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Citrus Research and Education Center, and Department of Plant Pathology, Institute of Food and Agricultural Sciences (IFAS), University of Florida, 700 Experiment Station Road, Lake Alfred, FL 33850, USA
Correspondence
Kuang-Ren Chung
krchung{at}ufl.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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The GenBank/EMBL/DDBJ accession numbers for the sequence data reported in this article are: EfHP1, EU414199; EfHP2, EU414200; EfHP3, EU414201; RDT1, EU401704; TSF1, EU401705; OXR1, EU401706; EfPKS1, EU086466; PRF1, EU401707; ECT1, EU414198; and EfHP4, EU414202.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). This fungal pathogen attacks a wide variety of citrus species and cultivars, including grapefruit, lemon, and many tangerines and their hybrids. Citrus scab is widely distributed in many humid citrus-growing areas of the world, reducing fruit values for the fresh-fruit market. Another Elsinoë species, E. australis Bitancourt & Jenkins, induces lesions only on the fruit of sweet orange and mandarin, but not on leaves, and is present largely in South America (Tan et al., 1996; Timmer et al., 1996). Isolates of E. fawcettii grow extremely slowly in culture and often form gummy colonies less than 10 mm in diameter after 4 to 5 weeks. Little is known about the molecular and genetic interactions between Elsinoë species and their hosts.
Many Elsinoë species produce red or orange pigments, collectively termed elsinochromes, in culture (Weiss et al., 1987). Elsinochromes consist of at least four interconvertible tautomers (A, B, C and D) that have a core phenolic quinone to which various side chains are attached, and they have remarkable structural similarity to many perylenequinone phytotoxins, such as cercosporin, stemphyltoxin, hypomycin, hypocrellin and phleichrome of fungal origin (Weiss et al., 1987; Daub et al., 2005). Elsinochrome A is readily converted to elsinochrome B or elsinochrome C in the presence of chromium trioxide (Lousberg et al., 1969); elsinochrome D, containing a methylenedioxy ring, is derived from elsinochrome C (Shirasugi & Misaki, 1992). The structures of elsinochromes were determined by NMR spectroscopic and X-ray crystallographic studies several decades ago (Weiss et al., 1965; Lousberg et al., 1969, 1970; Meille et al., 1989; Mebius et al., 1990). The biological function of elsinochromes as phytotoxins toward host and nonhost plant cells both in vitro and in vivo was recently documented to be light-dependent (Liao & Chung, 2008a). Their cellular toxicities were further shown to be mediated through production of reactive oxygen species such as superoxide and singlet oxygen, which may damage cell membranes and induce electrolyte leakage from the cells (Liao & Chung, 2008a). Further studies employing molecular and genetic approaches unambiguously confirmed a critical role for elsinochromes in lesion development and fungal pathogenesis. E. fawcettii mutants disrupted in the EfPKS1 gene, which encodes a fungal polyketide synthase, do not produce any detectable elsinochromes and have a drastically reduced ability to form lesions on citrus (Liao & Chung, 2008b).
Early studies of Elsinoë spp. fed radioisotope-labelled substrate revealed that elsinochromes are synthesized in a polyketide pathway by condensation of acetate and malonate monomers (Chen et al., 1966; Kurobane et al., 1981). To determine if any genes adjacent to EfPKS1 might also be involved in elsinochrome production in E. fawcettii, we carried out a DNA sequence analysis upstream and downstream of the EfPKS1 locus and identified nine new ORFs. In the work reported here, we characterized the function of one of these genes, TSF1, which encodes a polypeptide containing a Cys2His2-type zinc finger and a GAL4-like Zn2Cys6 binuclear cluster implicated in DNA binding, and evaluated its function in relation to gene regulation and elsinochrome production using a disruption strategy in E. fawcettii. This study provides the molecular foundation on which to elucidate the biosynthetic and regulatory networks leading to elsinochrome production in this important citrus pathogen.
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METHODS |
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). The genetically modified EfPKS1 disruptant (D4) and two complementation strains (C1 and C2) were reported in a previous study (Liao & Chung, 2008b). Fungal isolates were cultured on potato dextrose agar (PDA, Difco). Conidia were prepared using Fries medium (Fries, 1978) as described by Timmer et al. (1996). Other media used in this study were complete medium (CM), minimal medium (MM) and protoplast regeneration medium (RMM) (Jenns et al., 1989; Chung et al., 2002). The pH of media was adjusted with 0.1 M phosphate buffer as described elsewhere (You et al., 2007). For toxin production, fungal mycelium was ground with a sterile blender, spread on PDA, and incubated under continuous F15T8 fluorescent light (wavelength 436 nm) at an intensity of 3.5 J m–2 s–1 for 5 days at room temperature (
25 °C). The plates were placed approximately 45 cm away from the light source. When preparation of protoplasts was desired, fungal isolates were grown in 50 ml potato dextrose broth (PDB, Difco) for 7 days, ground, mixed with fresh PDB (200 ml), and incubated for an additional 15 h. For DNA or RNA isolation, fungal isolates were grown on media with a layer of sterile cellophane (Choquer et al., 2005). Screening of elsinochrome-deficient mutants was performed on thin PDA as previously described (Choquer et al., 2005; Liao & Chung, 2008a).
Extraction and analysis of elsinochrome toxin.
Elsinochromes were extracted from fungal cultures with 5 M KOH or acetone. Agar plugs bearing fungal mycelium were cut with a 6 mm sterilized cork borer, and soaked in 5 M KOH in the dark for at least 12 h. The A480 of the soaking solution was measured using a model Genesys 5 spectrophotometer (Spectronic Instruments). Since elsinochromes and cercosporin have a similar structure and molecular mass (Weiss et al., 1987), elsinochromes were quantified using a molar absorption coefficient of 23 300 l mol–1 cm–1 (Jenns et al., 1989) and expressed as nmoles per plug. For TLC analysis, elsinochromes were extracted twice from dried agar medium with fungal mycelia in acetone for 16 h, condensed and separated on a TLC plate coated with 60 F254 fluorescent silica gel (5x20 cm, Selecto Scientific) as previously described (Liao & Chung, 2008a, b). The concentration of elsinochromes was estimated by reference to a regression line that was established using pure cercosporin (Sigma) as a standard and expressed as cercosporin equivalents.
Chromosomal walking and sequence analysis.
Fungal DNA was isolated employing the DNeasy Plant Mini kit (Qiagen). A genomic library of E. fawcettii was constructed from DNA digested with DraI, EcoRI, PvuI and StuI and ligated to adaptors using the Universal GenomeWalker kit (BD Biosciences) according to the manufacturer's instructions. Primers were designed based on the known sequences and paired with adaptor primers to obtain DNA fragments harbouring unknown genomic regions using Titanium or Advantage 2 DNA polymerase (BD Biosciences). In some cases, DNA fragments were obtained by PCR with inverse primers from fungal DNA that was digested with restriction endonucleases and self-ligated as previously described (You et al., 2007). After being purified with a DNA-purification kit (Mo Bio Laboratories), the amplified DNA fragments were either directly sequenced or cloned into pGEM-T easy vector (Promega) for sequence analysis at Eton Bioscience. Oligonucleotides used for PCR and sequencing were synthesized by Integrated DNA Technologies and Allele Biotechnology and Pharmaceuticals. Similarity searches using the BLASTX program (Altschul et al., 1997) were performed at the National Center for Biotechnology Information. Prediction of ORFs and exon/intron junctions was first performed using the gene-finding software at http://www.softberry.com and further confirmed by comparing genomic and cDNA sequence. Functional domains were identified using the PROSITE database available through the ExPASy Molecular Biology Server (http://us.expasy.org) (Gasteiger et al., 2003) and the Motif/ProDom and Block programs (Henikoff et al., 2000) at http://motif.genome.jp/. Analysis of the promoter regions was conducted using regulatory sequence analysis tools (van Helden, 2003) at http://rsat.ulb.ac.be/rsat/. Palindrome searches were performed at http://bioweb.pasteur.fr/seqanal/interfaces/palindrome.html.
Targeted gene disruption and genetic complementation.
To disrupt the TSF1 gene in E. fawcettii, a 5.4 kb DNA fragment encompassing the TSF1 ORF and flanking sequences was amplified by PCR with primers efup11 (5'-catctcgcatatctggacccgtc-3') and efup28 (5'-cgggctattcttagagcagag-3'). The amplified DNA fragment was cloned into pGEM-T easy vector, creating pSTSF1128. A 2.1 kb fragment harbouring the hygromycin phosphotransferase B gene (HYG) cassette under the control of the Aspergillus nidulans trpC gene promoter and terminator was released from pUCATPH (Lu et al., 1994) by digestion with XbaI, blunt-ended with DNA polymerase I (New England BioLabs), and cloned into the NruI site of pSTSF1128, creating the disruption construct pTSF1128 (see Fig. 3). The NruI site is approximately 1200 nt downstream from the predicted TSF1 start codon. A split-marker strategy was applied to promote double crossing-over recombination as previously described (Choquer et al., 2005). Briefly, a 4.4 kb DNA fragment containing truncated 5' TSF1 fused with 3' HYG and a 4.2 kb fragment encompassing 3' TSF1 linked to 5' HYG were amplified with, respectively, primers efup11/hyg3 (5'-ggatgcctccgctcgaagta-3') and efup28/hyg4 (5'-cgttgcaagaactgcctgaa-3') from pTSF1128 using the Takara Ex Tar PCR system (Takara Bio). Fungal protoplasts were released from hyphae by a mixture of cell-wall-degrading enzymes as previously described (Chung et al., 2002). Fungal transformation using a CaCl2 and polyethylene glycol-mediated method was performed by mixing PCR fragments with 1x105 protoplasts ml–1 as previously described (Chung et al., 2002). The HYG gene cannot encode a functional protein until recombination occurs between the 800 nt overlapping portions of HYG sequence in the two PCR products. Fungal transformants were recovered from RMM medium containing 200 µg hygromycin ml–1 (Roche Applied Science) after 2 to 3 weeks and were screened for the loss of elsinochrome (red pigment) production on PDA.
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). Transformants were first identified from a medium supplemented with 50 µg bialaphos ml–1 (Gold Biotech) and tested for production of elsinochromes.
Pathogenicity tests.
Fungal pathogenicity was assessed on detached rough lemon (Citrus jambhiri Lush.) leaves inoculated with agar plugs bearing fungal mycelium as previously described (Liao & Chung, 2008b).
Manipulation of nucleic acids.
Plasmid DNA was propagated in Escherichia coli DH5
and purified using the Wizard DNA purification kit (Promega). Fungal RNA was purified by Trizol reagent according to the manufacturer's directions (Invitrogen). Single- and double-strand cDNA was prepared with a cDNA synthesis kit (BD Biosciences) and amplified by reverse transcriptase (RT)-PCR with gene-specific primers. The resulting fragments were purified and directly subjected to sequence analysis. Standard molecular techniques were used for endonuclease digestion of DNA, electrophoresis, and Southern- and Northern-blot hybridizations (Sambrook & Russell, 2001). DNA probes used for Southern- and Northern-blot hybridization were labelled by PCR to randomly incorporate digoxigenin (DIG)-11-dUTP (Roche Applied Science) into DNA with gene-specific primers. Procedures used for probe labelling, hybridization, post-hybridization washing and immunological detection of the probe using the CSPD (C18H19Cl2O7PNa2) chemofluorescent substrate for alkaline phosphatase were performed following the manufacturer's recommendations (Roche Applied Science).
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RESULTS |
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).
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). The ECT1 gene encodes a product with strong similarity to membrane transporters, with the highest hit to a nitrate transporter of Aspergillus terreus (Table 1). The TSF1 gene, which is located upstream of and transcribed divergently from EfPKS1, encodes a putative protein showing strong similarity to many zinc finger transcriptional factors of fungi. The RDT1 gene, located further upstream from TSF1, encodes a putative polypeptide similar to a wide range of reductases, with strong similarity to the 1,3,8-trihydroxynaphthalene (T3HN) reductase involved in melanin biosynthesis (Table 1). The OXR1 ORF is located between EfPKS1 and TSF1. The predicted OXR1 protein of unknown function is rich in proline. Three putative ORFs (designated EfHP1 to EfHP3) upstream of RDT1 encode hypothetical proteins with unknown functions. Sequence alignment revealed that EfHP1 and EfHP3 share 52 % similarity on both the nucleotide and amino acid level. A small ORF that defines the EfHP4 gene located downstream of ECT1 encodes a putative polypeptide, which is not involved in the production of any secondary metabolites. Sequencing beyond the EfHP1 and EfHP4 genes failed to produce reliable sequences after several attempts with different primers, probably due to the presence of inverted repeat sequences (data not shown).
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). Expression of EfHP3 appears to be constitutive as its transcript level remained unchanged under all conditions tested. Accumulation of the TSF1, EfPKS1, PRF1, ECT1 and EfHP2 gene transcripts increased considerably when the fungus was incubated under alkaline conditions (Fig. 2a). Under acidic conditions (pH 4), the RDT1 and EfHP1 gene transcripts accumulated to slightly but not significantly higher levels compared to those under alkaline conditions. By contrast, expression of EfHP3, EfHP4 and OXR1 was not affected by pH. It appears that EfPKS1, PRF1, ECT1 and OXR1 were preferentially expressed under conditions of high glucose concentration, whereas accumulation of the TSF1 gene transcript was repressed (Fig. 2a). Expression of RDT1, EfHP1, EfHP2 and EfHP4 was apparently not regulated by glucose.
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). Addition of ammonium nitrate, reducing the glucose concentration to 2 g l–1, or lowering the pH to acidic conditions (pH 4.0) completely suppressed elsinochrome production.
Targeted disruption of the TSF1 gene
To determine if the genes adjacent to EfPKS1 are also involved in elsinochrome biosynthesis and regulation, we first characterized the TSF1 gene, encoding a putative transcriptional activator, in detail. The TSF1 gene contains 3075 nt and is interrupted by four introns of 49, 50, 43 and 53 bp. The predicted 959 aa product of the TSF1 gene contains a Cys2His2-type zinc finger and a GAL4-like Zn2Cys6 binuclear cluster DNA-binding motif, and displays strong similarity to numerous conserved transcriptional regulators from the sequenced genomes of fungi. Analysis of the promoter region 1090 nt upstream from the predicated start codon of the TSF1 gene revealed the presence of three GCCARG consensus motifs, which bind the pH-responsive PACC regulator (Espeso et al., 1997), one GATA binding site that is recognized by the AREA nitrogen regulator (Marzluf, 1997), and the WC1/WC2 light-responsive regulator (Linden & Macino, 1997). Similarly, analysis of at least 528 bp of sequence upstream of the putative ATG translational start codon identified several consensus sequences in the RDT1, OXR1, EfPKS1, PRF1, ECT1 and EfHP1–4 promoters (Table 2). The consensus TATA motif was found in the promoters of all genes except OXR1, PRF1 and EfHP3. All the genes contain at least one CAAT or CCAAT consensus sequence in their promoter regions. The pH-regulatory PACC-binding sequence (5'-GCCA(A/G)G-3') was identified only in the promoter regions of TSF1, EfPKS1, ECT1 and EfHP2. All the genes, except EfHP1 and EfHP3, have one or multiple GATA consensus sequences potentially recognized and bound by the nitrogen-regulatory AREA protein or the light-regulatory transcriptional WC1/WC2 complex. However, none of the promoter regions has the binding sequence (5'-(G/C)PyGGGG-3') that is recognized by the CREA carbon repressor. In addition, the promoter regions of the TSF1, EfPKS1 and PRF1 genes each have a consensus sequence, 5'-(A/C)(A/G)AGGG(A/G)-3', that serves as a binding site for the conidial formation-related BRLA transcriptional activator in A. nidulans (Adams et al., 1998). The promoter regions of the RDT1, OXR1, EfPKS1, and ECT1 genes each have a consensus sequence, 5'-CATTC(C/T)-3', that acts a binding site for the ABAA transcriptional activator involved in conidiophore development in A. nidulans. Two palindromic sequences, 5'-TCG(N2–4)CGA-3' and 5'-CGG(N3–11)CCG-3', were also identified in the promoter regions of some but not all genes (Table 2).
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). All putative transformants recovered were then screened for production of red/orange pigments (elsinochromes). Of 20 transformants tested, four failed to accumulate the red/orange pigments and were considered to be TSF1 disruptants. Successful disruption of the TSF1 gene was confirmed by Southern-blot analysis. Genomic DNA digested with PvuII and hybridized to a DNA probe corresponding to TSF1 coding sequence resulted in a single 4.8 kb band in the wild-type and two bands of the expected sizes of 3.9 and 3.2 kb in transformants (Fig. 3b). Hybridization of genomic DNA cleaved with BglI and PvuII also yielded different banding patterns between wild-type and the disruptants as expected (Fig. 3c), indicating specific disruption at the TSF1 locus.
Northern-blot analysis was conducted to determine if the disrupted mutants accumulated the TSF1 transcript. As shown in Fig. 4(a), hybridization of total RNA identified a sole TSF1 transcript of 3.1 kb from wild-type, but not from three disruptants. Apparently, integration of the HYG cassette within the TSF1 ORF has completely abolished expression of the TSF1 gene, indicating that they are TSF1 null mutants. Compared to wild-type, the TSF1 disruptants failed to accumulate elsinochromes in axenic cultures, as assayed either spectrophotometrically after KOH extraction (Fig. 4b) or by TLC analysis (Fig. 4c). In addition to elsinochrome deficiency, the TSF1 disruptants were also defective in conidiation and fungal pathogenesis to rough lemon (Table 3), similar to the mutant phenotypes resulting from disruption of the EfPKS1 gene (Liao & Chung, 2008b).
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). Expression of a functional TSF1 in a null mutant only partially restored conidiation and virulence (Table 3). However, Southern- and Northern-blot analyses confirmed that the two complementation strains did acquire and express the TSF1 gene (Fig. 5b and data not shown).
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, disruption of the TSF1 gene resulted in a considerable reduction of the levels of TSF1, RDT1, EfPKS1, PRF and EfHP1 gene transcripts, but had little or no effect on expression of ECT1, OXR1, EfHP2 and EfHP3. Acquiring and expressing a functional copy of TSF1 in a null strain restored expression of the TSF1, RDT1, EfPKS1, PRF, ECT1 and EfHP1 genes to wild-type levels (Fig. 5b).
Gene expression in an EfPKS1 null mutant
Accumulation of the gene transcripts was also examined in an EfPKS1 null mutant (D4) that is defective in elsinochrome production (Liao & Chung, 2008b). Northern-hybridization analysis revealed a marked reduction of transcripts of the RDT1, TSF1, PRF1, ECT1 and EfHP1 genes in the D4 mutant (Fig. 6). Accumulation of the RDT1, TSF1, PRF1 and ECT1 gene transcripts was nearly restored to wild-type levels in strains (C1 and C2) expressing a functional copy of EfPKS1. In contrast, expression of the OXR1, EfHP2, and EfHP3 gene transcripts was apparently not affected by disruption of the EfPKS1 gene (Fig. 6). A regulatory network deciphering environmental cues, signal transduction, transcriptional activation and feedback suppression of the genes required for elsinochrome biosynthesis and conidial formation via TSF1 is proposed (Fig. 7).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The genes involved in the biosynthesis of polyketide compounds are often closely linked in fungal genomes (Hoffmeister & Keller, 2007), and this also appears to be the case for some of the genes governing biosynthesis of elsinochromes in E. fawcettii. In the present study, we sequenced the regions flanking EfPKS1 to obtain a 30 kb contig in which we detected nine additional ORFs. These new genes include RDT1, encoding a putative reductase, and TSF1, encoding a putative transcriptional activator; both are likely to be involved in elsinochrome biosynthesis. The ECT1 gene, encoding a probable membrane transporter, might function in toxin secretion. The role of the PRF1 gene, encoding a putative prefoldin protein, in relation to elsinochrome production remains unknown. Similarly, the remaining five ORFs (OXR1 and EfHP1 to EfHP4) encode polypeptides with low similarity to hypothetical proteins of unknown function in the database. Whether they are involved in elsinochrome production will require further investigation. Our analysis of gene expression patterns in EfPKS1- or TSF1-disrupted mutants suggests that at least some of the newly identified genes are needed for elsinochrome production. As was evident from Northern-blot analysis, all the putative ORFs identified in the cluster are expressed. However, accumulation of gene transcripts does not fully correspond to all the conditions conducive to elsinochrome production. Expression of all ORFs, apart from EfHP3, and accumulation of elsinochromes did occur under limited nitrogen, but such coordinate gene expression and elsinochrome accumulation was not observed in response to increased pH or glucose concentration. Northern-blot analysis showed that not all of the genes are coordinately controlled by the putative transcriptional regulator, TSF1. For example, disruption of the TSF1 gene does not cause marked alteration in the ECT1 gene transcript; accumulation of the RDT1 or EfHP1 gene transcript is severely reduced in one TSF1-disruptant but not another, implying ‘leaky’ expression or requirement of other transcriptional regulators. The fact that the TSF1-disrupted mutants fail to accumulate any detectable TSF1 transcript and elsinochromes indicates that TSF1 has an indispensable role in elsinochrome biosynthesis. Disruption of the TSF1 gene also resulted in considerably reduced expression of the RDT1, EfPKS1 and PRF1 genes but had little or no effect on ECT1 and OXR1. It is yet not clear whether the OXR1 and EfHP1–4 genes flanking EfPKS1 and TSF1 are also involved in elsinochrome biosynthesis, even though expression of some of them was regulated by nitrogen limitation and altered by disruption of EFPKS1 or TSF1.
The EfPKS1 gene, encoding a polyketide synthase, has previously been shown to be required for elsinochrome biosynthesis (Liao & Chung, 2008b). Disruption of this gene also effectively abolished expression of the RDT1, TSF1, PRF1 and ECT1 genes in the cluster. Expression of a copy of the functional EfPKS1 gene in an EfPKS1 null mutant restored expression of RDT1, TSF1, PRF1 and ECT1 to wild-type levels, further implicating the requirement of these genes in elsinochrome biosynthesis.
In a prior study, we observed that disruption of the EfPKS1 gene gave rise to mutants displaying a remarkable reduction in conidiation and deficient in elsinochrome production (Liao & Chung, 2008b). Similar to the EfPKS1-disrupted mutants, TSF1 null mutants are also severely defective in conidial production, further suggesting a close linkage between elsinochrome production and asexual sporulation in E. fawcettii. The connections between production of secondary metabolites and cell development and/or differentiation have also been investigated in other filamentous fungi (Adams & Yu, 1998; Calvo et al., 2002; Yu & Keller, 2005; Brodhagen & Keller, 2006). In aspergilli, both asexual sporulation and production of sterigmatocystin and aflatoxin mycotoxins are negatively regulated by a G-protein signalling pathway (Hicks et al., 1997). It is unknown whether expression of the TSF1 gene is regulated by a G-protein pathway. Since loss of elsinochrome production by disrupting TSF1 greatly reduced conidial formation, it appears that the proteins or intermediates involved in elsinochrome production might directly participate in asexual sporulation. In addition, examination of upstream sequences revealed the presence of consensus MRAGGGR motifs in the promoter regions of TSF1, which are presumably involved in binding the conidiation-specific Bristle (BRLA)-like transcriptional activator in A. nidulans (Adams et al., 1998). The consensus CATTCY motif (Y=pyrimidine) that is recognized by the ABAA transcriptional activator (Andrianopoulos & Timberlake, 1994), is also found in the RDT1, OXR1, EfPKS1 and ECT1 gene promoters (Table 2). ABAA is activated by BRLA and is involved in conidiphore development of A. nidulans (Andrianopoulos & Timberlake, 1994). To provide a regulatory framework for better understanding the elsinochrome biosynthesis and conidial formation in E. fawcettii, a hypothetical model describing intertwined regulatory controls via TSF1 is proposed (Fig. 7). In this model, environmental cues activate signalling transduction cascades mediated by cAMP, G-protein or MAP kinase, which in turn activates transcriptional regulators such as AREA, WC1/WC2 complex, PACC, BRLA and ABAA. These transcriptional regulators may directly or indirectly trigger expression of TSF1 and the other genes, which eventually lead to elsinochrome production and conidial formation. It appears that accumulation of the EfPKS1, TSF1, RDT1 and PRF1 gene transcripts is controlled by TSF1. By contrast, expression of ECT1, OXR1, EfHP1, EfHP2 and EfHP3 is probably regulated by different transcriptional activators because disruption of TSF1 has little effect on their expression patterns. We also observed that disruption of EfPKS1 leads to coordinate inhibition of expression of the RDT1, TSF1, PRF1 and ECT1 genes, suggesting the presence of a feedback inhibition mechanism, in which disruption of one of the biosynthetic genes in the pathway abolishes elsinochrome production and in turn abrogates expression of the other biosynthetic genes.
Disruption of the EfPKS1 or TSF1 gene in E. fawcettii yielded mutants with pleiotropic phenotypes. All mutant phenotypes resulting from inactivation of the EfPKS1 gene were restored to the wild-type by expressing a functional copy of the EfPKS1 gene cassette (Liao & Chung, 2008b). It is surprising that expressing a functional TSF1 gene cassette under control of its own promoter (including 1 kb of upstream sequence) failed to fully restore conidiation, elsinochrome production or fungal pathogenesis to the TSF1 null mutants. It is very unlikely that failure to completely reverse the mutated phenotypes is due to an extraneous mutation in the transformed TSF1 gene. Sequence analysis of the TSF1 cassette revealed no nucleotide substitution, deletion or insertion (data not shown). Moreover, Northern-blot analysis showed expression of the TSF1 gene in the recombinant transformants. This failure of full complementation may reveal a specific requirement for TSF1 gene regulation within the context of the elsinochrome biosynthetic gene cluster. Alternatively, ectopic insertion of the TSF1 cassette may have affected the functions of nearby genes.
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ACKNOWLEDGEMENTS |
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Edited by: S. D. Harris
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Received 9 April 2008;
revised 21 July 2008;
accepted 22 July 2008.
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