Fungal Genetics and Biology

The Agaricomycotina accounts for roughly a third of all described fungi. They are important due to their wide range of lifestyles and economic and environmental relevance. Certain agaricomycetes act as lignocellulose degraders, playing a significant role in forest ecosystems and bioremediation processes. These wood-decaying fungi have historically been classified as mostly white- or brown-rot based on their ability to degrade lignin, with white-rot fungi possessing a collection of lignocellulose-degrading enzymes, which are reduced or absent in brown-rot fungi. Here, we sequenced and annotated the genome of the agaricomycete Crepidotus cesatii CBS 511.95 and explored its genome and predicted enzymatic content in a comparative context. The 36.04 Mbp genome is in 235 scaffolds, with 3.34% repeat content and 12,891 predicted genes. We found that the PFAM distributions of identified orthogroups suggested that C. cesatii shows patterns more similar to white-rot fungi compared to brown-rot fungi. Additionally, C. cesatii contained multiple copies of CAZymes CBM1 and AA9 involved in hydrolysis of lignocellulose, similar to white-rot fungi. On the other hand, according to the Conserved Unique Peptide Patterns (CUPP) data for AA2 peroxidases, the key enzymes in lignin degradation, C. cesatii is more similar to brown-rot fungi. Based on our analyses we predict that C. cesatii is another representation of the continuum of wood decaying modes between white and brown rot fungi combining genetic features of both types of fungi.

Botrytis cinerea is a necrotrophic plant pathogen with an extremely wide host range. During invasion, the fungus induces rapid host cell death and proliferates in the necrotic tissue. The mechanisms of host killing are still incompletely understood, they involve secretion of lytic enzymes, phytotoxic metabolites and cell death inducing proteins (CDIPs). We have previously shown that the sequential knockout of up to 12 CDIPs leads to a substantial reduction of virulence of B. cinerea mutants. In this study, we have identified additional CDIPs and generated an extended mutant series culminating in a 29x mutant which is deficient in most currently known CDIPs and two phytotoxic metabolites. These mutants are strongly reduced in infection, but still induced necrosis and grey mould symptoms, demonstrating that additional determinants of host killing remain unidentified. Overexpression of the highly phytotoxic Nep1 in a 22-fold CDIP mutant failed to increase its virulence. Reevaluation of several CDIPs previously described as virulence factors revealed for most tested CDIPs no major contribution to pathogenesis. Together with the observation that none of the CDIPs are specific to B. cinerea, our data question a particular role of CDIPs for necrotrophic pathogenesis. In contrast, generation of a mutant lacking all six predicted endo-polygalacturonases confirmed their major but not exclusive role for tissue degradation and infection.

In fungi, hyphal fusion is beneficial within an individual, but fusion between individuals comes with the risks of infection or exploitation. To manage this risk, fungi have developed mechanisms to restrict sustained fusion to be within a genetic individual, called allorecognition. In Ascomycete fungi, this recognition is based on allelic identity at several polymorphic allorecognition genes, often triggering cell death. However, the genetic basis of allorecognition is unknown in basidiomycetes, the clade that includes mushroom-forming fungi. Here, we map the first locus for this trait, which we call somA, in the mushroom-forming fungus Coprinopsis cinerea. We combined F1 offspring phenotypes with independent backcross lines to identify a region on chromosome 5 linked with the production of a barrage zone, a classic allorecognition phenotype. Fine-mapping of this region resulted in a region with a set of kinases and NACHT domain proteins, flanked by a leucine-rich repeat (LRR) protein. While the NACHT and kinase proteins are diverse between the parents, the LRR-encoding protein shows signs of purifying selection. Additional C. cinerea genomes show that this region contains several highly divergent alleles, consistent with long-term balancing selection. These polymorphic alleles all contain a single monomorphic LRR, which may indicate a novel mechanism for fungal nonself recognition. Based on a phylogenetic survey of related Basidiomycetes, this specific locus architecture appears to be restricted to closely related species. This finding of a multiallelic locus may explain the general trend of few nonself recognition loci in basidiomycetes. These results provide a first understanding of how individuality is maintained in basidiomycetes.

Fungal plant pathogens can be affected by the bacteria they interact with in their environment, yet the characterization of these interactions beyond direct antagonism is lacking, especially in the case of endohyphal bacteria (EHB). Though limited in characterized examples, EHB can alter disease severity of their fungal host, providing either a potential tool or target for control. We screened isolates of Fusarium oxysporum f. sp. niveum (FON), an important soil-borne watermelon pathogen, using 16S PCR and fluorescence in situ hybridization microscopy to identify novel EHB. A symbiont of FON AS124 was identified to be a Paenibacillus sp. through genome sequencing and average nucleotide identity. To begin characterizing this relationship, we conducted watermelon infection assays using FON cured of its symbiont, the native association, and a coinoculation of fungi and bacteria. Disease severity was reduced in watermelon seedlings inoculated with the native association, though not in the coinoculation, and Paenibacillus sp. CB74 did not alone promote plant growth or inhibit fungal growth. This study shows an important functional outcome, reduced disease, for a novel symbiosis between FON and Paenibacillus sp. CB74, setting up further investigation into the mechanisms behind this outcome and the application of this interaction. ImportanceFungi pose a challenge in both the field and hospital as antifungal resistance rises and chemical control is increasingly scrutinized. In plant pathogenic fungi, endohyphal bacteria may present alternative targets or mechanisms of fungal control. These relationships are observed across diverse groups of fungi and bacteria, though few have been studied to the point of understanding impact. To contribute to the small but growing catalog of known endofungal bacterial relationships, we identified a novel symbiosis and began characterizing its functional outcomes with plant infection assays. The identified bacterial symbiont does alter disease severity of the fungal host offering a new system for both application and study of fungal pathogenesis.

Leptosphaeria maculans is a plant-pathogenic fungus that infects Brassica species, including Brassica napus (oilseed rape). Breeding oilseed rape varieties with genetic resistance is an efficient way to control the disease; however, L. maculans can adapt and overcome these resistances. Understanding the mechanisms that enable L. maculans to adapt is crucial for managing the emergence of better-adapted isolates. Brassica carinata, the Ethiopian Mustard, although closely related to B. napus, is considered a nonhost species of L. maculans because this fungus cannot infect it. Despite the extreme resistance of B. carinata, one natural L. maculans isolate has been identified as unable to infect B. napus, causing moderate and atypical symptoms on this species. We performed a cross between this isolate and an isolate adapted to B. napus, followed by a QTL analysis, which identified seven QTL, each encompassing candidate genes involved in L. maculans adaptation to B. carinata or B. napus. Additionally, we observed transgression in the progeny, wherein a few strains caused significantly more or less aggressive symptoms on both species of Brassica. We found that epistasis within the L. maculans genome contributes to the observed transgression. These initial findings provide further opportunities to study the adaptive capacities of L. maculans, as well as data to initiate analysis of the extreme resistance of B. carinata to L. maculans. HighlightsO_LISeven pathogenicity QTL identified, carrying several interesting candidate genes C_LIO_LITransgression of some progeny isolates on B. napus and B. carinata was reported C_LIO_LIEpistasis plays a significant role in the adaptation of L. maculans toward host and nonhost Brassica species C_LI

Trichoderma species are widely used as biological control agents due to their ability to parasitize plant pathogens. However, substantial variability in mycoparasitic performance exists among strains, even within the same species, and the underlying molecular mechanisms remain poorly understood. Here, we performed comparative genomic and transcriptomic analyses of six Trichoderma atroviride strains exhibiting contrasting mycoparasitic performance (weakly or highly parasitic; WP or HP) against Alternaria brassicicola, Rhizoctonia solani, and Globisporangium ultimum. Comparative genomics revealed limited strain-specific differences, mainly restricted to NLR (NOD-like receptor) repertoires, with certain NLR-coding genes absent from WP strain genomes compared to HP strains, while overall genomic variation remained low. In contrast, transcriptomic analyses revealed strong differences in gene expression dynamics between HP and WP strains. Co-expression network analysis identified two modules associated with mycoparasitic performance. The first was specifically induced in response to pathogen contact and was enriched in genes encoding cell wall-degrading enzymes, with stronger expression in HP strains. The second module was more broadly overexpressed in HP strains across all conditions and included genes involved in detoxification and defense-related pathways. In addition, this module encompassed genes involved in specialized metabolite biosynthesis and effector-like protein secretion, with WP and HP strains differentially expressing distinct gene subsets within these categories. Together, these results provide a comprehensive framework for identifying the molecular drivers of mycoparasitic performance in T. atroviride. This study deepens our understanding of the functional diversity within the species and establishes a robust foundation for the future development of molecular markers to predict strain efficiency.

Pseudocercospora fijiensis is the causal agent of Black Leaf Streak Disease (BLSD), also known as black Sigatoka, in banana. The disease affects many banana varieties, including the highly susceptible Cavendish banana that dominates global production and the export trade, and several cooking bananas that are a staple food for hundreds of millions of people worldwide. Currently, the disease is controlled using preventative fungicide treatments with up to 70 applications per year in Cavendish plantations, which accounts for approximately 30% of the production costs. Resistant cultivars are required for more sustainable production, but no resistance gene to BLSD has been identified. This is partly due to the poor genetic amenability of P. fijiensis and the lack of methods for functional gene analysis. To address these limitations, we developed a CRISPR/Cas9-mediated transformation system specifically optimized for P. fijiensis. We established a protocol to produce protoplasts, evaluated their capacity to regenerate into new colonies, and assessed antibiotic sensitivity. Subsequently, we confirmed the integration of foreign DNA, including resistance markers, using PEG-mediated transformation. We demonstrated targeted transformation using CRISPR-Cas9 to knockout the polyketide synthase gene PKS10-1, which is responsible for the production of the pigment melanin, and the mitogen-activated protein kinase (MAPK) gene Fus3. Following the successful generation of knockout mutants for these genes, achieving gene targeting efficiencies of respectively 96% and 58%, we subsequently generated knockout mutants of the renowned effector Avr4 in P. fijiensis. The resulting mutants exhibited no reduction in virulence on the susceptible banana cultivar Cavendish. In addition, we used the wild-type isolate and Avr4 knockout strains to test the resistant banana accession Calcutta 4. Contrary to a previous study, we demonstrate that Avr4 does not explain the resistance of Calcutta 4, suggesting that resistance is instead triggered by the recognition of other hitherto unknown effectors. The established CRISPR/Cas9-mediated disruption system is highly efficient and enables routine functional gene characterization, which will help to elucidate genes involved in banana-P. fijiensis interaction, thereby supporting the discovery of resistance genes against BLSD.

Domestication of molds for cheese production has repeatedly shaped Penicillium fungi, most notably giving rise to the emblematic P. camemberti lineage, derived from P. biforme. Here, we identified a new P. biforme lineage, named cheesy, likely selected from P. biforme for food fermentation, including cheese and sausage production. This lineage exhibits evidence of a severe bottleneck, with little nucleotide polymorphism and a single mating type. The cheesy lineage has evolved advantageous traits for cheesemaking: compared to other P. biforme strains and its wild relative P. fuscoglaucum, it displays faster growth on cheese, galactose and lactose media, higher sporulation and germination rates on cheese, elevated lipolytic activity, enhanced inhibition capacities, and produced specific volatile organic compounds. Additionally, P. camemberti and P. biforme cheesy differ in their content of Starship mobile elements, acquired through horizontal transfers. These elements carry cargo genes potentially relevant for adaptation to cheese. Notably, the cheesy lineage has acquired a 20 kb Starship element (Rattus), nested within a much larger 160 kb Starship (Bilge), and carrying cargo genes with predicted functions involved in antagonistic interactions among micro-organisms. SignificanceO_LIWe identified a new Penicillium biforme lineage, named cheesy, which was likely selected from P. biforme for food fermentation (cheese and sausage), genetically and phenotypically different from the emblematic P. camemberti lineage. This lineage suffered from a severe bottleneck, displaying very little nucleotide polymorphism and a single mating type. C_LIO_LIThis newly identified lineage has evolved advantageous traits for cheesemaking; compared to other P. biforme strains and its wild relative P. fuscoglaucum, it displayed faster growth on cheese, galactose and lactose media, higher sporulation and germination rates on cheese, higher lipolytic activity, better inhibition capacities and specific volatile organic compounds. C_LIO_LIPenicillium camemberti and P. biforme cheesy also differed by their content in Starship mobile elements, acquired through horizontal transfers, and carrying cargo genes potentially relevant for adaptation to cheese. C_LIO_LINotably, the cheesy lineage has acquired a specific 20 kb Starship nested within a much larger 160 kb Starship, and likely involved in antagonistic interactions. C_LI

Poly(ADP-ribose) polymerases (PARPs) catalyze ADP-ribosylation, a conserved post- translational modification involved in DNA repair, transcriptional regulation, and chromatin remodeling. Although extensively studied in animals, the evolution and diversification of PARPs across the fungal kingdom remain largely unexplored. Here, we present the first kingdom-wide comparative genomic analysis of PARP proteins across 534 fungal species spanning eight phyla. We identified two primary conserved fungal PARP protein types corresponding to human PARP1 and PARP6. Both exhibit highly dynamic evolutionary histories characterized by frequent independent gain and loss events. Ancestral state reconstruction supports the presence of PARP1 in the last common ancestor of fungi, whereas the PARP6-like family has undergone repeated lineage-specific gains and losses. Fungal PARP6-like proteins retain a compact PARP catalytic domain fused to a C-terminal E2 ubiquitin-conjugating domain, whereas the PARP1 family displays extensive structural diversification through domain shuffling and lineage-specific fusions associated with DNA metabolism, chromatin remodeling, and signal transduction. Reconstruction of ancestral catalytic motifs across fungi and other eukaryotes revealed convergent evolution of a non-canonical H-Y-Y catalytic triad, with multiple motif variants co-occurring within individual proteins, suggesting functional diversification. In the Fusarium oxysporum species complex, we identified a lineage-specific expansion of the PARP family, driven exclusively by accessory chromosomes. Genomes with expanded PARP1 repertoires exhibited elevated basal PARylation, increased resistance to DNA-damaging agents that induce single strand breaks, and DNA damage- induced expression of accessory Parp genes. These findings reveal fungal PARPs as evolutionarily dynamic proteins that likely contribute to genome stability, adaptation, and pathogenicity.

Rhizosphere microbial communities contribute to the growth and health of their host but may be altered by the incidence of soil-borne pathogens. In avocado, the oomycete Phytophthora cinnamomi, causal agent of Phytophthora root rot (PRR), has been shown to alter rhizosphere bacterial communities, although its effect on fungal communities has seldom been explored. Our objective was thus to determine whether P. cinnamomi induced shifts in diversity, composition and co-occurrence networks of fungal communities in the rhizosphere of avocado trees, and to identify potential antagonists of P. cinnamomi that could be further considered for disease management. Fungal communities associated with the rhizosphere of asymptomatic and PRR-symptomatic avocado trees were studied through ITS metabarcoding. Although -diversity metrics were not significantly different between asymptomatic and PRR-symptomatic trees, differences in {beta}-diversity of rhizosphere fungal communities were detected. Moreover, PRR led to the enrichment of saprotrophic taxa and opportunistic pathogens such as Fusarium, Cladosporium or Plectosphaerella in the avocado rhizosphere, which were possibly attracted by the release of resources from necrosed roots. Co-occurrence network analysis revealed that fungal networks in the rhizosphere of PRR-symptomatic trees were more complex and connected than those from asymptomatic trees, suggesting a response of fungal communities to the disturbance caused by the pathogen. Some connector taxa from the PRR-symptomatic networks (Gibellulopsis, Cladorrhinum or Mycenella) were also identified as members of the P. cinnamomi pathobiome. Their negative correlations with the pathogen indicate they may act as potential antagonists, which calls for further isolation efforts to confirm their biocontrol activity of PRR.

Seed transmission is a critical pathway for the dispersal of phytopathogenic bacteria. This transmission can occur through three main routes: floral, internal, and external. Yet the relative contribution of individual transmission routes remains poorly characterized. Using a pathosystem based on Xanthomonas citri pv. fuscans (Xcf) and common bean (Phaseolus vulgaris cv. Flavert), we quantified the efficiency of each route. Under our experimental conditions, the vascular route was the most efficient with 25% of contaminated seeds and population sizes averaging 107 CFU per contaminated seed. Deploying this experimental framework to ten seed-borne bacterial strains isolated from bean revealed that almost none transmitted to seeds through any route, or at best at low efficiency. However, most of the strains were capable of surviving and disseminating within the vascular system. A major bottleneck for seed transmission was identified for pod vascular organs colonization and the similar behavior of an Xcf mutant, deficient in the T3SS, suggested that plant immunity could be involved at this step. Co-inoculation of a consortium composed of the seed-borne strains with Xcf reduced the number of seeds contaminated by Xcf at the highest inoculum concentration, although other consortia members were never recovered from seeds. This suggests that the strains are recognized by the plant and trigger defense responses. These findings also raise questions about the mechanisms used by seed-associated bacteria to colonize seeds in situ.

Ada1 (All Development Altered-1) is a conserved but poorly characterised basic leucine zipper (bZIP) transcription factor found throughout filamentous fungi. In the wheat pathogen Parastagonospora nodorum, PnAda1 is required for full virulence and is transcriptionally associated with the virulence regulator PnPf2, but its biological functions remain unclear. Here, we combined comparative RNA sequencing with targeted phenotypic analyses to define the role of PnAda1 during vegetative growth and host infection. Deletion of PnAda1 did not abolish pathogenicity but delayed disease progression, with the PnAda1-deletion mutant transcriptome at 7 days post-inoculation resembling that of the wildtype SN15 at 3 days. This developmental delay was associated with impaired activation of early infection-associated genes, including putative carbohydrate-active enzymes, proteases, transporters and other host-colonisation factors. In contrast, expression of major necrotrophic effector genes was not reduced and instead remained elevated during later stages of infection, indicating that PnAda1 is required for the timely progression of infection-associated transcriptional regulation rather than direct activation of effector genes. Beyond virulence, transcriptomic and phenotypic analyses revealed roles for PnAda1 in nitrogen assimilation, carbon utilisation, abiotic stress responses and fungicide sensitivity. Notably, PnAda1 deletion increased sensitivity to succinate dehydrogenase inhibitor fungicides and reduced expression of succinate dehydrogenase subunit genes. Collectively, our findings identify PnAda1 as a broad regulator of developmental and infection-associated transitions in P. nodorum and expand current understanding of the transcriptional network underlying virulence, metabolism and stress adaptation in an important fungal wheat pathogen.

During mycological surveys in Yunnan Province, China, specimens of a fungus producing massive, upright stromata up to 50 cm high and individually 2.2 Kg in weight were sampled. Through an integrative taxonomic approach combining detailed morphology, multilocus phylogeny (ITS, LSU, RPB2, TUB2), and phylogenomic analyses, this fungus is proposed as the new species Dianjunus rex gen. et sp. nov., the type of the new family Dianjunaceae (Xylariales). Phylogenetic analyses robustly place Dianjunaceae as a distinct sister clade to Graphostromataceae. Divergence time estimation dates the origin of this family to the early Paleocene (~65 Mya), coinciding with the post-K-Pg extinction period, when an estimated 75% of all plant and animal species went extinct, and a significant ecological reorganization of life on earth happened. The stromata of D. rex represent the largest fructifications documented within the Ascomycota, significantly expanding the known morphological range of the Xylariales. The study provides a comprehensive description, including a nodulisporium-like anamorph with periconiella-like branching patterns, and discusses the taxon's phylogenetic placement, and distinctive morphology. This discovery highlights the unexplored fungal diversity in East Asian forests.

The fungal pathogen Pyricularia oryzae is notorious for causing blast disease in various important cereal crops, including wheat, rice, millet, and oat. Whole-genome-informed data on this pathogen are necessary to better understand the host adaptability of the fungus, including identifying key determinants of infection to enable more precise disease control. Here, we report highly contiguous genome sequences (using long-read PacBio technology) of two isolates from rice paddies in Portugal, M22.7 and T22.2, which exhibit distinctly aggressive symptoms in rice. Both mitochondrial and nuclear sequences were characterised in this study. The resulting nuclear genomes have assembly lengths of 46.4 Mb for M22.7 (198x coverage) and 46.3 Mb for T22.2 (163x coverage), with near-complete BUSCO completeness (98.8%) and a 0% contamination score (EukCC). Phenotypic analysis showed M22.7 to be more virulent than T22.2, which may be explained by the lower number of predicted effector genes and higher transposable element content in M22.7 relative to T22.2. This announcement represents the first genome resource for natural isolates of P. oryzae from Portugal in over 20 years, filling an important data gap from a major European rice-producing country that produces locally adapted rice varieties under specific agro-environmental conditions (near the Atlantic coast).

A highly contiguous and complete reference genome of Cercospora cf. flagellaris, the causal agent of foliar disease on many plant hosts including Cercospora leaf blight of soybean, was assembled using a combination of PacBio and Illumina sequencing reads. The genome assembly is 33.72 Mb in length and consists of 14 nuclear scaffolds and one mitochondrial contig. Four scaffolds have telomeric repeats on both ends and represent fully assembled chromosomes, while nine scaffolds represent partially assembled chromosomes with telomeric repeats on one end. The assembly has an N50 of 2.90 Mb and an L50 of 5 scaffolds. Genome annotation identified 11,268 genes, of which 947 and 360 were predicted to encode secreted proteins and effectors, respectively. Additionally, 512 genes were predicted to encode carbohydrate-active enzymes and 60 biosynthetic gene clusters were annotated. Taken together, this annotated genome assembly will be a valuable resource for genomics, host-pathogen interactions, and population biology research in this economically important pathosystem.

Colletotrichum graminicola causes significant losses of the staple crop maize worldwide. The fungus produces two distinct asexual spore types, oval and falcate conidia, which show unique processes in development and plant interaction. Based on genome resequencing of our laboratory strain (CgM2/ M1.001), we investigated the gene expression profiles of oval and falcate conidia during development and early leaf infection using RNA-seq. Our results reveal specific gene expression profiles between the two spore types, indicating fundamental differences in their developmental programs that reflect different modes of infection. We identified expression patterns discriminating both conidia types from mycelium and spore type-specific ones for genes encoding transcription factors, conserved fungal developmental genes, transporters, genes of secondary metabolite clusters, and pathogenicity-related functions, including effectors and carbohydrate-active enzymes (CAZymes). Our study shows that despite the identical genomic basis, oval and falcate conidia show unique transcriptomes across vegetative development and early plant interaction. Taking together, these results provide new insights into the molecular mechanisms determining the biology of C. graminicola and its interaction with the plant host.

Septoria nodorum blotch (SNB) and tan spot (TS) wheat diseases are caused by necrotrophic fungal pathogens Parastagonospora nodorum (Pn) and Pyrenophora tritici-repentis (Ptr), respectively. Although recognised as premier model pathosystems for our understanding of necrotrophic effectors, no resistance mechanism has been reported in both diseases. Here, two SNB and TS resistance wheat lines ( 56:ZWB11 and 105:ZIF14) derived from the Australian national germplasm evaluation programme (CAIGE) were used to develop a double haploid mapping population. Two Pn and Ptr isolates of different pathotypes, their respective culture filtrates and effector SnTox267 were evaluated on the population. Genetic analysis of Ptr conidial inoculation of race 1 and race 2 identified a major resistance quantitative trait locus (QTL) (QTs.cur-1B) on chromosome 1B, while resistance to SNB was explained by several minor QTL. SnTox267 sensitivity was mapped to six locations (2A2, 2A3, 2B1, 2D3, 5B and 7B1) with only one QTL co-localized to known corresponding gene Snn7. Sensitivity loci 5B and 7B1 also conferred SNB resistance at seedling and adult stages. Two QTL on chromosome 2D1 and 7B2 were common in both SNB and TS, associated with disease at seedling stage and culture filtrate bioactivity, respectively. Resistance responses of 56:ZWB11 and 105:ZIF14 were confirmed cytologically, however, distinct responses were observed on wounded leaves. The defence responses were more effective against Ptr, while resistance to Pn infection was likely a combination of lack of susceptibility and effective physical barriers. Overall results demonstrated the distinction between the underlying resistance mechanisms to TS and SNB.

The contribution of accessory or conditionally dispensable chromosomes to host-specific virulence was first demonstrated in members of the Fusarium solani species complex (FSSC) that are pathogens of garden pea, Pisum sativum L. The phenomenon has since been shown to exist in many fungal plant pathogens, including the closely related F. oxysporum species complex (FOSC). Genome analysis of members of the FSSC and FOSC pathogenic on pea revealed a diverse size range of the accessory genome of these fungi. Despite the ~65 million years of diverging time, regions on a chromosome known to carry host-specific virulence factors for pea, including the cytochrome P450 pisatin demethylase (PDA) and other pea pathogenicity (PEP) genes, were present in all genomes of these pea pathogens. Genes directly involved in virulence on pea - PEP2, PDA, and PEP5- were the most frequently clustered together. Transcriptome analysis of fungal mycelia treated with the pea phytoalexin pisatin, identified 1,155 differentially expressed genes where many were involved in cellular stress responses. As wilt pathogens that invade host xylem, members of the FOSC encode more putative effectors, when compared to those in the FSSC, and several FOSC effectors were identified to confer race specificity. The conservation of part of the accessory genomes across two evolutionarily diverged species complexes suggests a common origin. Horizontal transfer of accessory chromosomes containing genetic loci involved in pathogenesis for garden pea offers a parsimonious explanation of the polyphyletic origin of host specificity.

Spores are the primary means of fungal reproduction, contributing to genetic diversity, colonization, and adaptation. Although spore formation is a pivotal part of the fungal life cycle, its genetic underpinnings remain poorly known. In this study, we characterize transcriptomic changes from late meiosis to early basidiospore formation in the mushroom-forming fungus Coprinopsis cinerea, decipher several cellular processes, and identify novel genes involved in this process. We identify distinct trajectories of gene expression, each of which display different functional signals, corresponding to meiotic and morphogenetic processes and transitions between these. Our analyses identify diverse arrays of fungal cell wall modifying carbohydrate-active enzymes, ferritins, a putative catechol-melanin synthesis pathway, as well as components of the mitotic/meiotic apparatus. We present twelve highly conserved genes with roles specific to sexual sporulation in both budding yeast and C. cinerea, indicating deep conservation of the gene networks driving sexual spore formation. Reverse genetics identified three conserved but functionally poorly characterized genes conferring sporeless and spore-poor phenotypes that result from postmeiotic developmental arrests stemming from spore inflation and nuclear migration problems. Overall, this study provides novel insight into basidiomycete spore formation and highlights the cornucopia of novel functions encoded by functionally dark genes.

The coprophilous agaricomycete Coprinopsis cinerea is a model organism for antagonistic fungal-bacterial interactions. Previous studies showed that C. cinerea responds to antagonistic bacteria with strong induction of a set of genes encoding secreted antibacterial molecules. However, little is known about the elicitors of this response. Key open questions in this respect include whether individual antibacterial defence genes are induced by different bacteria and/or by specific bacterial soluble molecules. Here, we present a new C. cinerea reporter system to monitor antibacterial defence induction and address related outstanding issues with minimal hands-on time. In this system, the promoter of the endogenous bacterial-induced gene cclys1 drives the expression of cnluc, which encodes a secreted variant of the deep-sea shrimp luciferase Nluc. We show that cNluc allows to detect and quantify cclys1 induction by measuring luminescence directly in the culture medium of reporter strain colonies. Building on these features, we successfully leveraged the inducible cNluc reporter strain for the development of a novel 96-well plate assay that allows the high-throughput screening of antibacterial defence elicitors. As cNluc can be subject to degradation by secreted proteases of fungal or bacterial origin in the culture medium, we coupled this assay to confirmatory qRT-PCR. Testing this set-up by confronting the reporter strain with several different bacteria revealed that cclys1 induction occurs independently of the bacterial ecological niche. Based on these results, we also recommend qRT-PCR exclusively for validation of negative results. We conclude that cNluc offers significant advantages over cytoplasmic reporter proteins, especially for preliminary rapid screening of multiple conditions.