 understanding of the molecular mechanisms leading to their biosynthesis. To date, the most studied and well characterized mycotoxin biosynthesis pathways are for aflatoxin and sterigmatocystin (sterigmatocystin is the penultimate precursor of aflatoxin) [16]. Genes required for the synthesis of aflatoxin are well conserved among Aspergilli and are located in large gene clusters [17]. The relative order and transcriptional direction of some of the homologous gene pairs though are not conserved [18]. Thus far, most of the genes in the respective clusters have been shown to encode enzymes required for toxin biosynthesis [19], [20]. In addition, the cluster harbors a transcription factor, AflR, which controls the expression of many or all genes located in the aflatoxin gene cluster [21]. In addition, the expression control requires specific chromatin remodeling of the clusters and thus epigenetic control [22], [23]. This appears to be also true for other fungi [24].
Although much progress has been made on the molecular characterization of the genes involved in and the regulation of the biosynthesis of other mycotoxins like fumonisin or trichothecene, no report is yet available regarding alternariol biosynthesis. The genes involved in AOH biosynthesis have yet to be discovered, despite AOH being one of the major mycotoxins produced by Alternaria species such as A. alternata and despite the importance of AOH as contaminant of food and feed.
Here we present the first report on a polyketide synthase involved in the biosynthesis of alternariol (AOH) and alternariol-methyl-ether (AME). We used both, gene deletion and RNA-silencing strategies to knock-down the function of PKS and other genes in A. alternata, which are involved in the biosynthetic pathway.
Our aim was to identify the genes encoding enzymes involved in alternariol (AOH) biosynthesis. Because it is likely that the pathway contains a polyketide synthase as one of the central enzymes, we screened an A. alternata genome sequence for PKS-encoding genes. The A. alternata genome has been sequenced recently (Lawrence et al., unpublished) using 454 Titanium deep sequencing technology (Roche, Indianapolis, IN). An approximate 20× draft of the ∼32 Mb genome sequence was assembled using Newbler software (Roche, Indianapolis, IN) and was used to search for PKS genes in this species. The analysis of the complete genome sequence will be published elsewhere. Applying blast searches with amino acid sequences of 50 known polyketide synthases from different fungi identified 10 putative polyketide synthases in A. alternata. Intron-exon borders were predicted using FGENESH (softberry.com) trained on A. brassicicola gene models but have not yet been verified experimentally due to the rather large nature of PKS genes. Among these 10 PKS genes, the PKS involved in melanin biosynthesis was already known and named ALM (albino) [25]. We renamed ALM according to a three-letter standard abbreviation code for genes, as PksA, and the remaining ones as PksB to PksJ. As the architectures of fungal PKSs are very similar to each other, we first analyzed the domain structures of the PKSs of A. alternata with SMART, WoLFPSORT, epestfind, ELM, ScanProsite and InterPro Scan software tools using standard parameters (Fig. 2). Except pksE, all PKS genes encode iterative type I RD-PKSs. Typical RD-PKS conserved domains, such as KR, DH and ER, were identified in PksC, PksD, PksF, PksG, PksH and PksJ in addition to KS, AT and ACP except PksC and PksF, which lack the ACP domain. Surprisingly the pksI gene is predicted to encode a protein of only 1484 amino acids, containing a KS and an AT domain. PksE did not show any known domain of fungal polyketide synthases. However, the 599 aa long protein of pksE displays similarity to chalcone (CHS)/stilbene (STS) synthase and uracil DNA glycosylase like domains. The CHS/STS domain of PksE shares 40% identity to type III polyketide synthase of Sordaria macrospora [26]. Type III PKS are relatively small dimeric proteins (subunit sizes about 40–45 kDa) that usually carry out iterative condensation reactions with malonyl-CoA; the numbers can range from one to seven. CHS and STS are plant-specific polyketide synthases. CHS catalyzes the first step in the biosynthesis of a large number of biologically important substances, e.g. flavonoids (flower color) and phytoalexins (defense against pathogens). STS forms the backbone of the stilbene phytoalexins. However, these enzymes are rare in higher plants.
Figure 2. Architecture of PKSs of Alternaria alternata.
KS, β-ketoacyl synthase; AT, acyltransferase; DH, dehydratase; MT, methyltransferase; ER, enoyl reductase; KR, ketreductase; ACP, acyl carrier protein, CD, condensation domain; AA, Amino acid adenylation domain; CS:Chalon- and Stilben-Synthase (N)/(C); UDG: Uracil DNA Glycolase Superfamily; NAD, NAD binding domain. The sequences of the PKS loci are deposited under the following accession numbers: pksA (JX103636); pksB (JX103637); pksC (JX103638); pksD (JX103639); pksE (JX103640); pksF (JX103641); pksG (JX103642); pksH (JX103643); pksI (JX103644); pksJ (JX103645). The genbank accession numbers are given in brackets.
As mentioned above, genes encoding enzymes involved in the biosynthesis of a given secondary metabolite are usually clustered in the genome. Together with other biosynthetic enzymes, a regulatory transcription factor is often found within the cluster. In order to better understand the roles and organization of PKS biosynthetic clusters in A. alternata we analyzed the genome sequences up- and downstream of the PKSs. Indeed most of the gene clusters also contained putative transcription factors (Fig. 3).
Figure 3. Organization of polyketide biosynthesis gene clusters in Alternaria alternata.
Each arrow indicates the direction of transcription deduced from the analysis of the nucleotide sequences. The asterisks indicate the genes of the pksJ and pksH clusters, which have been silenced in addition to pksJ and pksH.
Secondary metabolites are often not produced during logarithmic growth of fungi, but rather in aging mycelium or in response to certain stimuli [27]. The induction of the biosynthesis is probably a result of differential gene expression under those conditions. Therefore, we determined temporal aspects of when individual PKS genes were expressed and correlated this expression pattern with the timing of AOH production. As a first step to identify the initial time point for AOH production, MCDB agar plates were inoculated with defined numbers of spores and incubated for 3 to 14 days at 28°C in the dark. Both, mycelium and agar medium were extracted with ethyl acetate and analyzed by thin layer chromatography (TLC). AOH was detected initially after 5 days in very small quantities and increased after 7 days of incubation (Fig. 4A). In order to determine the relative expression levels of the selected PKS genes, RNA was isolated from the same mycelium samples at three different time points, starting from day 7 and subjected to real time PCR analyses (Fig. 4B). Except pksE, all other PKS genes were expressed by 12 days, making them potential candidates for being involved in AOH biosynthesis.
Figure 4. Time-course expression analysis of different PKS genes.
(A) TLC analysis of AOH formation at different time points. Extracts from cultures grown on MCDB agar at 28°C in constant darkness for 3, 5, 7, 10, 12 and 14 days, respectively. For each time point three extractions were performed. The last lane contained the AOH standard. (B) Quantitative real time reverse-transcription polymerase chain reaction (RT-PCR) gene expression analysis of different PKS genes after 7, 12 and 14 days of post inoculation (dpi).
To initiate the molecular analysis of alternariol (AOH) production in A. alternata we aimed to identify the role of pksJ by gene deletion, as the pksJ gene showed the highest expression among all PKSs from day 7 onwards. Approximately, one kb upstream (left border) and downstream regions (right border) of the predicted pksJ ORF were PCR amplified and fused to the hygromycin B (hph) resistance cassette by fusion PCR [28]. The 5.7 kb long fusion PCR product was directly used for protoplast transformation and homologous replacement of the pksJ open reading frame. Transformants were analyzed by PCR and Southern blotting (data not shown). The integration of the construct was verified by PCR using primers derived from the hygromycin B cassette and primers outside the left or the right border sequences. Southern blot analysis with a probe for hygromycin B confirmed the results and demonstrated a single integration (data not shown). However, a wildtype copy of pksJ was still detectable by PCR and Southern blotting in the transformants. Even though we performed multiple rounds of single spore isolation on hygromycin-containing media, we anticipated that the transformants still harbored non-transformed wildtype nuclei as a heterokaryon. The