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Appl Environ Microbiol. 2008 December; 74(24): 7790–7801. Published online 2008 October 24. doi: 10.1128/AEM.01819-08. | PMCID: PMC2607190 |
Copyright © 2008, American Society for Microbiology Loss of Gibberellin Production in Fusarium verticillioides ( Gibberella fujikuroi MP-A) Is Due to a Deletion in the Gibberellic Acid Gene Cluster ![[down-pointing small open triangle]](/corehtml/pmc/pmcents/x25BF.gif) 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 August 6, 2008; Accepted October 20, 2008. 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. 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 GA 4 desaturase gene ( des) ( 53, 58, 61, 62, 63). Thirteen enzymatic steps are involved in the biosynthesis of gibberellic acid (GA 3) from geranylgeranyl diphosphate, indicating that most of the cluster genes encode multifunctional enzymes (Fig. ). 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). 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 (FfΔdes) 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. 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-Δorf3-T33 (FfΔ des) is a des deletion mutant of F. fujikuroi strain IMI58289, lacking GA 3 and GA 7 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 Fv des gene from strain A00149, encoding the GA 4 desaturase, and the rudimentary P450-4 gene, encoding ent-kaurene oxidase. For functional studies with Fv P450-4, vectors pFv-P450-4 (genomic gene copy with natural promoter region) and pFfP4 prom::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 Fv P450-4 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 Fv P450-4 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 Fv P450-4, generating plasmid pFfP4 prom::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% CaCO 3, 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% MgSO 4, 0.1% KH 2PO 4, and 0.5, 0.1, or 0% NH 4NO 3, 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]). 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 5× Denhardt's solution containing 5% dextran sulfate ( 36). Filters were washed at the same temperature used for hybridization in 1× SSPE (1× 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 × 10 8 spores were inoculated into 100 ml CM medium. For complementation experiments, 10 7 protoplasts (in 50 μl) of strain A00149, Ff-Δorf3-T33 (FfΔ 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 pFfP4 prom::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 (GA 3) 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]GA 4, cultures grown in 40% ICI were transferred to 0% ICI (10 ml) buffered at pH 3.0 and 300,000 dpm [ 14C]GA 4 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. 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 GA 4 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. , 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). 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. ). 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. ). 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 Fv P450-4 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. ). 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. ). 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. ). 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. 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 Ff P450-4 with the Fv P450-4 coding sequence (Ff P4prom::Fv P4). We complemented the F. fujikuroi mutant B1-41a ( 2) with the fusion construct and with the Fv P450-4 gene driven by the native promoter. Strain B1-41a produces significantly less GA 3 than the wild type due to a point mutation in the Ff P450-4 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 Ff P4prom::Fv P4 (Fig. ). Since the two types of P450-4 transcripts that accumulate in the transformed B1-41a, the misspliced native Ff P450-4 gene and the introduced Fv P450-4 genes, could not be distinguished by Northern analysis, we performed RT-PCR to differentiate between the longer misspliced Ff P450-4 transcripts and the shorter, spliced Fv P450-4 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. , lane 7). None of the transformants exhibited a restored GA production, as evidenced by similar GA 3 levels found in the cultures of B1-41a (0.71 μg/10 ml) and of the B1-41a Fv P450-4 (1.04 μg/10 ml) and B1-41a Ff P4prom::Fv P4 transformants (1.46 μg/10 ml). The B1-41a mutant produces only 2.5% of the amount of GA 3 produced by the wild-type GF-1a strain ( 2); thus, the above results demonstrate that Fv P450-4 does not encode a functional protein. In contrast to Fv P450-4, Fv des is expressed but at a low level in F. verticillioides. To determine if it encodes a functional GA 4 desaturase, Fv des 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 Ff des ( 63) (AJ417493, CAD10289), as well as 89% and 95% identity, respectively, to des from F. proliferatum (Fp des) ( 34) (AJ628021, CAF31351) ( 32). The Fv des gene was transformed into a des deletion strain of F. fujikuroi (FfΔ des). This mutant was shown to accumulate GA 1 and GA 4 instead of GA 7 and GA 3 ( 63) (Fig. ). 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 Fv des was highly expressed in some transformants (Fig. ). 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 NH 4NO 3 as nitrogen source), and the secreted GAs were analyzed by GC-MS. Transformants T3 and T4, which were shown to have high levels of Fv des expression, were able to produce GA 3 in addition to GA 1 (Fig. ). Therefore, Fv des of strain A00149 encodes a functional protein that fully complemented the biosynthetic block in the FfΔ des mutant. As shown in Fig. , the expression level of Fv des 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. 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. ). Therefore, the genomic region where the GA biosynthesis genes are located seems to play an important role in their expression level. 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. , , , and ). 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. , results for T1, T2, T4, and T5). 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. ). 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 Δ P450-3 mutants express the other GA cluster genes in a wild-type-like manner and produce large amounts of GA 7 ( 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. ). 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. We directly compared the enzyme activities of FvDES and FfDES in the F. fujikuroi background by transforming Fv des into the F. fujikuroi mutant SG139, which lacks the entire GA gene cluster ( 61). Transformants were screened for integration of the Fv des 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. ). The results of Northern blot analyses showed a high level of expression of Fv des in F. fujikuroi SG139, in contrast to its low level of expression in the F. verticillioides wild-type strain (Fig. ). Cultures of two SG139 transformants with Fv des (T4 and T10) and two previously described transformants with Ff des (T1 and T2) ( 63) were incubated with [ 14C]GA 4 for 3 days in nitrogen-free medium, and its conversion to [ 14C]GA 7 was investigated by GC-MS. The DES enzymes of both species revealed similar enzymatic activities: SG139 transformants with Fv des gave 12.7% conversion of [ 14C]GA 4 to [ 14C]GA 7, while SG139 with Ff des gave 19% conversion. Thus, Fv des 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 FfΔ des mutant by Fv des described above. 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., Fp P450-1 and Fp P450-4) or coding regions (e.g., Fp ggs2 and Fp cps/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. ). With Fv des, encoding GA 4 desaturase, and Fv P450-4, encoding a nonfunctional ent-kaurene oxidase, there are only two genes left in the genomes of the 10 F. verticillioides strains analyzed, but only Fv des 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. ). 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 Fv des 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 Fv des, as has been shown for the GA genes Fp P450-1 and Fp P450-4 in F. proliferatum ( 32). However, Fv des, with its slightly altered promoter region with respect to that of Ff des, 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 Δ des mutant. These results suggest that additional factors, such as the genomic environment, seem to be responsible for the low level of expression of Fv des. Indeed, by transforming Fv des 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 Fv des 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, Fv des, 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 GA 3 production in the F. fujikuroi Δ 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. 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. 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. [PubMed] 2. Bearder, J. R., J. MacMillan, M. Wels, M. B. Chaffey, and B. O. Phinney. 1974. 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