The PKS4 Gene of Fusarium graminearum Is Essential for Zearalenone Production

Strains and culture conditions.

F. graminearum 1104-14 (24) was obtained from the Fusarium collection at the National Veterinary Institute in Norway, and F. pseudograminearum IBT 1544 was obtained from the IBT culture collection at the Technical University of Denmark. Two aurofusarin-deficient F. pseudograminearum mutants, PK2.1 and KHt62.1, were used (28). All strains were maintained as conidial suspensions (1 × 10⁷ spores ml⁻¹) in 20% glycerol at −80°C or in sterile soil.

For genomic DNA extraction, the fungi were cultured with shaking at 28°C in liquid complete medium (25). For RNA isolation, the F. pseudograminearum strains were cultivated on the medium described by Bell et al. (2) with maltose (20 g/liter) as the C source and urea (140 mg/liter) as the N source for 2 weeks at room temperature. For RNA isolation and high-performance liquid chromatography (HPLC) analysis of ZON production, F. graminearum strains were cultivated on polished parboiled rice at 25°C in the dark for up to 2 weeks; 20 g of rice (Uncle Ben's long-grain white rice) and 20 ml distilled H₂O were mixed in a 500-ml Erlenmeyer flask, left at room temperature for 1 h, and then autoclaved at 121°C for 1 h. Each flask was inoculated with 1 × 10⁶ spores. For real-time gene expression studies the fungal mycelia were harvested after 4, 7, 11, and 14 days of growth, and for each strain and incubation time three replicate flasks were used. Escherichia coli strains Top10 (Invitrogen, Carlsbad, Calif.) and JM109 (Promega, Madison, Wis.) were used for plasmid manipulations and propagation. E. coli was grown at 37°C in liquid Luria-Bertani (LB) medium or on LB agar with or without 50 ppm kanamycin (Sigma, St. Louis, MO) and 5-bromo-4-chloro-3-indolyl-β-d-galactopyranosie (X-Gal)/isopropyl-β-d-thiogalactopyranoside (IPTG) (Invitrogen) as appropriate (46). Agrobacterium tumefaciens LBA4404 carrying pAL4404, a disarmed version of the octopine-type Ti plasmid pTiAch5 (18), was purchased from Invitrogen.

DNA procedures.

Fungal genomic DNA was extracted essentially as described by Malz et al. (28). The fungal genomic DNA used for Southern analysis of ΔPKS4 mutants was extracted by the method of Rolland et al. (45). For PCR screening of mutants, DNA extraction was performed with microwave treatment of fungal samples (50). PCR was carried out with a GeneAmp PCR 9700 system (Applied Biosystems, Foster City, Calif.) with Dynazyme II (Finnzymes, Espoo, Finland) DNA polymerase. Primers were designed on the basis of the genome sequence determined by the Broad Institute (http://www.broad.mit.edu), the annotated MIPS F. graminearum genome database (31) (http://mips.gsf.de/genre/proj/fusarium/), the program SEARCHPKS (58), and the previously published PKS gene sequences of Kroken et al. (23). All primers used in this work are listed in Table 1.

Generation of replacement mutants.

The A. tumefaciens vector pAg1-H3 (60) was used to create the PKS4 (FG12055) replacement vector. All gene designations are based on those used by Kroken et al. (23) and MIPS F. graminearum genome database (31). Genomic DNA of F. graminearum 1104-14, primers PKS4-1-PacI and PKS4-1-FseI, and primers PKS4-2-ApaI and PKS4-2-SacI were used in a PCR to produce the flanking regions PKS4-1 (2,092 bp) and PKS4-2 (1,808 bp). The PCR conditions were 35 cycles of denaturation at 94°C for 30 s (with a first-cycle hold for 5 min), annealing at 60°C for 1 min, and extension at 72°C for 2 min (with a last-cycle hold for 9 min). The fragments were cloned (TOPO TA cloning kit; Invitrogen), digested with the appropriate restriction enzymes (New England Biolabs, Ipswich, United Kingdom), gel purified (QIAquick gel extraction kit; QIAGEN), and ligated (T4 DNA ligase; Promega) into pAg1-H3 to create the replacement vector pAg1-H3-PKS4. This vector was used for homologous recombination and replacement of the central part of PKS4 with hygB.

The replacement vector pAgI-H3-PKS4 was introduced into A. tumefaciens LBA4404 by electroporation (Gene Pulser II; Bio-Rad, Hercules, Calif.) according to the manufacturer's instructions. A. tumefaciens-mediated transformation was carried out as described by Malz et al. (28). The number of transformed colonies was estimated, and 40 colonies were transferred to new DFM plates containing 150 ppm hygB. PKS4 replacement mutants were verified by PCR performed with primers PKS4-Hyg-A1 and PKS4-Hyg-A2 and primers PKS4-Hyg-B1 and PKS4-Hyg-B2 under the following conditions: 40 cycles of denaturation at 94°C for 30 s (with a first-cycle hold for 5 min), annealing at 60°C for 1 min, and extension at 72°C for 2 min (with a last-cycle hold for 9 min). The primers are located inside the hygB resistance gene and outside the replacement cassette in pAg1-H3-PKS4 but are within the flanking PKS4 sequence.

Southern blot analyses.

Genomic DNA (5 μg) used for Southern analysis of ΔPKS4 mutants was digested to completion with BglII, separated on a 0.8% agarose gel, and transferred to a GeneScreen Plus transfer membrane (NEN Life Science Products, Inc., Boston, Mass.) as described by Molenaar and Wilkins (34). The 1,684-bp hygB fragment used as a probe was obtained by digestion of plasmid pAg1-H3-PKS4 with BglII and AsiSI. The 794-bp fragment from the central region of PKS4, delPKS4 (located at positions 2635 to 3429 in GenBank accession number XM_382572) was obtained by performing PCR with primers PKS4-del-1 and PKS4-del-2 and with F. graminearum 1104-14 genomic DNA as the template. The 1,511-bp fragment from the PKS4-1 region was obtained by digestion of the same plasmid with BglII and XhoI. The probes were labeled by random priming with [α-³²P]dCTP (Ready-To-Go DNA labeling beads; Amersham Biosciences, Uppsala, Sweden). Prehybridization, hybridization, and washing were performed by using methods described in Current Protocols in Molecular Biology (1); the prehybridization and hybridization solutions contained 5× SSC, 5× Denhardt solution, 1% (wt/vol) sodium dodecyl sulfate, and 100 mg ml⁻¹ denatured salmon sperm DNA (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate), and the high-stringency wash solution used before autoradiography contained prewarmed (68°C) 0.1%× SSC and 0.1% sodium dodecyl sulfate.

Gene expression analysis.

RNA was purified from 50 mg fungal biomass by using a RNeasy plant mini kit (QIAGEN, Hilden, Germany) and was treated with RQ1 RNase-free DNase (Promega) or a Turbo DNA-free kit (Ambion, Austin, TX). For reverse transcriptase PCR total RNA was reverse transcribed with Superscript III (Invitrogen) and a random primer mixture of nine nucleotides. The first-strand cDNA product was then used in a PCR with the PKS primers and the following PCR conditions: 35 cycles of denaturation at 94°C for 30 s (with a first-cycle hold for 2 min), annealing at 57 to 59°C for 30 s, and extension at 72°C for 1 min (with a last-cycle hold for 11 min). The PCR products were compared by agarose gel electrophoresis.

Relative expression of the PKS4 and PKS13 (FG12014) genes was determined over time by real-time quantitative PCR using an Applied Biosystems 7900HT instrument (Applied Biosystems) with a standard 96-well block, SYBR Green PCR Master Mix, and SYBR Green reverse transcription-PCR reagents (Applied Biosystems). One-half of the inoculated rice was used for RNA extraction. The ZON content was determined by HPLC by using the second half of the culture. Housekeeping genes, including the genes encoding β-tubulin (FG09530), translation elongation factor 1α (FG08811), and ubiquitin conjugating enzyme (FG10805), were used as internal controls because the proteins belong to different protein families and were therefore unlikely to be coregulated. For the β-tubulin gene we used primers FGtubf and FGtubr (44). Specific primers for the other control genes and the target genes were designed by using Primer Select (DNAStar, Madison, WI). The specificity and efficiency of each primer pair were tested by constructing a standard curve over 5 logs, followed by a dissociation curve. All primer pairs had an efficiency of 1.91 to 1.93 and produced only a single amplicon. Target and internal control genes for each biological sample were examined on the same plate. A minimum of three PCRs were performed for each biological sample.

Housekeeping genes were stably expressed in this experiment, as determined by BestKeeper (43), and the BestKeeper Index was used to normalize target gene data. The comparative cycle threshold (C_T) method (User Bulletin 2; ABI PRISM 7700 sequence detection system; Applied Biosystems) was used to calculate the level of gene expression relative to the calibrator.

Analysis of ZON production.

ZON production was determined by HPLC. Chemical analyses were carried out with ΔPKS4 mutants and the wild-type strain cultivated on polished parboiled rice. Samples were freeze-dried, and 0.2 g of each sample was transferred to a 14-ml screw-cap glass vial. HPLC analysis was carried out as described by Nielsen and Smedsgaard (37), but the procedure was modified by using a photo diode array detector (DAD), a fluorescence detector (FLD) with excitation at 230 nm and with an emission cutoff at 450 nm, and a Luna C₁₈ II column (100 by 2 mm; inside diameter, 3 μm; Phenomenex, Torrance, Calif.). Samples (3 μl injected) were analyzed by using a water-CH₃CN gradient that began at 15% CH₃CN, increased linearly to 100% CH₃CN over 20 min, and then remained the same for 5 min. Both solvents were acidified with 50 ppm trifluoroacetic acid. The DAD signal range was 200 to 600 nm, and the FLD was set to excitation at 230 nm with an emission cutoff at 450 nm. ZON was detected by comparison to data obtained from an analysis of a ZON standard (Sigma), using retention times recorded by the DAD and FLD supported by the characteristic UV-visible spectrum.

Pathogenicity tests.

Seeds of barley (Hordeum vulgare cv. Alexis) were sterilized in 5% (vol/vol) NaOCl and 0.1% (vol/vol) Tween 20 essentially as described by Miller et al. (32). For assessment of barley stem blight and root rot, the seeds were incubated for 1 h in an aqueous spore suspension containing 5 × 10⁶ spores ml⁻¹ of wild-type F. graminearum 1104-14 or ΔPKS4-T9. Control seeds were incubated in sterile distilled H₂O. For each treatment, three replicates (replicates A, B, and C) were used. Each replicate contained three plants in a closed system. Inoculated and control seeds were germinated in prewetted sterile vermiculite and grown for 2 weeks at 15°C with a cycle consisting of 16 h of light and 8 h of darkness. The light intensity was 35,000 lx. A nutrient solution (38) was supplied after 2 days. Stems and leaves of the seedlings were scored for disease symptoms after 1 week and 2 weeks. Roots were examined for root rot after 2 weeks of growth.

Outcross experiments.

To test the genetic linkage of the ZON-deficient ΔPKS4-T9 mutant, we made crosses with the MAT1-1 deletion strain T39ΔM1-3 and with Z3639-M25, Z3643, and H-4 derivatives that had a green fluorescent protein sequence inserted into the MAT1-2 locus (26). The four heterothallic MAT deletion strains were used as females, and the ΔPKS4-T9 mutant and the 1104-14 wild-type strain were used as males in crosses on carrot agar, as described by Bowden and Leslie (6). Each outcross was performed on 10 carrot agar plates. Production of perithecia with emerging cirri containing viable ascopores was observed only with the ΔPKS4-T9 and 1104-14 controls.

Analysis of the PKS protein sequences with SEARCHPKS.

ZON is a reduced macrolide, and a PKS involved in the biosynthesis of ZON probably must have an ER domain (9). Analysis of the putative PKS protein sequences from the annotated MIPS F. graminearum genome database (31) with the program SEARCHPKS (58) revealed that 8 (PKS1 [FG10548], PKS2 [FG04694], PKS4 [FG12055], PKS5 [FG05794], PKS6 [FG12109], PKS7 [FG08795], PKS11 [FG01790], and FG03340) of the predicted 15 PKS protein sequences (23) had a potential ER domain. Primers for the PKSs were designed and tested with genomic DNA of F. graminearum 1104-14 and F. pseudograminearum IBT 1544. All of the primers gave a 400-bp amplification product, as expected (results not shown). These primers were used in reverse transcriptase PCR to identify a putative PKS involved in ZON biosynthesis.

PKS4 expression in the aurofusarin mutants and in the strain producing high levels of ZON.

Based on the HPLC analysis, ZON was produced by the aurofusarin-deficient mutants of F. pseudograminearum, PK2.1 and KHt62.1, but not by F. pseudograminearum wild-type strain IBT 1544 (28) under the culture conditions used. The PKS4 gene encoded the only PKS with an ER for which no transcript was detected by reverse transcriptase PCR in the wild type but which was detected in both mutants (Fig. 1A). For other PKSs (encoded by PKS3, PKS13, and PKS14) without an ER the gene expression patterns were similar to that of PKS4, with downregulation in the wild type compared to the aurofusarin mutants (Fig. 1B). The expression of the genes encoding most of the remaining PKSs without ER domains did not differ in the wild type and mutants (results not shown); the exception was PKS12, which is described elsewhere (28). PKS4 was expressed at a high level in F. graminearum wild-type strain 1104-14 after 7 and 11 days of growth on polished parboiled rice (Fig. 1C), which coincided with the accumulation of ZON.

A. tumefaciens-mediated replacement of the PKS4 gene.

Transformation of F. graminearum 1104-14 with pAg1-H3-PKS4 yielded approximately one hygB-resistant transformant per 1,000 fungal spores. Most of the transformants had the wild-type growth morphology (∼95%), but some exhibited restricted growth. Eight mutants with wild-type morphology were arbitrarily selected for further study. Homologous integration into the PKS4 gene was confirmed by PCR amplification with primers PKS4-Hyg-A1 and PKS4-Hyg-A2 and with primers PKS4-Hyg-B1 and PKS4-Hyg-B2. These primers were located inside the hygB gene and outside the replacement cassette, but they were within the flanking PKS4 sequence (Fig. 2) and yielded products only if homologous recombination occurred. The ΔPKS4 mutants T4, T5, T9, and T11 were double homologous recombinants that produced 2.4-kb and 2.6-kb amplification products with primers PKS4-Hyg-A1 and PKS4-Hyg-A2 and with primers PKS4-Hyg-B1 and PKS4-Hyg-B2 (Fig. 3). Mutants T7, T8, and T12 were single homologous recombinants; T7 and T8 produced amplification products with primers PKS4-Hyg-A1 and PKS4-Hyg-A2, and T12 produced an amplification product with primers PKS4-Hyg-B1 and PKS4-Hyg-B2. Mutant T6 produced no PCR product and was probably not a homologous recombinant.

Southern analysis confirmed the PCR results. In seven mutants, the hygB probe hybridized to a 4,100-bp fragment (Fig. 4A), as expected if the entire replacement cassette was inserted into the fungal genome. Additional hybridizing fragments from T7, T11, and T12 probably resulted from partial insertions of the hygB gene and the replacement cassette. Hybridization with the delPKS4 probe (Fig. 4B) showed that the central region of the PKS4 gene was deleted in mutants T4, T5, T9, and T11 but was present in the other four transformants. Hybridization with the PKS4-1 probe (Fig. 4C) resulted in identification of multiple hybridizing bands in mutants T7 and T12 but only a single hybridizing fragment in the other mutants. Based on the results of the Southern blot and PCR analyses, T4, T5, and T9 were mutants in which the central region of the PKS4 gene was replaced by a single copy of the hygB gene through double homologous recombination. In mutants T7, T8, and T12 the hygB gene was inserted into the PKS4 gene by a single homologous recombination event. In T7 and T8 the insertion was in PKS4-1, and in T12 it was in the PKS4-2 region. In mutants T7 and T12 a second nonhomologous recombination event also occurred. In mutant T11 the replacement cassette also was inserted into the PKS4 gene by a double homologous recombination event; in addition, this mutant had a second insert in which part of the cassette (hygB was present, but PKS4-1 was not present) was inserted by nonhomologous recombination.

Functional analysis of ΔPKS4 mutants.

The wild-type strain produced ZON, but neither ZON nor any of its derivatives was detected in the ΔPKS4 mutants tested (Fig. 5). There were no significant quantitative differences in auro- and rubrofusarin production between the wild-type strain and the mutants. In a pathogenicity test, ΔPKS4-T9 caused the same severe root rot symptoms in barley roots that the wild-type strain caused (Table 2). Treated and nontreated barley seeds germinated within 2 days. Seedlings coated with conidia of F. graminearum 1104-14 or ΔPKS4-T9 all had the typical bleaching and impaired growth of stems and leaves. After 2 weeks, the roots showed typical signs of root rot. The control seedlings were healthy. The mycelial morphology on potato dextrose agar, YES, and carrot agar was similar to that of the wild type.

Real-time gene expression analysis of PKS4 and PKS13.

In wild-type strain F. graminearum 1104-14, both PKS4 and PKS13 were expressed at low levels after 4 days of growth (Fig. 6). The relative gene expression then increased before it returned to the low level after 14 days of incubation. After 7 and 11 days of growth the level of expression of PKS13 was twice the level of expression of PKS4. In the gene replacement mutant ΔPKS4-T9 the expression of PKS13 was ∼1% that in the wild type after 11 days. The levels of PKS4 expression were similar in the wild type and ΔPKS4-T9 after 4 and 7 days of incubation. However, in ΔPKS4-T9 the level of expression of PKS4 was lower after 11 days and slightly higher after 14 days than the level of expression in the wild-type strain. The quantitative real-time PCR assays were highly reproducible, with standard deviations of <0.5 in replicate PCRs with the same RNA sample. More variation was observed in the results for replicates with RNA samples from different cultures, and the standard deviations of ΔC_T ranged from 0.35 to 5.16. High levels of ZON were produced under the culture conditions used. After 11 days the level in the culture inoculated with strain 1104-14 was 58 ppm.

PKS expression in the ZON mutant.

To determine if the lack of ZON altered the expression of other PKSs, we performed a reverse transcriptase PCR analysis of the wild type and ΔPKS4-T9 after 7 days of growth on rice. In ΔPKS4-T9 expression of PKS5, PKS11, and PKS12 increased and expression of PKS10 decreased (Fig. 7). For the eight other PKSs there was no clear difference between the wild type and ΔPKS4-T9 under these conditions.