-P450-4-RT-2 (P450-4), respectively, with RNA isolated from nitrogen-starved mycelium of F. verticillioides strain A00149. By this more sensitive method, a transcript was detected for the desaturase gene, and its identity was confirmed by sequencing the RT-PCR fragment (data not shown). However, no transcript was present for P450-4. Thus, only one of the two remaining GA cluster genes is expressed in F. verticillioides, which might encode a functional GA4 desaturase.


Figure 4
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  		FIG. 4. Northern blot analysis of F. verticillioides isolates and the F. fujikuroi wild-type strain IMI58289. Total RNA was hybridized with the probes as indicated. P450-3 is not present in the genome of F. verticillioides and was used as a negative control. Fv, F. verticillioides; Ff, F. fujikuroi.

Functional analysis of the P450-4 and des genes from F. verticillioides.
P450-4 catalyzes the first oxidation steps in GA biosynthesis, converting ent-kaurene in three steps to ent-kaurenoic acid (61). To determine if the corresponding gene from F. verticillioides encodes a functional protein despite it not being expressed, we fused the promoter of FfP450-4 with the FvP450-4 coding sequence (FfP4prom::FvP4). We complemented the F. fujikuroi mutant B1-41a (2) with the fusion construct and with the FvP450-4 gene driven by the native promoter. Strain B1-41a produces significantly less GA3 than the wild type due to a point mutation in the FfP450-4 gene that interferes with the splicing of intron 2 and thus translation but not transcription (61). The mutant was cotransformed with vector pNR1 carrying the nourseothricin resistance gene. The complete integration of the P450-4 constructs was confirmed by PCR using primer combinations Fv-P450-4-F1 and Fv-P450-4-Prom-R2 (original F. verticillioides gene) and Ff-P450-4-1-Prom-NcoI-R1 combined with Fv-P450-4-F1 (F. verticillioides coding sequence fused with the F. fujikuroi promoter). Northern experiments with five verified transformants each revealed a higher expression level than that of B1-41a, particularly for those which were transformed with the construct FfP4prom::FvP4 (Fig. 5A). Since the two types of P450-4 transcripts that accumulate in the transformed B1-41a, the misspliced native FfP450-4 gene and the introduced FvP450-4 genes, could not be distinguished by Northern analysis, we performed RT-PCR to differentiate between the longer misspliced FfP450-4 transcripts and the shorter, spliced FvP450-4 transcripts. By this means, we could show that the fusion of the F. verticillioides coding sequence to the F. fujikuroi promoter enabled the expression of FvP450-4 in the B1-41a background (Fig. 5B, lane 7). None of the transformants exhibited a restored GA production, as evidenced by similar GA3 levels found in the cultures of B1-41a (0.71 µg/10 ml) and of the B1-41a FvP450-4 (1.04 µg/10 ml) and B1-41a FfP4prom::FvP4 transformants (1.46 µg/10 ml). The B1-41a mutant produces only 2.5% of the amount of GA3 produced by the wild-type GF-1a strain (2); thus, the above results demonstrate that FvP450-4 does not encode a functional protein.


Figure 5
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  		FIG. 5. Analysis of ent-kaurene oxidase P450-4 expression. (A) Northern blot analysis of F. fujikuroi wild-type strain IMI58289, the F. fujikuroi mutant B1-41a carrying a point mutation in the gene P450-4, and transformants of B1-41a transformed (+) with the F. verticillioides ent-kaurene oxidase gene FvP450-4, driven either by its own promoter or the promoter of the F. fujikuroi P450-4 gene (FfP4prom::FvP4). (B) RT-PCR analysis of the P450-4 gene in different fusaria and mutant strains. Lanes: 1 and 10, markers; 2, F. verticillioides cDNA; 3, F. verticillioides genomic DNA; 4, F. fujikuroi cDNA; 5, B1-41a cDNA; 6, B1-41a FvP450-4 T6 cDNA; 7, B1-41a FfP4prom::FvP4 T3 cDNA; 8, F. fujikuroi cDNA; 9, F. fujikuroi genomic DNA. Ff, F. fujikuroi; Fv, F. verticillioides; WT, wild type.

In contrast to FvP450-4, Fvdes is expressed but at a low level in F. verticillioides. To determine if it encodes a functional GA4 desaturase, Fvdes was cloned from strain F. verticillioides A00149 by PCR with primers des-Prom and P450-4-GD1, and three independent clones were sequenced in both directions. The gene revealed 92% and 94% identity at the nucleotide and amino acid levels, respectively, to Ffdes (63) (AJ417493, CAD10289), as well as 89% and 95% identity, respectively, to des from F. proliferatum (Fpdes) (34) (AJ628021, CAF31351) (32). The Fvdes gene was transformed into a des deletion strain of F. fujikuroi (Ff{Delta}des). This mutant was shown to accumulate GA1 and GA4 instead of GA7 and GA3 (63) (Fig. 1). The results of Southern blot analysis of five transformants clearly showed that some contained multiple copies of the transformed DNA (data not shown), and the results of Northern analysis indicated that Fvdes was highly expressed in some transformants (Fig. 6A). To test the enzymatic activity of FvDES, five of the transformants were cultivated for 10 days in the synthetic ICI medium (1.0 g/liter NH4NO3 as nitrogen source), and the secreted GAs were analyzed by GC-MS. Transformants T3 and T4, which were shown to have high levels of Fvdes expression, were able to produce GA3 in addition to GA1 (Fig. 6B). Therefore, Fvdes of strain A00149 encodes a functional protein that fully complemented the biosynthetic block in the Ff{Delta}des mutant. As shown in Fig. 6A, the expression level of Fvdes is much higher in the genetic background of F. fujikuroi than in that of F. verticillioides. One of the transformants with a high expression level, T3, contains at least three gene copies, while for T4 only one copy was present (data not shown), suggesting that the integration site in the genome plays an important role in the expression level.


Figure 6
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  		FIG. 6. (A) Northern blot analysis of strains F. fujikuroi IMI58289, the F. fujikuroi Ff{Delta}des mutant, and transformants of the Ff{Delta}des mutant complemented (+) with Fvdes. Total RNA was hybridized with the probes as indicated. (B) GC-MS analysis of culture filtrates of Ff{Delta}des and of transformants of Ff{Delta}des complemented (+) with Fvdes (T2, T3, and T4). Total ion currents are shown for ethyl acetate extracts as methyl esters trimethylsilyl ethers. Components were identified by comparison of their mass spectra and GC retention times with published data (15). Peaks: 1, GA9; 2, GA25; 3, GA14; 4, GA20; 5, GA24; 6, 7β-hydroxykaurenolide; 7, GA4; 8, gibberellenic acid; 9, GA7; 10, GA40; 11, GA13; 12, fujenoic acid; 13, GA47; 14, GA16; 15, iso-GA3; 16, GA36; 17, GA1; 18, 7β,18-dihydroxykaurenolide; 19, GA3. Ff, F. fujikuroi; Fv, F. verticillioides; WT, wild type.

To investigate if the genomic environment may be responsible for the low expression level of Fvdes in F. verticillioides, we transformed Fvdes back into its original source strain. Transformants were shown to have integrated the des gene in copy numbers from 1 to 3 at different loci (data not shown). Some of these transformants (T2 with one copy, T6 with three copies, and T7 with one copy) indeed have a higher expression level than the recipient strain, F. verticillioides A00149 (Fig. 7). Therefore, the genomic region where the GA biosynthesis genes are located seems to play an important role in their expression level.


Figure 7
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  		FIG. 7. Northern blot analysis of F. fujikuroi wild-type strain IMI58289, F. verticillioides wild-type strain A00149, and A00149 transformed (+) with additional copies of the Fvdes gene from strain A00149. Total RNA was hybridized with Fvdes. Ff, F. fujikuroi; Fv, F. verticillioides; WT, wild type.

The GA regulatory network is functional in F. verticillioides.
The results of Northern blot analyses indicated that the expression of P450-4 and des in F. verticillioides remained absent or low regardless of the strain or culture medium (Fig. 4, 6A, 7, and 8B). However, the much higher expression level of Fvdes in F. fujikuroi than in F. verticillioides suggests that nucleotide differences in the Fvdes promoter region in comparison with the Ffdes promoter cannot be the only reason for the different expression levels in these genetic backgrounds. To investigate if the transcription factors that regulate the expression of GA biosynthesis genes are active in F. verticillioides, we transformed the cosmid pCos1, carrying the entire GA gene cluster from the GA-producing strain F. fujikuroi IMI58289, into F. verticillioides strain A00149. The results of Southern blot analysis revealed the integration of the entire GA gene cluster of F. fujikuroi (including the 5' and 3' neighborhood of the GA cluster) into pCos1 transformants T2, T5, T7, T8, and T9 of F. verticillioides, whereas transformant T4 did not contain P450-3, the right border of the GA gene cluster, and T1 had integrated none of the cluster genes (Fig. 8A, results for T1, T2, T4, and T5).


Figure 8
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  		FIG. 8. Transformation (+) of F. verticillioides A00149 with the cosmid (pCos1) carrying the entire GA gene cluster from F. fujikuroi. (A) Southern blot analysis of F. fujikuroi IMI58289, F. verticillioides A00149, and transformants of A00149 carrying pCos1. The genomic DNA was restricted with HindIII. The filter was probed with GA-biosynthetic genes from F. fujikuroi as indicated. (B) Northern blot analysis of F. fujikuroi IMI58289, F. verticillioides A00149, and transformants of strain A00149 with pCos1. The filter was probed with GA-biosynthetic genes from F. fujikuroi as indicated. Ff, F. fujikuroi; Fv, F. verticillioides; WT, wild type.

In contrast to the recipient strain F. verticillioides A00149, most of the transformants express the GA pathway genes at about the same level as F. fujikuroi IMI58289 (results shown for des, P450-3, cps/ks, and P450-1 in Fig. 8B). Six of the seven GA genes (except for P450-3) are found to be regulated by nitrogen: large amounts of glutamine or ammonium almost totally repressed gene expression, as for F. fujikuroi (40; data not shown). Surprisingly, transformant T4 does not express the GA genes, although six of the seven genes are integrated into the genome. However, it is not likely that the lack of P450-3 is the reason for the inhibition of gene expression since F. fujikuroi {Delta}P450-3 mutants express the other GA cluster genes in a wild-type-like manner and produce large amounts of GA7 (63).

In order to compare GA production, we performed GC-MS analysis of the culture extracts after incubating F. verticillioides A00149, three pCos1 transformants of A00149 (T2, T5, and T8) carrying the entire gene cluster, and the F. fujikuroi wild-type strain IMI58289 in synthetic ICI medium for 10 days (Fig. 9). The transformants were able to produce the full spectrum of GAs as in the wild-type F. fujikuroi strain, demonstrating that all essential regulators needed for GA gene expression are present in the genome of F. verticillioides.


Figure 9
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  		FIG. 9. GC-MS analysis of culture filtrates of wild-type strains F. fujikuroi IMI58289 and F. verticillioides A00149 and strains of A00149 transformed (+) with pCos1 carrying the entire F. fujikuroi GA gene cluster. Total ion current for ethyl acetate extracts after derivatization to methyl esters trimethylsilyl ethers is shown. Peak identities from comparison with published mass spectra (15) are as listed for Fig. 6B, with, additionally, GA42 (peak 20). Ff, F. fujikuroi; Fv, F. verticillioides.

We directly compared the enzyme activities of FvDES and FfDES in the F. fujikuroi background by transforming Fvdes into the F. fujikuroi mutant SG139, which lacks the entire GA gene cluster (61). Transformants were screened for integration of the Fvdes gene by diagnostic PCR using the primers des-Prom and des-2. The results of Southern blot analysis revealed that the gene was integrated with one to three copies (Fig. 10A). The results of Northern blot analyses showed a high level of expression of Fvdes in F. fujikuroi SG139, in contrast to its low level of expression in the F. verticillioides wild-type strain (Fig. 10B). Cultures of two SG139 transformants with Fvdes (T4 and T10) and two previously described transformants with Ffdes (T1 and T2) (63) were incubated with [14C]GA4 for 3 days in nitrogen-free medium, and its conversion to [14C]GA7 was investigated by GC-MS. The DES enzymes of both species revealed similar enzymatic activities: SG139 transformants with Fvdes gave 12.7% conversion of [14C]GA4 to [14C]GA7, while SG139 with Ffdes gave 19% conversion. Thus, Fvdes encodes a functional GA desaturase with an activity similar to that of the desaturase from F. fujikuroi, consistent with the rescue of the F. fujikuroi Ff{Delta}des mutant by Fvdes described above.


Figure 10
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  		FIG. 10. Heterologous expression of Fvdes in F. fujikuroi mutant SG139. The entire GA gene cluster is missing in strain SG139. (A) Southern blot analysis of F. fujikuroi IMI58289 (wild type), F. fujikuroi SG139, and transformants (+) of SG139 with the gene Fvdes. Genomic DNA was digested with HindIII and probed with the genomic fragment of gene Fvdes. (B) Northern blot analysis of strains F. fujikuroi IMI58289 (wild type) and F. fujikuroi SG139 and transformants of SG139 which were transformed (+) with the gene Fvdes. The filter was probed with the genomic fragment of gene Fvdes. Ff, F. fujikuroi; Fv, F. verticillioides; WT, wild type.


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DISCUSSION
 
The G. fujikuroi species complex is known for the substantial production of a broad variety of secondary metabolites (reviewed in reference 9). Differences in metabolic chemotypes were part of recent investigations to characterize different Fusarium strains of this species complex (27, 33, 47, 52). F. verticillioides (G. fujikuroi MP-A) isolates from various areas and host plants produce large amounts of the mycotoxin fumonisin (7, 11, 12, 25). Fumonisin production is not restricted to F. verticillioides, as several isolates of F. proliferatum (MP-D), Fusarium nygamai (MP-G), and F. fujikuroi (MP-C) were also reported to contain the fumonisin gene cluster and to produce significant levels of this group of mycotoxins (9, 49). Recently, a correlation between the production of fumonisins and the host specificity has been shown for F. verticillioides isolates. While members of a distinct population of F. verticillioides are pathogenic on bananas and nonpathogenic on maize and are not able to produce fumonisins, another F. verticillioides subgroup of isolates infect maize and produce fumonisins (41, 42). The banana strains were shown to have a deletion of the fumonisin biosynthesis gene cluster, with only parts of the two terminal FUM genes remaining, suggesting that the ability to produce fumonisins is not essential for banana infection. Transformation-mediated complementation of banana strains with the complete FUM gene cluster restored fumonisin production, as well as pathogenicity on maize seedlings (17). Thus, there seems to be a correlation between fumonisin production and host specificity. The ubiquitous presence of the FUM cluster in maize isolates on one hand and the apparent deletion of almost the entire FUM gene cluster in banana isolates on the other hand suggests that the discontinuous distribution of gene clusters in highly related strains and species is due to specialization to different host plants and infection strategies during evolution.

We found a similar situation for the presence of the GA gene cluster and the ability to produce GAs in the G. fujikuroi species complex. While we did not find any GA-nonproducing F. fujikuroi strain isolated from rice, almost all the other Fusarium species of the G. fujikuroi species complex with different host plants have lost the ability to produce GAs due to multiple mutations in some GA cluster genes (33). Other genes in these clusters still encode functional enzymes, as they were able to complement the corresponding mutants of F. fujikuroi and restore the GA biosynthesis capability (34).

For F. proliferatum, which is one of the closest relatives to F. fujikuroi, we have studied the reasons for the loss of GA production in more detail. Despite the overall high degree of sequence identity of GA biosynthesis genes in these species, several mutations accumulated in the 5' noncoding (e.g., FpP450-1 and FpP450-4) or coding regions (e.g., Fpggs2 and Fpcps/ks) of GA genes in F. proliferatum strain D02945. Complementation of this nonproducing strain with the ggs2 and cps/ks genes from F. fujikuroi restored the ability to produce GAs, demonstrating that the mutations in these genes were the main reason for the loss of GA production (32, 34). In contrast to these results, we recently isolated a GA-producing strain, F. proliferatum ET-1, from the roots of tropical orchids (56). Since F. fujikuroi and F. proliferatum are closely related species, we verified the identity of the latter strain by phylogenetic analysis with five genetic markers. These results clearly demonstrate that the capacity to synthesize GAs and the spectrum of GAs present may differ even between strains of one species.

A similar situation was found for the aflatoxin gene cluster in different species of the Aspergillus section Flavi, which includes species such as A. parasiticus, A. flavus, A. sojae, and A. oryzae. Some of the species produce aflatoxin, while others have lost this ability for different reasons. Recently, it was shown that several nonproducing strains have retained the entire gene cluster but have acquired mutations in the regulatory gene aflR or in the noncoding or coding regions of three other open reading frames encoding biosynthesis genes. Other strains harbored small or large (more than 1 to 2 kb) deletions in the gene cluster. In several cases, the lack of expression of pathway genes can be explained by mutations in the AreA, FacB, and AflR binding motifs in their promoters (54). The authors suggest that losses of aflatoxin biosynthesis in A. oryzae and A. sojae are the result of a long history of use in industrial brewing processes where this pathway is apparently not needed by the fungus.

In this paper, we show that F. verticillioides, a member of the G. fujikuroi species complex, has lost its ability to produce GAs due to a substantial deletion of the GA gene cluster (Fig. 3). With Fvdes, encoding GA4 desaturase, and FvP450-4, encoding a nonfunctional ent-kaurene oxidase, there are only two genes left in the genomes of the 10 F. verticillioides strains analyzed, but only Fvdes seems to encode a functional enzyme, albeit with slightly reduced activity. Interestingly, downstream of the two remaining genes there is a genomic region of about 50 kb that is not found in F. fujikuroi, followed by a region with about 90% sequence identity to the F. fujikuroi region immediately adjacent to the GA gene cluster, though with the opposite orientation (Fig. 3). These data demonstrate that not only deletions but also genome rearrangements took place during the evolution of the G. fujikuroi species complex. A similar phenomenon of intercalary inversions, as well as other chromosome rearrangements, has been described from interlineage crosses of Gibberella zeae (18) and from interspecies crosses between isolates of Gibberella fujikuroi and Gibberella intermedia (27).

Recently, similar events of gene duplications, multiple losses, and chromosome inversions were shown for the Magnaporthe grisea ACEI cluster (22). A complete ACEI cluster was found in the genomes of only 4 of the 23 sequenced Pezizomycotina genomes. The authors showed that the main part of the cluster has been distributed from an ancestor of M. grisea to other species by horizontal gene transfer but that evolutionary constraints acted to maintain this cluster in only a few species, probably because of an important role of the yet-unknown product for their pathogenic life style (22). Similarly, highly conserved GA gene clusters might have been distributed by horizontal gene transfer from an ancestor species to other ascomycetes, but only some distantly related species, such as Sphaceloma manihoticola (6) and Phaeosphaeria sp. (19) maintained these clusters and produce GAs. Even among the closely related species of the G. fujikuroi species complex, only some are still able to produce GAs, while the others have lost this ability due to mutations in the GA gene cluster and/or to losses of one or more genes (32, 33, 34). F. verticillioides is an example for both GA gene losses and mutations. The remaining two genes are very poorly expressed. There might be different reasons for this. First, the loss of five GA cluster genes could result in low levels of expression of the remaining genes. However, this is not very likely as no gene(s) coding for pathway-specific transcription factors are located in the GA gene cluster in F. fujikuroi or any other species. In this case we would have expected a coordinated expression of all cluster genes, but in fact Fvdes is highly expressed in the F. fujikuroi mutant SG139, which has lost the entire gene cluster by UV mutation. Second, the accumulation of point mutations, e.g., in some of the GATA sequence elements, could be one reason for the reduced expression level of Fvdes, as has been shown for the GA genes FpP450-1 and FpP450-4 in F. proliferatum (32). However, Fvdes, with its slightly altered promoter region with respect to that of Ffdes, is expressed in the genetic background of F. fujikuroi much more strongly than in F. verticillioides and was able to complement the GA production defect of the F. fujikuroi {Delta}des mutant. These results suggest that additional factors, such as the genomic environment, seem to be responsible for the low level of expression of Fvdes. Indeed, by transforming Fvdes back into its original source strain, we revealed several transformants with different numbers of gene copies that have a higher expression level than the recipient strain F. verticillioides A00149.

To check if F. verticillioides has the regulatory network for the synthesis of GAs, we transformed a cosmid with the entire GA gene cluster from F. fujikuroi strain IMI58289 into F. verticillioides A00149. Surprisingly, the seven GA-biosynthetic genes were expressed in some transformants at almost the same high level as in F. fujikuroi. This is only possible if all the necessary transcriptional regulators, which might act in addition to AreA, exist and are active in F. verticillioides. These results clearly corroborate our previous findings in F. proliferatum, where the general transcription regulator AreA and other yet-unknown GA-specific regulator(s) were found to be functional (34).

Recent data from work with Aspergillus nidulans have demonstrated that the chromosomal position of gene clusters appears to affect transcription (5). We could show that the integration of Fvdes into different loci in the F. verticillioides genome can significantly increase the expression level, confirming that the differing histone structure caused by the deletion of the GA gene cluster in F. verticillioides might indeed be very important.

To summarize, we showed that the genomic region containing the remnant GA cluster in F. verticillioides has undergone two rearrangements, a sequence deletion and an inversion of a 50-kb fragment relative to that in the GA cluster of F. fujikuroi. Of the two remaining genes, only one, Fvdes, is still expressed (although at a low level) and encodes a functional enzyme, which has slightly lower activity than the corresponding F. fujikuroi enzyme. The activity was high enough to fully restore GA3 production in the F. fujikuroi {Delta}des mutant. We suggest that the presence of an active GA gene cluster in all F. fujikuroi strains studied so far and in some F. konzum (33) and F. proliferatum (56) isolates on one hand and the loss of GA production capacity in other species of the G. fujikuroi species complex on the other hand might be correlated with the specialization of the latter to different host plants and/or to pathogenic versus endophytic life styles.


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ACKNOWLEDGMENTS
 
We thank J. F. Leslie for providing F. verticillioides strains, J. Avalos (University of Seville, Seville, Spain) for F. fujikuroi strains IMI58289 and SG139, and J. MacMillan (University of Bristol, Bristol, United Kingdom) for providing F. fujikuroi mutant strain B1-41a.

This work was supported by the Deutsche Forschungsgemeinschaft (DFG; Tu101/9-5, SPP 1152 Evolution of Metabolic Diversity) and Fondo Nacional de Desarrollo Cientifico y Tecnologico (grant 1061127). Furthermore, we thank CONICYT/DFG (project 105-2007) for their grant of short-term scholarships (D/06/47017). Rothamsted Research receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the United Kingdom.


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FOOTNOTES
 
* Corresponding author. Mailing address: Westfälische Wilhelms-Universität Münster, Institut für Botanik, Schlossgarten 3, 48149 Münster, Germany. Phone: (49)251 8324801. Fax: (49)251 8323823. E-mail: Bettina.Tudzynski{at}uni-muenster.de Back

{triangledown} Published ahead of print on 24 October 2008. Back


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REFERENCES
 
    1
  1. Bacon, C. W., J. K. Porter, W. P. Norred, and J. F. Leslie. 1996. Production of fusaric acid by Fusarium species. Appl. Environ. Microbiol. 62:4039-4043.[Abstract]
    2. 2
  2. Bearder, J. R., J. MacMillan, M. Wels, M. B. Chaffey, and B. O. Phinney. 1974. Position of the metabolic block for gibberellin biosynthesis in mutant B1-41a of Gibberella fujikuroi. Phytochemistry 13:911-917.[CrossRef]
    3. 3
  3. Bezuidenhout, S. C., W. C. A. Gelderblom, C. P. Gorst-Allman, R. M. Horak, W. F. O. Marasas, G. Spiteller, and R. Vleggaar. 1988. Structure elucidation of the fumonisins, mycotoxins from Fusarium moniliforme. J. Chem. Soc. 1988:743-745.
    4. 4
  4. Bojja, R. S., R. L. Cerny, R. H. Proctor, and L. C. Du. 2004. Determining the biosynthetic sequence in the early steps of the fumonisin pathway by use of three gene-disruption mutants of Fusarium verticillioides. J. Agr. Food Chem. 52:2855-2860.[CrossRef]
    5. 5
  5. Bok, J. W., and N. P. Keller. 2004. LaeA, a regulator of secondary metabolism in Aspergillus spp. Eukaryot. Cell 3:527-535.[Abstract/Free Full Text]
    6. 6
  6. Bömke, C., M. C. Rojas, F. Gong, P. Hedden, and B. Tudzynski. 2008. Isolation and functional characterization of the gibberellin biosynthetic gene cluster in Sphaceloma manihoticola. Appl. Environ. Microbiol. 74:5325-5339.[Abstract/Free Full Text]
    7. 7
  7. Chulze, S. N., M. L. Ramirez, M. Pascale, and A. Visconti. 1998. Fumonisin production by, and mating populations of, Fusarium section Liseola isolates from maize in Argentina. Mycol. Res. 102:141-144.[CrossRef]
    8. 8
  8. Church, G. M., and W. Gilbert. 1984. Genomic sequencing. Proc. Natl. Acad. Sci. USA 81:1991-1995.[Abstract/Free Full Text]
    9. 9
  9. Desjardins, A. E. 2003. Gibberella from A (venaceae) to Z (eae). Annu. Rev. Phytopathol. 41:177-198.[CrossRef][Medline]
    10. 10
  10. Desjardins, A. E., and R. D. Plattner. 2000. Fumonisin B-1-nonproducing strains of Fusarium verticillioides cause maize (Zea mays) ear infection and ear rot. J. Agr. Food Chem. 48:5773-5780.[CrossRef]
    11. 11
  11. Desjardins, A. E., R. D. Plattner, T. C. Nelsen, and J. F. Leslie. 1995. Genetic analysis of fumonisin production and virulence of Gibberella fujikuroi mating population A (Fusarium moniliforme) on maize (Zea mays) seedlings. Appl. Environ. Microbiol. 61:79-86.[Abstract]
    12. 12
  12. Desjardins, A. E., H. K. Manandhar, R. D. Plattner, G. G. Manandhar, S. M. Poling, and C. M. Maragos. 2000. Fusarium species from Nepalese rice and production of mycotoxins and gibberellic acid by selected species. Appl. Environ. Microbiol. 66:1020-1025.[Abstract/Free Full Text]
    13. 13
  13. Doyle, J. J., and J. L. Doyle. 1990. Isolation of plant DNA from fresh tissue. Focus 12:13-15.[Medline]
    14. 14
  14. Fernández-Martín, R., F. Reyes, C. E. Domenech, E. Cabrera, P. M. Bramley, A. F. Barrero, J. Avalos, and E. Cerdá-Olmedo. 1995. Gibberellin biosynthesis in gib mutants of Gibberella fujikuroi. J. Biol. Chem. 270:14970-14974.[Abstract/Free Full Text]
    15. 15
  15. Gaskin, P., and J. MacMillan. 1992. GC-MS of the gibberellins and related compounds: methodology and a library of spectra. Cantock's Enterprises, Bristol, United Kingdom.
    16. 16
  16. Geissman, T. A., A. J. Verbiscar, B. O. Phinney, and G. Cragg. 1966. Studies on the biosynthesis of gibberellins from (–)–kaurenoic acid in cultures of Gibberella fujikuroi. Phytochemistry 5:933-947.[CrossRef]
    17. 17
  17. Glenn, A. E., N. C. Zitomer, A. M. Zimeri, L. D. Williams, R. T. Riley, and R. H. Proctor. 2008. Transformation-mediated complementation of a FUM gene cluster deletion in Fusarium verticillioides restores both fumonisin production and pathogenicity on maize seedlings. Mol. Plant Microbe Interact. 21:87-97.[CrossRef][Medline]
    18. 18
  18. Jurgenson, J. E., R. L. Bowden, K. A. Zeller, J. F. Leslie, N. J. Alexander, and R. D. Plattner. 2002. A genetic map of Gibberella zeae (Fusarium graminearum). Genetics 160:1451-1460.[Abstract/Free Full Text]
    19. 19
  19. Kawaide, H. 2006. Biochemical and molecular analyses of gibberellin biosynthesis in fungi. Biosci. Biotechnol. Biochem. 70:583-590.[CrossRef][Medline]
    20. 20
  20. Keller, N. P., and T. M. Hohn. 1997. Metabolic pathway gene cluster in filamentous fungi. Fungal Genet. Biol. 21:17-29.[CrossRef][Medline]
    21. 21
  21. Kerényi, Z., K. Zeller, L. Hornok, and J. F. Leslie. 1999. Standardization of mating type terminology in the Gibberella fujikuroi species complex. Appl. Environ. Microbiol. 65:4071-4076.[Abstract/Free Full Text]
    22. 22
  22. Khaldi, N., J. Collemare, M. H. Lebrun, and K. H. Wolfe. 2008. Evidence for horizontal transfer of a secondary metabolite gene cluster between fungi. Genome Biol. 9:R18.[CrossRef][Medline]
    23. 23
  23. Krügel, H., G. Fiedler, C. Smithand, and S. Baumberg. 1993. Sequence and transcriptional analysis of the nourseothricin acetyltransferase-encoding gene nat1 from Streptomyces noursei. Gene 127:127-131.[CrossRef][Medline]
    24. 24
  24. Leslie, J. F. 1995. Gibberella fujikuroi: available populations and variable traits. Can. J. Bot. 73:282-291.[CrossRef]
    25. 25
  25. Leslie, J. F., W. F. O. Marasas, G. S. Shephard, E. W. Sydenham, S. Stockenström, and P. G. Thiel. 1996. Duckling toxicity and the production of fumonisin and moniliformin by isolates in the A and F mating populations of Gibberella fujikuroi (Fusarium moniliforme). Appl. Environ. Microbiol. 62:1182-1187.[Abstract]
    26. 26
  26. Leslie, J. F., K. A. Zeller, A. Logrieco, G. Mule, A. Moretti, and A. Ritieni. 2004. Species diversity of and toxin production by Gibberella fujikuroi species complex strains isolated from native prairie grasses in Kansas. Appl. Environ. Microbiol. 70:2254-2262.[Abstract/Free Full Text]
    27. 27
  27. Leslie, J. F., K. Zeller, M. Wohler, and B. Summerell. 2004. Interfertility of two mating populations in the Gibberella fujikuroi species complex. Eur. J. Plant Pathol. 110:611-618.[CrossRef]
    28. 28
  28. Linnemannstöns, P., T. Voß, P. Hedden, P. Gaskin, and B. Tudzynski. 1999. Deletions in the gibberellin biosynthesis gene cluster of Gibberella fujikuroi by restriction enzyme-mediated integration and conventional transformation-mediated mutagenesis. Appl. Environ. Microbiol. 65:2558-2564.[Abstract/Free Full Text]
    29. 29
  29. Linnemannstöns, P., J. Schulte, M. del Mar Prado, R. H. Proctor, J. Avalos, and B. Tudzynski. 2002. The polyketide synthase gene pks4 from Gibberella fujikuroi encodes a key enzyme in the biosynthesis of the red pigment bikaverin. Fungal Genet. Biol. 37:134-148.[CrossRef][Medline]
    30. 30
  30. Logrieco, A., A. Moretti, G. Castella, M. Kostecki, P. Golinski, A. Ritieni, and J. Chelkowski. 1998. Beauvericin production by Fusarium species. Appl. Environ. Microbiol. 64:3084-3088.[Abstract/Free Full Text]
    31. 31
  31. Malonek, S., M. C. Rojas, P. Hedden, P. Gaskin, P. Hopkins, and B. Tudzynski. 2004. The NADPH:cytochrome P450 reductase gene from Gibberella fujikuroi is essential for gibberellin biosynthesis. J. Biol. Chem. 279:25075-25084.[Abstract/Free Full Text]
    32. 32
  32. Malonek, S., M. C. Rojas, P. Hedden, P. Gaskin, P. Hopkins, and B. Tudzynski. 2005. Functional characterization of two cytochrome P450 monooxygenase genes, P450-1 and P450-4, of the gibberellic acid gene cluster in Fusarium proliferatum (Gibberella fujikuroi MP-D). Appl. Environ. Microbiol. 71:1462-1472.[Abstract/Free Full Text]
    33. 33
  33. Malonek, S., C. Bömke, E. Bornberg-Bauer, M. C. Rojas, P. Hedden, P. Hopkins, and B. Tudzynski. 2005. Distribution of gibberellin biosynthetic genes and gibberellin production in the Gibberella fujikuroi species complex. Phytochemistry 66:1296-1311.[CrossRef][Medline]
    34. 34
  34. Malonek, S., M. C. Rojas, P. Hedden, P. Hopkins, and B. Tudzynski. 2005. Restoration of gibberellin production in Fusarium proliferatum by functional complementation of enzymatic blocks. Appl. Environ. Microbiol. 71:6014-6025.[Abstract/Free Full Text]
    35. 35
  35. Malz, S., M. N. Grell, C. Thrane, F. J. Maier, P. Rosager, A. Felk, K. S. Albertsen, S. Salomon, L. Bohn, W. Schäfer, and H. Giese. 2005. Identification of a gene cluster responsible for the biosynthesis of aurofusarin in the Fusarium graminearum species complex. Fungal Genet. Biol. 42:420-443.[CrossRef][Medline]
    36. 36
  36. Maniatis, T., J. Sambrook, and E. F. Fritsch. 1982. Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
    37. 37
  37. Marasas, W. F. O. 2001. Discovery and occurrence of the fumonisins: a historical perspective. Environ. Health Perspect. 109:239-243.[Medline]
    38. 38
  38. Marasas, W. F. O., P. G. Thiel, C. J. Rabie, P. E. Nelson, and T. A. Toussoun. 1986. Moniliformin production in Fusarium section Liseola. Mycologia 78:242-247.[CrossRef]
    39. 39
  39. Marasas, W. F. O., T. S. Kellerman, W. C. A. Gelderblom, J. A. W. Coetzer, P. G. Thiel, and J. J. Vanderlugt. 1988. Leukoencephalomalacia in a horse induced by fumonisin-B1 isolated from Fusarium moniliforme. Onderstepoort J. Vet. Res. 55:197-203.[Medline]
    40. 40
  40. Mihlan, M., V. Homann, T.-W. D. Liu, and B. Tudzynski. 2003. AreA directly mediates nitrogen regulation of gibberellin biosynthesis in Gibberella fujikuroi, but its activity is not affected by NMR. Mol. Microbiol. 47:975-991.[CrossRef][Medline]
    41. 41
  41. Mirete, S., C. Vázquez, G. Mulè, M. Jurado, and M. T. González-Jaén. 2004. Differentiation of Fusarium verticillioides from banana fruits by IGS and EF-1{alpha} sequence analyses. Eur. J. Plant Pathol. 110:515-523.[CrossRef]
    42. 42
  42. Moretti, A., G. Mulè, A. Susca, M. T. González-Jaén, and A. Logrieco. 2004. Toxin profile, fertility and AFLP analysis of Fusarium verticillioides from banana fruits. Eur. J. Plant Pathol. 110:601-609.[CrossRef]
    43. 43
  43. Nelson, P. E., A. E. Desjardins, and R. D. Plattner. 1993. Fumonisins, mycotoxins produced by Fusarium species: biology, chemistry, and significance. Annu. Rev. Phytopathol. 31:233-252.[CrossRef][Medline]
    44. 44
  44. Nirenberg, H. I., and K. O'Donnell. 1998. New Fusarium species and combinations within the Gibberella fujikuroi species complex. Mycologia 90:434-458.[CrossRef]
    45. 45
  45. O'Donnell, K., E. Cigelnik, and H. I. Nirenberg. 1998. Molecular systematics and phylogeography of the Gibberella fujikuroi species complex. Mycologia 90:465-493.[CrossRef]
    46. 46
  46. Pontecorvo, G. V., J. A. Poper, L. M. Hemmonns, K. D. MacDonald, and A. W. J. Buften. 1953. The genetics of Aspergillus nidulans. Adv. Genet. 141:141-238.
    47. 47
  47. Proctor, R. H., A. E. Desjardins, and R. D. Plattner. 1999. Biosynthetic and genetic relationships of B-series fumonisins produced by Gibberella fujikuroi mating population A. Nat. Toxins 7:251-258.[CrossRef][Medline]
    48. 48
  48. Proctor, R. H., D. W. Brown, R. D. Plattner, and A. E. Desjardins. 2003. Co-expression of 15 contiguous genes delineates a fumonisin biosynthetic gene cluster in Gibberella moniliformis. Fungal Genet. Biol. 38:237-249.[CrossRef][Medline]
    49. 49
  49. Proctor, R. H., R. D. Plattner, D. W. Brown, J. A. Seo, and Y. W. Lee. 2004. Discontinuous distribution of fumonisin biosynthetic genes in the Gibberella fujikuroi species complex. Mycol. Res. 108:815-822.[CrossRef][Medline]
    50. 50
  50. Punt, P. J., R. P. Oliver, M. A. Dingenanse, P. H. Puwels, and C. A. M. J. J. van den Hondel. 1987. Transformation of Aspergillus nidulans based on the hygromycin B resistance marker from Escherichia coli. Gene 56:117-124.[CrossRef][Medline]
    51. 51
  51. Reynoso, M. M., A. M. Torres, and S. N. Chulze. 2004. Fusaproliferin, beauvericin and fumonisin production by different mating populations among the Gibberella fujikuroi complex isolated from maize. Mycol. Res. 108:154-160.[CrossRef][Medline]
    52. 52
  52. Rheeder, J. P., W. F. O. Marasas, and H. F. Vismer. 2002. Production of fumonisin analogs by Fusarium species. Appl. Environ. Microbiol. 68:2101-2105.[Free Full Text]
    53. 53
  53. Rojas, M. C., P. Hedden, P. Gaskin, and B. Tudzynski. 2001. The P450-1 gene of Gibberella fujikuroi encodes a multifunctional enzyme in gibberellin biosynthesis. Proc. Natl. Acad. Sci. USA 98:5838-5843.[Abstract/Free Full Text]
    54. 54
  54. Tominaga, M., Y. H. Lee, R. Hayashi, Y. Suzuki, O. Yamada, K. Sakamoto, K. Gotoh, and O. Akita. 2006. Molecular analysis of an inactive aflatoxin biosynthesis gene cluster in Aspergillus oryzae RIB strains. Appl. Environ. Microbiol. 72:484-490.[Abstract/Free Full Text]
    55. 55
  55. Torres, A. M., M. M. Reynoso, F. G. Rojo, M. L. Ramirez, and S. N. Chulze. 2001. Fusarium species (section Liseola) and its mycotoxins in maize harvested in northern Argentina. Food Addit. Contam. 18:836-843.[CrossRef][Medline]
    56. 56
  56. Tsavkelova, E. A., C. Bömke, A. I. Netrusov, J. Weiner, and B. Tudzynski. 2008. Production of gibberellic acids by an orchid-associated Fusarium proliferatum strain. Fungal Genet. Biol. 45:1393-1403.[CrossRef][Medline]
    57. 57
  57. Tudzynski, B. 2005. Gibberellin biosynthesis in fungi: genes, enzymes, evolution, and impact on biotechnology. Appl. Microbiol. Biotechnol. 66:597-611.[CrossRef][Medline]
    58. 58
  58. Tudzynski, B., and K. Hölter. 1998. The gibberellin biosynthetic pathway in Gibberella fujikuroi: evidence for a gene cluster. Fungal Genet. Biol. 25:157-170.[CrossRef][Medline]
    59. 59
  59. Tudzynski, B., K. Mende, K.-M. Weltring, S. E. Unkles, and J. R. Kinghorn. 1996. The Gibberella fujikuroi niaD gene encoding nitrate reductase: isolation, sequence, homologous transformation and electrophoretic karyotype location. Microbiology 142:533-539.[Abstract/Free Full Text]
    60. 60
  60. Tudzynski, B., V. Homann, B. Feng, and G. A. Marzluf. 1999. Isolation, characterization and disruption of the areA nitrogen regulatory gene of Gibberella fujikuroi. Mol. Gen. Genet. 261:106-114.[CrossRef][Medline]
    61. 61
  61. Tudzynski, B., P. Hedden, E. Carrera, and P. Gaskin. 2001. The P450-4 gene of Gibberella fujikuroi encodes ent-kaurene oxidase in the gibberellin biosynthetic pathway. Appl. Environ. Microbiol. 67:3514-3522.[Abstract/Free Full Text]
    62. 62
  62. Tudzynski, B., M. C. Rojas, P. Gaskin, and P. Hedden. 2002. The Gibberella fujikuroi gibberellin 20-oxidase is a multifunctional monooxygenase. J. Biol. Chem. 277:21246-21253.[Abstract/Free Full Text]
    63. 63
  63. Tudzynski, B., M. Mihlan, M. C. Rojas, P. Linnemannstöns, P. Gaskin, and P. Hedden. 2003. Characterization of the final two genes of the gibberellin biosynthesis gene cluster of Gibberella fujikuroi: des and P450-3 encode GA4 desaturase and the 13-hydroxylase, respectively. J. Biol. Chem. 278:28635-28643.[Abstract/Free Full Text]
    64. 64
  64. Voß, T., J. Schulte, and B. Tudzynski. 2001. A new MFS-transporter gene next to the gibberellin biosynthesis gene cluster of Gibberella fujikuroi is not involved in gibberellin secretion. Curr. Genet. 39:377-383.[CrossRef][Medline]
    65. 65
  65. Zeller, K. A., B. A. Summerell, S. Bullock, and J. F. Leslie. 2003. Gibberella konza (Fusarium konzum) sp. nov. from prairie grasses, a new species in the Gibberella fujikuroi species complex. Mycologia 95:943-954.[Abstract/Free Full Text]

Applied and Environmental Microbiology, December 2008, p. 7790-7801, Vol. 74, No. 24
0099-2240/08/$08.00+0     doi:10.1128/AEM.01819-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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