Aspergillus flavus pangenome (AflaPan) uncovers novel aflatoxin and secondary metabolite associated gene clusters.

BACKGROUND: Aspergillus flavus is an important agricultural and food safety threat due to its production of carcinogenic aflatoxins. It has high level of genetic diversity that is adapted to various environments. Recently, we reported two reference genomes of A. flavus isolates, AF13 (MAT1-2 and highly aflatoxigenic isolate) and NRRL3357 (MAT1-1 and moderate aflatoxin producer). Where, an insertion of 310 kb in AF13 included an aflatoxin producing gene bZIP transcription factor, named atfC. Observations of significant genomic variants between these isolates of contrasting phenotypes prompted an investigation into variation among other agricultural isolates of A. flavus with the goal of discovering novel genes potentially associated with aflatoxin production regulation. Present study was designed with three main objectives: (1) collection of large number of A. flavus isolates from diverse sources including maize plants and field soils; (2) whole genome sequencing of collected isolates and development of a pangenome; and (3) pangenome-wide association study (Pan-GWAS) to identify novel secondary metabolite cluster genes. RESULTS: Pangenome analysis of 346 A. flavus isolates identified a total of 17,855 unique orthologous gene clusters, with mere 41% (7,315) core genes and 59% (10,540) accessory genes indicating accumulation of high genomic diversity during domestication. 5,994 orthologous gene clusters in accessory genome not annotated in either the A. flavus AF13 or NRRL3357 reference genomes. Pan-genome wide association analysis of the genomic variations identified 391 significant associated pan-genes associated with aflatoxin production. Interestingly, most of the significantly associated pan-genes (94%; 369 associations) belonged to accessory genome indicating that genome expansion has resulted in the incorporation of new genes associated with aflatoxin and other secondary metabolites. CONCLUSION: In summary, this study provides complete pangenome framework for the species of Aspergillus flavus along with associated genes for pathogen survival and aflatoxin production. The large accessory genome indicated large genome diversity in the species A. flavus, however AflaPan is a closed pangenome represents optimum diversity of species A. flavus. Most importantly, the newly identified aflatoxin producing gene clusters will be a new source for seeking aflatoxin mitigation strategies and needs new attention in research.

Development of a telomere vector-based approach to overcome limitations caused by lethal phenotypes in the study of essential genes in Magnaporthe oryzae.

Reverse genetic approaches are common tools in genomics for elucidating gene functions, involving techniques such as gene deletion followed by screening for aberrant phenotypes. If the generation of gene deletion mutants fails, the question arises whether the failure stems from technical issues or because the gene of interest (GOI) is essential, meaning that the deletion causes lethality. In this report, we introduce a novel method for assessing gene essentiality using the phytopathogenic ascomycete Magnaporthe oryzae. The method is based on the observation that telomere vectors are lost in transformants during cultivation without selection pressure. We tested the hypothesis that essential genes can be identified in deletion mutants co-transformed with a telomere vector. The M. oryzae gene MoPKC, described in literature as essential, was chosen as GOI. Using CRISPR/Cas9 technology transformants with deleted GOI were generated and backed up by a telomere vector carrying a copy of the GOI and conferring fenhexamid resistance. Transformants in which the GOI deletion in the genome was not successful lost the telomere vector on media without fenhexamid. In contrast, transformants with confirmed GOI deletion retained the telomere vector even in absence of fenhexamid selection. In the latter case, the maintenance of the telomere indicates that the GOI is essential for the surveillance of the fungi, as it would have been lost otherwise. The method presented here allows to test for essentiality of genes when no mutants can be obtained from gene deletion approaches, thereby expanding the toolbox for studying gene function in ascomycetes.

Selection of stable reference genes for qPCR expression of Colletotrichum lindemuthianum, the bean anthracnose pathogen.

Phaseolus vulgaris L., commonly known as the common bean, is a highly nutritious crop often called the "poor man's meat". However, it is susceptible to various diseases throughout the cropping season, with anthracnose caused by Colletotrichum lindemuthianum being a significant threat that leads to substantial losses. There is still a lack of understanding about the molecular basis of C. lindemuthianum pathogenicity. The first step in understanding this is to identify pathogenicity genes that express more during infection of common beans. A reverse transcription quantitative real-time PCR (qPCR) method can be used for virulence gene expression. However, this approach requires selecting appropriate reference genes to normalize relative gene expression data. Currently, there is no reference gene available for C. lindemuthianum. In this study, we selected eight candidate reference genes from the available genome of C. lindemuthianum to bridge the gap. These genes were ACT (Actin), ß-tub (ß-tubulin), EF (Elongation Factor), Cyt C (Cytochrome C), His H3 (Histone H3), CHS1 (Chitin synthetase), GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) and abfA (Alpha-l-Arabinofuranosidase A). The primers for these candidate reference genes were able to amplify cDNA only from the pathogen, demonstrating their specificity. The qPCR efficiency of the primers ranged from 80% to 103%. We analyzed the stability of gene expression in C. lindemuthianum by exposing the mycelium to nine different stress conditions. We employed algorithms, such as GeNorm, NormFinder, BestKeeper, and RefFinder tools, to identify the most stable gene. The analysis using these tools revealed that EF, GAPDH, and ß-tub most stable genes, while ACT and CHS1 showed relatively low expression stability. A large number of potential effector genes have been identified through bioinformatics analysis in C. lindemuthianum. The stable genes for qPCR (EF and GAPDH) discovered in this study will aid the scientific community in determining the relative expression of C. lindemuthianum effector genes.

The Narrow Footprint of Ancient Balancing Selection Revealed by Heterokaryon Incompatibility Genes in Aspergillus fumigatus.

In fungi, fusion between individuals leads to localized cell death, a phenomenon termed heterokaryon incompatibility. Generally, the genes responsible for this incompatibility are observed to be under balancing selection resulting from negative frequency-dependent selection. Here, we assess this phenomenon in Aspergillus fumigatus, a human pathogenic fungus with a very low level of linkage disequilibrium as well as an extremely high crossover rate. Using complementation of auxotrophic mutations as an assay for hyphal compatibility, we screened sexual progeny for compatibility to identify genes involved in this process, called het genes. In total, 5/148 (3.4%) offspring were compatible with a parent and 166/2,142 (7.7%) sibling pairs were compatible, consistent with several segregating incompatibility loci. Genetic mapping identified five loci, four of which could be fine mapped to individual genes, of which we tested three through heterologous expression, confirming their causal relationship. Consistent with long-term balancing selection, trans-species polymorphisms were apparent across several sister species, as well as equal allele frequencies within A. fumigatus. Surprisingly, a sliding window genome-wide population-level analysis of an independent dataset did not show increased Tajima's D near these loci, in contrast to what is often found surrounding loci under balancing selection. Using available de novo assemblies, we show that these balanced polymorphisms are restricted to several hundred base pairs flanking the coding sequence. In addition to identifying the first het genes in an Aspergillus species, this work highlights the interaction of long-term balancing selection with rapid linkage disequilibrium decay.

FGCD: a database of fungal gene clusters related to secondary metabolism.

Fungal secondary metabolites are not necessary for growth, but they are important for fungal metabolism and ecology because they provide selective advantages for competition, survival and interactions with the environment. These various metabolites are widely used as medicinal precursors and insecticides. Secondary metabolism genes are commonly arranged in clusters along chromosomes, which allow for the coordinate control of complete pathways. In this study, we created the Fungal Gene Cluster Database to store, retrieve, and visualize secondary metabolite gene cluster information across fungal species. The database was created by merging data from RNA sequencing, Basic Local Alignment Search Tool, genome browser, enrichment analysis and the R Shiny web framework to visualize and query putative gene clusters. This database facilitated the rapid and thorough examination of significant gene clusters across fungal species by detecting, defining and graphically displaying the architecture, organization and expression patterns of secondary metabolite gene clusters. In general, this genomic resource makes use of the tremendous chemical variety of the products of these ecologically and biotechnologically significant gene clusters to our further understanding of fungal secondary metabolism. Database URL: https://www.hebaubioinformatics.cn/FungalGeneCluster/.

Identification of two genes essential for basidiospore formation during the postmeiotic stages in Pleurotus ostreatus.

A sporeless strain is an important breeding target in the mushroom industry. However, basidiospore production in the oyster mushroom Pleurotus ostreatus has been shown to be impaired by single-gene mutations in only two meiosis-related genes, mer3 and msh4. This study proposed a strategy for identifying the genes essential for basidiospore formation after meiotic division to determine new targets for molecular breeding. RNA-seq analysis was performed to identify P. ostreatus genes that are specifically expressed in the gill tissue of fruiting bodies, where basidiospore formation occurs. Transcriptome data during fruiting development of Coprinopsis cinerea, in which the meiotic steps progress synchronously, were then used to identify genes that are active in the postmeiotic stages. Based on these comparative analyses, five P. ostreatus genes were identified. Plasmids containing expression cassettes for hygromycin B-resistance screening, Cas9, and single-guide RNA targeting each gene were introduced into the protoplasts of dikaryotic strain, PC9×#64, to generate dikaryotic gene disruptants. Among the obtained transformants, three dikaryotic pcl1 disruptants and two cro6c disruptants did not produce basidiospores. Microscopic analyses indicated that spore formation was arrested at particular stages in these gene disruptants. These results indicate that these two genes are essential for mature spore formation in this fungus.

Functional and Evolutionary Integration of a Fungal Gene With a Bacterial Operon.

Siderophores are crucial for iron-scavenging in microorganisms. While many yeasts can uptake siderophores produced by other organisms, they are typically unable to synthesize siderophores themselves. In contrast, Wickerhamiella/Starmerella (W/S) clade yeasts gained the capacity to make the siderophore enterobactin following the remarkable horizontal acquisition of a bacterial operon enabling enterobactin synthesis. Yet, how these yeasts absorb the iron bound by enterobactin remains unresolved. Here, we demonstrate that Enb1 is the key enterobactin importer in the W/S-clade species Starmerella bombicola. Through phylogenomic analyses, we show that ENB1 is present in all W/S clade yeast species that retained the enterobactin biosynthetic genes. Conversely, it is absent in species that lost the ent genes, except for Starmerella stellata, making this species the only cheater in the W/S clade that can utilize enterobactin without producing it. Through phylogenetic analyses, we infer that ENB1 is a fungal gene that likely existed in the W/S clade prior to the acquisition of the ent genes and subsequently experienced multiple gene losses and duplications. Through phylogenetic topology tests, we show that ENB1 likely underwent horizontal gene transfer from an ancient W/S clade yeast to the order Saccharomycetales, which includes the model yeast Saccharomyces cerevisiae, followed by extensive secondary losses. Taken together, these results suggest that the fungal ENB1 and bacterial ent genes were cooperatively integrated into a functional unit within the W/S clade that enabled adaptation to iron-limited environments. This integrated fungal-bacterial circuit and its dynamic evolution determine the extant distribution of yeast enterobactin producers and cheaters.

Analysis of five near-complete genome assemblies of the tomato pathogen Cladosporium fulvum uncovers additional accessory chromosomes and structural variations induced by transposable elements effecting the loss of avirulence genes.

BACKGROUND: Fungal plant pathogens have dynamic genomes that allow them to rapidly adapt to adverse conditions and overcome host resistance. One way by which this dynamic genome plasticity is expressed is through effector gene loss, which enables plant pathogens to overcome recognition by cognate resistance genes in the host. However, the exact nature of these loses remains elusive in many fungi. This includes the tomato pathogen Cladosporium fulvum, which is the first fungal plant pathogen from which avirulence (Avr) genes were ever cloned and in which loss of Avr genes is often reported as a means of overcoming recognition by cognate tomato Cf resistance genes. A recent near-complete reference genome assembly of C. fulvum isolate Race 5 revealed a compartmentalized genome architecture and the presence of an accessory chromosome, thereby creating a basis for studying genome plasticity in fungal plant pathogens and its impact on avirulence genes. RESULTS: Here, we obtained near-complete genome assemblies of four additional C. fulvum isolates. The genome assemblies had similar sizes (66.96 to 67.78 Mb), number of predicted genes (14,895 to 14,981), and estimated completeness (98.8 to 98.9%). Comparative analysis that included the genome of isolate Race 5 revealed high levels of synteny and colinearity, which extended to the density and distribution of repetitive elements and of repeat-induced point (RIP) mutations across homologous chromosomes. Nonetheless, structural variations, likely mediated by transposable elements and effecting the deletion of the avirulence genes Avr4E, Avr5, and Avr9, were also identified. The isolates further shared a core set of 13 chromosomes, but two accessory chromosomes were identified as well. Accessory chromosomes were significantly smaller in size, and one carried pseudogenized copies of two effector genes. Whole-genome alignments further revealed genomic islands of near-zero nucleotide diversity interspersed with islands of high nucleotide diversity that co-localized with repeat-rich regions. These regions were likely generated by RIP, which generally asymmetrically affected the genome of C. fulvum. CONCLUSIONS: Our results reveal new evolutionary aspects of the C. fulvum genome and provide new insights on the importance of genomic structural variations in overcoming host resistance in fungal plant pathogens.

Genomic insights into Penicillium chrysogenum adaptation to subseafloor sedimentary environments.

BACKGROUND: Penicillium chrysogenum is a filamentous fungal species with diverse habitats, yet little is known about its genetics in adapting to extreme subseafloor sedimental environments. RESULTS: Here, we report the discovery of P. chrysogenum strain 28R-6-F01, isolated from deep coal-bearing sediments 2306 m beneath the seafloor. This strain possesses exceptional characteristics, including the ability to thrive in extreme conditions such as high temperature (45 °C), high pressure (35 Mpa), and anaerobic environments, and exhibits broad-spectrum antimicrobial activity, producing the antibiotic penicillin at a concentration of 358 µg/mL. Genome sequencing and assembly revealed a genome size of 33.19 Mb with a GC content of 48.84%, containing 6959 coding genes. Comparative analysis with eight terrestrial strains identified 88 unique genes primarily associated with penicillin and aflatoxins biosynthesis, carbohydrate degradation, viral resistance, and three secondary metabolism gene clusters. Furthermore, significant expansions in gene families related to DNA repair were observed, likely linked to the strain's adaptation to its environmental niche. CONCLUSIONS: Our findings provide insights into the genomic and biological characteristics of P. chrysogenum adaptation to extreme anaerobic subseafloor sedimentary environments, such as high temperature and pressure.

Whole genome sequencing and annotation of Aspergillus flavus JAM-JKB-B HA-GG20.

Groundnuts are mostly contaminated with the mold Aspergillus flavus which produces a carcinogenic mycotoxin called as aflatoxin. It is very important to understand the genetic factors underlying its pathogenicity, regulation, and biosynthesis of secondary metabolites and animal toxicities, but it still lacks useful information due to certain gaps in the era of modern technology. Therefore, the present study was considered to determine the key genes and metabolites involved in the biosynthesis of aflatoxin by using a molecular approach in a virulent strain of Aspergillus. The whole genome sequence of highly toxic and virulent Aspergillus isolates JAM-JKB-B HA-GG20 revealed 3,73,54,834 bp genome size, 2, 26, 257 number of contigs with N50 value of 49,272 bp, 12,400 genes and 48.1% of GC contained respectively. The genome sequence was compared with other known aflatoxin producing and non-producing genome of Aspergillus spp. and 61 secondary metabolite (SM) gene clusters were annotated with the toxic strain JAM-JKB-BHA-GG20 which showed similarity with other Aspergillus spp. A total number of eight genes (ver-1, AflR, pksA, uvm8, omt1, nor-1, Vha and aflP) were identified related to biosynthesis of aflatoxin and ochratoxin. Also, 69 SSR with forward and reverse primers and 137 di and tri nucleotide motifs were identified in the nucleotide sequence region related to aflatoxin gene pathway. The genes and putative metabolites identified in this study are potentially involved in host invasion and pathogenicity. As such, the genomic information obtained in this study is helpful in understanding aflatoxin gene producing pathway in comparison to other Aspergillus spp. and predicted presence of other secondary metabolites clusters viz. Nrps, T1pks etc. genes associated with a biosynthesis of OTA mycotoxin.

Large-scale population survey of Leptosphaeria maculans in France highlights both on-going breakdowns and potentially effective resistance genes in oilseed rape.

BACKGROUND: Leptosphaeria maculans, the cause of stem canker of oilseed rape, develops gene-for-gene interactions with its host and shows a high evolutionary potential to 'break down' novel resistance genes (R, Rlm) deployed in cultivars over large areas. For optimal management of R genes, updated knowledge of the population structure of the pathogen is needed. In France, large-scale surveys have been done at 10-year intervals since 2000. Here we report the characterization of a large L. maculans population collected in France in 2019-2020. RESULTS: A total of 844 isolates were collected from 11 sites in ten French departments and were phenotyped for their virulence against nine Brassica napus R genes. All isolates were virulent toward Rlm2 and Rlm9. Very few isolates were avirulent on Rlm1 (1.8%) and Rlm4 (0.6%). Avirulent isolates toward Rlm7 ('AvrLm7') varied from 67% to 11.3%, depending on the site sampled, illustrating the ongoing breakdown of Rlm7. The decrease of AvrLm7 isolates (29.2% at the national level) compared to the 2010 survey (96.5%) was accompanied by an increase of avirulent isolates on Rlm3 (0% in 2010; 54% in 2019-2020). However, virulent isolates on both Rlm3 and Rlm7, previously rarely detected, were found in all sites with a frequency of 17.3%. Finally, most or all isolates were avirulent on Rlm11 (96.1%), LepR2 (RlmS, 99.8%), and Rlm6 (100%), suggesting these three genes still effectively control the disease. CONCLUSION: These data will help guide strategies for breeding and deploying resistant oilseed rape varieties against L. maculans in France. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Multifunctionality of AsCFEM6 and AsCFEM12 effectors from the potato early blight pathogen Alternaria solani.

Plant pathogens secrete fungal-specific common in several fungal extracellular membrane (CFEM) effectors to manipulate host immunity and contribute to their virulence. Little is known about effectors and their functions in Alternaria solani, the necrotrophic fungal pathogen causing potato early blight. To identify candidate CFEM effector genes, we mined A. solani genome databases. This led to the identification of 12 genes encoding CFEM proteins (termed AsCFEM1-AsCFEM12) and 6 of them were confirmed to be putative secreted effectors. In planta expression revealed that AsCFEM6 and AsCFEM12 have elicitor function that triggers plant defense response including cell death in different botanical families. Targeted gene disruption of AsCFEM6 and AsCFEM12 resulted in a change in spore development, significant reduction of virulence on potato and eggplant susceptible cultivars, increased resistance to fungicide stress, variation in iron acquisition and utilization, and the involvement in 1,8-dihydroxynaphthalene (DHN) melanin biosynthesis pathway. Using maximum likelihood method, we found that positive selection likely caused the polymorphism within AsCFEM6 and AsCFEM12 homologs in different Alternaria spp. Site-directed mutagenesis analysis indicated that positive selection sites within their CFEM domains are required for cell death induction in Nicotiana benthamiana and are critical for response to abiotic stress in yeast. These results demonstrate that AsCFEM effectors possess additional functions beyond their roles in host plant immune response and pathogen virulence.

Complexity of fungal polyketide biosynthesis and function.

Where does one draw the line between primary and secondary metabolism? The answer depends on the perspective. Microbial secondary metabolites (SMs) were at first believed not to be very important for the producers because they are dispensable for growth under laboratory conditions. However, such compounds become important in natural niches of the organisms, and some are of prime importance for humanity. Polyketides are an important group of SMs with aflatoxin as a well-known and well-characterized example. In Aspergillus spp., all 34 afl genes encoding the enzymes for aflatoxin biosynthesis are located in close vicinity on chromosome III in a so-called gene cluster. This led to the assumption that most genes required for polyketide biosynthesis are organized in gene clusters. Recent research, however, revealed an enormous complexity of the biosynthesis of different polyketides, ranging from individual polyketide synthases to a gene cluster producing several compounds, or to several clusters with additional genes scattered in the genome for the production of one compound. Research of the last decade furthermore revealed a huge potential for SM biosynthesis hidden in fungal genomes, and methods were developed to wake up such sleeping genes. The analysis of organismic interactions starts to reveal some of the ecological functions of polyketides for the producing fungi.

A rapid PCR-based method to determine the Neurospora crassa mating type.

So far mating type determination in Neurospora crassa requires test crosses with strains of known mating type. We present a simple, quick, and reliable polymerase chain reaction-based method for mating type determination in N. crassa.

Lineage-specific genes are clustered with HET-domain genes and respond to environmental and genetic manipulations regulating reproduction in Neurospora.

Lineage-specific genes (LSGs) have long been postulated to play roles in the establishment of genetic barriers to intercrossing and speciation. In the genome of Neurospora crassa, most of the 670 Neurospora LSGs that are aggregated adjacent to the telomeres are clustered with 61% of the HET-domain genes, some of which regulate self-recognition and define vegetative incompatibility groups. In contrast, the LSG-encoding proteins possess few to no domains that would help to identify potential functional roles. Possible functional roles of LSGs were further assessed by performing transcriptomic profiling in genetic mutants and in response to environmental alterations, as well as examining gene knockouts for phenotypes. Among the 342 LSGs that are dynamically expressed during both asexual and sexual phases, 64% were detectable on unusual carbon sources such as furfural, a wildfire-produced chemical that is a strong inducer of sexual development, and the structurally-related furan 5-hydroxymethyl furfural (HMF). Expression of a significant portion of the LSGs was sensitive to light and temperature, factors that also regulate the switch from asexual to sexual reproduction. Furthermore, expression of the LSGs was significantly affected in the knockouts of adv-1 and pp-1 that regulate hyphal communication, and expression of more than one quarter of the LSGs was affected by perturbation of the mating locus. These observations encouraged further investigation of the roles of clustered lineage-specific and HET-domain genes in ecology and reproduction regulation in Neurospora, especially the regulation of the switch from the asexual growth to sexual reproduction, in response to dramatic environmental conditions changes.

Building a eukaryotic chromosome arm by de novo design and synthesis.

The genome of an organism is inherited from its ancestor and continues to evolve over time, however, the extent to which the current version could be altered remains unknown. To probe the genome plasticity of Saccharomyces cerevisiae, here we replace the native left arm of chromosome XII (chrXIIL) with a linear artificial chromosome harboring small sets of reconstructed genes. We find that as few as 12 genes are sufficient for cell viability, whereas 25 genes are required to recover the partial fitness defects observed in the 12-gene strain. Next, we demonstrate that these genes can be reconstructed individually using synthetic regulatory sequences and recoded open-reading frames with a "one-amino-acid-one-codon" strategy to remain functional. Finally, a synthetic neochromsome with the reconstructed genes is assembled which could substitute chrXIIL for viability. Together, our work not only highlights the high plasticity of yeast genome, but also illustrates the possibility of making functional eukaryotic chromosomes from entirely artificial sequences.

Extending the reach of homology by using successive computational filters to find yeast pheromone genes.

The mating of fungi depends on pheromones that mediate communication between two mating types. Most species use short peptides as pheromones, which are either unmodified (e.g., α-factor in Saccharomyces cerevisiae) or C-terminally farnesylated (e.g., a-factor in S. cerevisiae). Peptide pheromones have been found by genetics or biochemistry in a small number of fungi, but their short sequences and modest conservation make it impossible to detect homologous sequences in most species. To overcome this problem, we used a four-step computational pipeline to identify candidate a-factor genes in sequenced genomes of the Saccharomycotina, the fungal clade that contains most of the yeasts: we require that candidate genes have a C-terminal prenylation motif, are shorter than 100 amino acids long, and contain a proteolytic-processing motif upstream of the potential mature pheromone sequence and that closely related species contain highly conserved homologs of the potential mature pheromone sequence. Additional manual curation exploits the observation that many species carry more than one a-factor gene, encoding identical or nearly identical pheromones. From 332 Saccharomycotina genomes, we identified strong candidate pheromone genes in 241 genomes, covering 13 clades that are each separated from each other by at least 100 million years, the time required for evolution to remove detectable sequence homology among small pheromone genes. For one small clade, the Yarrowia, we demonstrated that our algorithm found the a-factor genes: deleting all four related genes in the a-mating type of Yarrowia lipolytica prevents mating.

A fungal sesquiterpene biosynthesis gene cluster critical for mutualist-pathogen transition in Colletotrichum tofieldiae.

Plant-associated fungi show diverse lifestyles from pathogenic to mutualistic to the host; however, the principles and mechanisms through which they shift the lifestyles require elucidation. The root fungus Colletotrichum tofieldiae (Ct) promotes Arabidopsis thaliana growth under phosphate limiting conditions. Here we describe a Ct strain, designated Ct3, that severely inhibits plant growth. Ct3 pathogenesis occurs through activation of host abscisic acid pathways via a fungal secondary metabolism gene cluster related to the biosynthesis of sesquiterpene metabolites, including botrydial. Cluster activation during root infection suppresses host nutrient uptake-related genes and changes mineral contents, suggesting a role in manipulating host nutrition state. Conversely, disruption or environmental suppression of the cluster renders Ct3 beneficial for plant growth, in a manner dependent on host phosphate starvation response regulators. Our findings indicate that a fungal metabolism cluster provides a means by which infectious fungi modulate lifestyles along the parasitic-mutualistic continuum in fluctuating environments.

The Bcvic1 and Bcvic2 vegetative incompatibility genes in Botrytis cinerea encode proteins with domain architectures involved in allorecognition in other filamentous fungi.

Vegetative incompatibility is a fungal allorecognition system characterised by the inability of genetically distinct conspecific fungal strains to form a viable heterokaryon and is controlled by multiple polymorphic loci termed vic (vegetative incompatibility) or het (heterokaryon incompatibility). We have genetically identified and characterised the first vic locus in the economically important, plant-pathogenic, necrotrophic fungus Botrytis cinerea. A bulked segregant approach coupled with whole genome Illumina sequencing of near-isogenic lines of B. cinerea was used to map a vic locus to a 60-kb region of the genome. Within that locus, we identified two adjacent, highly polymorphic open reading frames, Bcvic1 and Bcvic2, which encode predicted proteins that contain domain architectures implicated in vegetative incompatibility in other filamentous fungi. Bcvic1 encodes a predicted protein containing a putative serine esterase domain, a NACHT family of NTPases domain, and several Ankyrin repeats. Bcvic2 encodes a putative syntaxin protein containing a SNARE domain; such proteins typically function in vesicular transport. Deletion of Bcvic1 and Bcvic2 individually had no effect on vegetative incompatibility. However, deletion of the region containing both Bcvic1 and Bcvic2 resulted in mutant lines that were severely restricted in growth and showed loss of vegetative incompatibility. Complementation of these mutants by ectopic expression restored the growth and vegetative incompatibility phenotype, indicating that Bcvic1 and Bcvic2 are controlling vegetative incompatibility at this vic locus.