Andy M. Bailey

Finished Genome of the Fungal Wheat Pathogen Mycosphaerella graminicola Reveals Dispensome Structure, Chromosome Plasticity, and Stealth Pathogenesis

The plant-pathogenic fungus Mycosphaerella graminicola (asexual stage: Septoria tritici) causes septoria tritici blotch, a disease that greatly reduces the yield and quality of wheat. This disease is economically important in most wheat-growing areas worldwide and threatens global food production. Control of the disease has been hampered by a limited understanding of the genetic and biochemical bases of pathogenicity, including mechanisms of infection and of resistance in the host. Unlike most other plant pathogens, M. graminicola has a long latent period during which it evades host defenses. Although this type of stealth pathogenicity occurs commonly in Mycosphaerella and other Dothideomycetes, the largest class of plant-pathogenic fungi, its genetic basis is not known. To address this problem, the genome of M. graminicola was sequenced completely. The finished genome contains 21 chromosomes, eight of which could be lost with no visible effect on the fungus and thus are dispensable. This eight-chromosome dispensome is dynamic in field and progeny isolates, is different from the core genome in gene and repeat content, and appears to have originated by ancient horizontal transfer from an unknown donor. Synteny plots of the M. graminicola chromosomes versus those of the only other sequenced Dothideomycete, Stagonospora nodorum, revealed conservation of gene content but not order or orientation, suggesting a high rate of intra-chromosomal rearrangement in one or both species. This observed “mesosynteny” is very different from synteny seen between other organisms. A surprising feature of the M. graminicola genome compared to other sequenced plant pathogens was that it contained very few genes for enzymes that break down plant cell walls, which was more similar to endophytes than to pathogens. The stealth pathogenesis of M. graminicola probably involves degradation of proteins rather than carbohydrates to evade host defenses during the biotrophic stage of infection and may have evolved from endophytic ancestors.

Genome sequence of the button mushroom Agaricus bisporus reveals mechanisms governing adaptation to a humic-rich ecological niche

Agaricus bisporus is the model fungus for the adaptation, persistence, and growth in the humic-rich leaf-litter environment. Aside from its ecological role, A. bisporus has been an important component of the human diet for over 200 y and worldwide cultivation of the “button mushroom” forms a multibillion dollar industry. We present two A. bisporus genomes, their gene repertoires and transcript profiles on compost and during mushroom formation. The genomes encode a full repertoire of polysaccharide-degrading enzymes similar to that of wood-decayers. Comparative transcriptomics of mycelium grown on defined medium, casing-soil, and compost revealed genes encoding enzymes involved in xylan, cellulose, pectin, and protein degradation are more highly expressed in compost. The striking expansion of heme-thiolate peroxidases and β-etherases is distinctive from Agaricomycotina wood-decayers and suggests a broad attack on decaying lignin and related metabolites found in humic acid-rich environment. Similarly, up-regulation of these genes together with a lignolytic manganese peroxidase, multiple copper radical oxidases, and cytochrome P450s is consistent with challenges posed by complex humic-rich substrates. The gene repertoire and expression of hydrolytic enzymes in A. bisporus is substantially different from the taxonomically related ectomycorrhizal symbiont Laccaria bicolor. A common promoter motif was also identified in genes very highly expressed in humic-rich substrates. These observations reveal genetic and enzymatic mechanisms governing adaptation to the humic-rich ecological niche formed during plant degradation, further defining the critical role such fungi contribute to soil structure and carbon sequestration in terrestrial ecosystems. Genome sequence will expedite mushroom breeding for improved agronomic characteristics.

Biosynthesis of the 2-pyridone Tenellin in the insect pathogenic fungus Beauveria bassiana

Genomic DNA from the insect pathogenic fungus Beauveria bassiana was used as a template in a PCR with degenerate primers designed to amplify a fragment of a C‐methyl transferase (CMeT) domain from a highly reduced fungal polyketide synthase (PKS). The resulting 270‐bp PCR product was homologous to other fungal PKS CMeT domains and was used as a probe to isolate a 7.3‐kb fragment of genomic DNA from a BamH1 library. Further library probing and TAIL‐PCR then gave a 21.9‐kb contig that encoded a 12.9‐kb fused type I PKS–NRPS ORF together with ORFs encoding other oxidative and reductive enzymes. A directed knockout experiment with a BaR cassette, reported for the first time in B. bassiana, identified the PKS–NRPS as being involved in the biosynthesis of the 2‐pyridone tenellin. Other fungal PKS–NRPS genes are known to be involved in the formation of tetramic acids in fungi, and it thus appears likely that related compounds are precursors of 2‐pyridones in fungi. B. bassiana tenellin KO and WT strains proved to be equally pathogenic towards insect larvae; this indicated that tenellin is not involved in insect pathogenesis.

Authentic Heterologous Expression of the Tenellin Iterative Polyketide Synthase Nonribosomal Peptide Synthetase Requires Coexpression with an Enoyl Reductase

The tenS gene encoding tenellin synthetase (TENS), a 4239‐residue polyketide synthase nonribosomal‐peptide synthetase (PKS‐NRPS) from Beauveria bassiana, was expressed in Aspergillus oryzae M‐2‐3. This led to the production of three new compounds, identified as acyl tetramic acids, and numerous minor metabolites. Consideration of the structures of these compounds indicates that the putative C‐terminal thiolester reductase (R) domain does not act as a reductase, but appears to act as a Dieckmann cyclase (DKC). Expression of tenS in the absence of a trans‐acting ER component encoded by orf3 led to errors in assembly of the polyketide component, giving clues to the mode of programming of highly reducing fungal PKS. Coexpression of tenS with orf3 from the linked gene cluster led to the production of a correctly elaborated polyketide. The NRPS adenylation domain possibly shows the first identified fungal signature sequences for tyrosine selectivity.

A single amino-acid substitution in the iron-sulphur protein subunit of succinate dehydrogenase determines resistance to carboxin in Mycosphaerella graminicola

Abstract A gene encoding the iron-sulphur protein (Ip) subunit of succinate dehydrogenase (Sdh, EC 1.3.99.1) from Mycosphaerella graminicola (Septoria tritici) has been cloned and sequenced. The deduced amino-acid sequence exhibited a high degree of homology to Ip subunits of Sdh from other organisms; three cysteine-rich clusters associated with the iron-sulphur centres involved in electron transport were particularly conserved. Expression studies using a synthetic green fluorescent protein (SGFP) expression vector demonstrated that the cloned DNA also contained a functional promoter region and confirmed that the deduced initiation codon could act as a translational start site. Mutants resistant to the fungicide carboxin (Cbx), a known inhibitor of Sdh, were found to contain a single amino-acid substitution in the third cysteine-rich domain of the Ip protein. These mutations resulted in the conversion of a highly conserved His residue, located in a region of the protein associated with the [3Fe-4 S] high-potential non-heme iron sulphur-redox (S3) centre, to either Tyr or Leu. An Ip gene containing the His → Tyr mutation was constructed and shown to confer Cbx resistance following co-transformation into the Cbx-sensitive wild-type strain. This confirmed that the mutation identified by sequence analysis was responsible for determining Cbx resistance.

Late stage oxidations during the biosynthesis of the 2-pyridone tenellin in the entomopathogenic fungus Beauveria bassiana.

Late stage oxidations during the biosynthesis of the 2-pyridone tenellin in the insect pathogenic fungus Beauveria bassiana were investigated by a combination of gene knockout, antisense RNA, and gene coexpression studies. Open reading frames (ORF) 3 and 4 of the tenellin biosynthetic gene cluster were previously shown to encode a trans-acting enoyl reductase and a hybrid polyketide synthase nonribosomal peptide synthetase (PKS-NRPS), respectively, which together synthesize the acyltetramic acid pretenellin-A. In this work, we have shown that ORF1 encodes a cytochrome P450 oxidase, which catalyzes an unprecedented oxidative ring expansion of pretenellin-A to form the 2-pyridone core of tenellin and related metabolites, and that this enzyme does not catalyze the formation of a hydroxylated precursor. Similar genes appear to be associated with PKS-NRPS genes in other fungi. ORF2 encodes an unusual cytochrome P450 monooxygenase required for the selective N-hydroxylation of the 2-pyridone which is incapable of N-hydroxylation of acyltetramic acids.

Rational Domain Swaps Decipher Programming in Fungal Highly Reducing Polyketide Synthases and Resurrect an Extinct Metabolite

The mechanism of programming of iterative highly reducing polyketide synthases remains one of the key unsolved problems of secondary metabolism. We conducted rational domain swaps between the polyketide synthases encoding the biosynthesis of the closely related compounds tenellin and desmethylbassianin. Expression of the hybrid synthetases in Aspergillus oryzae led to the production of reprogrammed compounds in which the changes to the methylation pattern and chain length could be mapped to the domain swaps. These experiments reveal for the first time the origin of programming in these systems. Domain swaps combined with coexpression of two cytochrome P450 encoding genes from the tenellin biosynthetic gene cluster led to the resurrection of the extinct metabolite bassianin.

Genetic, molecular, and biochemical basis of fungal tropolone biosynthesis

A gene cluster encoding the biosynthesis of the fungal tropolone stipitatic acid was discovered in Talaromyces stipitatus (Penicillium stipitatum) and investigated by targeted gene knockout. A minimum of three genes are required to form the tropolone nucleus: tropA encodes a nonreducing polyketide synthase which releases 3-methylorcinaldehyde; tropB encodes a FAD-dependent monooxygenase which dearomatizes 3-methylorcinaldehyde via hydroxylation at C-3; and tropC encodes a non-heme Fe(II)-dependent dioxygenase which catalyzes the oxidative ring expansion to the tropolone nucleus via hydroxylation of the 3-methyl group. The tropA gene was characterized by heterologous expression in Aspergillus oryzae, whereas tropB and tropC were successfully expressed in Escherichia coli and the purified TropB and TropC proteins converted 3-methylorcinaldehyde to a tropolone in vitro. Finally, knockout of the tropD gene, encoding a cytochrome P450 monooxygenase, indicated its place as the next gene in the pathway, probably responsible for hydroxylation of the 6-methyl group. Comparison of the T. stipitatus tropolone biosynthetic cluster with other known gene clusters allows clarification of important steps during the biosynthesis of other fungal compounds including the xenovulenes, citrinin, sepedonin, sclerotiorin, and asperfuranone.

First Heterologous Reconstruction of a Complete Functional Fungal Biosynthetic Multigene Cluster

Fungal natural products include antibiotics such as the penicillins, antirejection drugs such as cyclosporins and cholesterollowering drugs such as the statins. High productivity of these pharmaceutically important compounds is desirable and has regularly been pursued by strain improvement and metabolic engineering. Recently partial and full genome sequencing has revealed clusters of genes encoding the biosynthesis of these compounds in fungi. Surprisingly it has been found that while rich in bioactive compounds, fungi are even richer in biosynthetic gene clusters. For example, in the well-characterised species Aspergillus nidulans, there are at least 54 secondary metabolite gene clusters, only half of which are characterised at a chemical level. Fungal secondary metabolite gene clusters can be readily identified from key genes within the clusters, such as nonribosomal peptide synthetases (NRPS), terpene cyclases or polyketide synthases (PKS). Although it is relatively easy to identify such gene clusters, it is currently almost impossible to predict the chemical product synthesised, as the programming of the synthase cannot be predicted from sequence information alone. This is further compounded by the presence of genes for many tailoring enzymes, which usually act after the core synthase has completed synthesis of the skeleton, further modifying the resulting compound, making it extremely difficult to elucidate the likely metabolite from gene sequence data alone. A number of different strategies have been employed to decipher fungal gene clusters. These include: chemical profiling of cultures produced under a range of different conditions; over-expression of pathway regulatory genes (where present) ; manipulation of transcriptional activators (e.g. , LaeA) ; manipulation of the pH regulatory system (e.g. , PacC) ; use of chromatin modifying (i.e. , epigenetic) chemicals ; and cofermentation of bacteria and fungi. Unfortunately all of these techniques are restricted in their general utility. Many of the biosynthetic clusters do not include specific transcriptional regulators, many pathways do not respond to LaeA or PacC regulation and chromatin modifiers only activate a small subset of the gene clusters within a genome. In addition, many fungi are difficult to cultivate on a large scale and are often recalcitrant to molecular techniques, so these approaches cannot be applied. We and others have used another approach to investigate the biosynthesis of biologically active compounds in fungi that of heterologous gene expression. This method involves transfer of a gene of interest from a donor strain to a suitable host. In principle a bacterial host such as E. coli could be used, but several problems are usually encountered. First, bacterial hosts are unable to process eukaryotic introns and so these must be removed. Second, expression of eukaryotic genes in bacteria can be problematic, especially if there is a significant codon bias. Third, bacteria can experience difficulty in correctly folding fungal polypeptides. Fourth, proteins such as PKS and NRPS require selective post-translational phosphopantetheinylation for them to be active in vivo. Finally, bacteria may not supply specific metabolites for biosynthesis. While each of these problems can be overcome in isolation, cumulative effects can make the use of bacteria as expression hosts for fungal genes troublesome and inefficient. This is illustrated by the recent expression of the beauvericin NRPS (bbBeas) from

PEG-mediated and Agrobacterium -mediated transformation in the mycopathogen Verticillium fungicola

Verticillium fungicola, a severe mycopathogen of the cultivated mushroom Agaricus bisporus, was successfully transformed using both PEG-mediated and Agrobacterium-mediated techniques. PEG-mediated co-transformation was successful with hygromycin B resistance (hph), uidA (β-glucuronidase GUS), and green fluorescent protein (GFP) genes. Agrobacterium-mediated transformation was successful with the hph gene. Transformation frequencies of up to 102 transformants per μg DNA and 4068 transformants per 105 conidia were obtained for PEG-mediated and Agrobacterium-mediated transformation respectively. Expression of integrated genes in co-transformants was stable after 18 months of successive sub-culturing on non-selective medium, and following storage at -80 °C in glycerol. Molecular analysis of PEG-mediated transformants showed integration of the transforming genes into the target genome. Molecular analysis of Agrobacterium-mediated transformants showed integration of transforming DNA as single copies within the target genome. Co-transformants exhibited symptoms of disease in inoculation experiments and were at least as virulent as the wild-type fungus. GFP and GUS expression were observed in-vivo with the GFP-tagged strain showing great potential as a tool in epidemiological and host-pathogen interaction studies. The development of transformation systems for V. fungicola will allow in-depth molecular studies of the interaction of this organism with A. bisporus.