Chapter 8

The Secondary Metabolite Toxin, Sirodesmin PL, and Its Role in Virulence of the Blackleg Fungus
Barbara J. Howlett, Ellen M. Fox, Anton J. Cozijnsen, Angela P. Van de Wouw, and Candace E. Elliott

8.1 Sirodesmin PL, an Epipolythiodioxopiperazine Toxin

Sirodesmin PL is a non-host specific toxin produced by the ascomycete Leptosphaeria maculans, which causes blackleg (phoma stem canker) of oilseed rape (Brassica napus), the most damaging disease of this crop worldwide (Fitt etal. 2006). As well as causing chlorotic (yellow) lesions on leaves, sirodesmin PL has antibacterial and antiviral properties (Rouxel etal. 1988). Sirodesmin PL is a member of the epipolythiodioxopiperazine (ETP) class of fungal secondary metabolites, which are characterised by a sulphur-bridged dioxopiperazine ring synthesised from two amino acids (Fig.8.1a, b). The disulphide bridge confers toxicity by enabling ETPs to cross-link proteins via cysteine residues, and to generate reactive oxygen species through redox cycling. At least 14 different ETPs are known, and all are produced by ascomycetes (for review see Gardiner et al. 2005). The best-characterised ETP, gliotoxin (Fig.8.1c), is produced by the opportunistic human pathogen Aspergillus fumigatus, as well as A. terreus, A. flavus, A .niger, Penicillium terlikowskii and Trichoderma virens, a fungus that controls root diseases of plants. Other ETPs with roles in animal diseases include sporidesmins, produced by Pithomyces chartarum which infects grasses and causes facial eczema and liver diseases of grazing animals. Chaetomin and chaetocin are produced by Chaetomium globosum, a systemic pathogen of immunocompromised humans. The cytotoxicity of some ETPs has made them attractive as potential anticancer agents and accordingly interest in these molecules is increasing.

B.J. Howlett (*), E.M. Fox, A.J. Cozijnsen, A.P. Wouw, and C.E. Elliott School of Botany, The University of Melbourne, Vic 3010, Australia e-mail: bhowlett@unimelb.edu.au

R.N. Strange and M.L. Gullino (eds.), The Role of Plant Pathology in Food Safety and Food Security, Plant Pathology in the 21st Century 3, DOI 10.1007/978-1-4020-8932-9_8, Springer Science+Business Media B.V. 2010

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Fig. 8.1 Structures of epithiodioxopiperazines. (a) Core epithiodioxopiperazine (ETP) moiety; (b) sirodesmin PL; (c) gliotoxin

8.2 Biosynthesis of Sirodesmin PL

Sirodesmin PL is derived from serine and tyrosine. Labelling experiments and analysis of putative intermediates have been used to deduce the biosynthetic pathway (Ferezou et al. 1980a, b; BuLock and Clough 1992). A prenyl transferase is predicted to catalyse the addition of a dimethylallyl group to either the dipeptide cyclo-l-tyrosyll-serine or free tyrosine, before condensation with serine, to produce the intermediate cyclic dipeptide, phomamide. The genes responsible for the biosynthesis of sirodesmin PL in L. maculans were identified 4 years ago by an approach that took advantage of the clustering of genes encoding biosynthetic enzymes for secondary metabolites in fungal genomes (Gardiner etal. 2004). A homologue of prenyl transferase (a dimethyl tryptophan synthetase) from the endophyte Neotyphodium coenphialum was identified from an L. maculans Expressed Sequence Tag (EST) library. This EST was used to probe an L. maculans cosmid DNA library and a cosmid (35 kb) containing the prenyl transferase was sequenced. Regions flanking this prenyl transferase gene (named sirD) contained a cluster of 18 genes which, based on best matches to genes in databases, could be assigned roles in sirodesmin PL biosynthesis. These included a two module non-ribosomal peptide synthetase (sirP), acetyl transferase (sirH), methyl transferases (sirM and sirN), an ATP-binding cassette (ABC) type transporter (sirA), responsible for toxin efflux, and a member of the zinc binuclear cluster (Zn(II)2Cys6) family (sirZ) (Fig.8.2). Of the nine cluster genes tested, all were co-regulated and timing of expression was consistent with the production of sirodesmin PL in culture, suggesting the gene cluster is involved in sirodesmin PL biosynthesis. Furthermore, the disruption of the peptide synthetase, sirP, resulted in a mutant isolate unable to produce sirodesmin PL, confirming that this gene is essential for the synthesis of this toxin, and that the biosynthetic gene cluster was correctly identified (Fox and Howlett 2008a).

8.3 Regulation of Sirodesmin PL Biosynthesis

Members of the zinc binuclear cluster (Zn(II)2Cys6) family of transcription factors are unique to fungi (Todd and Andrianopoulos 1997). Genes in this family control production of other secondary metabolites such as aflatoxins, therefore,

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Fig.8.2 The Leptosphaeria maculans sirodesmin PL (a) and Aspergillus fumigatus gliotoxin (b) biosynthetic gene clusters. Common ETP moiety genes (white text on black background) include those with best matches to non-ribosomal peptide synthetase (P), thioredoxin reductase (T), methyl transferases (M and N), glutathione-S-transferase (G) and cytochrome P450 mono-oxygenase (C), amino cyclopropane carboxylate synthase (ACCS) (I), dipeptidase (J), as well as a transcriptional regulator (Z) and a transporter (A). Other genes (black text on white background) do not have obvious homologues in the other cluster and are thought to be involved in modification of the side chains of the core ETP moiety. These encode cytochrome P450 mono-oxygenases (F, B and E), a prenyl transferase (D), an acetyl transferase (H), epimerases (Q, S and R), an oxidoreductase (O) and a hypothetical protein (K) (Gardiner and Howlett 2005). Genes shaded in grey encode proteins with best matches to proteins with no potential roles in ETP biosynthesis. The forward slash marks represent a 17 kb region of repetitive DNA (reproduced from Fox and Howlett (2008a), with permission from Elsevier)

SirZ was an obvious candidate for the regulation of sirodesmin PL. RNAiinduced silencing of this gene led to minimal production of sirodesmin PL and very low expression of several of the biosynthetic genes (Fox et al. 2008). Binding sites for Zn(II)2Cys6 transcription factors consist of conserved terminal trinucleotides, usually in a symmetrical configuration, spaced by an internal variable sequence of defined length (Todd and Andrianopoulos 1997). Such sequences are present in the promoters of most of the sirodesmin PL biosynthetic genes, and it is likely that these are binding sites for the pathway specific transcription factor, sirZ (Fox etal. 2008). In order to identify regulators of sirodesmin PL biosynthesis upstream of sirZ, a library of L. maculans insertional mutants has been screened for lack of sirodesmin PL production, using an assay that relies on the antibacterial properties of sirodesmin (E.M. Fox and B.J. Howlett, unpublished, 2009). The insertional mutants were created via Agrobacterium tumefaciens-mediated transformation whereby T-DNA is inserted randomly in the genome (Elliott and Howlett 2006). Ten-day-old colonies of individual L. maculans mutants growing on an agar plate were assayed for loss of the ability to produce a ring of clearing when overlaid with molten agar containing Bacillus subtilis. Five sirodesmin-deficient mutants have been isolated; four of which have insertions in genes with best matches to transcription factors, whilst the other is a hypothetical gene (E.M. Fox and B.J. Howlett, unpublished, 2009). One of the transcription factors was cpcA, a general amino acid transcriptional regulator in fungi including A. fumigatus (Krappmann etal. 2004). When amino acids are unavailable, fungi activate a complex regulatory network that allows the coordinated expression of a whole suite of genes required for amino acid biosynthesis. The central control element of this network is CpcA. The role of cpcA in the regulation of sirodesmin PL biosynthesis is currently being assessed.

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8.4 The Gliotoxin Gene Cluster in Aspergillus fumigatus

Upon completion of the genome sequence of A. fumigatus the gliotoxin biosynthetic gene cluster was identified in this fungus. Ten clustered genes were identified, eight of which were in common with genes in the sirodesmin gene cluster. These genes had an expression pattern consistent with the timing of production of gliotoxin (Gardiner and Howlett 2005). The identity of this cluster was confirmed as the gliotoxin cluster by several research groups via the disruption of the two module non-ribosomal peptide synthetase, gliP. The resultant mutant was unable to make gliotoxin (Cramer et al. 2006; Kupfahl etal. 2006), confirming that this indeed was the gliotoxin biosynthetic cluster. Additionally, disruption of gliZ, encoding a Zn(II)2Cys6 transcription factor like sirZ, resulted in a mutant isolate unable to produce gliotoxin (Bok etal. 2006). Both the L. maculans and A. fumigatus ETP biosynthetic clusters encode proteins predicted to be involved in the efflux of the toxin from the cell, SirA and GliA respectively. However, while SirA is an ATP-binding cassette (ABC) type transporter, GliA is a Major Facilitator Superfamily transporter (Gardiner etal. 2005).

8.5 Origin and Distribution of ETP Gene Clusters in Ascomycetes

With the increasing number of sequenced genomes becoming available for fungi, the distribution and heredity of gene clusters such as those encoding ETPs can be investigated. Putative ETP gene clusters with most or all of the common eight genes in the sirodesmin and gliotoxin gene clusters have been found in Magnaporthe grisea, Neosartorya fischeri, Penicillium lilacinoechinulatum and Trichoderma reesei (Patron etal. 2007). Whether these fungi produce ETPs is unknown. Numerous other ascomycetes, including Saccharomyces cerevisiae and Schizosaccharomyces pombe, as well as all basidiomycetes that have been sequenced so far lack ETP-like clusters. Phylogenetic analysis of individual cluster genes has shown ETP gene clusters appear to have a common origin and the cluster has been inherited relatively intact, rather than assembling independently in the different ascomycete lineages (Patron et al. 2007). Although a mechanism describing this complex heredity cannot be conclusively identified, movement of entire clusters by horizontal gene transfer is the most parsimonious explanation of the discontinuous distribution of clusters.

8.6 Role of Sirodesmin PL in Virulence of Leptosphaeria maculans on Oilseed Rape

The contribution of sirodesmin PL to virulence of L. maculans on oilseed rape (Brassica napus) has now been determined unequivocally, due to exploitation of a defined sirodesmin-deficient L. maculans mutant, that had a DNA insertion in the

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non-ribosomal peptide synthetase gene (sirP). When the sirP mutant was inoculated onto cotyledons of B. napus, it caused similar-sized lesions as the wild type isolate, indicating that sirodesmin PL was not a virulence factor at this stage of infection. Subsequently, the mutant caused fewer stem lesions and was half as effective as the wild type in colonising stems, as shown by quantitative PCR analyses (Elliott etal. 2007) (Fig.8.3). Thus sirodesmin PL contributes to virulence in B. napus stems. The expression of two cluster genes, the peptide synthetase, sirP and an ABC transporter, sirA, was also studied during infection. Fungal isolates containing fusions of the green fluorescent protein gene (GFP) with the promoters of these genes fluoresced 10 days post-inoculation (dpi). This expression pattern was consistent with the distribution of sirodesmin PL in both cotyledons and stems, as revealed by mass spectrometry experiments (Elliott etal. 2007). It is intriguing to speculate as to why sirodesmin PL contributes to colonisation of stems but not to generation of the necrotic symptoms of lesions in the cotyledons. Sirodesmin PL may suppress plant defences during the biotrophic growth down the stem, or it might play a role in competition between L. maculans and other microorganisms in planta, or even on stubble during the saprophytic stage of its life cycle. Germination of several fungal species including Fusarium graminaerum and L. biglobosa brassicae was inhibited in the presence of a 13 day old colony of the wild type sirodesmin PL-producing isolate when grown invitro, illustrating the antifungal activity of this molecule (Elliott etal. 2007). The role that secondary metabolites play in the biology of fungi is elusive. In many cases fungi producing toxins do not rely on growth on a host to complete their life cycle. The most likely advantage of secondary metabolites to organisms that produce such molecules is that they enhance survival. Many such organisms live saprophytically in the soil where they are exposed to a harsh environment with a plethora of competing organisms. Fungal virulence has been proposed to have evolved to protect fungi in such an environment against amoebae, nematodes or other invertebrates that

Fig. 8.3 Quantification of fungal biomass in infected stems of Brassica napus cv. Monty. Cotyledons and first leaves of B. napus cv. Monty were inoculated with wild type (wt) or the sirodesmin-deficient mutant, sirP and were analysed at 33 dpi. Genomic DNA was used as a template for quantitative PCR. The relative amount of fungal biomass was calculated as the ratio of the amplification of a fragment of fungal actin to that of plant actin. Each bar represents the average of three replicate PCRs on three independent sets of infected stems. The asterisk indicates that fungal biomass of wild type is significantly different from that of the mutant

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can feed on fungi (Mylonakis et al. 2007); Fox and Howlett (2008b). Secondary metabolite toxins could play a role in such behaviour. The recent availability of defined mutants in the biosynthesis of secondary metabolites will enable this hypothesis to be tested for some molecules and their producing-organisms.
Acknowledgements We thank the Australian Research Council and the Grains Research and Development Corporation for funding our research.

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