The bZIP transcription factor BIP1 of the rice blast fungus is essential for infection and regulates a specific set of appressorium genes.

The rice blast fungus Magnaporthe oryzae differentiates specialized cells called appressoria that are required for fungal penetration into host leaves. In this study, we identified the novel basic leucine zipper (bZIP) transcription factor BIP1 (B-ZIP Involved in Pathogenesis-1) that is essential for pathogenicity. BIP1 is required for the infection of plant leaves, even if they are wounded, but not for appressorium-mediated penetration of artificial cellophane membranes. This phenotype suggests that BIP1 is not implicated in the differentiation of the penetration peg but is necessary for the initial establishment of the fungus within plant cells. BIP1 expression was restricted to the appressorium by both transcriptional and post-transcriptional control. Genome-wide transcriptome analysis showed that 40 genes were down regulated in a BIP1 deletion mutant. Most of these genes were specifically expressed in the appressorium. They encode proteins with pathogenesis-related functions such as enzymes involved in secondary metabolism including those encoded by the ACE1 gene cluster, small secreted proteins such as SLP2, BAS2, BAS3, and AVR-Pi9 effectors, as well as plant cuticle and cell wall degrading enzymes. Interestingly, this BIP1 network is different from other known infection-related regulatory networks, highlighting the complexity of gene expression control during plant-fungal interactions. Promoters of BIP1-regulated genes shared a GCN4/bZIP-binding DNA motif (TGACTC) binding in vitro to BIP1. Mutation of this motif in the promoter of MGG_08381.7 from the ACE1 gene cluster abolished its appressorium-specific expression, showing that BIP1 behaves as a transcriptional activator. In summary, our findings demonstrate that BIP1 is critical for the expression of early invasion-related genes in appressoria. These genes are likely needed for biotrophic invasion of the first infected host cell, but not for the penetration process itself. Through these mechanisms, the blast fungus strategically anticipates the host plant environment and responses during appressorium-mediated penetration.

Evolutionary relationships of adenylation domains in fungi.

Non-ribosomal peptide synthetases (NRPSs) and NRPS-like enzymes are abundant in microbes as they are involved in the production of primary and secondary metabolites. In contrast to the well-studied NRPSs, known to produce non-ribosomal peptides, NRPS-like enzymes exhibit more diverse activities and their evolutionary relationships are unclear. Here, we present the first in-depth phylogenetic analysis of fungal NRPS-like A domains from functionally characterized pathways, and their relationships to characterized A domains found in fungal NRPSs. This study clearly differentiated amino acid reductases, including NRPSs, from CoA/AMP ligases, which could be divided into 10 distinct phylogenetic clades that reflect their conserved domain organization, substrate specificity and enzymatic activity. In particular, evolutionary relationships of adenylate forming reductases could be refined and explained the substrate specificity difference. Consistent with their phylogeny, the deduced amino acid code of A domains differentiated amino acid reductases from other enzymes. However, a diagnostic code was found for α-keto acid reductases and clade 7 CoA/AMP ligases only. Comparative genomics of loci containing these enzymes revealed that they can be independently recruited as tailoring genes in diverse secondary metabolite pathways. Based on these results, we propose a refined and clear phylogeny-based classification of A domain-containing enzymes, which will provide a robust framework for future functional analyses and engineering of these enzymes to produce new bioactive molecules.

The histone code of the fungal genus Aspergillus uncovered by evolutionary and proteomic analyses.

Chemical modifications of DNA and histone proteins impact the organization of chromatin within the nucleus. Changes in these modifications, catalysed by different chromatin-modifying enzymes, influence chromatin organization, which in turn is thought to impact the spatial and temporal regulation of gene expression. While combinations of different histone modifications, the histone code, have been studied in several model species, we know very little about histone modifications in the fungal genus Aspergillus, whose members are generally well studied due to their importance as models in cell and molecular biology as well as their medical and biotechnological relevance. Here, we used phylogenetic analyses in 94 Aspergilli as well as other fungi to uncover the occurrence and evolutionary trajectories of enzymes and protein complexes with roles in chromatin modifications or regulation. We found that these enzymes and complexes are highly conserved in Aspergilli, pointing towards a complex repertoire of chromatin modifications. Nevertheless, we also observed few recent gene duplications or losses, highlighting Aspergillus species to further study the roles of specific chromatin modifications. SET7 (KMT6) and other components of PRC2 (Polycomb Repressive Complex 2), which is responsible for methylation on histone H3 at lysine 27 in many eukaryotes including fungi, are absent in Aspergilli as well as in closely related Penicillium species, suggesting that these lost the capacity for this histone modification. We corroborated our computational predictions by performing untargeted MS analysis of histone post-translational modifications in Aspergillus nidulans. This systematic analysis will pave the way for future research into the complexity of the histone code and its functional implications on genome architecture and gene regulation in fungi.

A genomic journey in the secondary metabolite diversity of fungal plant and insect pathogens: from functional to population genomics.

Fungal pathogens produce a broad array of secondary metabolites (SMs), which allow the fungus to thrive in its natural habitat and gain competitive advantage. Analysis of the genetically encoded blueprints for SM assembly highlighted that only a small portion of the SMs these fungi are capable of producing are known, and even fewer have been investigated for their natural function. Using molecular tools, a lot of progress has been made recently in identifying the blueprint products and linking them to their ecological purpose such as the peptide virulence factor fusaoctaxin A released by Fusarium graminearum during infection of wheat or the F. oxysporum polyketide bikaverin that provides competitive advantage against bacteria in tomato. In addition, population genomics have given particularly important insights into the species-specific plasticity of the SM blueprint arsenal, showcasing the ongoing evolution and adaptation of fungal pathogens. This approach holds promise in inferring roles in pathogenicity of many more fungal SMs.

Characterization of NRPS and PKS genes involved in the biosynthesis of SMs in Alternaria dauci including the phytotoxic polyketide aldaulactone.

Alternaria dauci is a Dothideomycete fungus, causal agent of carrot leaf blight. As a member of the Alternaria genus, known to produce a lot of secondary metabolite toxins, A. dauci is also supposed to synthetize host specific and non-host specific toxins playing a crucial role in pathogenicity. This study provides the first reviewing of secondary metabolism genetic basis in the Alternaria genus by prediction of 55 different putative core genes. Interestingly, aldaulactone, a phytotoxic benzenediol lactone from A. dauci, was demonstrated as important in pathogenicity and in carrot partial resistance to this fungus. As nothing is known about aldaulactone biosynthesis, bioinformatic analyses on a publicly available A. dauci genome data set that were reassembled, thanks to a transcriptome data set described here, allowed to identify 19 putative secondary metabolism clusters. We exploited phylogeny to pinpoint cluster 8 as a candidate in aldaulactone biosynthesis. This cluster contains AdPKS7 and AdPKS8, homologs with genes encoding a reducing and a non-reducing polyketide synthase. Clusters containing such a pair of PKS genes have been identified in the biosynthesis of resorcylic acid lactones or dihydroxyphenylacetic acid lactones. AdPKS7 and AdPKS8 gene expression patterns correlated with aldaulactone production in different experimental conditions. The present results highly suggest that both genes are responsible for aldaulactone biosynthesis.

A Bioinformatics Workflow for Investigating Fungal Biosynthetic Gene Clusters.

Predicting secondary metabolite biosynthetic gene clusters is a routine analysis performed for each newly sequenced fungal genome. Yet, the usefulness of such predictions remains restricted as they provide total numbers of biosynthetic pathways with only very limited biological significance. In this chapter, we describe a workflow to predict and analyze biosynthetic gene clusters in fungal genomes. It relies on similarity networking and phylogeny to perform genetic dereplication and to prioritize candidate gene clusters that potentially produce new compounds. This basic workflow includes the generation of high-quality figures for publication.

Evolution-Informed Discovery of the Naphthalenone Biosynthetic Pathway in Fungi.

Fungi produce a wide diversity of secondary metabolites with interesting biological activities for the health, industrial, and agricultural sectors. While fungal genomes have revealed an unexpectedly high number of biosynthetic pathways that far exceeds the number of known molecules, accessing and characterizing this hidden diversity remain highly challenging. Here, we applied a combined phylogenetic dereplication and comparative genomics strategy to explore eight lichenizing fungi. The determination of the evolutionary relationships of aromatic polyketide pathways resulted in the identification of an uncharacterized biosynthetic pathway that is conserved in distant fungal lineages. The heterologous expression of the homologue from Aspergillus parvulus linked this pathway to naphthalenone compounds, which were detected in cultures when the pathway was expressed. Our unbiased and rational strategy generated evolutionary knowledge that ultimately linked biosynthetic genes to naphthalenone polyketides. Applied to many more genomes, this approach can unlock the full exploitation of the fungal kingdom for molecule discovery. IMPORTANCE Fungi have provided us with life-changing small bioactive molecules, with the best-known examples being the first broad-spectrum antibiotic penicillin, immunosuppressive cyclosporine, and cholesterol-lowering statins. Since the 1980s, exploration of chemical diversity in nature has been highly reduced. However, the genomic era has revealed that fungal genomes are concealing an unexpected and largely unexplored chemical diversity. So far, fungal genomes have been exploited to predict the production potential of bioactive compounds or to find genes that control the production of known molecules of interest. But accessing and characterizing the full fungal chemical diversity require rational and, thus, efficient strategies. Our approach is to first determine the evolutionary relationships of fungal biosynthetic pathways in order to identify those that are already characterized and those that show a different evolutionary origin. This knowledge allows prioritizing the choice of the pathway to functionally characterize in a second stage using synthetic-biology tools like heterologous expression. A particular strength of this strategy is that it is always successful: it generates knowledge about the evolution of bioactive-molecule biosynthesis in fungi, it either yields novel molecules or links the studied pathway to already known molecules, and it reveals the chemical diversity within a given pathway, all at once. The strategy is very powerful to avoid studying the same pathway again and can be used with any fungal genome. Functional characterization using heterologous expression is particularly suitable for fungi that are difficult to grow or not genetically tractable. Thanks to the decreasing cost of gene synthesis, ultimately, only the genome sequence is needed to identify novel pathways and characterize the molecules that they produce. Such an evolution-informed strategy allows the efficient exploitation of the chemical diversity hidden in fungal genomes and is very promising for molecule discovery.

Hybridizations Between formae speciales of Venturia inaequalis Pave the Way for a New Biocontrol Strategy to Manage Fungal Plant Pathogens.

Hybridization and adaptation to new hosts are important mechanisms of fungal disease emergence. Evaluating the risk of emergence of hybrids with enhanced virulence is then key to develop sustainable crop disease management. We evaluated this risk in Venturia inaequalis, the fungus responsible for the common and serious scab disease on Rosaceae hosts, including apple, pyracantha, and loquat. Field isolates from these three hosts and progenies obtained from five crosses between formae speciales isolates collected from pyracantha (f. sp. pyracantha) and apple (f. sp. pomi) were tested for their pathogenicity on the three hosts. We confirmed a strict host specificity between isolates from apple and pyracantha and showed that most isolates were able to cause disease on loquat. None of the 251 progeny obtained from five crosses between V. inaequalis f. sp. pyracantha and V. inaequalis f. sp. pomi could infect apple. If confirmed on more crosses, the inability of the hybrids to infect apple could lead to a novel biocontrol strategy based on a sexual hijacking of V. inaequalis f. sp. pomi by a massive introduction of V. inaequalis f. sp. pyracantha in apple orchards. This strategy, analogous to the sterile insect approach, could lead to the collapse of the population size of V. inaequalis and dramatically reduce the use of chemicals in orchards.

Developing fungal heterologous expression platforms to explore and improve the production of natural products from fungal biodiversity.

Natural products from fungi represent an important source of biologically active metabolites notably for therapeutic agent development. Genome sequencing revealed that the number of biosynthetic gene clusters (BGCs) in fungi is much larger than expected. Unfortunately, most of them are silent or barely expressed under laboratory culture conditions. Moreover, many fungi in nature are uncultivable or cannot be genetically manipulated, restricting the extraction and identification of bioactive metabolites from these species. Rapid exploration of the tremendous number of cryptic fungal BGCs necessitates the development of heterologous expression platforms, which will facilitate the efficient production of natural products in fungal cell factories. Host selection, BGC assembly methods, promoters used for heterologous gene expression, metabolic engineering strategies and compartmentalization of biosynthetic pathways are key aspects for consideration to develop such a microbial platform. In the present review, we summarize current progress on the above challenges to promote research effort in the relevant fields.

MIBiG 2.0: a repository for biosynthetic gene clusters of known function.

Fueled by the explosion of (meta)genomic data, genome mining of specialized metabolites has become a major technology for drug discovery and studying microbiome ecology. In these efforts, computational tools like antiSMASH have played a central role through the analysis of Biosynthetic Gene Clusters (BGCs). Thousands of candidate BGCs from microbial genomes have been identified and stored in public databases. Interpreting the function and novelty of these predicted BGCs requires comparison with a well-documented set of BGCs of known function. The MIBiG (Minimum Information about a Biosynthetic Gene Cluster) Data Standard and Repository was established in 2015 to enable curation and storage of known BGCs. Here, we present MIBiG 2.0, which encompasses major updates to the schema, the data, and the online repository itself. Over the past five years, 851 new BGCs have been added. Additionally, we performed extensive manual data curation of all entries to improve the annotation quality of our repository. We also redesigned the data schema to ensure the compliance of future annotations. Finally, we improved the user experience by adding new features such as query searches and a statistics page, and enabled direct link-outs to chemical structure databases. The repository is accessible online at https://mibig.secondarymetabolites.org/.

The High-Quality Complete Genome Sequence of the Opportunistic Fungal Pathogen Candida vulturna CBS 14366T.

Candida vulturna is a new member of the Candida haemulonii species complex that recently received much attention as it includes the emerging multidrug-resistant pathogen Candida auris. Here, we describe the high-quality genome sequence of C. vulturna type strain CBS 14366T to cover all genomes of pathogenic C. haemulonii species complex members.

Chromatin-dependent regulation of secondary metabolite biosynthesis in fungi: is the picture complete?

Fungal secondary metabolites are small molecules that exhibit diverse biological activities exploited in medicine, industry and agriculture. Their biosynthesis is governed by co-expressed genes that often co-localize in gene clusters. Most of these secondary metabolite gene clusters are inactive under laboratory conditions, which is due to a tight transcriptional regulation. Modifications of chromatin, the complex of DNA and histone proteins influencing DNA accessibility, play an important role in this regulation. However, tinkering with well-characterised chemical and genetic modifications that affect chromatin alters the expression of only few biosynthetic gene clusters, and thus the regulation of the vast majority of biosynthetic pathways remains enigmatic. In the past, attempts to activate silent gene clusters in fungi mainly focused on histone acetylation and methylation, while in other eukaryotes many other post-translational modifications are involved in transcription regulation. Thus, how chromatin regulates the expression of gene clusters remains a largely unexplored research field. In this review, we argue that focusing on only few well-characterised chromatin modifications is significantly hampering our understanding of the chromatin-based regulation of biosynthetic gene clusters. Research on underexplored chromatin modifications and on the interplay between different modifications is timely to fully explore the largely untapped reservoir of fungal secondary metabolites.

Population Genome Sequencing of the Scab Fungal Species Venturia inaequalis, Venturia pirina, Venturia aucupariae and Venturia asperata.

The Venturia genus comprises fungal species that are pathogens on Rosaceae host plants, including V. inaequalis and V. asperata on apple, V. aucupariae on sorbus and V. pirina on pear. Although the genetic structure of V. inaequalis populations has been investigated in detail, genomic features underlying these subdivisions remain poorly understood. Here, we report whole genome sequencing of 87 Venturia strains that represent each species and each population within V. inaequalis We present a PacBio genome assembly for the V. inaequalis EU-B04 reference isolate. The size of selected genomes was determined by flow cytometry, and varied from 45 to 93 Mb. Genome assemblies of V. inaequalis and V. aucupariae contain a high content of transposable elements (TEs), most of which belong to the Gypsy or Copia LTR superfamilies and have been inactivated by Repeat-Induced Point mutations. The reference assembly of V. inaequalis presents a mosaic structure of GC-equilibrated regions that mainly contain predicted genes and AT-rich regions, mainly composed of TEs. Six pairs of strains were identified as clones. Single-Nucleotide Polymorphism (SNP) analysis between these clones revealed a high number of SNPs that are mostly located in AT-rich regions due to misalignments and allowed determining a false discovery rate. The availability of these genome sequences is expected to stimulate genetics and population genomics research of Venturia pathogens. Especially, it will help understanding the evolutionary history of Venturia species that are pathogenic on different hosts, a history that has probably been substantially influenced by TEs.

Draft Genome Sequence of the Wood-Staining Ascomycete Chlorociboria aeruginascens DSM 107184.

Chlorociboria aeruginascens DSM 107184 is a wood-decomposing ascomycetous fungus known to produce the bluish-green dimeric naphthoquinone derivate xylindein. Here, we present the first draft genome sequence, which contains 588 contigs with a total length of 33.1 Mb. Altogether, 8,648 protein-coding genes were predicted.

Nonproteinaceous effectors: the terra incognita of plant-fungal interactions.

Molecular plant-fungal interaction studies have mainly focused on small secreted protein effectors. However, accumulating evidence shows that numerous fungal secondary metabolites are produced at all stages of plant colonization, especially during early asymptomatic/biotrophic phases. The discovery of fungal small RNAs targeting plant transcripts has expanded the fungal repertoire of nonproteinaceous effectors even further. The challenge now is to develop specific functional methods to fully understand the biological roles of these effectors. Studies on fungal extracellular vesicles are also needed because they could be the universal carriers of all kinds of fungal effectors. With this review, we aim to stimulate the nonproteinaceous effector research field to move from descriptive to functional studies, which should bring a paradigm shift in plant-fungal interactions.

Evolutionary Histories of Type III Polyketide Synthases in Fungi.

Type III polyketide synthases (PKSs) produce secondary metabolites with diverse biological activities, including antimicrobials. While they have been extensively studied in plants and bacteria, only a handful of type III PKSs from fungi has been characterized in the last 15 years. The exploitation of fungal type III PKSs to produce novel bioactive compounds requires understanding the diversity of these enzymes, as well as of their biosynthetic pathways. Here, phylogenetic and reconciliation analyses of 522 type III PKSs from 1,193 fungal genomes revealed complex evolutionary histories with massive gene duplications and losses, explaining their discontinuous distribution in the fungal tree of life. In addition, horizontal gene transfer events from bacteria to fungi and, to a lower extent, between fungi, could be inferred. Ancestral gene duplication events have resulted in the divergence of eight phylogenetic clades. Especially, two clades show ancestral linkage and functional co-evolution between a type III PKS and a reducing PKS genes. Investigation of the occurrence of protein domains in fungal type III PKS predicted gene clusters highlighted the diversity of biosynthetic pathways, likely reflecting a large chemical landscape. Type III PKS genes are most often located next to genes encoding cytochrome P450s, MFS transporters and transcription factors, defining ancestral core gene clusters. This analysis also allowed predicting gene clusters for the characterized fungal type III PKSs and provides working hypotheses for the elucidation of the full biosynthetic pathways. Altogether, our analyses provide the fundamental knowledge to motivate further characterization and exploitation of fungal type III PKS biosynthetic pathways.

Down-regulation of cladofulvin biosynthesis is required for biotrophic growth of Cladosporium fulvum on tomato.

Fungal biotrophy is associated with a reduced capacity to produce potentially toxic secondary metabolites (SMs). Yet, the genome of the biotrophic plant pathogen Cladosporium fulvum contains many SM biosynthetic gene clusters, with several related to toxin production. These gene clusters are, however, poorly expressed during the colonization of tomato. The sole detectable SM produced by C. fulvum during in vitro growth is the anthraquinone cladofulvin. Although this pigment is not detected in infected leaves, cladofulvin biosynthetic genes are expressed throughout the pre-penetration phase and during conidiation at the end of the infection cycle, but are repressed during the biotrophic phase of tomato colonization. It has been suggested that the tight regulation of SM gene clusters is required for C. fulvum to behave as a biotrophic pathogen, whilst retaining potential fitness determinants for growth and survival outside its host. To address this hypothesis, we analysed the disease symptoms caused by mutant C. fulvum strains that do not produce or over-produce cladofulvin during the biotrophic growth phase. Non-producers infected tomato in a similar manner to the wild-type, suggesting that cladofulvin is not a virulence factor. In contrast, the cladofulvin over-producers caused strong necrosis and desiccation of tomato leaves, which, in turn, arrested conidiation. Consistent with the role of pigments in survival against abiotic stresses, cladofulvin protects conidia against UV light and low-temperature stress. Overall, this study demonstrates that the repression of cladofulvin production is required for C. fulvum to sustain its biotrophic lifestyle in tomato, whereas its production is important for survival outside its host.

Assignment of a dubious gene cluster to melanin biosynthesis in the tomato fungal pathogen Cladosporium fulvum.

Pigments and phytotoxins are crucial for the survival and spread of plant pathogenic fungi. The genome of the tomato biotrophic fungal pathogen Cladosporium fulvum contains a predicted gene cluster (CfPKS1, CfPRF1, CfRDT1 and CfTSF1) that is syntenic with the characterized elsinochrome toxin gene cluster in the citrus pathogen Elsinoë fawcettii. However, a previous phylogenetic analysis suggested that CfPks1 might instead be involved in pigment production. Here, we report the characterization of the CfPKS1 gene cluster to resolve this ambiguity. Activation of the regulator CfTSF1 specifically induced the expression of CfPKS1 and CfRDT1, but not of CfPRF1. These co-regulated genes that define the CfPKS1 gene cluster are orthologous to genes involved in 1,3-dihydroxynaphthalene (DHN) melanin biosynthesis in other fungi. Heterologous expression of CfPKS1 in Aspergillus oryzae yielded 1,3,6,8-tetrahydroxynaphthalene, a typical precursor of DHN melanin. Δcfpks1 deletion mutants showed similar altered pigmentation to wild type treated with DHN melanin inhibitors. These mutants remained virulent on tomato, showing this gene cluster is not involved in pathogenicity. Altogether, our results showed that the CfPKS1 gene cluster is involved in the production of DHN melanin and suggests that elsinochrome production in E. fawcettii likely involves another gene cluster.

Specific Hypersensitive Response-Associated Recognition of New Apoplastic Effectors from Cladosporium fulvum in Wild Tomato.

Tomato leaf mold disease is caused by the biotrophic fungus Cladosporium fulvum. During infection, C. fulvum produces extracellular small secreted protein (SSP) effectors that function to promote colonization of the leaf apoplast. Resistance to the disease is governed by Cf immune receptor genes that encode receptor-like proteins (RLPs). These RLPs recognize specific SSP effectors to initiate a hypersensitive response (HR) that renders the pathogen avirulent. C. fulvum strains capable of overcoming one or more of all cloned Cf genes have now emerged. To combat these strains, new Cf genes are required. An effectoromics approach was employed to identify wild tomato accessions carrying new Cf genes. Proteomics and transcriptome sequencing were first used to identify 70 apoplastic in planta-induced C. fulvum SSPs. Based on sequence homology, 61 of these SSPs were novel or lacked known functional domains. Seven, however, had predicted structural homology to antimicrobial proteins, suggesting a possible role in mediating antagonistic microbe-microbe interactions in planta. Wild tomato accessions were then screened for HR-associated recognition of 41 SSPs, using the Potato virus X-based transient expression system. Nine SSPs were recognized by one or more accessions, suggesting that these plants carry new Cf genes available for incorporation into cultivated tomato.