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Applied and Environmental Microbiology, September 2008, p. 5325-5339, Vol. 74, No. 17
0099-2240/08/$08.00+0     doi:10.1128/AEM.00694-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Isolation and Characterization of the Gibberellin Biosynthetic Gene Cluster in Sphaceloma manihoticola ^,

Christiane Bömke,¹ Maria Cecilia Rojas,² Fan Gong,³ Peter Hedden,³ and Bettina Tudzynski^1*

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 25 March 2008/ Accepted 14 June 2008

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Gibberellins (GAs) are tetracyclic diterpenoid phytohormones that were first identified as secondary metabolites of the fungus Fusarium fujikuroi (teleomorph, Gibberella fujikuroi). GAs were also found in the cassava pathogen Sphaceloma manihoticola, but the spectrum of GAs differed from that in F. fujikuroi. In contrast to F. fujikuroi, the GA biosynthetic pathway has not been studied in detail in S. manihoticola, and none of the GA biosynthetic genes have been cloned from the species. Here, we present the identification of the GA biosynthetic gene cluster from S. manihoticola consisting of five genes encoding a bifunctional ent-copalyl/ent-kaurene synthase (CPS/KS), a pathway-specific geranylgeranyl diphosphate synthase (GGS2), and three cytochrome P450 monooxygenases. The functions of all of the genes were analyzed either by a gene replacement approach or by complementing the corresponding F. fujikuroi mutants. The cluster organization and gene functions are similar to those in F. fujikuroi. However, the two border genes in the Fusarium cluster encoding the GA₄ desaturase (DES) and the 13-hydroxylase (P450-3) are absent in the S. manihoticola GA gene cluster, consistent with the spectrum of GAs produced by this fungus. The close similarity between the two GA gene clusters, the identical gene functions, and the conserved intron positions suggest a common evolutionary origin despite the distant relatedness of the two fungi.

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Although they are ubiquitous phytohormones, gibberellins (GAs) were first identified as secondary metabolites of the fungus Fusarium fujikuroi (teleomorph, Gibberella fujikuroi mating population C [MP-C]). GAs are also present in other fungi, including Sphaceloma manihoticola (teleomorph, Elsinoë brasiliensis) (42) and Phaeosphaeria sp. strain L487 (47), and several bacteria, such as Rhizobium phaseoli (5) and some Bacillus species (17). In recent years, studies of GA biosynthesis in plants and F. fujikuroi have revealed significant differences at the chemical (pathway), biochemical (enzyme), and genetic levels, indicating that plants and Fusarium have evolved their complex biosynthetic pathways to GAs independently and that corresponding genes have not been transferred to the fungus by horizontal gene transfer (reviewed in reference 20). Although higher plants (Arabidopsis thaliana) and Fusarium produce structurally identical GAs, there are profound differences in the sequences of the biosynthetic steps (reviewed in references 20 and 51). A major difference is the stage at which the hydroxyl groups are introduced. Whereas 3β-hydroxylation occurs at an early stage in the fungus, with GA₁₂-aldehyde being converted to GA₁₄-aldehyde (Fig. 1) (44), in Arabidopsis, 3β-hydroxylation of GA₉ and GA₂₀ are the final reactions in the formation of the biologically active end products, GA₄ and GA₁, respectively. The final biosynthetic step in F. fujikuroi is the 13-hydroxylation of GA₇ to form GA₃ (58), whereas in plants, 13-hydroxylation of GA₁₂ to GA₅₃ is a relatively early step (20). The members of the two kingdoms also differ in the nature of the enzymes utilized for GA biosynthesis. The formation of plant GAs requires both membrane-bound cytochrome P450 monooxygenases and soluble 2-oxoglutarate-dependent dioxygenases (18, 19), whereas in fungal GA biosynthesis, only monooxygenases have been shown to participate in the same reactions. Interestingly, in F. fujikuroi, as well as in the GA-producing fungus Phaeosphaeria sp. strain L487, the two-step cyclization of geranylgeranyl diphosphate (GGDP) to ent-kaurene via ent-copalyl diphosphate (CDP) is catalyzed by a fungal-type bifunctional cyclase (CPS/KS) (Fig. 1) (54), whereas in plants, two independent diterpene cyclases catalyze these reactions (48, 54, 62). In contrast to Arabidopsis and other plants, where the GA biosynthetic genes are dispersed throughout the genome (20), in F. fujikuroi the genes are physically linked in a gene cluster (29, 52). The gene cluster in F. fujikuroi includes a GA pathway-specific GGDP synthase-encoding gene (ggs2), whereas a second gene, ggs1, encodes the enzyme responsible for GGDP synthesis for primary metabolism (35). The ggs2 gene shares a bidirectional promoter with the ent-copalyl diphosphate or ent-kaurene synthase gene, cps/ks. In addition to four cytochrome P450 monooxygenase genes (P450-1 to P450-4), which encode multifunctional enzymes, the GA gene cluster also contains a GA₄ desaturase-encoding gene, des (58).

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FIG. 1. Biosynthetic pathway for GAs and related diterpenoids in F. fujikuroi. The major pathway is indicated by boldface arrows.

Recently, the GA biosynthetic gene cluster has been identified in a second GA-producing fungus, Phaeosphaeria sp. strain L487 (23). In contrast to F. fujikuroi, this fungus produces GA₁, rather than GA₃. Aside from the Phaeosphaeria sp. strain L487 cps/ks gene (Phcps/ks), the GA gene cluster of Phaeosphaeria sp. strain L487 contains four cytochrome P450 monooxygenase genes, three of which are similar to the F. fujikuroi monooxygenase genes P450-1, P450-2, and P450-4. Surprisingly, the Phaeosphaeria cluster does not contain a pathway-specific GGDP synthase gene equivalent to ggs2 in F. fujikuroi nor genes with homology to the 13-hydroxylase-encoding gene P450-3 or the desaturase-encoding gene des (23).

At present, only a few GA-producing fungi are known. Besides F. fujikuroi (G. fujikuroi MP-C), Fusarium konzum (G. fujikuroi MP-I) (32), and Phaeosphaeria sp. strain L487, the ability to produce GAs has been described for several Sphaceloma species, mainly for S. manihoticola (reviewed in references 30, 42, and 43). In contrast to F. fujikuroi, Sphaceloma spp. produce GA₄ rather than GA₃ as the main end product of the GA pathway (8, 38, 42, 43), indicating that the desaturase, for conversion of GA₄ to GA₇, and the 13-hydroxylase, for conversion of GA₇ to GA₃, are inactive or missing in these fungi. However, GA biosynthesis in Sphaceloma has not yet been subjected to molecular genetic studies.

In this paper, we focus on the identification of the GA biosynthetic gene cluster in S. manihoticola, a pathogen of the tropical starchy root crop cassava (Manihot esculenta) that causes the hyperelongation of the internodes as a result of GA secretion. The cluster consists of five genes that share high similarity with the corresponding F. fujikuroi homologues. However, the cluster contains neither a desaturase-encoding gene (des) nor a P450-3 homologue, consistent with the inability to produce GA₇, GA₃, and GA₁. The S. manihoticola cluster genes fully restored GA production in the corresponding Fusarium mutants, confirming that the new gene cluster is indeed responsible for GA biosynthesis. Despite some differences in the cluster organization, we suggest that the pathway genes in both fungi have a common phylogenetic origin.

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Fungal strains.
The S. manihoticola wild-type strain Lu949 (ATCC 44292) was donated by W. Rademacher (BASF Agricultural Center, Limburgerhof, Germany). IMI58289 (CABI Biosciences, Kew, United Kingdom) is a GA-producing wild-type strain of F. fujikuroi (teleomorph, G. fujikuroi MP-C). Strain Ff-cps/ks-GD-T22 (54) is an F. fujikuroi IMI58289 disruption mutant that does not produce GAs or precursors. The GA-defective strain SG139 is an F. fujikuroi UV mutant that lacks the whole GA gene cluster (14, 56). It was kindly provided by E. Cerda-Olmedo and J. Avalos (University of Seville, Seville, Spain). Mutant B1-41a, obtained by UV irradiation of F. fujikuroi strain GF-1a (7), was donated by J. MacMillan (University of Bristol, Bristol, United Kingdom). The strain was shown to contain a point mutation in the P450-4 locus and is GA deficient (56).

Media and culture conditions.
For DNA isolation, Fusarium strains were grown on cellophane sheets (Alba Gewürze, Bielefeld, Germany) placed on CM agar (39) for 3 days at 28°C. S. manihoticola Lu949 was grown on potato dextrose agar plates for 10 days under the same conditions. The mycelium was harvested, frozen in liquid nitrogen, and lyophilized for 24 h. For RNA isolation, Fusarium strains were grown in 100%, 20%, or 0% ICI medium (16) containing 8% glucose, 0.5% MgSO₄, 0.1% KH₂PO₄, and 5.0, 1.0, or 0 g/liter NH₄NO₃, respectively. S. manihoticola was grown in optimized production medium (OPM) containing 6% sunflower oil, 0.05% (NH₄)₂SO₄, 1.5% corn steep solids (Sigma-Aldrich, Taufkirchen, Germany), and 0.1% KH₂PO₄. For shift experiments, the Sphaceloma wild-type strain Lu949 was grown as indicated below. For GA production, Fusarium strains were grown for 7 to 10 days and S. manihoticola for about 44 days on a rotary shaker (190 rpm) at 28°C in 300-ml Erlenmeyer flasks containing 100 ml of either 20% ICI medium or OPM. S. manihoticola was also cultivated for 30 days on 10 g brown rice that had been autoclaved with 15 ml H₂O in 300-ml Erlenmeyer flasks. ICI medium for Sphaceloma cultures was prepared with 8% maltose instead of glucose. All fungal strains were precultivated in Darken medium (11) for 3 (Fusarium) or 7 (Sphaceloma) days.

Bacterial strains and plasmids.
Escherichia coli strain Top10F' (Invitrogen, Groningen, The Netherlands) was used for plasmid propagation. The vectors pUC19 (Fermentas, St. Leon-Rot, Germany) and pBluescript II SK(–) (Stratagene, La Jolla, CA) were used to clone DNA fragments carrying the S. manihoticola Lu949 cluster genes and gene fragments. The plasmid pSm-cps/ks was used for complementation of the F. fujikuroi cps/ks-GD-T22 strain. To obtain the plasmid pSm-cps/ks, a 5,276-kb SacI fragment of clone 13.1 was cloned into pUC19/SacI. The S. manihoticola cps/ks (Smcps/ks) gene replacement vector pSm-cps/ks was obtained by amplifying two flanking sequences of Smcps/ks by PCR, introducing a KpnI (flank 1) restriction site and HindIII and BamHI (flank 2) restriction sites into the oligonucleotides. For the amplified flank 1, a genomic SalI site could be used (see Fig. 3). The flanking sequences (flank1, 1.2 kb; flank 2, 1 kb) were cloned into the corresponding sites of the vector pUCH2-8 (1) carrying a hygromycin resistance cassette. The 4.7-kb replacement fragment was obtained by restriction with KpnI and BamHI. For heterologous expression of S. manihoticola GA genes in the GA-deficient F. fujikuroi mutants SG139 and B1-41a, the three P450 genes were isolated by PCR approaches and cloned into the vector pCR2.1TOPO, creating the vectors pSm-P450-1, pSm-P450-2, and pSm-P450-4. For expression of the Sphaceloma P450 genes under the control of the corresponding F. fujikuroi promoter, the single genes were amplified with introduced or genomic (SmP450-1) NcoI sites at the translation start codon, cloned into pCR2.1TOPO, excised with EcoRI, and ligated into pUC19. The corresponding Fusarium promoters were generated by PCR, introducing NcoI sites at the 5' and 3' ends. They were ligated into the NcoI-digested pUC19 containing the appropriate Sphaceloma genes, creating the vectors pFfP1_prom::SmP1, pFfP2_prom::SmP2, and pFfP4_prom::SmP4, respectively. The cosmid pCos1, derived from a cosmid library based on strain m567, contains the entire GA gene cluster, including the 5' and 3' regions (an 40-kb insert) (P. Linnemannstöns and B. Tudzynski, unpublished data). In cotransformation experiments, pNR1 (nourseothricin resistance) (27, 31) or pAN7-1 (hygromycin resistance) (41) was used for selection of resistant transformants.

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FIG. 3. Targeted replacement of the Smcps/ks gene. (A) Replacement strategy showing physical maps of the gene replacement fragment Sm-cps/ks and the Smcps/ks wild type (WT) and the gene locus from an Sm-cps/ks knockout mutant showing the components of the hygromycin resistance cassette (hyg; black boxes), restriction sites used for generating the replacement fragment, and digestion of the genomic DNA for Southern blot analysis (panel B) (see the text). The small arrows indicate the positions of primers used for cloning the 5' and 3' flanks and scrutinizing the correct integration of the replacement cassette (see Materials and Methods). The sizes of hybridizing fragments are also indicated. Note that the drawing is not to scale. (B) Southern blot analysis of genomic DNA of the WT and some homokaryotic single protoplast isolates of Sm-cps/ks deletion mutants T1 (T1-1), T2 (T2-1, T2-2, and T2-3), and T12 (T12-3, T12-5, and T12-7). DNA was digested with HindIII, electrophoresed, blotted, and hybridized with two different probes (panel A) (see the text). The hybridizing patterns demonstrate the successful deletion of Smcps/ks in all transformants, although some additional ectopic integrations appear to indicate the WT fragment. (C) Northern blot analysis of the S. manihoticola cps/ks deletion mutants T1-1, T2-1, and T12-3. Total RNA was hybridized to the probes as indicated. The hybridization patterns confirm the deletion of Smcps/ks in all transformants, although rRNA appears in the background with approximately the same size as Smcps/ks and Smggs2 transcripts. All mutants showed downregulation of the expression of the other GA cluster genes, probably due to the integration of the larger replacement fragment and not because of feedback regulation, since single genes are expressed at a high level in F. fujikuroi SG139, a mutant lacking the whole GA gene cluster (Fig. 6).

Construction and screening of a genomic library.
A DASH II library (Stratagene) from genomic DNA of S. manihoticola strain Lu949 was prepared following the manufacturer's instructions. About 35,000 recombinant phage were plated and transferred to nylon membranes (Whatman GmbH, Dassel, Germany). Hybridization was performed at high stringency (65°C) in 5x Denhardt's solution containing 5% dextran sulfate. The blots were washed under hybridization conditions with 0.1x SSC (1x SSC is 0.15 M NaCl, 0.015 M sodium citrate), 0.1% sodium dodecyl sulfate (SDS). Positive recombinant phages were used for a second round of plaque purification.

DNA isolation.
Genomic DNA was isolated from lyophilized mycelium as described previously by Doyle and Doyle (13). Plasmid DNA was extracted using the Qiagen plasmid extraction kit (Qiagen, Hilden, Germany). Lambda DNA from positive lambda phages was prepared according to the method of Maniatis et al. (34).

Southern blot and Northern blot analyses.
For Southern analysis, genomic, plasmid, or phage DNA was digested to completion with appropriate restriction enzymes (Fermentas, St. Leon-Rot, Germany), fractionated in 1.0% (wt/vol) agarose gels, and transferred to Hybond N⁺ filters (Amersham Pharmacia, Freiburg, Germany). Hybridization was carried out in 6x SSC, 5x Denhardt's solution, 0.1% SDS, and 50 mM phosphate buffer, pH 6.6, at 65°C in the presence of a random-primed [-³²P]dCTP-labeled probe. Membranes were washed once (2x SSPE [1x SSPE is 0.18 M NaCl, 10 mM NaH₂PO₄, and 1 mM EDTA {pH 7.7}], 0.1% SDS) before being exposed to autoradiographic film. Total fungal RNA was isolated using the RNAgents total-RNA isolation kit (Promega, Mannheim, Germany). Northern blot hybridizations were done as previously described by Church and Gilbert (10).

DNA sequencing and sequence homology searching.
DNA sequencing of recombinant plasmid clones was accomplished by the dideoxy chain termination method (46) with an automatic sequencer, LI-COR 4200 (MWG, Munich, Germany). The two strands of overlapping subclones obtained from the genomic DNA clones were sequenced using the universal and the reverse primers. DNA and protein sequence alignments were done using DNA Star (DNA Star Inc., Madison, WI).

Sequence homology searches were performed using the NCBI database server. Protein homology was based on BlastX searches (2). For further investigations, the programs of DNA Star Inc. (Madison, WI) were used.

PCR and reverse transcription (RT)-PCR.
PCR was performed as previously described (32). Heterologous PCR based on the two known fungal diterpene cyclase sequences of F. fujikuroi (FfCPS/KS) and Phaeosphaeria sp. strain L487 (PhCPS/KS) to identify Smcps/ks was performed with the primer combination cps/ks-Sm-F2 and Sm-cps/ks-5R1 (Table 1) . For analysis of the GA gene cluster organization in S. manihoticola, the following primers (Table 1) were synthesized on the basis of the determined sequences of lambda clones 12.1, 13.1, and 14.1. They were applied in different combinations to obtain the whole cluster sequence.

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TABLE 1. Primers used for GA gene cluster isolation, vector construction, and diagnostic PCR

For amplifying the 5' and 3' flanks of the gene replacement vector pSm- cps/ks, the following primers were used (Table 1): Sm-GRP-cpsks-KpnI-F1 and Sm-GRP-cpsks-LF-R1 (flank 1) and Sm-GRP-cpsks-HindIII-F1 combined with Sm-GRP-cpsks-BamHI-R1 (flank 2). The analysis of putative Smcps/ks knockout transformants was performend with the diagnostic primers Sm-cps/ks-GR-K-F1 and PUCH-P for integration in the 5' region of Smcps/ks, and Sm-cps/ks-GR-K-R1 and PUCH-T for integration in the 3' region of Smcps/ks (Table 1). The deletion of the wild-type Smcps/ks gene copy was demonstrated by diagnostic PCR using the primers Sm-cps/ks-nF3 and Sm-cps/ks-nR1. The primers used for generation of vectors for heterologous gene expression are shown in Table 1.

For RT-PCR, 1 µg of total RNA of nitrogen-starved wild-type cells served as a template, and the nonspecific oligo(dT)₁₈ primer (Fermentas) was used to create cDNA by reverse transcription.

Fungal transformations.
Preparation of protoplasts of F. fujikuroi was performed as previously described (53). For S. manihoticola, the following modifications were used: Lu949 was precultivated for 7 to 10 days on potato dextrose agar and used for inoculation of 100 ml of Darken medium. After 5 days on a rotary shaker at 28°C and 190 rpm, 1 ml of this culture was transferred to 100 ml of CM medium for 24 h. Sphaceloma protoplasts were obtained with the same mixture of lysing enzymes as Fusarium, with an additional 250 mg/100 ml mutanase (InterSpex Products Inc., San Mateo, CA). Protoplasts (10⁸) of each strain were transformed with up to 13 µg of the respective complementation vectors or pCos1 and, if necessary, with 7 µg of the nourseothricin resistance-mediating vector pNR1 or the hygromycin resistance cassette-containing vector pAN7-1, respectively. For deletion of Smcps/ks, 10 µg of the KpnI/BamHI fragment of the gene replacement vector pSm-cps/ks was used for transformation of S. manihoticola strain Lu949. Transformed protoplasts were regenerated at 28°C in a complete regeneration agar (0.7 M sucrose, 0.05% yeast extract, 0.1% Casamino Acids) containing 120 µg/ml hygromycin B (InvivoGen, San Diego, CA) or 120 µg/ml nourseothricin (Werner BioAgents, Jena, Germany) for 6 or 7 days. Complementation mutants of the hygromycin-resistant Fusarium strain Ff-cps/ks-GD-T22 were generated by cotransformation with the nourseothricin resistance-mediating vector pNR1. For purification of the Smcps/ks deletion mutants, single protoplasts were regenerated from hygromycin-resistant transformants and used for DNA isolation, PCR, and Southern and Northern blot analyses.

GA analysis.
GA₃, GA₄, and GA₇ in culture fluids of all strains were analyzed by high-performance liquid chromatography (HPLC) (6) with a Merck HPLC system with a UV detector and a Lichrospher 100 RP18 column (5 µm; 250 by 4 mm). These GAs were also analyzed by thin-layer chromatography (TLC) on silica gels eluted with ethyl acetate-chloroform-acetic acid (90:60:7.5). Gas chromatography-mass spectrometry (GC-MS) using a GCQ system (ThermoFinnigan) as described by Troncoso et al. (50) was used to determine the complete GA complements produced by the different strains after extraction from the culture fluid and to identify the products from incubations with ¹⁴C-labeled substrates after HPLC or TLC separation. Identifications were based on comparison of mass spectra with published data (15).

Labeled substrates.
ent-7-hydroxy[¹⁴C₄]kaurenoic acid, [¹⁴C₄]GA₁₂-aldehyde, ent-6,7-diOH[¹⁴C₄]kaurenoic acid, and ent-[¹⁴C₄]kauradienoic acid were prepared from R-[2-¹⁴C]mevalonic acid (Amersham) by incubation with a cell-free system from Cucurbita maxima endosperm in the presence of ATP, MgCl₂, and NADPH (59). [¹⁴C₁]GA₁₄ was prepared from [¹⁴C₁]GA₁₂ by incubation with cultures of an SG139-P450-1 F. fujikuroi transformant (44). ent-[17-¹⁴C]kaurenoic acid, [17-¹⁴C]GA₁₂, [17-¹⁴C]GA₁₅, and[17-¹⁴C]GA₂₄ were obtained from L. Mander (Australian National University, Canberra, Australia).

Incubations with radiolabeled GA precursors.
Cultures of SG139-Sm-P450-1 or SG139-Sm-P450-2 transformants grown in 40% ICI medium for 3 days at 28°C were harvested, and the mycelia were washed with 0% ICI medium (16). After resuspension of the mycelia in 0% ICI, 1-ml aliquots were transferred to 25-ml flasks containing 10 ml of the same medium. Radiolabeled substrates were added as methanol solutions (300,000 dpm to 500,000 dpm per flask), and the cultures were incubated for 3 days on an orbital shaker at 28°C. After incubation, the culture was filtered and the culture fluid was acidified to pH 3.0 and partitioned against ethyl acetate. The organic phase was evaporated, and the residue was dissolved in 20% MeOH/H₂O, pH 3.0. This solution was applied to C₁₈ cartridges (Bakerbond; Baker), eluted in 2 ml MeOH, and separated by HPLC on a C₁₈ column. A 30-min linear gradient from 60 to 100% MeOH/H₂O, pH 3.0, was utilized to separate the products formed from the radiolabeled substrates, except for [¹⁴C₄]kaurenolides, which were separated from ent-[¹⁴C₄]kauradienoic acid in a 15-min gradient from 75 to 100% MeOH/H₂O, pH 3.0, followed by 100% MeOH for 15 min. The flow rate was 1 ml/min. Fractions (1 ml) were collected, and the radioactivity was measured by scintillation counting. [¹⁴C₄]fujenoic acids were separated from ent-6,7-diOH[¹⁴C₄]kaurenoic acid by TLC on silica gel G plates developed with hexane-ethyl acetate (3:7) containing drops of formic acid. The silica was scraped from the plate and eluted with methanol, and the radioactivity was quantified by liquid scintillation counting.

Nucleotide sequence accession numbers.
The gene sequences of SmP450-1, SmP450-4, SmP450-2, Smggs2, and Smcps/ks of S. manihoticola Lu949 have been deposited in the GenBank database under accession numbers AM886288, AM886289, AM886290, AM886291, and AM886292, respectively.

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Isolation and organization of the GA gene cluster in S. manihoticola.
In order to identify the GA biosynthetic genes in the Sphaceloma genome, we constructed a lambda DASH II genomic library from the S. manihoticola wild-type strain Lu949. Degenerate primers were designed from different conserved domains of the two known fungal CPS/KS enzymes from F. fujikuroi and Phaeosphaeria sp. strain L487. Only the combination of primers encoding DVDDTAK (Sm-cps/ks-5R1) and TSCQ(I/V)YDTAW(V/A)A (cps/ks-Sm-F2) yielded a single DNA fragment of the expected size, approximately 1 kb, at an annealing temperature of 47°C. Sequence analysis revealed significant similarity (51% identity at the amino acid level) to both FfCPS/KS and PhCPS/KS. This PCR product was used as a probe to screen the genomic library. Subcloning of different hybridizing fragments into the vectors pUC19 and pBluescript II SK(–) and subsequent sequencing revealed several overlapping clones carrying the cps/ks homologous gene, a putative GGDP synthase gene, and parts of a cytochrome P450 monooxygenase gene. The sequences possess close similarity to F. fujikuroi GA biosynthetic genes, suggesting that the cloned genes are part of the GA gene cluster in S. manihoticola. Subsequent chromosome walking resulted in identification of two additional P450 monooxygenase-encoding genes upstream of the first one. Sequencing of cDNA clones of the predicted GA genes and comparisons with the genomic sequences revealed their coding regions and intron positions (Table 2).

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TABLE 2. Gibberellin biosynthetic gene cluster in S. manihoticola

Neither an FfP450-3 homologue nor a desaturase-encoding gene was detected at the right and left borders, respectively, indicating that the putative GA biosynthesis gene cluster in S. manihoticola consists of only five genes (Fig. 2 and Table 2). The lack of these enzymes is consistent with the accumulation of GA₄ and GA₉, rather than GA₃ and GA₁, as final products (43) (Fig. 1). Furthermore, there are no homologues of genes defined immediately outside of the F. fujikuroi GA gene cluster present at the borders in S. manihoticola. The putative gene cluster is flanked by two open reading frames, a chitinase-encoding gene (orf1) upstream and a methyltransferase-encoding gene (orf2; pfam05148 family) downstream, which probably do not belong to the GA gene cluster (Fig. 2).

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FIG. 2. Comparison of the GA gene clusters in S. manihoticola and F. fujikuroi. Homologous genes are shown in the same colors. Fusarium genes without homologues in the Sphaceloma gene cluster are shown in black. The crosses indicate gene inversions. The arrows indicate the orientations of transcription. Genes not belonging to the cluster are shown in gray (without indication of intron positions). clones used for isolation and sequencing of the cluster are displayed above the S. manihoticola cluster.

The structure of the gene cluster in S. manihoticola Lu949 is very similar to that in F. fujikuroi. The most striking similarity is that both clusters contain a pathway-specific GGDP synthase gene that is physically linked to the cps/ks homologous gene sharing the same promoter region (Fig. 2). As in F. fujikuroi, two of the cytochrome P450 monooxygenase genes with the highest levels of sequence similarity to FfP450-1 (GA₁₄ synthase) (44) and FfP450-4 (ent-kaurene oxidase) (56) are also physically linked and form a transcriptional unit by sharing a bidirectional promoter. Compared with the situation in F. fujikuroi, the latter complex is inverted in the Sphaceloma genome. The third P450 monooxygenase gene, SmP450-2, which revealed the highest degree of sequence similarity to the GA 20 oxidase gene FfP450-2 (57), is also inverted with respect to the cluster in F. fujikuroi (Fig. 2).

Knockout of Smcps/ks.
To confirm that the identified genes are indeed responsible for GA biosynthesis in S. manihoticola, we performed a knockout of the putative ent-copalyl diphosphate/ent-kaurene synthase-encoding gene (Smcps/ks). In F. fujikuroi, Ffcps/ks encodes a bifunctional enzyme that catalyzes two specific steps of the GA biosynthetic pathway, the cyclization of GGDP via CDP to ent-kaurene. Knockout mutants would be characterized by the total lack of GAs and their diterpenoid precursors. For this experiment, we needed to establish a transformation system for S. manihoticola, which was achieved by modifying the transformation procedure used for F. fujikuroi. Transformation of the wild-type strain, Lu949, with the 4.8-kb KpnI/BamHI fragment of the Smcps/ks replacement vector (Fig. 3A) resulted in about 300 hygromycin-resistant transformants, 13 of which had integrated the replacement fragment at the cps/ks locus, as shown by diagnostic PCR using the primer pairs pUCH-P and Sm-cps/ks-GR-K-F1, and pUCH-T and Sm-cps/ks-GR-K-R1, respectively (Fig. 3A). The heterokaryotic transformants were purified to homokaryons by protoplast isolation. Three of the transformants, T1, T2, and T12, did not contain an intact wild-type Smcps/ks gene, as demonstrated by diagnostic PCR using primers Sm-cps/ks-nF3 and Sm-cps/ks-nR1 and by Southern blot hybridization of HindIII-digested genomic DNAs of the transformants and the wild-type Lu949 using flank 1 as a probe (probe 1) (Fig. 3B; shown for T1 and T12). Due to the homologous integration of the replacement cassette, the hybridizing 3.9-kb wild-type fragment was replaced by a larger (5.3-kb) band in the transformants. Transformants T2 and T12 showed one or two additional hybridizing fragments resulting from ectopic integrations of the cassette or parts of it, whereas T1 contained only one copy of the replacement fragment. This was also demonstrated by probing with an internal cps/ks fragment (probe 2), which was replaced by the resistance marker in the mutants (Fig. 3B). The successful deletion of the Smcps/ks gene copy in these transformants was confirmed by Northern blot analysis. Only the wild-type strain Lu949 produced the correct 2.9-kb Smcps/ks transcript (Fig. 3C). Furthermore, the purified deletion mutants expressed none of the other cluster genes or the flanking genes orf1 and orf2 (data not shown). Possible reasons for this are discussed below.

To show if the deletion of the Smcps/ks gene affects GA biosynthesis, two purified clones of transformants T1 and T12, as well as the wild-type strain, Lu949, were cultivated under GA production conditions for 44 days in the synthetic ICI medium containing maltose instead of glucose and on autoclaved brown rice kernels. The culture extracts were analyzed by GC-MS. No GAs or any intermediates were found, consistent with SmCPS/KS acting as a bifunctional ent-kaurene synthase (Fig. 4).

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FIG. 4. GC-MS analysis of culture filtrates from wild-type S. manihoticola and of the cps/ks disruption mutant Sm-cps/ks. Total ion current chromatograms are shown for methyl esters trimethylsilyl ethers. Compounds identified in the wild type by comparison of the mass spectra with published spectra (15) are as follows: 1, GA14; 2, GA4; 3, fujenoic diacid; 4, GA13; 5, GA36; 6, 7β,18-dihydroxykaurenolide; and 7, 7β,13-dihydroxykarenolide.

Restoration of GA production in F. fujikuroi by complementation with Smcps/ks.
To confirm the function of Smcps/ks, we complemented an F. fujikuroi cps/ks disruption strain with the homologous gene from S. manihoticola. We transformed the plasmid pSm-cps/ks, carrying the entire S. manihoticola cps/ks gene copy with its endogenous promoter, into Ff-cps/ks-GD-T22, an F. fujikuroi cps/ks disruption mutant, which was shown to be unable to produce GAs or precursors (54). Cotransformation of pSm-cps/ks with the vector pNR1 carrying the nourseothricin resistance cassette yielded 40 resistant transformants, 26 of which were shown to contain the Smcps/ks gene in the F. fujikuroi genome by PCR analysis using the primer pair Sm-cpsks-K-F1 and Sm-cpsks-K-R1. Heterologous expression of the transgene was confirmed by Northern blot analysis with a 1.1-kb SalI fragment of Smcps/ks as a probe (Fig. 5A). To confirm the restoration of GA biosynthesis in the complemented mutants, we cultivated three transformants (aT2, aT3, and aT4), the F. fujikuroi wild-type strain IMI58289, and the F. fujikuroi cps/ks mutant under GA production conditions for 7 days in 20% ICI medium and analyzed the culture media by GC-MS (Fig. 5B). Production of GA₃ and some precursors was restored in the complemented strains aT2 and aT3, but not in aT4. These results are consistent with the observation that only transformants aT2 and aT3 express Smcps/ks at a detectable level (Fig. 5A).

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FIG. 5. Heterologous expression of Smcps/ks in F. fujikuroi. (A) Northern blot analysis of the heterologous expression of Smcps/ks in the F. fujikuroi disruption mutant Ff-cps/ks-GD-T22. Total RNA of S. manihoticola and F. fujikuroi wild-type (WT) strains, Ff-cps/ks-GD-T22, and the complemented mutants Ff-cps/ks-Sm-C-aT2, -aT3, -aT4, -T1, -3, -4, -6, -14, -16, -19, -20, and -22 was hybridized with Ffcps/ks and Smcps/ks (indicated by cps/ks) as probes. (B) GC-MS analysis of culture filtrates of wild-type F. fujikuroi (strain IMI58389), the F. fujikuroi disruption mutant Ff-cps/ks-GD-T22 (cps/ks), and the complemented strains Ff-cps/ks-Sm-C-aT2, -aT3, and -aT4. Total ion current chromatograms are shown for methyl esters trimethylsilyl ethers. Compounds identified in the wild type and strains Ff-cps/ks-Sm-C-aT2 and -aT3 by comparison of the mass spectra with published spectra (15) are as follows: 1, GA7; 2, fujenal diacid; 3, fujenoic triacid; 4, GA13; 5, isoGA3; 6, 7β,18-dihydroxykaurenolide; and 7, GA3.

Functional analysis of the three P450 monooxygenase genes.
The deduced amino acid sequences of the proteins encoded by the three cytochrome P450 genes in the Sphaceloma cluster were found to be similar to those of the F. fujikuroi enzymes P450-1, P450-2, and P450-4 (Table 2). To demonstrate that they possessed the same activities as the F. fujikuroi homologues, we transformed the Sphaceloma P450 genes into different F. fujikuroi mutant strains (see below). In order to ensure high expression levels of the Sphaceloma genes in the F. fujikuroi background, transformations were also carried out with the Sphaceloma P450 genes driven by the corresponding Fusarium promoters (pFfP1_prom::SmP1, pFfP2_prom::SmP2, and pFfP4_prom::SmP4) (Fig. 6).

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FIG. 6. Northern blot analysis of heterologously expressed Sphaceloma GA genes in Fusarium SG139 (a mutant lacking the entire GA gene cluster) or B1-41a (P450-4 mutant) background driven by their own and corresponding Fusarium promoters (as described in the text) hybridized to probes as indicated. (A) Expression of SmP450-1 in F. fujikuroi SG139. (B) Expression of SmP450-2 in F. fujikuroi SG139. (C) Expression of SmP450-4 in F. fujikuroi B1-41a.

To find out which of the two physically linked Sphaceloma genes SmP450-4 and SmP450-1 encodes ent-kaurene oxidase, each of the genes was transformed into the F. fujikuroi B1-41a mutant, which lacks ent-kaurene oxidase activity due to a mutation in FfP450-4 (7, 56). Ethyl acetate extracts of the culture fluids from the transformants were analyzed by TLC and GC-MS to determine GA production (Fig. 7). The extracts of B1-41a transformants complemented with the vectors pSm-P450-4 (T3) and pFfP4 _prom::SmP4 (T4) contained high levels of GA₃ and its precursors GA₄ and GA₇ in contrast to the recipient strain, B1-41a, which contained only traces of GA₃ (Fig. 7). Transformation of B1-41a with SmP450-1 did not restore GA production (Fig. 7), although the gene was expressed in the F. fujikuroi background (data not shown). These results confirm that SmP450-4 indeed encodes ent-kaurene oxidase, which catalyzes the three-step oxidation of ent-kaurene to ent-kaurenoic acid.

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FIG. 7. Analysis of F. fujikuroi culture filtrates. (A) TLC analysis of the wild type (WT), the P450-4 mutant B1-41a and B1-41a complemented with SmP450-1, and SmP450-4 driven by its own or the F. fujikuroi P450-4 promoter. (B) Total ion current chromatograms from GC-MS analysis of B1-41a, B1-41a complemented with SmP450-4 driven by its own promoter (SmP450-4), or the F. fujikuroi P450-4 promoter (FfP4 prom::SmP4) and SmP450-1. The positions of elution of gibberellenic acid (1), GA13 (2), isoGA3 (3), GA1 (4), and GA3 (5) are marked.

SmP450-1 and SmP450-2 were transformed into the F. fujikuroi SG139 mutant, which lacks the entire GA gene cluster and thus allows investigation of the catalytic functions of GA biosynthetic enzymes in isolation, as described previously for the F. fujikuroi enzymes (44, 57, 58). The reactions catalyzed by SmP450-1 and SmP450-2 were demonstrated by adding ¹⁴C-labeled GA precursors to cultures of SG139, complemented with the gene SmP450-1 or SmP450-2 driven by its own promoter or the promoter of the corresponding F. fujikuroi gene. The reaction products were separated by HPLC and identified by GC-MS (see Tables S1 and S2 in the supplemental material). SG139-Sm-P450-1 (T5) and SG139-FfP1_prom::SmP1 (T1) efficiently converted ent-[¹⁴C₁]kaurenoic acid, ent-7-hydroxy [¹⁴C₄]kaurenoic acid, or [¹⁴C₄]GA₁₂-aldehyde into one main labeled product identified as the 3β-hydroxylated GA [¹⁴C]GA₁₄ (see Table S1 in the supplemental material). SmP450-1 also catalyzed 3β-hydroxylation of [¹⁴C₁]GA₁₂, which was completely converted into [¹⁴C₁]GA₁₄ (see Table S1 in the supplemental material). Thus, SmP450-1 is indeed the functional homologue of the F. fujikuroi GA₁₄ synthase FfP450-1 (44), catalyzing the four oxidation steps from ent-kaurenoic acid to GA₁₄ via ent-7-hydroxykaurenoic acid and GA₁₂-aldehyde. Two additional products were formed from ent-[¹⁴C₁]kaurenoic acid, identified as 7β,18-dihydroxy[¹⁴C₁]kaurenolide and 3β,7β-dihydroxy[¹⁴C₁]-kaurenolide (see Table S1 in the supplemental material; Fig. 8). These, as well as 7β-hydroxy[¹⁴C₄]kaurenolide, were formed from ent-[¹⁴C₄]kauradienoic acid, which indicates that, besides catalyzing GA₁₄ synthesis, SmP450-1 catalyzes kaurenolide production through the same reaction sequence described for F. fujikuroi (45). Finally, the reactions of the branch pathway to fujenoic acids were demonstrated in SG139 complemented with SmP450-1 by incubation with ent-6,7-diOH-[¹⁴C₄]kaurenoic acid. This precursor was completely converted into [¹⁴C₄]fujenal diacid and [¹⁴C₄]fujenoic triacid (see Table S1 in the supplemental material; Fig. 8). Therefore, SmP450-1 catalyzes oxidation at several carbon centers of ent-kaurenoic acid (C-7, C-6, C-3, and C-18), resulting in the formation of GA₁₄, as well as kaurenolides and fujenoic acids, as previously shown for the F. fujikuroi homologue (44, 45).

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FIG. 8. Structures of products formed from labeled precursors. (A) Kaurenolides. (B) Fujenoic acids. (C) C-20 oxidation intermediates.

Functional analysis of SmP450-2 was performed with cultures of SG139 transformed with SmP450-2 driven by the F. fujikuroi P450-2 promoter (pFfP2_prom::SmP2). Transformants T2 and T4 oxidized the C-20 methyl precursors [¹⁴C₁]GA₁₂ and [¹⁴C₁]GA₁₄ to give several C-20 oxidation products that were identified by GC-MS (see Table S2 in the supplemental material). The non-3β-hydroxylated precursor [¹⁴C₁]GA₁₂ gave mainly the C-20 aldehyde product [¹⁴C₁]GA₂₄ (Fig. 8) plus a smaller amount of the 19,10--lactone [¹⁴C₁]GA₉, while the 3β-hydroxylated substrate [¹⁴C₁]GA₁₄ was completely converted into the 19,10--lactone [¹⁴C₁]GA₄. The C-20 alcohol and C-20 carboxylic acid products, [¹⁴C₁]GA₁₅ and [¹⁴C₁]GA₂₅, were also detected in incubations with [¹⁴C₁]-GA₁₂, the latter as a minor product (see Table S2 in the supplemental material). C-20 alcohol and C-20 aldehyde GAs would be intermediates in the C-20 oxidation sequence catalyzed by SmP450-2. Added [¹⁴C₁]GA₁₅ (C-20 alcohol) was not utilized and was recovered unconverted in the culture fluid, while [¹⁴C₁]GA₂₄ showed only a trace conversion into the C-20 carboxylic acid product [¹⁴C₁]GA₂₅ (see Table S2 in the supplemental material). Thus, GA C-20 oxidation to form the biologically active C₁₉-GA GA₄ is catalyzed by a P450 monooxygenase in S. manihoticola, as in F. fujikuroi (57).

Regulation of GA gene expression.
In F. fujikuroi, GA biosynthesis is mainly controlled by the general transcription regulator AreA, so that expression of six of the seven genes (with FfP450-3 as the exception) is strictly repressed by large amounts of nitrogen (e.g., ammonium, glutamine, glutamate, and nitrate) in the culture medium (36, 55). AreA directly binds to GATA motifs in the promoter regions of the Fusarium GA biosynthetic genes, triggering transcription under nitrogen starvation conditions (36). To determine whether GA biosynthesis was regulated similarly in Sphaceloma, the effect of a nitrogen source on the expression of the GA biosynthetic genes was examined by Northern blot analysis. After growing the S. manihoticola wild-type strain Lu949 in liquid OPM for 7 days, equal amounts of the washed mycelium were transferred to nitrogen-free ICI medium (0% ICI) or to ICI medium with 100 mM NH₄NO₃ (100% ICI) for 20 h. Hybridization experiments showed that the expression of the GA genes in S. manihoticola also depends on the level of nitrogen (Fig. 9), but to a lesser extent than in F. fujikuroi. This is consistent with the lower number of putative AreA binding sites in the Sphaceloma promoter regions than in the Fusarium GA gene promoters (only 3 to 6 GATA sequence elements compared to 6 to 12 in Fusarium). As in F. fujikuroi, the expression of the GA genes was not affected by the level of the carbon source or phosphate or the pH value (data not shown). However, there was a detectable difference in the expression levels of the S. manihoticola genes driven by their original or the corresponding Fusarium promoters. In the Fusarium background, expression was always better if the gene was under the control of the F. fujikuroi promoter (Fig. 6). TLC and GC-MS analyses also revealed higher GA production in these transformants (Fig. 7).

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FIG. 9. Effects of various nitrogen concentrations on the expression levels of the S. manihoticola GA genes cps/ks, P450-2, and ggs2.

Complementation of S. manihoticola Lu949 with the genes P450-3 and des from F. fujikuroi.
In order to determine whether GA₃ production could be restored in Sphaceloma, we transformed the fungus with the cosmid pCos1 containing the entire GA gene cluster of F. fujikuroi (Linnemannstöns and Tudzynski, unpublished), or with vectors carrying the two single Fusarium GA genes des (porf3-Sal) and P450-3 (p450-3-GC) (58), which are absent in the Sphaceloma gene cluster. If the fungus was able to express the heterologous genes from the F. fujikuroi gene cluster, pCos1 transformants of Sphaceloma would be able to produce GA₇ and GA₁ in addition to GA₃, while Sphaceloma transformants carrying the F. fujikuroi P450-3 gene should produce GA₁, and des transformants should form GA₇ (Fig. 1). The Fusarium genes were shown by Northern analyses and RT-PCR to be expressed in Sphaceloma (data not shown). Furthermore, GC-MS analyses of the culture fluids of the transformants confirmed GA₃ synthesis in pCos1 transformants while des transformants formed GA₇ and P450-3 transformants produced GA₁, as postulated (Fig. 10).

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FIG. 10. GC-MS analysis of culture filtrates of S. manihoticola wild type and of the wild type transformed with the F. fujikuroi des gene (strain T1), the F. fujikuroi P450-3 gene (strain T4), or the complete F. fujikuroi GA gene cluster (pCos1) (strain T1). Total ion current chromatograms are shown for methyl esters trimethylsilyl ethers. Compounds were identified by comparison of their mass spectra with published spectra (15).

Top ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Sphaceloma manihoticola was the second fungus after F. fujikuroi shown to produce GAs, mainly GA₄ (42). The fungus is only distantly related to F. fujikuroi, making it especially interesting to compare its GA biosynthetic genes, their organization in the genome, their regulation, and the functions of the encoded enzymes with those in F. fujikuroi. However, nothing was known about the genetic background of GA biosynthesis in S. manihoticola. In order to undertake such studies, it was first necessary to establish standard molecular methods, such as transformation and nucleic acid preparation, for the fungus.

S. manihoticola grows extremely slowly, as already described by Zeigler et al. in 1980 (64). The strain used in our study, Lu949, takes about 5 days to build colonies 0.5 cm in diameter and is not able to overgrow the agar plate if only a piece of mycelium is transferred as the inoculum. This has also been described for isolates of the related species Sphaceloma fawcettii, which forms <10-mm colonies in 30 days (28). The fungus synthesizes large amounts of exopolysaccharides, making it difficult to separate the mycelium from the culture medium. RNA isolation, in particular, is hindered by this gelatinous matrix, so that expression studies were technically difficult. The low growth rate correlates with the small amount of detectable GAs even after 30 days on autoclaved rice kernels or after 44 days of cultivation in liquid culture. Compared with the Fusarium wild-type strain, IMI58289, which produces about 1 g/liter GA₃ after 10 days (36), S. manihoticola Lu949 forms only about 20 mg/liter GA₄ after 14 days (43) when grown under optimal conditions. Low catalytic efficiency of the S. manihoticola enzymes could also explain the low levels of GAs found.

The small amount of biologically active GAs produced by S. manihoticola could explain the fact that only severely infected cassava plants show the hyperelongation of young internodes as a secondary symptom. The primary symptoms are, inter alia, necrotic leaf spots and cankers on stalks and petioles (3, 64). Nevertheless, the superelongation disease of cassava is still economically important, causing crop losses of more than 80% in many areas of South America and the Caribbean (4).

Cloning and functional characterization of the S. manihoticola GA genes.
In this work, we have described the isolation and functional characterization of the GA biosynthetic gene cluster of S. manihoticola Lu949. We have shown that in contrast to the F. fujikuroi cluster, which contains seven biosynthetic genes (reviewed in reference 51), the newly identified cluster consists of only five GA-specific genes. Homologues of the 13-hydroxylase-encoding gene, P450-3, and the GA₄-desaturase gene, des, are not present at the borders of the Sphaceloma GA gene cluster, as they are in Fusarium (Fig. 2). This is entirely consistent with the GA spectrum in Sphaceloma, which produces GA₄, but not GA₃, GA₇, and GA₁ (43, 64), the formation of which requires the activities of the desaturase (DES) and 13-hydroxylase (P450-3). As in the F. fujikuroi GA cluster, there is no pathway-specific transcription factor in the Sphaceloma cluster. In contrast, genes for pathway-specific regulators are common in several other fungal gene clusters, e.g., AflR, which upregulates the expression of the aflatoxin cluster genes in Aspergillus flavus and Aspergillus parasiticus (9, 61); AurR1, which is part of the aurofusarin gene cluster in the Fusarium graminearum species complex (33); and TRI6, the unusual Cys₂ His₂-like regulator of the thrichothecene gene cluster in Fusarium sporotrichioides (40).

Based on amino acid alignments of the deduced proteins, we were able to isolate a homologous gene in S. manihoticola by heterologous PCR. BlastP searches revealed homology to the known fungal-type cyclases involved in GA biosynthesis (FfCPS/KS, 44% identity; PhCPS/KS, 44% identity), as well as to other bifunctional fungal terpene cyclases, such as the aphidicolan-16β-ol synthase (ACS; BAB62102) of Phoma betae (35% identity), which is responsible for the cyclization of GGDP to form the diterpene skeleton in the biosynthesis of aphidicolin (37, 49).

Therefore, in contrast to plant (angiosperm) GA biosynthesis, in which two enzymes, CPS and KS, are involved in the successive cyclization of GGDP via CDP to ent-kaurene (20), all fungal-type GA cyclases known so far are bifunctional enzymes, with both CPS and KS activities (references 22 and 54 and this paper). In addition, genes probably encoding bifunctional terpene cyclases are present in published fungal genome databases, e.g., in Magnaporthe grisea (MGG_01949, 43% identity; MGG_00027, 35% identity) and Aspergillus nidulans (three homologous hypothetical proteins, AN1594.2, AN3252.2, and AN9314.2; 30 to 36% identity). Although homology searches also revealed similarity to the bifunctional ent-kaurene synthase from the moss Physcomitrella patens (PpCPS/PpKS; BAF61135) and abietadiene synthase from the gymnosperm Abies grandis (Agggabi; Q38710), similarity could be observed only to the CPS domain, but not the KS domain, of the fungal CPS/KS proteins. Furthermore, the KS domain of SmCPS/KS also exhibits no significant similarity to any published angiosperm KS protein.

We used several approaches to confirm that the genes in the newly identified cluster were indeed involved in GA biosynthesis: gene replacement of the putative Smcps/ks, functional complementation of F. fujikuroi mutants, and expression of single Sphaceloma genes in the well-established background of the F. fujikuroi mutant SG139, which lacks the entire GA gene cluster (56).

The function of SmCPS/KS as the GA-specific diterpene cyclase was confirmed by targeted disruption of the Smcps/ks gene and by expression of this gene in an F. fujikuroi cps/ks deletion mutant. Both approaches demonstrated that Smcps/ks is the S. manihoticola orthologue of Ffcps/ks: the deletion led to the total loss of GAs and precursors in the Sphaceloma cps/ks mutant, and the complementation of F. fujikuroi cps/ks with Smcps/ks restored GA biosynthesis in the mutant.

Most steps of GA biosynthesis in S. manihoticola are catalyzed by P450 monooxygenases, as described for F. fujikuroi (20, 51), and in contrast to plant systems, which utilize soluble dioxygenases, as well as P450 enzymes (20). Three P450 monooxygenase-encoding genes were identified in the S. manihoticola GA gene cluster: SmP450-1, SmP450-2, and SmP450-4; their annotation was based on their close sequence similarity to the F. fujikuroi GA genes, placing them in the same CYP group. It was, however, important to confirm their catalytic functions, particularly since there was an inversion of the P450-1-P450-4 unit in comparison with the F. fujikuroi cluster (Fig. 2). After complementation of the F. fujikuroi mutant B1-41a, which has a point mutation in the P450-4 gene (56), with SmP450-1 or SmP450-4, only SmP450-4 restored GA biosynthesis in the mutant, and it is therefore the orthologue of the F. fujikuroi gene FfP450-4 (Fig. 7). Thus, SmP450-4 encodes ent-kaurene oxidase, which oxidizes the C-19 methyl of ent-kaurene to a carboxylic acid to give ent-kaurenoic acid.

By heterologous expression of SmP450-1 in the clusterless mutant F. fujikuroi SG139, we could demonstrate that SmP450-1 is a multifunctional enzyme involved in GA₁₄ synthesis from ent-kaurenoic acid. As for FfP450-1, the intermediacy of ent-7-hydroxykaurenoic acid and GA₁₂-aldehyde was indicated by their efficient conversion into GA₁₄. GA₁₂ was also converted by SG139-SmP450-1 into GA₁₄, although it is not a main intermediate in GA₁₄ synthesis, since mycelial microsomal fractions convert [¹⁴C]GA₁₂-aldehyde, but not [¹⁴C]-GA₁₂, into [¹⁴C]GA₁₄ in the presence of NADPH (unpublished results). This indicates that GA biosynthesis in S. manihoticola involves 3β-hydroxylation of GA₁₂ aldehyde, as it does in F. fujikuroi (44). This contrasts with the situation in the GA₁-producing fungus Phaeosphaeria sp., in which 3β-hydroxylation occurs late in the pathway after formation of C₁₉ GAs (22). It was suggested that PhP450-1 (ent-kaurenoic acid oxidase) lacked 3β-hydroxylase activity (23), although this has not been demonstrated experimentally, and the enzyme responsible for 3β-hydroxylation in Phaeosphaeria has not yet been characterized.

SmP450-2 was shown to function as a 20-oxidase converting GA₁₄ to GA₄ without accumulation of intermediates. The nonhydroxylated substrate GA₁₂ was oxidized with lower efficiency, as shown by accumulation of the C-20 aldehyde product GA₂₄, together with the C-20 alcohol GA_15. This differs from the Fusarium enzyme FfP450-2, which oxidizes both GA₁₄ and GA₁₂ efficiently to give GA₄ and GA₉, respectively, without accumulation of intermediates (57).

Regulation of gene expression.
Northern blot analysis revealed that the Sphaceloma GA genes are already highly expressed after 7 days of incubation in liquid media. However, GA accumulation could be demonstrated only after 44 days in liquid culture, perhaps due to difficulty in extracting GAs from the slimy culture fluid at an earlier stage.

Expression of the Sphaceloma GA genes is regulated by nitrogen, although to a lesser extent than has been demonstrated for the Fusarium genes (36, 55). The promoters of the Sphaceloma GA genes contain far fewer potential AreA binding sites (double GATA motifs) than the Fusarium genes. For example, there are only three GATA motifs in the bidirectional promoter shared by SmP450-1 and SmP450-4, and none of them as a double GATA motif, which have been demonstrated to be the most important AreA binding sites in the corresponding promoter region of F. fujikuroi (36). Nevertheless, shift experiments revealed that large amounts of ammonium nitrate (100 mM) in the culture medium led to reduced expression levels of the GA genes, compared to nitrogen-free medium, also in S. manihoticola (Fig. 9). Since the large amounts of exopolysaccharides also complicate shift experiments, impeding accurate purification of the mycelium from the precultivation broth, the cultivation conditions have to be optimized in order to investigate regulation mechanisms in more detail.

In general, the main regulatory elements appear to be conserved between Fusarium and Sphaceloma, as Sphaceloma GA genes are expressed and the encoded enzymes are functional in the Fusarium background and vice versa. Moreover, we showed that the Fusarium GA genes P450-3 and des, whose homologues are missing in Sphaceloma, can be expressed in S. manihoticola and can generate the complete biosynthetic pathway to the main F. fujikuroi end product, GA₃. It is particularly noteworthy that the Fusarium gene P450-3 is expressed and the encoded 13-hydroxylase is functional in the genetic background of this distantly related fungus, but it was not expressed at detectable levels when transformed into the Fusarium mutant strain SG139, lacking the entire GA gene cluster, and no 13-hydroxylase activity has been obtained in these transformants (36, 58). Furthermore, although fusion to the strong F. fujikuroi glnA promoter enabled expression of P450-3 in SG139, enzyme functionality could not be demonstrated (C. Bömke and B. Tudzynski, unpublished data). Another peculiarity of P450-3 is its distinct regulation. It is the only Fusarium GA cluster gene whose expression is not nitrogen repressed and therefore seems to be controlled by at least one additional transcription factor, other than AreA. This difference might point to an origin of this gene different from those of the other GA cluster genes and could explain the lack of a homologous gene in Sphaceloma. It is also possible that the corresponding gene was lost in Sphaceloma ancestors during evolution, although the possibility that a homologue is located at another locus in the Sphaceloma genome, though probably in an inactive form, cannot be excluded.

An interesting observation is the downregulation of the other GA cluster genes, as well as the flanking genes orf1 and orf2, as a consequence of the knockout of the Smcps/ks gene. This effect is more likely due to the integration of the larger replacement fragment than to a positive feedback regulation of the GA genes, because the single Sphaceloma (and Fusarium) P450 monooxygenase genes are highly expressed in the genetic background of F. fujikuroi SG139, which lacks the entire GA gene cluster (Fig. 6).

Evolution of fungal diterpene gene clusters.
The different cluster organization in Sphaceloma (inversion of the P450-1-P450-4 transcriptional unit and P450-2), as well as the lack of the border genes of the Fusarium GA cluster (Ffdes and FfP450-3), raises questions about the evolution of the GA gene clusters in fungi and GA biosynthesis in general. Previous comparisons of GA biosynthesis in plants and F. fujikuroi disproved the hypothesis of horizontal gene transfer between higher plants and fungi and suggested independent evolution of GA biosynthesis pathways (20).

The organizations of different fungal diterpene gene clusters are very similar, suggesting a common origin. It is particularly noticeable that a large number of diterpene gene clusters contain a pathway-specific GGDP synthase-encoding gene, for example, Ffggs2 and Smggs2 in the GA biosynthetic gene clusters, PbGGS in the aphidicolin gene cluster (49), and paxG, ltmG, and atmG in the indole-diterpene gene clusters for paxilline (63), lolitrem (63a), and aflatrem (65) biosynthesis in Penicillium paxilli, Neotyphodium lolii, and A. flavus, respectively. The identification of the pathway-specific ggs gene in the S. manihoticola GA gene cluster supports the hypothesis that the presence of two copies of GGDP synthase genes may be a molecular signature for diterpene biosynthesis (65). Remarkably, the unit of a diterpene cyclase (MGG_00027) and a GGDP synthase gene (MGG_14382) is also conserved in a putative rudimentary diterpene cluster in M. grisea. An exception is the GA gene cluster in Phaeosphaeria sp. strain L487, which does not contain a GGDP synthase gene (23). Interestingly, a couple of diterpenoid gene clusters have been identified in Streptomyces species that also contain a GGDP synthase-encoding gene next to cyclase genes (21, 24). However, phylogenetic studies with bacterial, plant, fungal, and animal GGDP synthases and diterpene cyclases clearly show that the bacterial genes are very distant from fungal genes and share a much higher similarity with the plant enzymes (Bömke and Tudzynski, unpublished).

In addition to the similar cluster organizations of fungal diterpene gene clusters, the conserved intron positions, especially in fungal cps/ks genes (Ffcps/ks and Smcps/ks), as well as the P. betae homologue PbACS and both Magnaporthe cps/ks homologous genes, MGG_00027 and MGG_01949, is a further indication of a common origin. The conservation of some intron positions can also be observed in the P450 monooxygenase genes and ggs2, although fungal P450 genes in general exhibit low conservation of intron-exon structure (12). Since only the mRNA sequence of Phcps/ks (AB003395) and that of only one monooxygenase (GA 20-oxidase)-encoding gene (AB106677) of Phaeosphaeria sp. strain L487 are available, it is not possible to include these genes in this comparison.

How gene clusters are formed during evolution and their evolutionary advantage are unknown, but there are several hypotheses concerning cluster assembly and the putative benefits of clustering genes in genomes. The "selfish-gene" hypothesis was established to be the driving force of comobilization of these genes within species through mating or into other species by horizontal gene transfer (60). The occurrence of horizontal gene transfer in fungi was recently demonstrated for the fungal ACEI gene cluster (25), and it represents a convincing explanation for the existence of shared gene clusters in distantly related fungi, such as the GA gene clusters in F. fujikuroi and S. manihoticola. However, other genome events, such as gene duplications and differential gene losses, are alternative explanations for the discontinuous distribution of fungal gene clusters (26). Thus, it remains unclear if the GA gene cluster of Sphaceloma ancestors has lost flanking genes homologous to des and P450-3 or if an ancient Fusarium species acquired a basic GA gene cluster via horizontal gene transfer and it was coincidentally inserted next to a P450 monooxygenase-encoding gene, P450-3, that could contribute to GA biosynthesis. The increasing number of sequenced genomes might help to advance the theory of the evolution of gene clusters, especially those for diterpene biosynthesis, such as the GA gene cluster, in fungi.

Conclusions.
In this paper, we describe the identification and functional characterization of all genes involved in GA biosynthesis in the cassava pathogen S. manihoticola. There are several features, such as gene structure, their organization in a gene cluster, the presence of a pathway-specific GGDP synthase-encoding gene transcribed from the same promoter as the cps/ks gene, and the order of enzymatic steps, that are similar to those in F. fujikuroi, suggesting a common origin of GA clusters in F. fujikuroi and S. manihoticola. The Sphaceloma genes fully complement the corresponding F. fujikuroi mutants, demonstrating that general regulatory features, including nitrogen regulation, are similar in these fungi. On the other hand, there are only five genes in this gene cluster, because the flanking genes of the F. fujikuroi gene cluster are absent in Sphaceloma. As a consequence, the biosynthetic pathway ends with GA₄ instead of being further converted to GA₇, GA₁, and GA₃, as occurs in F. fujikuroi (Fig. 11).

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FIG. 11. End products of the GA biosynthetic pathways in F. fujikuroi and S. manihoticola.

 
We thank W. Rademacher (BASF Agricultural Center, Limburgerhof, Germany) for providing the strain S. manihoticola Lu949, J. Avalos (University of Seville, Seville, Spain) for providing F. fujikuroi strains IMI58289 and SG139, and J. MacMillan (University of Bristol, Bristol, United Kingdom) for providing the F. fujikuroi mutant strain B1-41a.

The 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). Rothamsted Research receives grant-aided support from the Biotechnology and Biological Sciences Research Council of the United Kingdom. Furthermore, we thank the DAAD (Deutscher Akademischer Austausch Dienst) and CONICYT/DFG (project 105-2007) for granting short-term scholarships (D/06/47017).

 
* 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

Published ahead of print on 20 June 2008.

Supplemental material for this article may be found at http://aem.asm.org/.

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