Introduction

Cercospora species are important phytopathogenic fungi that have been reported to cause leaf spots on more than 100 plant species (Daub and Ehrenshaft 2000; Daub et al. 2005). Many Cercospora species produce a light-activated, nonspecific phytotoxin, cercosporin, which is required for full virulence to invade their hosts and for lesion development (Choquer et al. 2005).

Cercosporin is synthesized by polypeptides of eight co-regulated and clustering genes (designated CTB1-8), encoding a polyketide synthase (CTB1), two O-methyltransferases (CTB2 and CTB3 N terminus), a monooxygenase (CTB3 C terminus), an MFS transporter, three oxidoreductases (CTB5, CTB6, and CTB7) and a Zn(II) Cys6 transcription regulator (CTB8) (Chen et al. 2007a, b; Dekkers et al. 2007). Expression of the CTB genes is in part regulated by CRG1 zinc finger transcriptional regulator (Chung et al. 2003a, b). However, the CRG1 coding sequence is unlinked to the CTB gene cluster. Biosynthesis of cercosporin is influenced by light and numerous environmental cues such as ions, nitrogen and carbon sources (You et al. 2008). In addition, considerable research has been devoted to understanding the mechanisms of cellular antioxidant defense that are operated by Cercospora fungi to avoid the toxicity of cercosporin and reactive oxygen species (Daub et al. 1992; Sollod et al. 1992; Ehrenshaft et al. 1998, 1999; Chung et al. 1999; Daub and Ehrenshaft 2000). As the CRG1 transcriptional factor regulates genes involved in cercosporin resistance and biosynthesis (Chung et al. 2003a), suppressive subtractive hybridization was used to recover several hundred genes that are differentially expressed between the wild type and a crg1 null mutant of C. nicotianae (Herrero et al. 2007). Determination of the biological functions of those identified genes will be accelerated by the availability of an efficient gene inactivation system.

Genetic transformation and targeted gene disruption or replacement provides essential tools to analyze gene functions in filamentous fungi. Numerous fungi have been successfully transformed over the last two decades, due mainly to a great improvement in molecular methodologies for the delivery of DNA constructs carrying various selectable markers. However, gene disruption in some fungal species can be problematic largely due to the predominance of ectopic integrations and the ineffectiveness of homologous recombination (Pratt and Aramayo 2002). Unlike yeasts, integration of foreign genes is considered a rare event in filamentous fungi and presumably influenced by a nonhomologous end joining (NHEJ) mechanism (Ninomiya et al. 2004). To successfully disrupt a gene in filamentous fungi, it often requires at least 0.5–2 kb homologous DNA sequence (Nelson et al. 2003). Some fungal species, such as Cladosporium fulvum and Leptosphaeria maculans require a minimum of 5–7 kb of flanking DNA to obtain rare disruptants (Segers et al. 2001; Idnurm et al. 2003). Furthermore, integration of foreign genes in filamentous fungi often occurs ectopically and, thus, identification of the desired mutants often requires a large number of transformants to be screened. Impairment of the NHEJ machinery by disrupting the Ku70- and Ku80-coding genes has been shown to significantly increase homologous recombination in fungi (da Silva Ferreira et al. 2006; Haarmann et al. 2008). However, for organisms without genome sequence data, cloning the Ku70- or Ku80-coding gene and creating a parental strain that is defective only in the NHEJ pathway is time consuming and labor intensive.

Cercospora species can be genetically transformed with plasmid vectors containing a selectable marker conferring resistance to antibiotics or herbicides. In Cercospora species, the disruption frequency is often lower than 1% among transformants recovered (Ehrenshaft et al. 1998, 1999; Chung et al. 1999; Ehrenshaft and Daub 2001; Chung et al. 2003a, b, c; Wetzel et al. 2004), which has greatly hampered functional analysis of cloned genes in these species. To disrupt or replace a given gene in fungi, one must construct a plasmid harboring a selectable marker gene, such as the hygromycin phosphotransferase B gene, cloned within the ORF of the targeted gene (Pratt and Aramayo 2002). However, the efficiency of gene disruption varies considerably among species, strains and even among isolates of a given species, and systems developed for one microorganism may not be suitable for another. To facilitate targeted gene disruption, a split-marker disruption strategy (Fu et al. 2006) fusing the targeted DNA fragments with truncated, but overlapping, within the selectable marker gene has been successfully adapted in filamentous fungi. This strategy was originally developed for rapid, gap repaired-mediated cloning in Saccharomyces cerevisiae (Fairhead et al. 1996). In theory, transformants will not grow on a medium containing the selection agent unless homologous recombination occurs between the overlapping regions of the selectable marker gene. Since the frequency of ectopic integration is decreased markedly, a high frequency of targeted gene disruption via homologous recombination can be achieved by screening fewer transformants. In this study, we evaluated the frequency of targeted gene disruption for four CTB genes required for cercosporin biosynthesis in C. nicotianae (Chen et al. 2007a). Interruption of any of the CTB genes in C. nicotianae gave rise to a mutant strain completely lacking the production of the red-pigmented cercosporin (Choquer et al. 2005; Chen et al. 2007a, b; Dekkers et al. 2007). Thus, we took advantage of an easily visualized phenotype of the CTB disruptants and the simple extraction method for cercosporin to dissect genetic elements that may impede targeted gene disruption in C. nicotianae.

Materials and methods

Microorganisms, culture conditions and cercosporin analysis

Cercospora nicotianae wild-type strain ATCC18366 was used as the DNA recipient host for targeted gene disruption throughout the study. Fungal strains were cultured in potato dextrose agar (PDA, Difco, Sparks, MD). Cercosporin-deficiency mutants (cr−) were identified by the lack of production of a red pigment on thin PDA plates as previously described (Chung 2003). Cercosporin was extracted from agar plugs with fungal hyphae with 5 N KOH as described previously (Chung 2003). Cercosporin in the KOH extracts was detected using the spectrophotometer at 480 nm.

Targeted gene disruption

Disruption constructs, pCTB115, p∆ctb5 and p∆ctb7, harboring an HYG gene under the Aspergillus nidulans trpC promoter conferring resistance to hygromycin B, and pCTB3/Bar6 containing a BAR gene under the trpC promoter conferring bialaphos resistance, were created in the previous studies (Choquer et al. 2005; Chen et al. 2007a; Dekkers et al. 2007). The HYG or BAR gene cassette flanked with different lengths of the CTB gene sequence was amplified by PCR with the CTB gene-specific primers (Table 1). The resulting PCR products were directly transformed into the protoplasts prepared from the wild type. Disruption frequency (%) is calculated by dividing the number of disruptants by the total number of transformants recovered.

Table 1 Oligonucleotide primers used in the study
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Preparation of fungal protoplasts and transformation

Preparation of fungal protoplasts (>107 ml−1) and transformation using polyethylene glycol (PEG)/CaCl2 were performed as described previously (Chung et al. 2002). Transformants were selected in a regeneration medium containing 250 μg ml−1 hygromycin (Roche Applied Science) or 50 μg ml−1 bialaphos (Phytotechnology Lab., Lenexa, KS), and tested for cercosporin production.

Molecular analysis

Standard protocols were used to perform endonuclease digestion, electrophoresis, Southern blotting and hybridization of DNA. Fungal DNA was isolated using a DNeasy Plant Mini kit (Qiagen, Valencia, CA); plasmid DNA was purified using a Wizard DNA purification kit (Promega, Madison, WI). DNA hybridization probes were synthesized by PCR with gene-specific primers to integrate digoxigenin-11-dUTP (Roche Applied Science, Indianapolis, IN) as previously described (Chung et al. 2003b, c). Immunological detection of the probe using a CSPD lumigenic substrate for alkaline phosphatase was performed following the manufacturer’s instructions (Roche).

Results and discussion

In the present study, we disrupted four CTB genes that reside in a cluster in C. nicotianae (Fig. 1a) to evaluate how genetic loci and their sizes will affect efficiency of homologous recombination. The minimum flanking sequence required for efficient homologous integration has never been determined in C. nicotianae. In each experiment, we were able to repeatedly identify the cercosporin non-producing mutants (cr −) after transforming a wild-type strain with most of the constructs, providing an opportunity to determine the genetic factors that might have a profound effect on targeted gene disruption in this fungal species. All putative cr − mutants were streaked three times for single colony on medium to eliminate false positive. All cr − mutants recovered were very stable and no spontaneously reverted strains were identified for the duration of the experiment.

Fig. 1
figure 1

a Physical map of the cercosporin toxin biosynthetic (CTB1–CTB8) gene cluster in C. nicotianae. b Production of the red-pigmented cercosporin and other pigments by strains of C. nicotianae on potato dextrose agar plates. c Targeted gene disruption of the CTB1 gene in C. nicotianae. The disruption plasmid, pCTB115, was constructed by inserting a hygromycin phosphotransferase B gene (HYG) into CTB1. DNA fragments with various flanking sequence on the left and right border, separated by the slash, were obtained by PCR with primers as indicated. The split-marker fragments contain a mixture of DNA with overlapping, but truncated, HYG gene. The symbol (X) represents homologous recombination between the two DNA fragments. The number of cercosporin-deficient transformants identified and the total number of transformants recovered are indicated for each construct

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Disruption of the CTB1 gene

The CTB gene cluster contains eight genes (Fig. 1a) that have been previously shown to be essential for the production and accumulation of cercosporin (Chen et al. 2007a). As described below, successful disruption of the CTB genes in the wild type was identified for the strains that failed to accumulate the red pigment, exemplified by the CTB1, CTB3 and CTB7 disruptants (Fig. 1b).

CTB1 contains a 7,036-bp ORF that encodes a putative fungal type-I polyketide synthase in C. nicotianae (Choquer et al. 2005). To disrupt the CTB1 gene, the pCTB115 plasmid was used as a template in PCR to generate all DNA fragments with various lengths of homologous sequence (Fig. 1c). The pCTB115 plasmid contains the HYG gene cassette under the control of the A. nidulanstrpC gene promoter that is flanked with a 2.5-kb fragment representing the 5′ end region of CTB1 and a 1.6-kb fragment of 3′CTB1 region. It was demonstrated that the addition of restriction endonucleases enhances the rate of recovery of transformants in C. nicotianae (Chung et al. 2003b). To determine whether or not endonucleases will promote targeted gene disruption, transformation of the wild-type strain of C. nicotianae with circular pCTB115 plasmid alone or with 10 U NruI endonuclease yielded low frequency of cr− mutants among transformants; transformation of the C. nicotianae wild type with pCTB115 mixed with an XbaI endonuclease gave rise to an enhanced efficiency for recovery of cr− mutants. The restriction endonuclease in storage buffer was added directly into transformation cocktail. There is no restriction site for either NruI or XbaI within the pCTB115 plasmid. Transformation of the C. nicotianae wild type with a linear construct comprised 1.3-kb CTB1 sequence flanking near either ends of the HYG cassette (construct 1-d) resulted in high frequency for recovery of cr− mutants (Fig. 1c).

A novel liner minimal element (LME) construct, containing a selectable marker gene fused with partial target gene sequence at only one end, was developed to inactivate genes with an incredibly high frequency in Alternaria brassicicola (Cho et al. 2006). Transformation of the C. nicotianae wild type with the 5′-end CTB1 fused with the HYG fragment (construct 1-e) yielded very few cr − mutants, whereas transformation of the HYG fused with the 3′-end CTB1 fragment (construct 1-f) produced numerous cr − mutants.

DNA fragments from constructs (1-g) to (1-l) contain the split-HYG gene marker flanked with asymmetric lengths of the truncated CTB1 DNA fragment (Fig. 1c). In all constructs, the 5′ CTB1 was fused with the 3′ HYG fragment and the 5′ HYG was fused with the 3′ CTB1 fragment. All DNA fragments were amplified by PCR and directly transformed into the wild type. The results revealed that transformation of the C. nicotianae wild type with two split, but overlapping, DNA fragments yielded cr − mutants with varied frequencies, depending on the lengths of homologous DNA at either end of HYG (Fig. 1c). It appears that disruption frequency increased as the lengths of flanking CTB1 sequence on one end or both ends increased (Fig. 1c).

Disruption of the CTB7 gene

The CTB7 gene (1,401 bp) encodes an FAD/FMN-dependent oxidoreductase for the cercosporin biosynthesis (Chen et al. 2007b). The p∆ctb7 plasmid contains the HYG gene cassette surrounded by truncated 2.5- and 1.6-kb fragments of CTB7 (construct 7-a). Transformation of the C. nicotianae wild type with a DNA fragment, harboring the HYG cassette flanked with various lengths of the truncated CTB7 DNA fragment (constructs 7-b–7-e), resulted in cr − mutants at varied frequencies, which ranged from 0 to 10% (Fig. 2). Reducing the flanking sequence apparently decreased the disruption efficacy. No transformants were identified when the lengths of the flanking region were both reduced to 0.6 kb. DNA fragments containing homologous CTB7 sequence at only one end (construct 7-f or 7-g) did not result in any cr − mutants. Co-transformation of the C. nicotianae wild type with two split-HYG fragments flanked with the truncated CTB7 near either end (construct 7-h) resulted in abundant cr − mutants, whereas disruption frequency dropped sharply as the flanking CTB7 sequence on both ends was reduced (construct 7-i to 7-k). Transformation of the C. nicotianae wild type with two split-HYG fragments in which one contains a 0.7-kb 5′CTB7 and the other contains a 1.6-kb 3′CTB7 (construct 7-l) failed to yield any cr − mutants (Fig. 2).

Fig. 2
figure 2

Targeted gene disruption of the CTB7 gene in C. nicotianae. The disruption plasmid, p∆ctb7, was constructed by inserting a hygromycin phosphotransferase B gene (HYG) under the trpC promoter into CTB7. The split-marker fragments contain a mixture of DNA with overlapping but truncated HYG gene. nd Not determined. The number of cercosporin-deficient transformants identified and the total number of transformants recovered are indicated for each experiment

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Disruption of the CTB3 and CTB5 genes

The CTB3 gene (2,731 bp) encoding a polypeptide with dual O-methyltransferase/monooxygenase domains is also required for cercosporin production in C. nicotianae (Dekkers et al. 2007). To disrupt the CTB3 gene, the pCTB3/Bar6 plasmid (construct 3-a), harboring the BAR gene cassette responsible for bialaphos herbicide resistance under control of the A. nidulans trpC promoter was constructed (Fig. 3a). Transformation of the C. nicotianae wild type with the split BAR fragments, in which one contains a 2.1-kb flanking sequence of the 5′-CTB3 and the other contains a 0.3-kb flanking sequence of the 3′-CTB3 (construct 3-b) failed to obtain any cr − mutants. In contrast, transformation of the wild type with the split BAR fragments containing 1-kb flanking sequence of CTB3 on both ends (construct 3-c) generated cr − mutants at a frequency as high as 32% (Fig. 3a).

Fig. 3
figure 3

a, b Targeted gene disruption of the CTB3 and CTB5 genes in C. nicotianae. The disruption plasmids, pCTB3/Bar6 and p∆ctb5, were constructed, respectively, by inserting a phosphinothricin acetyltransferase gene (BAR) under the trpC promoter into CTB3 and a hygromycin phosphotransferase B gene (HYG) into CTB5. The split-marker fragments contain a mixture of DNA with overlapping, but truncated, BAR or HYG gene

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The p∆ctb5 plasmid, containing 1.2-kb flanking sequence on both ends of HYG, was prepared for disruption of the CTB5 gene (1,380 bp) encoding a putative NADPH-dependent oxidoreductase for cercosporin biosynthesis in C. nicotianae (Fig. 3b). Transformation of the C. nicotianae wild type with circular p∆ctb5 alone or with a BamHI endonuclease at 10 U generated cr − mutants at low frequency. Transformation of the wild type using p∆ctb5 mixed with an NheI endonuclease increased the overall disruption efficiency. There is no restriction site for either BamHI or NheI within the p∆ctb5 plasmid. Transformation of the C. nicotianae wild type with a linear DNA fragment containing a functional HYG gene cassette within the CTB5 ORF resulted in low frequency for recovery of cr − mutants. Transformation of the C. nicotianae wild type with the split-HYG fragments with 1.2-kb flanking sequence of CTB5 on both ends yielded cr − mutants at high frequency (Fig. 3b).

The split-marker approach decreases ectopic integration

To determine if integration of split-marker fragments occurred specifically at the targeted CTB1 gene locus and to assess if the split-marker approach would reduce ectopic integration (non-homologous integration), ten putative cr − mutants were randomly selected from transformants transformed with split-HYG fragments (construct 1-c). Hybridization of wild-type genomic DNA cleaved with EcoRV and NcoI to a 5′ CTB1 probe resulted in two expected hybridizing bands of 4.1 and 1.4 kb in sizes (Fig. 4a). In contrast, hybridization of the EcoRV/NcoI-digested genomic DNA from cr − mutants also displayed two expected bands of 2.1 and 1.4 kb due to insertion of the HYG gene within CTB1. Of the 10 cr − mutants examined, four of them were targeted gene disruption mutants clearly derived from homologous recombination specifically at the CTB1 gene, whereas the other six mutants had multiple hybridizing bands in addition to the 2.1 and 1.4-kb bands (Fig. 4a). The transforming DNA in the latter was likely resulting from ectopic integration or tandem insertion at the integration site. Similar analyses were conducted to examine ten randomly selected cr − mutants that were transformed with a whole PCR fragment containing partial CTB1 at either end of HYG (construct 1-d), revealing that only one cr − mutant (#1 in Fig. 4b) was derived from homologous integration. The other nine mutants, displaying multiple hybridizing signals larger or smaller than 2.1 kb, had ectopic or tandem integrations (Fig. 4b). Southern-blot analysis of genomic DNA in randomly selected CTB3, CTB5 or CTB7 disruptants also revealed that at least two cr − disruptants displayed integration profiles clearly resulted from homologous recombination after transformation of the C. nicotianae wild type with the split-marker approach, whereas other disruptants had profiles from both ectopic and homologous integrations (Fig. 4c–e).

Fig. 4
figure 4

a–e Southern-blot analyses of genomic DNA from the cercosporin non-producing mutants of Cercospora nicotianae, obtained from transformation experiments with the CTB1 split-marker DNA fragments (a), the entire PCR fragment with truncated CTB1 (b), or with the CTB7 (c), CTB3 (d), and CTB5 (e) split-marker fragments as indicated on the top of each panel, WT wild-type strain

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As it was evidenced from the present study, the frequency of CTB disruptants differed greatly among the transforming DNA constructs, highly depending on the gene targeted and the length of the homologous sequences. Although the size of the sequence being disrupted varied among constructs, it appears that the longer homologous sequences within the construct often resulted in higher frequencies of disruption in C. nicotianae even though the CTB clustering genes are involved in cercosporin biosynthesis. It also appears that circular plasmid constructs resulted in a low rate of disruption. Transformation of plasmid constructs with certain, but not all, restriction endonucleases slightly elevated the disruption frequency, as evidenced in the disruption of both CTB1 and CTB5 genes. Transformation with linear DNA fragments, obtained from disruption constructs by PCR (whole PCR fragments), improved disruption frequency as tested in CTB1 and CTB7, but not CTB5, genes. As shown in the disruption of the CTB1 and CTB7 genes, the length of the homologous DNA sequence present in the whole PCR fragment affected the disruption frequency variably. When the split-marker approach was used for disruption, recovery of cr − mutants markedly increased for all CTB genes tested. As the homologous DNA sequence was decreased from one or both fragments, disruption frequency deceased to varied degrees, depending on the gene of the target. Thus, it is essential to have sufficient lengths of the flanking DNA sequence (at least 0.8 kb on both ends are needed) when using the split-marker approach for targeted gene disruption in C. nicotianae. Although the split-marker approach also led to ectopic or tandem integrations in addition to the gene-specific disruption, we were able to identify cr − mutants with a clean disruption at the target gene allele by screening fewer transformants in each case. Thus, it appears that the split-marker approach led to an increase in the homologous integration frequency.

Compared to the disruption targeting at CTB1, disruption of the CTB5 or CTB7 gene yielded lower disruption frequencies, indicating an allele-dependent disruption. It also suggests that the size of the targeting gene may influence disruption frequency, as the larger genes such as CTB1 (7 kb) have higher rates of disruption than other smaller genes such as CTB5 and CTB7 (1.4 kb). Further, it was demonstrated that the split-marker approach led to a decrease in ectopic integration as evident by Southern-blot analysis, thereby promoting gene-specific disruption. Efficient gene disruption strategies along with the other molecular techniques available for manipulating C. nicotianae shall facilitate functional genomic analysis for this important fungal pathogen.