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.

Update on the state of research to manage Fusarium head blight.

Fusarium head blight (FHB) is one of the most devastating diseases of cereal crops, causing severe reduction in yield and quality of grain worldwide. In the United States, the major causal agent of FHB is the mycotoxigenic fungus, Fusarium graminearum. The contamination of grain with mycotoxins, including deoxynivalenol and zearalenone, is a particularly serious concern due to its impact on the health of humans and livestock. For the past few decades, multidisciplinary studies have been conducted on management strategies designed to reduce the losses caused by FHB. However, effective management is still challenging due to the emergence of fungicide-tolerant strains of F. graminearum and the lack of highly resistant wheat and barley cultivars. This review presents multidisciplinary approaches that incorporate advances in genomics, genetic-engineering, new fungicide chemistries, applied biocontrol, and consideration of the disease cycle for management of FHB.

Endophytic Fungi as a Promising Biocontrol Agent to Protect Wheat from Fusarium graminearum Head Blight.

The search for beneficial endophytes that can be part of a constructed microbial community has increased in recent years. We characterized three endophytic fungi previously isolated from wheat for their in vitro and in planta antagonism toward the Fusarium head blight pathogen, Fusarium graminearum. The endophytes were phylogenetically characterized and shown to be Alternaria destruens, Fusarium commune, and Fusarium oxysporum. Individual fungal endophytes significantly increased seed weight and lowered the accumulation of the mycotoxin deoxynivalenol compared with F. graminearum-infected wheat heads without endophyte pretreatment. Investigation into the mechanism of competition in vitro showed that endophytes competitively excluded F. graminearum by preemptive colonization and possible inhibition over a distance. Investigations on the use of these endophytes in the field are in progress. Identification of these three endophytes highlights a common quandary in searching for beneficial microbes to use in agriculture: species definitions often do not separate individual isolates' lifestyles. A greater understanding of the risks in using intraspecies variants for biocontrol is needed and should be examined in the context of the ecology of the individuals being investigated.

The ancestral levels of transcription and the evolution of sexual phenotypes in filamentous fungi.

Changes in gene expression have been hypothesized to play an important role in the evolution of divergent morphologies. To test this hypothesis in a model system, we examined differences in fruiting body morphology of five filamentous fungi in the Sordariomycetes, culturing them in a common garden environment and profiling genome-wide gene expression at five developmental stages. We reconstructed ancestral gene expression phenotypes, identifying genes with the largest evolved increases in gene expression across development. Conducting knockouts and performing phenotypic analysis in two divergent species typically demonstrated altered fruiting body development in the species that had evolved increased expression. Our evolutionary approach to finding relevant genes proved far more efficient than other gene deletion studies targeting whole genomes or gene families. Combining gene expression measurements with knockout phenotypes facilitated the refinement of Bayesian networks of the genes underlying fruiting body development, regulation of which is one of the least understood processes of multicellular development.

Structure and Chemical Analysis of Major Specialized Metabolites Produced by the Lichen Evernia prunastri.

We performed comparative profiling of four specialized metabolites in the lichen Evernia prunastri, collected at three different geographic locations, California and Maine, USA, and Yoshkar Ola, Mari El, Russia. Among the compounds produced at high concentrations that were identified in all three specimens, evernic acid, usnic acid, lecanoric acid and chloroatranorin, evernic acid was the most abundant. Two depsidones, salazinic acid and physodic acid, were detected in the Yoshkar-Ola collection only. The crystalline structure of evernic acid (2-hydroxy-4-[(2-hydroxy-4-methoxy-6-methylbenzoyl)oxy]-6-methylbenzoate) (hmb) revealed two crystallographically and conformationally distinct hmb anions, along with two monovalent sodium atoms. One hmb moiety contained an exotetradentate binding mode to sodium, whereas the other exhibited an exohexadentate binding mode to sodium. Embedded edge-sharing {Na2 O8 }n sodium-oxygen chains connected the hmb anions into the full three-dimensional crystal structure of the title compound. The crystal used for single-crystal X-ray diffraction exhibited non-merohedral twinning. The data suggest the importance of the acetyl-polymalonyl pathway products to processes of maintaining integrity of the lichen holobiont community.

Light sensing by opsins and fungal ecology: NOP-1 modulates entry into sexual reproduction in response to environmental cues.

Understanding the genetic basis of the switch from asexual to sexual lifestyles in response to sometimes rapid environmental changes is one of the major challenges in fungal ecology. Light appears to play a critical role in the asexual-sexual switch-but fungal genomes harbour diverse light sensors. Fungal opsins are homologous to bacterial green-light-sensory rhodopsins, and their organismal functions in fungi have not been well understood. Three of these opsin-like proteins were widely distributed across fungal genomes, but homologs of the Fusarium opsin-like protein CarO were present only in plant-associated fungi. Key amino acids, including potential retinal binding sites, functionally diverged on the phylogeny of opsins. This diversification of opsin-like proteins could be correlated with life history-associated differences among fungi in their expression and function during morphological development. In Neurospora crassa and related species, knockout of the opsin NOP-1 led to a phenotype in the regulation of the asexual-sexual switch, modulating response to both light and oxygen conditions. Sexual development commenced early in ∆nop-1 strains cultured in unsealed plates under constant blue and white light. Furthermore, comparative transcriptomics showed that the expression of nop-1 is light-dependent and that the ∆nop-1 strain abundantly expresses genes involved in oxidative stress response, genes enriched in NAD/NADP binding sites, genes with functions in proton transmembrane movement and catalase activity, and genes involved in the homeostasis of protons. Based on these observations, we contend that light and oxidative stress regulate the switch via light-responsive and ROS pathways in model fungus N. crassa and other fungi.

Fusarium pathogenomics.

Fusarium is a genus of filamentous fungi that contains many agronomically important plant pathogens, mycotoxin producers, and opportunistic human pathogens. Comparative analyses have revealed that the Fusarium genome is compartmentalized into regions responsible for primary metabolism and reproduction (core genome), and pathogen virulence, host specialization, and possibly other functions (adaptive genome). Genes involved in virulence and host specialization are located on pathogenicity chromosomes within strains pathogenic to tomato (Fusarium oxysporum f. sp. lycopersici) and pea (Fusarium 'solani' f. sp. pisi). The experimental transfer of pathogenicity chromosomes from F. oxysporum f. sp. lycopersici into a nonpathogen transformed the latter into a tomato pathogen. Thus, horizontal transfer may explain the polyphyletic origins of host specificity within the genus. Additional genome-scale comparative and functional studies are needed to elucidate the evolution and diversity of pathogenicity mechanisms, which may help inform novel disease management strategies against fusarial pathogens.

Compression tests of Fusarium graminearum ascocarps provide insights into the strength of the perithecial wall and the quantity of ascospores.

The plant pathogenic ascomycete Fusarium graminearum produces perithecia on corn and small grain residues. These perithecia forcibly discharge ascospores into the atmosphere. Little is known about the relationship among the strength of the perithecial wall, the age of the perithecium, and the quantity of ascospores produced. We used a mechanical compression testing instrument to examine the structural failure rate of perithecial walls from three different strains of F. graminearum (two wild type strains, and a mutant strain unable to produce asci). The force required to compress a perithecium by one micrometer (the mean perithecium compression constant, MPCC) was used to determine the strength of the perithecial wall. Over the course of perithecial maturation (5-12days after the initiation of perithecial development), the MPCC was compared to the number of ascospores contained inside the perithecia. The MPCC increased as perithecia matured, from 0.06Nµm-1 at 5d to 0.12Nµm-1 at 12d. The highest number of ascospores was found in older perithecia (12d). The results indicated that for every additional day of perithecial aging, the perithecia become more resilient to compression forces. Every additional day of perithecial aging resulted in ∼900 more ascospores. Knowledge of how perithecia respond to external forces may provide insight into the development of ascospores and the accumulation of turgor pressure. In the future, compression testing may provide a unique method of determining perithecial age in the field, which could extend to management practices that are informed by knowledge of ascospore release and dispersal.

Global gene expression and focused knockout analysis reveals genes associated with fungal fruiting body development in Neurospora crassa.

Fungi can serve as highly tractable models for understanding genetic basis of sexual development in multicellular organisms. Applying a reverse-genetic approach to advance such a model, we used random and multitargeted primers to assay gene expression across perithecial development in Neurospora crassa. We found that functionally unclassified proteins accounted for most upregulated genes, whereas downregulated genes were enriched for diverse functions. Moreover, genes associated with developmental traits exhibited stage-specific peaks of expression. Expression increased significantly across sexual development for mating type gene mat a-1 and for mat A-1 specific pheromone precursor ccg-4. In addition, expression of a gene encoding a protein similar to zinc finger, stc1, was highly upregulated early in perithecial development, and a strain with a knockout of this gene exhibited arrest at the same developmental stage. A similar expression pattern was observed for genes in RNA silencing and signaling pathways, and strains with knockouts of these genes were also arrested at stages of perithecial development that paralleled their peak in expression. The observed stage specificity allowed us to correlate expression upregulation and developmental progression and to identify regulators of sexual development. Bayesian networks inferred from our expression data revealed previously known and new putative interactions between RNA silencing genes and pathways. Overall, our analysis provides a fine-scale transcriptomic landscape and novel inferences regarding the control of the multistage development process of sexual crossing and fruiting body development in N. crassa.

Role of Fig1, a component of the low-affinity calcium uptake system, in growth and sexual development of filamentous fungi.

The function of Fig1, a transmembrane protein of the low-affinity calcium uptake system (LACS) in fungi, was examined for its role in the growth and development of the plant pathogen Fusarium graminearum. The Δfig1 mutants failed to produce mature perithecia, and sexual development was halted prior to the formation of perithecium initials. The loss of Fig1 function also resulted in a reduced vegetative growth rate. Macroconidium production was reduced 70-fold in the Δfig1 mutants compared to the wild type. The function of the high-affinity calcium uptake system (HACS), comprised of the Ca(2+) channels Mid1 and Cch1, was previously characterized for F. graminearum. To better understand the roles of the LACS and the HACS, Δfig1 Δmid1, Δfig1 Δcch1, and Δfig1 Δmid1 Δcch1 double and triple mutants were generated, and the phenotypes of these mutants were more severe than those of the Δfig1 mutants. Pathogenicity on wheat was unaffected for the Δfig1 mutants, but the Δfig1 Δmid1, Δfig1 Δcch1, and Δfig1 Δmid1 Δcch1 mutants, lacking both LACS and HACS functions, had reduced pathogenicity. Additionally, Δfig1 mutants of Neurospora crassa were examined and did not affect filamentous growth or female fertility in a Δfig1 mating type A strain, but the Δfig1 mating type a strain failed to produce fertile fruiting bodies. These results are the first report of Fig1 function in filamentous ascomycetes and expand its role to include complex fruiting body and ascus development.

Comparative Transcriptomics of Fusarium graminearum and Magnaporthe oryzae Spore Germination Leading up To Infection.

For fungal plant pathogens, the germinating spore provides the first interaction with the host. Spore germlings move across the plant surface and use diverse penetration strategies for ingress into plant surfaces. Penetration strategies include pressurized melanized appressoria, which facilitate physically punching through the plant cuticle, and nonmelanized appressoria, which penetrate with the help of enzymes or cuticular damage to breach the plant surface. Two well-studied plant pathogens, Fusarium graminearum and Magnaporthe oryzae, are typical of these two modes of penetration. We applied comparative transcriptomics to Fusarium graminearum and Magnaporthe oryzae to characterize the genetic programming of the early host-pathogen interface. Four sequential stages of development following spore localization on the plant surface, from spore swelling to appressorium formation, were sampled for each species on culture medium and on barley sheaths, and transcriptomic analyses were performed. Gene expression in the prepenetration stages in both species and under both conditions was similar. In contrast, gene expression in the final stage was strongly influenced by the environment. Appressorium formation involved the greatest number of differentially expressed genes. Laser-dissection microscopy was used to perform detailed transcriptomics of initial infection points by F. graminearum. These analyses revealed new and important aspects of early fungal ingress in this species. Expression of the trichothecene genes involved in biosynthesis of deoxynivalenol by F. graminearum implies that toxisomes are not fully functional until after penetration and indicates that deoxynivalenol is not essential for penetration under our conditions. The use of comparative gene expression of divergent fungi promises to advance highly effective targets for antifungal strategies. IMPORTANCE Fusarium graminearum and Magnaporthe oryzae are two of the most important pathogens of cereal grains worldwide. Despite years of research, strong host resistance has not been identified for F. graminearum, so other methods of control are essential. The pathogen takes advantage of multiple entry points to infect the host, including breaches in the florets due to senescence of flower parts and penetration of the weakened trichome bases to breach the epidermis. In contrast, M. oryzae directly punctures leaves that it infects, and resistant cultivars have been characterized. The threat of either pathogen causing a major disease outbreak is ever present. Comparative transcriptomics demonstrated its potential to reveal novel and effective disease prevention strategies that affect the initial stages of disease. Shedding light on the basis of this diversity of infection strategies will result in development of increasingly specific control strategies.

The Sordariomycetes: an expanding resource with Big Data for mining in evolutionary genomics and transcriptomics.

Advances in genomics and transcriptomics accompanying the rapid accumulation of omics data have provided new tools that have transformed and expanded the traditional concepts of model fungi. Evolutionary genomics and transcriptomics have flourished with the use of classical and newer fungal models that facilitate the study of diverse topics encompassing fungal biology and development. Technological advances have also created the opportunity to obtain and mine large datasets. One such continuously growing dataset is that of the Sordariomycetes, which exhibit a richness of species, ecological diversity, economic importance, and a profound research history on amenable models. Currently, 3,574 species of this class have been sequenced, comprising nearly one-third of the available ascomycete genomes. Among these genomes, multiple representatives of the model genera Fusarium, Neurospora, and Trichoderma are present. In this review, we examine recently published studies and data on the Sordariomycetes that have contributed novel insights to the field of fungal evolution via integrative analyses of the genetic, pathogenic, and other biological characteristics of the fungi. Some of these studies applied ancestral state analysis of gene expression among divergent lineages to infer regulatory network models, identify key genetic elements in fungal sexual development, and investigate the regulation of conidial germination and secondary metabolism. Such multispecies investigations address challenges in the study of fungal evolutionary genomics derived from studies that are often based on limited model genomes and that primarily focus on the aspects of biology driven by knowledge drawn from a few model species. Rapidly accumulating information and expanding capabilities for systems biological analysis of Big Data are setting the stage for the expansion of the concept of model systems from unitary taxonomic species/genera to inclusive clusters of well-studied models that can facilitate both the in-depth study of specific lineages and also investigation of trait diversity across lineages. The Sordariomycetes class, in particular, offers abundant omics data and a large and active global research community. As such, the Sordariomycetes can form a core omics clade, providing a blueprint for the expansion of our knowledge of evolution at the genomic scale in the exciting era of Big Data and artificial intelligence, and serving as a reference for the future analysis of different taxonomic levels within the fungal kingdom.

Differential impact of nutrition on developmental and metabolic gene expression during fruiting body development in Neurospora crassa.

Fungal fruiting body size and form are influenced by the ecology of the species, including diverse environmental stimuli. Accordingly, nutritional resources available to the fungus during development can be vital to successful production of fruiting bodies. To investigate the effect of nutrition, perithecial development of Neurospora crassa was induced on two different media, a chemically sparsely nutritive Synthetic Crossing Medium (SCM) and a natural Carrot Agar (CA). Protoperithecia were collected before crossing, and perithecia were collected at 2, 24, 48, 72, 96, 120, and at full maturity 144 h after crossing. No differences in fruiting body morphology were observed between the two media at any time point. A circuit of microarray hybridizations comparing cDNA from all neighboring stages was performed. For a majority of differentially expressed genes, expression was higher in SCM than in CA, and expression of core metabolic genes was particularly affected. Effects of nutrition were highest in magnitude before crossing, lowering in magnitude during early perithecial development. Interestingly, metabolic effects of the media were also large in magnitude during late perithecial development, at which stage the lower expression in CA presumably reflected the continued intake of diverse complex initial compounds, diminishing the need for expression of anabolic pathways. However, for genes with key regulatory roles in sexual development, including pheromone precursor ccg-4 and poi2, expression patterns were similar between treatments. When possible, a common nutritional environment is ideal for comparing transcriptional profiles between different fungi. Nevertheless, the observed consistency of the developmental program across media, despite considerable metabolic differentiation is reassuring. This result facilitates comparative studies that will require different nutritional resources for sexual development in different fungi.

Transcriptome analyses during fruiting body formation in Fusarium graminearum and Fusarium verticillioides reflect species life history and ecology.

Fusarium graminearum and F. verticillioides are devastating cereal pathogens with very different life history and ecological characteristics. F. graminearum is homothallic, and sexual spores are an important component of its life cycle, responsible for disease initiation. F. verticilloides is heterothallic, and produces only modest numbers of fruiting bodies, which are not a significant source of inoculum. To identify corresponding differences in the transcriptional program underlying fruiting body development in the two species, comparative expression was performed, analyzing six developmental stages. To accompany the transcriptional analysis, detailed morphological characterization of F. verticillioides development was performed and compared to a previous morphological analysis of F. graminearum. Morphological development was similar between the two species, except for the observation of possible trichogynes in F. verticillioides ascogonia, which have not been previously reported for any Fusarium species. Expression of over 9000 orthologous genes were measured for the two species. Functional assignments of highly expressed orthologous genes at each time-point revealed the majority of highly expressed genes fell into the "unclassified proteins" category, reflecting the lack of characterization of genes for sexual development in both species. Simultaneous examination of morphological development and stage-specific gene expression suggests that degeneration of the paraphyses during sexual development is an apoptotic process. Expression of mating type genes in the two species differed, possibly reflecting the divergent roles they play in sexual development. Overall, the differences in gene expression reflect the greater role of fruiting bodies in the life cycle and ecology of F. graminearum versus F. verticillioides.

Mid1, a mechanosensitive calcium ion channel, affects growth, development, and ascospore discharge in the filamentous fungus Gibberella zeae.

The role of Mid1, a stretch-activated ion channel capable of being permeated by calcium, in ascospore development and forcible discharge from asci was examined in the pathogenic fungus Gibberella zeae (anamorph Fusarium graminearum). The Δmid1 mutants exhibited a >12-fold reduction in ascospore discharge activity and produced predominately abnormal two-celled ascospores with constricted and fragile septae. The vegetative growth rate of the mutants was ∼50% of the wild-type rate, and production of macroconidia was >10-fold lower than in the wild type. To better understand the role of calcium flux, Δmid1 Δcch1 double mutants were also examined, as Cch1, an L-type calcium ion channel, is associated with Mid1 in Saccharomyces cerevisiae. The phenotype of the Δmid1 Δcch1 double mutants was similar to but more severe than the phenotype of the Δmid1 mutants for all categories. Potential and current-voltage measurements were taken in the vegetative hyphae of the Δmid1 and Δcch1 mutants and the wild type, and the measurements for all three strains were remarkably similar, indicating that neither protein contributes significantly to the overall electrical properties of the plasma membrane. Pathogenicity of the Δmid1 and Δmid1Δcch1 mutants on the host (wheat) was not affected by the mutations. Exogenous calcium supplementation partially restored the ascospore discharge and vegetative growth defects for all mutants, but abnormal ascospores were still produced. These results extend the known roles of Mid1 to ascospore development and forcible discharge. However, Neurospora crassa Δmid1 mutants were also examined and did not exhibit defects in ascospore development or in ascospore discharge. In comparison to ion channels in other ascomycetes, Mid1 shows remarkable adaptability of roles, particularly with regard to niche-specific adaptation.

Biofilm Formation and Structure in the Filamentous Fungus Fusarium graminearum, a Plant Pathogen.

Biofilms are protective structures for pathogens of plants and animals, in which cells are shielded from host defense responses and antimicrobial treatments. Although biofilms are well studied in bacterial pathogens, their development and structure in filamentous fungi, as well as their role in pathogenicity, are poorly understood. We show that the economically important plant pathogen Fusarium graminearum, a filamentous fungus, forms biofilms in vitro, which adhere to polystyrene, a hydrophobic surface. The biofilms have complex hyphal structures surrounded by a polymeric matrix that consists primarily of polysaccharides and extracellular nucleic acids, and lack lipids. Pellicles are formed in liquid cultures, floating biofilm masses that are common in bacterial biofilms, and noted but undescribed in filamentous fungal biofilms. Commonly, F. graminearum grows as hyphal colonies; however, on media which lack electron acceptors, an altered morphology is formed with predominantly short, bulbous hyphae embedded in the matrix. Supplementation of the biofilm-inducing medium with an electron acceptor restores the filamentous hyphal morphology, demonstrating that the formation of bulbous hyphae is due, at least in part, to oxidative stress. Plant hosts infected with pathogens generally respond by producing reactive oxygen species, commonly produced as a defense response. Thus, the formation of biofilms strongly suggests a role in protecting cells from host responses during the course of plant disease. IMPORTANCE Fusarium graminearum is a filamentous fungal pathogen that causes Fusarium head blight (FHB) in cereal crops, leading to devastating crop losses. We have demonstrated the ability of this pathogen to form biofilms. Biofilms are likely to be important in the disease cycle of F. graminearum and other plant pathogens, protecting cells from plant defenses and environmental conditions. Towards this end, we have characterized the formation of biofilms in F. graminearum in vitro, which, together with ongoing characterization of their association with host plants, provides a basis for understanding the functionality of biofilms in the pathogen disease cycle.

Secondary Metabolism Gene Clusters Exhibit Increasingly Dynamic and Differential Expression during Asexual Growth, Conidiation, and Sexual Development in Neurospora crassa.

Secondary metabolite clusters (SMCs) encode the machinery for fungal toxin production. However, understanding their function and analyzing their products requires investigation of the developmental and environmental conditions in which they are expressed. Gene expression is often restricted to specific and unexamined stages of the life cycle. Therefore, we applied comparative genomics analyses to identify SMCs in Neurospora crassa and analyzed extensive transcriptomic data spanning nine independent experiments from diverse developmental and environmental conditions to reveal their life cycle-specific gene expression patterns. We reported 20 SMCs comprising 177 genes-a manageable set for investigation of the roles of SMCs across the life cycle of the fungal model N. crassa-as well as gene sets coordinately expressed in 18 predicted SMCs during asexual and sexual growth under three nutritional and two temperature conditions. Divergent activity of SMCs between asexual and sexual development was reported. Of 126 SMC genes that we examined for knockout phenotypes, al-2 and al-3 exhibited phenotypes in asexual growth and conidiation, whereas os-5, poi-2, and pmd-1 exhibited phenotypes in sexual development. SMCs with annotated function in mating and crossing were actively regulated during the switch between asexual and sexual growth. Our discoveries call for attention to roles that SMCs may play in the regulatory switches controlling mode of development, as well as the ecological associations of those developmental stages that may influence expression of SMCs. IMPORTANCE Secondary metabolites (SMs) are low-molecular-weight compounds that often mediate interactions between fungi and their environments. Fungi enriched with SMs are of significant research interest to agriculture and medicine, especially from the aspects of pathogen ecology and environmental epidemiology. However, SM clusters (SMCs) that have been predicted by comparative genomics alone have typically been poorly defined and insufficiently functionally annotated. Therefore, we have investigated coordinate expression in SMCs in the model system N. crassa, and our results suggest that SMCs respond to environmental signals and to stress that are associated with development. This study examined SMC regulation at the level of RNA to integrate observations and knowledge of these genes in various growth and development conditions, supporting combining comparative genomics and inclusive transcriptomics to improve computational annotation of SMCs. Our findings call for detailed study of the function of SMCs during the asexual-sexual switch, a key, often-overlooked developmental stage.

Transcriptional Divergence Underpinning Sexual Development in the Fungal Class Sordariomycetes.

Gene expression divergence through evolutionary processes is thought to be important for achieving programmed development in multicellular organisms. To test this premise in filamentous fungi, we investigated transcriptional profiles of 3,942 single-copy orthologous genes (SCOGs) in five related sordariomycete species that have morphologically diverged in the formation of their flask-shaped perithecia. We compared expression of the SCOGs to inferred gene expression levels of the most recent common ancestor of the five species, ranking genes from their largest increases to smallest increases in expression during perithecial development in each of the five species. We found that a large proportion of the genes that exhibited evolved increases in gene expression were important for normal perithecial development in Fusarium graminearum. Many of these genes were previously uncharacterized, encoding hypothetical proteins without any known functional protein domains. Interestingly, the developmental stages during which aberrant knockout phenotypes appeared largely coincided with the elevated expression of the deleted genes. In addition, we identified novel genes that affected normal perithecial development in Magnaporthe oryzae and Neurospora crassa, which were functionally and transcriptionally diverged from the orthologous counterparts in F. graminearum. Furthermore, comparative analysis of developmental transcriptomes and phylostratigraphic analysis suggested that genes encoding hypothetical proteins are generally young and transcriptionally divergent between related species. This study provides tangible evidence of shifts in gene expression that led to acquisition of novel function of orthologous genes in each lineage and demonstrates that several genes with hypothetical function are crucial for shaping multicellular fruiting bodies. IMPORTANCE The fungal class Sordariomycetes includes numerous important plant and animal pathogens. It also provides model systems for studying fungal fruiting body development, as its members develop fruiting bodies with a few well-characterized tissue types on common growth media and have rich genomic resources that enable comparative and functional analyses. To understand transcriptional divergence of key developmental genes between five related sordariomycete fungi, we performed targeted knockouts of genes inferred to have evolved significant upward shifts in expression. We found that many previously uncharacterized genes play indispensable roles at different stages of fruiting body development, which have undergone transcriptional activation in specific lineages. These novel genes are predicted to be phylogenetically young and tend to be involved in lineage- or species-specific function. Transcriptional activation of genes with unknown function seems to be more frequent than ever thought, which may be crucial for rapid adaption to changing environments for successful sexual reproduction.

Non-target impacts of fungicide disturbance on phyllosphere yeasts in conventional and no-till management.

Fungicides reduce fungal pathogen populations and are essential to food security. Understanding the impacts of fungicides on crop microbiomes is vital to minimizing unintended consequences while maintaining their use for plant protection. However, fungicide disturbance of plant microbiomes has received limited attention, and has not been examined in different agricultural management systems. We used amplicon sequencing of fungi and prokaryotes in maize and soybean microbiomes before and after foliar fungicide application in leaves and roots from plots under long-term no-till and conventional tillage management. We examined fungicide disturbance and resilience, which revealed consistent non-target effects and greater resiliency under no-till management. Fungicides lowered pathogen abundance in maize and soybean and decreased the abundance of Tremellomycetes yeasts, especially Bulleribasidiaceae, including core microbiome members. Fungicide application reduced network complexity in the soybean phyllosphere, which revealed altered co-occurrence patterns between yeast species of Bulleribasidiaceae, and Sphingomonas and Hymenobacter in fungicide treated plots. Results indicate that foliar fungicides lower pathogen and non-target fungal abundance and may impact prokaryotes indirectly. Treatment effects were confined to the phyllosphere and did not impact belowground microbial communities. Overall, these results demonstrate the resilience of no-till management to fungicide disturbance, a potential novel ecosystem service provided by no-till agriculture.