Summary: The D othideomycetes represents a large and diverse array of fungi in which prominent plant pathogens are over‐represented. Species within the C ochliobolus , A lternaria , P yrenophora and M ycosphaerella (amongst others) all cause diseases that threaten food security in many parts of the world. Significant progress has been made over the past decade in understanding how some of these pathogens cause disease at a molecular level. It is reasonable to suggest that much of this progress can be attributed to the increased availability of genome sequences. However, together with revealing mechanisms of pathogenicity, these genome sequences have also highlighted the capacity of the D othideomycetes to produce an extensive array of secondary metabolites, far greater than originally thought. Indeed, it is now clear that we appear to have only scratched the surface to date in terms of the identification of secondary metabolites produced by these fungi. In the first half of this review, we examine the current status of secondary metabolite research in the D othideomycetes and highlight the diversity of the molecules discovered thus far, in terms of both structure and biological activity. In the second part of this review, we survey the emerging techniques and technologies that will be required to shed light on the vast array of secondary metabolite potential that is encoded within these genomes. Experimental design, analytical chemistry and synthetic biology are all discussed in the context of how they will contribute to this field.

Pyrenophora teres, P. teres f. teres (PTT) and P. teres f. maculata (PTM) cause significant diseases in barley, but little is known about the large-scale genomic differences that may distinguish the two forms. Comprehensive genome assemblies were constructed from long DNA reads, optical and genetic maps. As repeat masking in fungal genomes influences the final gene annotations, an accurate and reproducible pipeline was developed to ensure comparability between isolates. The genomes of the two forms are highly collinear, each composed of 12 chromosomes. Genome evolution in P. teres is characterized by genome fissuring through the insertion and expansion of transposable elements (TEs), a process that isolates blocks of genic sequence. The phenomenon is particularly pronounced in PTT, which has a larger, more repetitive genome than PTM and more recent transposon activity measured by the frequency and size of genome fissures. PTT has a longer cultivated host association and, notably, a greater range of host–pathogen genetic interactions compared to other Pyrenophora spp., a property which associates better with genome size than pathogen lifestyle. The two forms possess similar complements of TE families with Tc1/Mariner and LINE-like Tad-1 elements more abundant in PTT. Tad-1 was only detectable as vestigial fragments in PTM and, within the forms, differences in genome sizes and the presence and absence of several TE families indicated recent lineage invasions. Gene differences between P. teres forms are mainly associated with gene-sparse regions near or within TE-rich regions, with many genes possessing characteristics of fungal effectors. Instances of gene interruption by transposons resulting in pseudogenization were detected in PTT. In addition, both forms have a large complement of secondary metabolite gene clusters indicating significant capacity to produce an array of different molecules. This study provides genomic resources for functional genetics to help dissect factors underlying the host–pathogen interactions.

Genome mining facilitated by heterologous systems is an emerging approach to access the chemical diversity encoded in basidiomycete genomes. In this study, three sesquiterpene synthase genes, GME3634, GME3638, and GME9210, which were highly expressed in the sclerotium of the medicinal mushroom Lignosus rhinocerotis, were cloned and heterologously expressed in a yeast system. Metabolite profile analysis of the yeast culture extracts by GC–MS showed the production of several sesquiterpene alcohols (C15H26O), including cadinols and germacrene D-4-ol as major products. Other detected sesquiterpenes include selina-6-en-4-ol, β-elemene, β-cubebene, and cedrene. Two purified major compounds namely (+)-torreyol and α-cadinol synthesised by GME3638 and GME3634 respectively, are stereoisomers and their chemical structures were confirmed by 1H and 13C NMR. Phylogenetic analysis revealed that GME3638 and GME3634 are a pair of orthologues, and are grouped together with terpene synthases that synthesise cadinenes and related sesquiterpenes. (+)-Torreyol and α-cadinol were tested against a panel of human cancer cell lines and the latter was found to exhibit selective potent cytotoxicity in breast adenocarcinoma cells (MCF7) with IC50 value of 3.5 ± 0.58 μg/ml while α-cadinol is less active (IC50 = 18.0 ± 3.27 μg/ml). This demonstrates that yeast-based genome mining, guided by transcriptomics, is a promising approach for uncovering bioactive compounds from medicinal mushrooms.

Alternariol (AOH) is an important mycotoxin from the Alternaria fungi. AOH was detected for the first time in the wheat pathogen Parastagonospora nodorum in a recent study. Here, we exploited reverse genetics to demonstrate that SNOG_15829 ( SnPKS19 ), a close homolog of Penicillium aethiopicum norlichexanthone (NLX) synthase gene gsfA , is required for AOH production. We further validate that SnPKS19 is solely responsible for AOH production by heterologous expression in Aspergillus nidulans . The expression profile of SnPKS19 based on previous P. nodorum microarray data correlated with the presence of AOH in vitro and its absence in planta . Subsequent characterization of the Δ SnPKS19 mutants showed that SnPKS19 and AOH are not involved in virulence and oxidative stress tolerance. Identification and characterization of the P. nodorum SnPKS19 cast light on a possible alternative AOH synthase gene in Alternaria alternata and allowed us to survey the distribution of AOH synthase genes in other fungal genomes. We further demonstrate that phylogenetic analysis could be used to differentiate between AOH synthases and the closely related NLX synthases. This study provides the basis for studying the genetic regulation of AOH production and for development of molecular diagnostic methods for detecting AOH-producing fungi in the future.