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ABSTRACT At human body temperature, the fungal pathogen Candida albicans can transition from yeast to filamentous morphologies in response to host-relevant cues. Additionally, elevated temperatures encountered during febrile episodes can independently induce C. albicans filamentation. However, the underlying genetic pathways governing this developmental transition in response to elevated temperatures remain largely unexplored. Here, we conducted a functional genomic screen to unravel the genetic mechanisms orchestrating C. albicans filamentation specifically in response to elevated temperature, implicating 45% of genes associated with the spliceosome or pre-mRNA splicing in this process. Employing RNA-Seq to elucidate the relationship between mRNA splicing and filamentation, we identified greater levels of intron retention in filaments compared to yeast, which correlated with reduced expression of the affected genes. Intriguingly, homozygous deletion of a gene encoding a spliceosome component important for filamentation (PRP19) caused even greater levels of intron retention compared with wild type and displayed globally dysregulated gene expression. This suggests that intron retention is a mechanism for fine-tuning gene expression during filamentation, with perturbations of the spliceosome exacerbating this process and blocking filamentation. Overall, this study unveils a novel biological process governing C. albicans filamentation, providing new insights into the complex regulation of this key virulence trait.IMPORTANCEFungal pathogens such as Candida albicans can cause serious infections with high mortality rates in immunocompromised individuals. When C. albicans is grown at temperatures encountered during human febrile episodes, yeast cells undergo a transition to filamentous cells, and this process is key to its virulence. Here, we expanded our understanding of how C. albicans undergoes filamentation in response to elevated temperature and identified many genes involved in mRNA splicing that positively regulate filamentation. Through transcriptome analyses, we found that intron retention is a mechanism for fine-tuning gene expression in filaments, and perturbation of the spliceosome exacerbates intron retention and alters gene expression substantially, causing a block in filamentation. This work adds to the growing body of knowledge on the role of introns in fungi and provides new insights into the cellular processes that regulate a key virulence trait in C. albicans.

ABSTRACT Alternaria alternata is a ubiquitous soil-borne fungus capable of causing diseases in a variety of plants and occasionally in humans. While populations of A. alternata from infected plants have received significant attention, relatively little is known about its soil populations, including its population genetic structure and antifungal susceptibilities. In addition, over the last two decades, greenhouses have become increasingly important for food and ornamental plant production throughout the world, but how greenhouses might impact microbial pathogens such as A. alternata populations remains largely unknown. Different from open crop fields, greenhouses are often more intensively cultivated, with each greenhouse being a relatively small and isolated space where temperature and humidity are higher than surrounding environments. Previous studies have shown that greenhouse populations of two common molds, Aspergillus fumigatus and A. alternata, within a small community in southwestern China were variably differentiated. However, the relative contribution of physical separation among local greenhouses to the large-scale population structure remains unknown. Here, we isolated strains of A. alternata from seven greenhouses in Shijiazhuang, northeast China. Their genetic diversity and triazole susceptibilities were analyzed and compared with each other and with 242 isolates from nine greenhouses in Kunming, southwest China. Results showed that the isolation of greenhouses located

Parastagonospora nodorum is a necrotrophic pathogen of wheat that is particularly destructive in major wheat-growing regions of the United States, northern Europe, Australia, and South America. P. nodorum secretes necrotrophic effectors that target wheat susceptibility genes to induce programmed cell death (PCD), resulting in increased colonization of host tissue and, ultimately, sporulation to complete its pathogenic life cycle. Intensive research over the last two decades has led to the functional characterization of five proteinaceous necrotrophic effectors, SnTox1, SnToxA, SnTox267, SnTox3, and SnTox5, and three wheat susceptibility genes, Tsn1, Snn1, and Snn3D-1. Functional characterization has revealed that these effectors, in addition to inducing PCD, have additional roles in pathogenesis, including chitin binding that results in protection from wheat chitinases, blocking defense response signaling, and facilitating plant colonization. There are still large gaps in our understanding of how this necrotrophic pathogen is successfully manipulating wheat defense to complete its life cycle. This review summarizes our current knowledge, identifies knowledge gaps, and provides a summary of well-developed tools and resources currently available to study the P. nodorum–wheat interaction, which has become a model for necrotrophic specialist interactions. Further functional characterization of the effectors involved in this interaction and work toward a complete understanding of how P. nodorum manipulates wheat defense will provide fundamental knowledge about this and other necrotrophic interactions. Additionally, a broader understanding of this interaction will contribute to the successful management of Septoria nodorum blotch disease on wheat. [Graphic: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

ABSTRACTAlthough fungi have been important model organisms for solving genetic, molecular, and ecological problems, recently, they are also becoming an important source of infectious disease. Despite their high medical burden, fungal pathogens are understudied, and relative to other pathogenic microbes, less is known about how their gene functions contribute to disease. This is due, in part, to a lack of powerful genetic tools to study these organisms. In turn, this has resulted in inappropriate treatments and diagnostics and poor disease management. There are a variety of reasons genetic studies were challenging in pathogenic fungi, but in recent years, most of them have been overcome or advances have been made to circumvent these barriers. In this minireview, we highlight how recent advances in genetic studies in fungal pathogens have resulted in the discovery of important biology and potential new antifungals and have created the tools to comprehensively study these important pathogens.

ABSTRACTCandida albicans, one of the most prevalent human fungal pathogens, causes diverse diseases extending from superficial infections to deadly systemic mycoses. Currently, only three major classes of antifungal drugs are available to treat systemic infections: azoles, polyenes, and echinocandins. Alarmingly, the efficacy of these antifungals against C. albicans is hindered both by basal tolerance toward the drugs and the development of resistance mechanisms such as alterations of the drug’s target, modulation of stress responses, and overexpression of efflux pumps. Thus, the need to identify novel antifungal strategies is dire. To address this challenge, we screened 3,049 structurally-diverse compounds from the Boston University Center for Molecular Discovery (BU-CMD) chemical library against a C. albicans clinical isolate and identified 17 molecules that inhibited C. albicans growth by >80% relative to controls. Among the most potent compounds were CMLD013360, CMLD012661, and CMLD012693, molecules representing two distinct chemical scaffolds, including 3-hydroxyquinolinones and a xanthone natural product. Based on structural insights, CMLD013360, CMLD012661, and CMLD012693 were hypothesized to exert antifungal activity through metal chelation. Follow-up investigations revealed all three compounds exerted antifungal activity against non-albicans Candida, including Candida auris and Candida glabrata, with the xanthone natural product CMLD013360 also displaying activity against the pathogenic mould Aspergillus fumigatus. Media supplementation with metallonutrients, namely ferric or ferrous iron, rescued C. albicans growth, confirming these compounds act as metal chelators. Thus, this work identifies and characterizes two chemical scaffolds that chelate iron to inhibit the growth of the clinically relevant fungal pathogen C. albicansIMPORTANCEThe worldwide incidence of invasive fungal infections is increasing at an alarming rate. Systemic candidiasis caused by the opportunistic pathogen Candida albicans is the most common cause of life-threatening fungal infection. However, due to the limited number of antifungal drug classes available and the rise of antifungal resistance, an urgent need exists for the identification of novel treatments. By screening a compound collection from the Boston University Center for Molecular Discovery (BU-CMD), we identified three compounds representing two distinct chemical scaffolds that displayed activity against C. albicans. Follow-up analyses confirmed these molecules were also active against other pathogenic fungal species including Candida auris and Aspergillus fumigatus. Finally, we determined that these compounds inhibit the growth of C. albicans in culture through iron chelation. Overall, this observation describes two novel chemical scaffolds with antifungal activity against diverse fungal pathogens.

Fusarium head blight (FHB), caused by the hemibiotrophic fungus Fusarium graminearum, is one of the major threats to global wheat productivity. A wheat pore-forming toxin-like (PFT) protein was previously reported to underlie Fhb1, the most widely used quantitative trait locus in FHB breeding programs worldwide. In the present work, wheat PFT was ectopically expressed in the model dicot plant Arabidopsis. Heterologous expression of wheat PFT in Arabidopsis provided a broad-spectrum quantitative resistance to fungal pathogens including F. graminearum, Colletotrichum higginsianum, Sclerotinia sclerotiorum, and Botrytis cinerea. However, there was no resistance to bacterial or oomycete pathogens Pseudomonas syringae and Phytophthora capsici, respectively in the transgenic Arabidopsis plants. To explore the reason for the resistance response to, exclusively, the fungal pathogens, purified PFT protein was hybridized to a glycan microarray having 300 different types of carbohydrate monomers and oligomers. It was found that PFT specifically hybridized with chitin monomer, N-acetyl glucosamine (GlcNAc), which is present in fungal cell walls but not in bacteria or oomycete species. This exclusive recognition of chitin may be responsible for the specificity of PFT-mediated resistance to fungal pathogens. Transfer of the atypical quantitative resistance of wheat PFT to a dicot system highlights its potential utility in designing broad-spectrum resistance in diverse host plants. [Graphic: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

BackgroundCryptococcosis and cryptococcal meningitis, caused by Cryptococcus neoformans infections, lead to approximately 180,000 deaths per year, primarily in developing countries. Individuals with compromised immune systems, e.g., due to HIV infection (AIDS) or chemotherapy, are particularly vulnerable. Conventional treatment options are often limited and can cause severe side effects. Therefore, this study aimed to investigate the antifungal effect of insect-derived proline-rich antimicrobial peptides (PrAMPs) against C. neoformans. These peptides are known for their low toxicity and their high efficacy in murine infection models, making them a promising alternative for treatment.ResultsA preliminary screening of the minimal inhibitory concentrations (MICs) of 20 AMPs, including the well-known PrAMPs Onc112, Api137, and Chex1Arg20 as well as the cathelicidin CRAMP against the C. neoformans strains 1841, H99, and KN99α revealed promising results, with MICs as low as 1.6 μmol/L. Subsequent investigations of selected peptides, determining their influence on fungal colony-forming units, confirmed their strong activity. The antifungal activity was affected by factors such as peptide net charge and sequence, with stronger effects at higher net charges probably due to better intracellular uptake confirmed by confocal laser scanning microscopy. Inactive scrambled peptides suggest a specific intracellular target, although scanning electron microscopy showed that PrAMPs also damaged the cell exterior for a low proportion of the cells. Possible pore formation could facilitate entry into the cytosol.

ABSTRACTInnate immune cells, especially neutrophils, play key roles in protecting against infection by Candida albicans and other fungal pathogens. A distinct aspect of neutrophils compared to other phagocytes is that they make much higher levels of myeloperoxidase, which converts H2O2 generated during the oxidative burst into highly reactive hypochlorous acid (HOCl). The effects of HOCl on C. albicans are not well defined, so comparative studies were used to show that HOCl kills C. albicans at lower doses than H2O2 and in a manner that correlates with permeabilization of the plasma membrane. Mutants with defects in plasma membrane organization and antioxidant pathways showed differential sensitivity to these oxidants, consistent with their distinct chemical properties. Transcriptional responses to HOCl and H2O2 were also quite different. Although they induced a common set of genes, most differentially regulated genes were unique. Testing the roles of genes that were predicted to be important for counteracting the types of thiol oxidation that are preferentially caused by HOCl showed that Mxr1 (methionine-S-sulfoxide reductase), Srx1 (sulfiredoxin that reduces cysteine-sulfinic acid groups), and Trx1 (thioredoxin that acts to reduce disulfide bonds) all promoted resistance to HOCl. Altogether, these results suggest that an effective way to help promote the killing of C. albicans by neutrophils would be to perturb the C. albicans plasma membrane, rather than targeting one specific antioxidant pathway.IMPORTANCEHypochlorous acid (HOCl), commonly known as bleach, is generated during the respiratory burst by phagocytes and is a key weapon used to attack Candida albicans and other microbial pathogens. However, the effects of hypochlorous acid on C. albicans have been less well studied than H2O2, a different type of oxidant produced by phagocytes. HOCl kills C. albicans more effectively than H2O2 and results in disruption of the plasma membrane. HOCl induced a very different transcriptional response than H2O2, and there were significant differences in the susceptibility of mutant strains of C. albicans to these oxidants. Altogether, these results indicate that HOCl has distinct effects on cells that could be targeted in novel therapeutic strategies to enhance the killing of C. albicans and other pathogens.

Candida auris is a human fungal pathogen classified as an urgent threat to the delivery of health care due to its extensive antimicrobial resistance and the high mortality rates associated with invasive infections. Global outbreaks have occurred in health care facilities, particularly, long-term care hospitals and nursing homes. Skin is the primary site of colonization for C. auris. To accelerate research studies, we developed microbiome sequencing protocols, including amplicon and metagenomic sequencing, directly from patient samples at health care facilities with ongoing C. auris outbreaks. We characterized the skin mycobiome with a database optimized to classify Candida species and C. auris to the clade level. While Malassezia species were the predominant skin-associated fungi, nursing home residents also harbored Candida species, including C. albicans, and C. parapsilosis. Amplicon sequencing was concordant with culturing studies to identify C. auris-colonized patients and provided further resolution that distinct clades of C. auris are colonizing facilities in New York and Illinois. Shotgun metagenomic sequencing from a clinical sample with a high fungal bioburden generated a skin-associated profile of the C. auris genome. Future larger scale clinical studies are warranted to more systematically investigate the effects of commensal microbes and patient risk factors on the colonization and transmission of C. auris. IMPORTANCE Candida auris is a human pathogen of high concern due to its extensive antifungal drug resistance and high mortality rates associated with invasive infections. Candida auris skin colonization and persistence on environmental surfaces make this pathogen difficult to control once it enters a health care facility. Residents in long-term care hospitals and nursing homes are especially vulnerable. In this study, we developed microbiome sequencing protocols directly from surveillance samples, including amplicon and metagenomic sequencing, demonstrating concordance between sequencing results and culturing.

Coccidioidomycosis, or Valley fever, is caused by two species of dimorphic fungi. Based on molecular phylogenetic evidence, the genus Coccidioides contains two reciprocally monophyletic species: C. immitis and C. posadasii. However, phenotypic variation between species has not been deeply investigated. We therefore explored differences in growth rate under various conditions. A collection of 39 C. posadasii and 46 C. immitis isolates, representing the full geographical range of the two species, was screened for mycelial growth rate at 37 °C and 28 °C on solid media. The radial growth rate was measured for 16 days on yeast extract agar. A linear mixed effect model was used to compare the growth rate of C. posadasii and C. immitis at 37 °C and 28 °C, respectively. C. posadasii grew significantly faster at 37 °C, when compared to C. immitis; whereas both species had similar growth rates at 28 °C. These results indicate thermotolerance differs between these two species. As the ecological niche has not been well-described for Coccidioides spp., and disease variability between species has not been shown, the evolutionary pressure underlying the adaptation is unclear. However, this research reveals the first significant phenotypic difference between the two species that directly applies to ecological research.

The pathogenic fungus Phomopsis longicolla causes numerous plant diseases, such as Phomopsis seed decay, pod and stem blight, and stem canker, which seriously affect the yield and quality of soybean production worldwide. Because of a lack of technology for efficient manipulation of genes for functional genomics, understanding of P. longicolla pathogenesis is limited. Here, we developed an efficient polyethylene glycol–mediated protoplast transformation system in P. longicolla that we used to characterize the functions of two genes involved in the cell wall integrity (CWI) pathway of the mitogen-activated protein kinase (MAPK) cascade, including PlMkk1, which encodes MAPK kinase, and its downstream gene PlSlt2, which encodes MAPK. Both gene knockout mutants ΔPlMkk1 and ΔPlSlt2 displayed a reduced growth rate, fragile aerial hyphae, abnormal polarized growth and pigmentation, defects in sporulation, inadequate CWI, enhanced sensitivity to abiotic stress agents, and significant deficiencies in virulence, although there were some differences in degree. The results suggest that PlMkk1 and PlSlt2 are crucial for a series of growth and development processes as well as pathogenicity. The developed transformation system will be a useful tool for additional gene function research and will aid in the elucidation of the pathogenic mechanisms of P. longicolla. [Graphic: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

ABSTRACTDuring a field survey of cultivated Morchella mushroom diseases, Diploöspora longispora and Paecilomyces penicillatus, causal agents of pileus rot or white mould disease were detected, which resulted in up to 80% of yield losses. Multi-locus phylogenic analysis revealed that the fungi were affiliated in a distinct clade in Hypocreales. We further constructed a phylogenetic tree with broader sampling in Hypocreales and estimated the divergence times. The D. longispora and P. penicillatus clades were estimated to have diverged from Hypocreaceae around 129 MYA and Pseudodiploösporeaceae fam. nov is herein proposed to accommodate species in this clade. Two new genera, i.e. Pseudodiploöspora and Zelopaecilomyceswere, were introduced based on morphological characteristics and phylogenic relationships of Diploöspora longispora and Paecilomyces penicillatus, respectively. Five new combinations – Pseudodiploöspora cubensis, P. longispora, P. fungicola, P. zinniae, and Zelopaecilomyces penicillatus – were proposed.

ABSTRACT The incidence of invasive aspergillosis caused by Aspergillus fumigatus has risen steadily over the past few decades due to the limited effective treatment options and the emergence of antifungal-resistant isolates. In clinic-isolated A. fumigatus, the azole resistance mechanism is primarily caused by mutations of the drug target and/or overexpression of drug efflux pumps. However, knowledge about how drug efflux pumps are transcriptionally regulated is limited. In this study, we found that loss of a C2H2 transcription factor ZfpA (zinc finger protein) results in the marked upregulation of a series of drug efflux pump-encoding genes, especially atrF, which contributes to azole drug resistance in A. fumigatus. CrzA is a previously identified positive transcription factor for genes of drug efflux pumps, and ZfpA transcriptionally inhibits expressions of drug efflux pumps in a CrzA-dependent way. Under the treatment of azoles, both ZfpA and CrzA transfer to nuclei and coregulate the expression of multidrug transporters and then keep normal drug susceptibility in fungal cells. Findings in this study demonstrated that ZfpA is not only involved in fungal growth and virulence potential but also negatively regulates antifungal drug susceptibility. IMPORTANCE Conserved across all kingdoms of life, ABC transporters comprise one of the largest protein families. They are associated with multidrug resistance, affecting aspects such as resistance to antimicrobials or anticancer drugs. Despite the importance of ABC transporters in multidrug resistance, the understanding of their regulatory network is still limited in A. fumigatus. Here, we found that the loss of the transcription factor ZfpA induces the expression of the ABC transporter gene atrF, altering azole susceptibility in A. fumigatus. ZfpA, coordinately with CrzA, affects the azole susceptibility by regulating the expression of the ABC transporter gene atrF. These findings reveal the regulatory mechanism of the ABC transporter gene atrF in A. fumigatus.

ABSTRACT Autophagy is a conserved degradation and recycling pathway in eukaryotes and is important for their normal growth and development. An appropriate status of autophagy is crucial for organisms which is tightly regulated both temporally and continuously. Transcriptional regulation of autophagy-related genes (ATGs) is an important layer in autophagy regulation. However, the transcriptional regulators and their mechanisms are still unclear, especially in fungal pathogens. Here, we identified Sin3, a component of the histone deacetylase complex, as a transcriptional repressor of ATGs and negative regulator of autophagy induction in the rice fungal pathogen Magnaporthe oryzae. A loss of SIN3 resulted in upregulated expression of ATGs and promoted autophagy with an increased number of autophagosomes under normal growth conditions. Furthermore, we found that Sin3 negatively regulated the transcription of ATG1, ATG13, and ATG17 through direct occupancy and changed levels of histone acetylation. Under nutrient-deficient conditions, the transcription of SIN3 was downregulated, and the reduced occupancy of Sin3 from those ATGs resulted in histone hyperacetylation and activated their transcription and in turn promoted autophagy. Thus, our study uncovers a new mechanism of Sin3 in modulating autophagy through transcriptional regulation. IMPORTANCE Autophagy is an evolutionarily conserved metabolic process and is required for the growth and pathogenicity of phytopathogenic fungi. The transcriptional regulators and precise mechanisms of regulating autophagy, as well as whether the induction or repression of ATGs is associated with autophagy level, are still poorly understood in M. oryzae. In this study, we revealed that Sin3 acts as a transcriptional repressor of ATGs to negatively regulate autophagy level in M. oryzae. Under the nutrient-rich conditions, Sin3 inhibits autophagy with a basal level through directly repressing the transcription of ATG1-ATG13-ATG17. Upon nutrient-deficient treatment, the transcriptional level of SIN3 would decrease and dissociation of Sin3 from those ATGs associates with histone hyperacetylation and activates their transcriptional expression and in turn contributes to autophagy induction. Our findings are important as we uncover a new mechanism of Sin3 for the first time to negatively modulate autophagy at the transcriptional level in M. oryzae.

The brown planthopper (BPH), Nilaparvata lugens, is one of the most destructive pests of rice. Given the threats posed by insecticide resistance to its control, eco-friendly strategies based on microbial pathogens emerged as a promising biocontrol alternative. In the present study, we isolated a native fungal pathogen against BPH from infected BPH cadavers and preliminarily identified as a strain of Aspergillus fumigatus based on morphological and molecular methods. Laboratory bioassay revealed that this fungal strain was highly virulent to BPH both at nymphal and adult stages, with the median lethal times (LT50) of 7.5 and 5.8 days under high conidial concentration of 1 × 109 conidia mL–1. A genome-wide view of gene expressions in BPH against fungal attack was analyzed by transcriptomic sequencing and consequently a large number of differentially expressed genes that mainly involved in host immune defense and cell detoxification were found. RNAi-mediated knockdown of an upregulated gene encoding a serine protease (NlSPN) could cause a significant decrease in BPH survival. Combination of dsRNA injection and fungal infection showed an additive effect on BPH mortality, which provided clues to develop new pest management strategies against BPH.

Ambiviruses are hybrid infectious elements encoding the hallmark gene of RNA viruses, the RNA-dependent RNA polymerase, and self-cleaving RNA ribozymes found in many viroids. Ambiviruses are thought to be pathogens of fungi, although the majority of reported genomes have been identified in metatranscriptomes. Here, we present a comprehensive screen for ambiviruses in more than 46,500 fungal transcriptomes from the Sequence Read Archive (SRA). Our data-driven virus discovery approach identified more than 2,500 ambiviral sequences across the kingdom Fungi with a striking expansion in members of the phylum Basidiomycota representing the most complex fungal organisms. Our study unveils a large diversity of unknown ambiviruses with as little as 27% protein sequence identity to known members and sheds new light on the evolution of this distinct class of infectious agents with RNA genomes. No evidence for the presence of ambiviruses in human microbiomes was obtained from a comprehensive screen of respective metatranscriptomes available in the SRA.

ABSTRACT The ability to transition between yeast and filamentous growth states is critical for virulence of the leading human fungal pathogen Candida albicans. Large-scale genetic screens have identified hundreds of genes required for this morphological switch, but the mechanisms by which many of these genes orchestrate this developmental transition remain largely elusive. In this study, we characterized the role of Ent2 in governing morphogenesis in C. albicans. We showed that Ent2 is required for filamentous growth under a wide range of inducing conditions and is also required for virulence in a mouse model of systemic candidiasis. We found that the epsin N-terminal homology (ENTH) domain of Ent2 enables morphogenesis and virulence and does so via a physical interaction with the Cdc42 GTPase-activating protein (GAP) Rga2 and regulation of its localization. Further analyses revealed that overexpression of the Cdc42 effector protein Cla4 can overcome the requirement for the ENTH-Rga2 physical interaction, indicating that Ent2 functions, at least in part, to enable proper activation of the Cdc42-Cla4 signaling pathway in the presence of a filament-inducing cue. Overall, this work characterizes the mechanism by which Ent2 regulates hyphal morphogenesis in C. albicans, unveils the importance of this factor in enabling virulence in an in vivo model of systemic candidiasis and adds to the growing understanding of the genetic control of a key virulence trait. IMPORTANCE Candida albicans is a leading human fungal pathogen that can cause life-threatening infections in immunocompromised individuals, with mortality rates of ~40%. The ability of this organism to grow in both yeast and filamentous forms is critical for the establishment of systemic infection. Genomic screens have identified many genes required for this morphological transition, yet our understanding of the mechanisms that regulate this key virulence trait remains incomplete. In this study, we characterized Ent2 as a core regulator of C. albicans morphogenesis. We show that Ent2 regulates hyphal morphogenesis through an interaction between its ENTH domain and the Cdc42 GAP, Rga2, which signals through the Cdc42-Cla4 signaling pathway. Finally, we show that the Ent2 protein, and specifically its ENTH domain, is required for virulence in a mouse model of systemic candidiasis. Overall, this work identifies Ent2 as a key regulator of filamentation and virulence in C. albicans.

Rhizosphere-competent microbes often interact with plant roots and exhibit beneficial effects on plant performance. Numerous bacterial and fungal isolates are able to prime host plants for fast adaptive responses against pathogen attacks. Combined action of fungi and bacteria may lead to synergisms exceeding effects of single strains. Individual beneficial fungi and bacteria have been extensively studied in Arabidopsis thaliana, but little is known about their concerted actions in the Brassicaceae. Here, an in-vitro system with oilseed rape (Brassica napus) was established. Roots of two different cultivars were inoculated with well-characterized fungal (Trichoderma harzianum OMG16) and bacterial (Bacillus velezensis FZB42) isolates alone or in combination. Microscopic analysis confirmed that OMG16 hyphae entered root hairs through root hair tips and formed distinct intracellular structures. Quantitative PCR revealed that root colonization of OMG16 increased up to 10-fold in the presence of FZB42. Relative transcript levels of the ethylene- and jasmonic acid–responsive genes PDF1.2, ERF2, and AOC3 were recorded in leaves by quantitative reverse transcription PCR to measure induced systemic resistance in tissues distant from the roots. Combined action of OMG16 and FZB42 induced transcript abundances more efficiently than single inoculation. Importantly, microbial priming reduced Verticillium longisporum root infection in rapeseed by approximately 100-fold compared with nonprimed plants. Priming also led to faster and stronger systemic responses of the defense genes PDF1.2, ERF2, AOC3, and VSP2.[Graphic: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Fungal pathogen contamination is one of the most important factors affecting the postharvest quality and shelf life of wolfberry fruits. Therefore, the prevention and control of fungal pathogens that cause fruit rot has become particularly important. Volatile antifungal agents of biological origin have broad application prospects. They may be safer and more efficient than traditional physical and chemical methods. Four pathogenic fungi were isolated and purified from rotting wolfberry. These pathogenic fungi were determined to be Mucor circinelloides LB1, Fusarium arcuatisporum LB5, Alternaria iridiaustralis LB7, and Colletotrichum fioriniae LB8. In vitro fumigation experiments showed that 2,3-butanedione can effectively inhibit the mycelial growth, spore germination, and sporulation ability of pathogenic fungi. The scanning electron microscope (SEM) showed morphological changes in hyphae. Propidium iodide (PI) Staining and leakage of 260 and 280 nm-absorbing increased, suggesting damage to cell membranes. Furthermore, 2,3-butanedione was found to significantly improve fruit firmness, soluble solid, total phenol, flavonoid, and soluble sugar content, as well as higher SOD enzyme activity and lower PPO and POD enzyme activity in the treated fruit, indicating that 2,3-butanedione can effectively reduce the adverse effects of pathogenic fungi in wolfberry. Based on these results, we conclude that 2,3-butanedione is effective against infection by pathogenic fungi in post-harvest wolfberry. 2,3-butanedione should be considered a viable substitute for conventional fungicides that are currently used to control rot in wolfberry.

Understanding of how intracellular pathogens survive in their host cells is important to improve management of their diseases. This has been fruitful for intracellular bacteria, but it is an understudied area in fungal pathogens. Here we start elucidating and characterizing the strategies used by one of the commonest fungal pathogens, Cryptococcus neoformans, to survive intracellularly. The ability of the fungus to survive inside host cells is one of the main drivers of disease progression, yet it is unclear whether C. neoformans resides in a fully acidified, partially acidic, or neutral phagosome. Using a dye that only fluoresce under acidic conditions to stain C. neoformans, a hypha-defective Candida albicans mutant, and the nonpathogenic Saccharomyces cerevisiae, we characterized the fungal behaviors in infected macrophages by live microscopy. The main behavior in the C. albicans mutant strain and S. cerevisiae-phagosomes was rapid acidification after internalization, which remained for the duration of the imaging. In contrast, a significant number of C. neoformans-phagosomes exhibited alternative behaviors distinct from the normal phagosomal maturation: some phagosomes acidified with subsequent loss of acidification, and other phagosomes never acidified. Moreover, the frequency of these behaviors was affected by the immune status of the host cell. We applied the same technique to a flow cytometry analysis and found that a substantial percentage of C. neoformans-phagosomes showed impaired acidification, whereas almost 100% of the S. cerevisiae-phagosomes acidify. Lastly, using a membrane-damage reporter, we show phagosome permeabilization correlates with acidification alterations, but it is not the only strategy that C. neoformans uses to manipulate phagosomal acidification. The different behaviors described here provide an explanation to the confounding literature regarding cryptococcal-phagosome acidification and the methods can be applied to study other intracellular fungal pathogens.

Alternaria brown spot (ABS), caused by Alternaria alternata, is an economically important fungal disease of citrus worldwide. The ABS pathogen A. alternata tangerine pathotype can produce a host-specific ACT toxin, which is regulated by ACT toxin gene cluster located in the conditionally dispensable chromosome (CDC). Previously, we have assembled a draft genome of A. alternata tangerine pathotype strain Z7, which comprises 165 contigs. In this study, we report a chromosome-level genome assembly of A. alternata Z7 through the combination of Oxford Nanopore sequencing and Illumina sequencing technologies. The assembly of A. alternata Z7 had a total size of 34.28 Mb, with a GC content of 51.01% and contig N50 of 3.08 Mb. The genome is encompassed 12,067 protein-coding genes, 34 ribosomal RNAs, and 107 transfer RNAs. Interestingly, A. alternata Z7 is composed of 10 essential chromosomes and 2 CDCs, which is consistent with the experimental evidences of pulsed-field gel electrophoresis. To our best knowledge, this is the first chromosome-level genome assembly of A. alternata. In addition, a database for citrus-related Alternaria genomes has been established to provide public resources for the sequences, annotation and comparative genomics data of Alternaria spp. The improved genome sequence and annotation at the chromosome level is a significant step toward a better understanding of the pathogenicity of A. alternata. The database will be updated regularly whenever the genomes of newly isolated Alternaria spp. are available. The citrus-related Alternaria genomes database is open accessible through the Citrus Fungal Disease Database.[Graphic: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Venturia carpophila, the causal agent of scab disease on peach, is a host-specific fungus that is widely distributed around the world, including China. In our previous study, samples were collected from 14 provinces in China, and 750 isolates were obtained by single-spore separation. Here, we reported the first highly contiguous whole-genome sequence (35.87 Mb) of the V. carpophila isolate ZJHZ1-1-1, which included 33 contigs with N50 value of 2.01 Mb and maximum contig length of 3.39 Mb. The high-quality genome sequence and annotation resource will be useful to study the fungal biology, pathogen-host interaction, fungicide resistance, characterization of important genes, population genetic diversity, and development of molecular markers for genotyping and species identification.[Graphic: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

ABSTRACT Candida albicans is a pathobiont fungus that can colonize multiple niches in the human body but is also a frequent cause of both mucosal and systemic disease. Despite its clinical importance, a paucity of dominant selectable markers has hindered the development of tools for genetic manipulation of the species. One factor limiting the utilization of dominant selectable markers is that C. albicans is inherently more resistant to antibiotics used for selection in other species. Here, we showed that the inclusion of suitable adjuvants can enable the use of two aminoglycoside antibiotics, hygromycin B and G418, for positive selection in C. albicans. Combining these antibiotics with an adjuvant, such as quinine or molybdate, substantially suppressed the background growth of C. albicans, thereby enabling transformants expressing CaHygB or CaKan markers to be readily identified. We verified that these adjuvants were not mutagenic to C. albicans and that CaHygB and CaKan markers were orthogonal to the existing marker NAT1/SAT1, and so provide complementary tools for the genetic manipulation of C. albicans strains. Our study also established that adjuvant-based approaches can enable the use of selectable markers that would otherwise be limited by high background growth from susceptible cells. IMPORTANCE Only a single dominant selectable marker has been widely adopted for use in the opportunistic fungal pathogen Candida albicans. This is in stark contrast to model fungi where a repertoire of dominant markers is readily available. A limiting factor for C. albicans has been the high levels of background growth obtained with multiple antibiotics, thereby limiting their use for distinguishing cells that carry an antibiotic-resistance gene from those that do not. Here, we demonstrated that the inclusion of adjuvants can reduce background growth and enable the robust use of both CaHygB and CaKan markers for genetic selection in C. albicans.

Anthracnose is a fungal disease caused by Colletotrichum species and has detrimental effects on many crops, including red pepper. This study used Bacillus tequilensis GYUN-300 (GYUN-300), which exhibit antagonistic activity against the fungal pathogen, Colletotrichum acutatum. This pathogen causes anthracnose that manifests primarily as a fruit rot in red pepper. There have been little efforts to identify antagonistic bacteria from mushrooms; this strain of bacteria was identified as B. tequilensis using BIOLOG and 16S rDNA sequencing analysis. The genetic mechanism underpinning the biocontrol traits of GYUN-300 was characterized using the complete genome sequence of GYUN-300, which was closely compared to related strains. GYUN-300 inhibited mycelial growth and spore germination of C. acutatum under in vitro conditions. Important antagonistic traits, such as siderophore production, solubilization of insoluble phosphate, and production of lytic enzymes (cellulase, protease, and amylase), were observed in GYUN-300, These trains promoted growth in terms of seed germination and vigorous seedling growth compared to the non-treated control. When red pepper fruits were treated with GYUN-300, the preventive and curative effects were 66.6 and 38.3% effective, respectively, in wounded red pepper fruits; there was no difference between the preventive and curative effects in non-wounded red pepper fruits. Furthermore, GYUN-300 was resistant to several commercial fungicides, indicating that GYUN-300 bacterial cells may also be used synergistically with chemical fungicides to increase biocontrol efficiency. Based on in vitro results, GYUN-300 played a role to control anthracnose disease effectively in field conditions when compared to other treatments and non-treated controls. The results from this study provide a better understanding of the GYUN-300 strain as an effective biocontrol agent against red pepper anthracnose; this form of biocontrol provides an environment-friendly alternative to chemical fungicides.

Verticillium dahliae is a widespread fungal pathogen that causes Verticillium wilt on many economically important crops and ornamentals worldwide. Populations of V. dahliae have been divided into two distinct races based upon differential host responses in tomato and lettuce. Recently, the contemporary race 2 isolates were further divided into an additional race in tomato. Herein, we provide a high-quality reference genome for the race 1 strain VdLs.16 isolated from lettuce in California, U.S.A. This resource will contribute to ongoing research that aims to elucidate the genetic basis of V. dahliae pathogenicity and population genomic diversity.

The fungus Myriosclerotinia sulcatula is a close relative of the notorious polyphagous plant pathogens Botrytis cinerea and Sclerotinia sclerotiorum but exhibits a host range restricted to plants from the Carex genus (Cyperaceae family). To date, there are no genomic resources available for fungi in the Myriosclerotinia genus. Here, we present a chromosome-scale reference genome assembly for M. sulcatula. The assembly contains 24 contigs with a total length of 43.53 Mbp, with scaffold N50 of 2,649.7 kbp and N90 of 1,133.1 kbp. BRAKER-predicted gene models were manually curated using WebApollo, resulting in 11,275 protein-coding genes that we functionally annotated. We provide a high-quality reference genome assembly and annotation for M. sulcatula as a resource for studying evolution and pathogenicity in fungi from the Sclerotiniaceae family.

The role and impact of commensal and pathogenic fungi in different parts of the human body are being increasingly appreciated, unveiling the importance of such microorganisms in human health. A key function is the involvement of the mycobiota in cross-kingdom interactions within the microbiome. Any disturbance in the functionality of the microbiota could alter metabolic reactions, have a negative impact on homeostasis or induce diseases. The association of fungi with cancer development is the focus of this review. Several studies have reported direct or indirect involvement of fungal pathogens and mycobiome dysbiosis in induction of carcinogenesis. Most studies focused on cancers of the gastrointestinal tract. However, researchers are now investigating other organs, such as the skin, where the significant results obtained confirm the involvement of fungal pathogens and administration of antifungal drugs in development of cancer. This review gives an overview of the different organs affected and describes the mechanisms used by these eukaryotes or antifungals to induce oncogenesis.

Ascochyta versabilis is the fungal pathogen that causes the severe leaf spot disease of Pseudostellaria heterophylla (Miq.) Pax, a vital Chinese herbal plant. Here, we deployed PacBio single-molecule real-time long-read sequencing technology to generate a near-complete genome assembly for the A. versabilis KC1 strain and obtained a total of 9.80 Gb raw reads. These reads were processed into a 41.05 Mb genome assembly containing 95 contigs with N50 of 1.70 Mb and a maximum length of 3.93 Mb. A total of 10,457 gene models, of which 1,004 encode putatively secreted proteins, were identified in the genome. This high-quality genome assembly and gene annotation resource will facilitate the institution of functional genetic studies aimed at providing a better insight into the infection mechanisms of A. versabilis to support the development of effective control strategies for leaf spot disease of P. heterophylla.

Olive leaf scab, also known as peacock spot disease, caused by Venturia oleaginea (syn. Spilocaea oleaginea and Fusicladium oleagineum) is the most widespread and economically important fungal disease attacking olive in production countries. Here, we report the first highly contiguous whole-genome sequence (46.08 Mb) of one isolate, YUN35, of V. oleaginea. The described genome sequence and annotation resource will be useful to study the fungal biology, pathogen-host interaction, characterization of genes of interest, and population genetic diversity.

Coccidioides immitis and Coccidioides posadasii contribute to the development of Valley Fever. The ability of these fungal pathogens to evade the host immune system creates difficulty in recognition and treatment of this debilitating infection. In this review, we describe the current knowledge of Valley Fever and approaches to improve prevention, detection, and treatment.

ABSTRACT Protein kinase A (PKA) signaling plays a critical role in the growth and development of all eukaryotic microbes. However, few direct targets have been characterized in any organism. The fungus Aspergillus fumigatus is a leading infectious cause of death in immunocompromised patients, but the specific molecular mechanisms responsible for its pathogenesis are poorly understood. We used this important pathogen as a platform for a comprehensive and multifaceted interrogation of both the PKA-dependent whole proteome and phosphoproteome in order to elucidate the mechanisms through which PKA signaling regulates invasive microbial disease. Employing advanced quantitative whole-proteomic and phosphoproteomic approaches with two complementary phosphopeptide enrichment strategies, coupled to an independent PKA interactome analysis, we defined distinct PKA-regulated pathways and identified novel direct PKA targets contributing to pathogenesis. We discovered three previously uncharacterized virulence-associated PKA effectors, including an autophagy-related protein, Atg24; a CCAAT-binding transcriptional regulator, HapB; and a CCR4-NOT complex-associated ubiquitin ligase, Not4. Targeted mutagenesis, combined with in vitro kinase assays, multiple murine infection models, structural modeling, and molecular dynamics simulations, was employed to characterize the roles of these new PKA targets in growth, environmental and antimicrobial stress responses, and pathogenesis in a mammalian system. We also elucidated the molecular mechanisms of PKA regulation for these effectors by defining the functionality of phosphorylation at specific PKA target sites. We have comprehensively characterized the PKA-dependent phosphoproteome and validated PKA targets as direct regulators of infectious disease for the first time in any pathogen, providing new insights into PKA signaling and control over microbial pathogenesis. IMPORTANCE PKA is essential for the virulence of eukaryotic human pathogens. Understanding PKA signaling mechanisms is therefore fundamental to deciphering pathogenesis and developing novel therapies. Despite its ubiquitous necessity, specific PKA effectors underlying microbial disease remain unknown. To address this fundamental knowledge gap, we examined the whole-proteomic and phosphoproteomic impacts of PKA on the deadly fungal pathogen Aspergillus fumigatus to uncover novel PKA targets controlling growth and virulence. We also defined the functional consequences of specific posttranslational modifications of these target proteins to characterize the molecular mechanisms of pathogenic effector regulation by PKA. This study constitutes the most comprehensive analysis of the PKA-dependent phosphoproteome of any human pathogen and proposes new and complex roles played by PKA signaling networks in governing infectious disease.

ABSTRACT Polysaccharides are key components of both the fungal cell wall and biofilm matrix. Despite having distinct assembly and regulation pathways, matrix exopolysaccharide and cell wall polysaccharides share common substrates and intermediates in their biosynthetic pathways. It is not clear, however, if the biosynthetic pathways governing the production of these polysaccharides are cooperatively regulated. Here, we demonstrate that cell wall stress promotes production of the exopolysaccharide galactosaminogalactan (GAG)-depend biofilm formation in the major fungal pathogen of humans Aspergillus fumigatus and that the transcription factor SomA plays a crucial role in mediating this process. A core set of SomA target genes were identified by transcriptome sequencing and chromatin immunoprecipitation coupled to sequencing (ChIP-Seq). We identified a novel SomA-binding site in the promoter regions of GAG biosynthetic genes agd3 and ega3, as well as its regulators medA and stuA. Strikingly, this SomA-binding site was also found in the upstream regions of genes encoding the cell wall stress sensors, chitin synthases, and β-1,3-glucan synthase. Thus, SomA plays a direct regulation of both GAG and cell wall polysaccharide biosynthesis. Consistent with these findings, SomA is required for the maintenance of normal cell wall architecture and compositions in addition to its function in biofilm development. Moreover, SomA was found to globally regulate glucose uptake and utilization, as well as amino sugar and nucleotide sugar metabolism, which provides precursors for polysaccharide synthesis. Collectively, our work provides insight into fungal adaptive mechanisms in response to cell wall stress where biofilm formation and cell wall homeostasis were synchronously regulated. IMPORTANCE The cell wall is essential for fungal viability and is absent from human hosts; thus, drugs disrupting cell wall biosynthesis have gained more attention. Caspofungin is a member of a new class of clinically approved echinocandin drugs to treat invasive aspergillosis by blocking β-1,3-glucan synthase, thus damaging the fungal cell wall. Here, we demonstrate that caspofungin and other cell wall stressors can induce galactosaminogalactan (GAG)-dependent biofilm formation in the human pathogen Aspergillus fumigatus. We further identified SomA as a master transcription factor playing a dual role in both biofilm formation and cell wall homeostasis. SomA plays this dual role by direct binding to a conserved motif upstream of GAG biosynthetic genes and genes involved in cell wall stress sensors, chitin synthases, and β-1,3-glucan synthase. Collectively, these findings reveal a transcriptional control pathway that integrates biofilm formation and cell wall homeostasis and suggest SomA as an attractive target for antifungal drug development.

ABSTRACT Worldwide increases in antibiotic resistance and the dearth of new antibiotics have created a global crisis in the treatment of infectious diseases. These concerns highlight the pressing need for novel antimicrobial agents. Natural clay minerals have a long history of therapeutic and biomedical applications and have lately received specific attention for their potent antimicrobial properties. In particular, Kisameet clay (KC) has strong antibacterial activity against a variety of multidrug-resistant (MDR) bacterial pathogens in vitro. Here, we have extended the known spectrum of activity of KC by demonstrating its efficacy against two major fungal pathogens, Candida albicans and Cryptococcus neoformans. In addition, KC also exhibits potent activity against the opportunistic bacterial pathogen Mycobacterium marinum, a model organism for M. ulcerans infection. Moreover, aqueous KC leachates (KC-L) exhibited broad-spectrum antibacterial activity, eradicated Gram-negative and Gram-positive biofilms, and prevented their formation. The mechanism(s) underlying KC antibacterial activity appears to be complex. Adjusting KC-L to neutral pH rendered it inactive, indicating a contribution of pH, although low pH alone was insufficient for its antibacterial activity. Treatment of KC minerals with cation-chelating agents such as EDTA, 2,2′-bipyridyl, and deferoxamine reduced the antibacterial activity, while supplementation of KC-L with these chelating agents eliminated the inhibitory activity. Together, the data suggest a positive role for divalent and trivalent cations, including iron and aluminum, in bacterial inhibition by KC. Collectively, these studies demonstrate the range of KC bioactivity and provide a better understanding of the mechanism underlying its antibacterial effects. IMPORTANCE The escalating emergence of multidrug-resistant (MDR) bacteria, together with the paucity of novel antimicrobial agents in antibiotic development, is recognized as a worldwide public health crisis. Kisameet clay (KC), found in British Columbia (BC), Canada, is a clay mineral with a long history of therapeutic applications among people of the First Nations. We previously reported the antibacterial activity of KC against a group of MDR clinical pathogens. Here, we demonstrate its activity against two major human-pathogenic fungal species, as well as against bacterial biofilms, which underlie many recalcitrant bacterial infections. In these studies, we also identified several geochemical characteristics of KC, such as metal ions and low pH, which are involved in its antibacterial activity. These findings provide a better understanding of the components of KC antibacterial activity and a basis for developing defined preparations of this clay mineral for therapeutic applications.

Histoplasma and Paracoccidioides are related thermally dimorphic fungal pathogens that cause deadly mycoses (i.e., histoplasmosis and paracoccidioidomycosis, respectively) primarily in North, Central, and South America. Mammalian infection results from inhalation of conidia and their subsequent conversion into pathogenic yeasts. Macrophages in the lung are the first line of defense, but are generally unable to clear these fungi. Instead, Histoplasma and Paracoccidioides yeasts survive and proliferate within the phagosomal compartment of host macrophages. Growth within macrophages requires strategies for acquisition of sufficient nutrients (e.g., carbon, nitrogen, and essential trace elements and co-factors) from the nutrient-depleted phagosomal environment. We review the transcriptomic and recent functional genetic studies that are defining how these intracellular fungal pathogens tune their metabolism to the resources available in the macrophage phagosome. In addition, recent studies have shown that the nutritional state of the macrophage phagosome is not static, but changes upon activation of adaptive immune responses. Understanding the metabolic requirements of these dimorphic pathogens as they thrive within host cells can provide novel targets for therapeutic intervention.

ABSTRACT The origins and maintenance of the rich fungal diversity have been longstanding issues in evolutionary biology. To investigate how differences in expression regulation contribute to divergences in development and ecology among closely related species, transcriptomes were compared between Chaetomium globosum, a homothallic pathogenic fungus thriving in highly humid ecologies, and Neurospora crassa, a heterothallic postfire saprotroph. Gene expression was quantified in perithecia at nine distinct morphological stages during nearly synchronous sexual development. Unlike N. crassa, expression of all mating loci in C. globosum was highly correlated. Key regulators of the initiation of sexual development in response to light stimuli—including orthologs of N. crassa sub-1, sub-1-dependent gene NCU00309, and asl-1—showed regulatory dynamics matching between C. globosum and N. crassa. Among 24 secondary metabolism gene clusters in C. globosum, 11—including the cochliodones biosynthesis cluster—exhibited highly coordinated expression across perithecial development. C. globosum exhibited coordinately upregulated expression of histidine kinases in hyperosmotic response pathways—consistent with gene expression responses to high humidity we identified in fellow pathogen Fusarium graminearum. Bayesian networks indicated that gene interactions during sexual development have diverged in concert with the capacities both to reproduce asexually and to live a self-compatible versus self-incompatible life cycle, shifting the hierarchical roles of genes associated with conidiation and heterokaryon incompatibility in N. crassa and C. globosum. This divergence supports an evolutionary history of loss of conidiation due to unfavorable combinations of heterokaryon incompatibility in homothallic species. IMPORTANCE Fungal diversity has amazed evolutionary biologists for decades. One societally important aspect of this diversity manifests in traits that enable pathogenicity. The opportunistic pathogen Chaetomium globosum is well adapted to a high-humidity environment and produces numerous secondary metabolites that defend it from predation. Many of these chemicals can threaten human health. Understanding the phases of the C. globosum life cycle in which these products are made enables better control and even utilization of this fungus. Among its intriguing traits is that it both is self-fertile and lacks any means of propagule-based asexual reproduction. By profiling genome-wide gene expression across the process of sexual reproduction in C. globosum and comparing it to genome-wide gene expression in the model filamentous fungus N. crassa and other closely related fungi, we revealed associations among mating-type genes, sexual developmental genes, sexual incompatibility regulators, environmentally responsive genes, and secondary metabolic pathways.

ABSTRACT Most known examples of horizontal gene transfer (HGT) between eukaryotes are ancient. These events are identified primarily using phylogenetic methods on coding regions alone. Only rarely are there examples of HGT where noncoding DNA is also reported. The gene encoding the wheat virulence protein ToxA and the surrounding 14 kb is one of these rare examples. ToxA has been horizontally transferred between three fungal wheat pathogens (Parastagonospora nodorum, Pyrenophora tritici-repentis, and Bipolaris sorokiniana) as part of a conserved ∼14 kb element which contains coding and noncoding regions. Here we used long-read sequencing to define the extent of HGT between these three fungal species. Construction of near-chromosomal-level assemblies enabled identification of terminal inverted repeats on either end of the 14 kb region, typical of a type II DNA transposon. This is the first description of ToxA with complete transposon features, which we call ToxhAT. In all three species, ToxhAT resides in a large (140-to-250 kb) transposon-rich genomic island which is absent in isolates that do not carry the gene (annotated here as toxa−). We demonstrate that the horizontal transfer of ToxhAT between P. tritici-repentis and P. nodorum occurred as part of a large (∼80 kb) HGT which is now undergoing extensive decay. In B. sorokiniana, in contrast, ToxhAT and its resident genomic island are mobile within the genome. Together, these data provide insight into the noncoding regions that facilitate HGT between eukaryotes and into the genomic processes which mask the extent of HGT between these species. IMPORTANCE This work dissects the tripartite horizontal transfer of ToxA, a gene that has a direct negative impact on global wheat yields. Defining the extent of horizontally transferred DNA is important because it can provide clues to the mechanisms that facilitate HGT. Our analysis of ToxA and its surrounding 14 kb suggests that this gene was horizontally transferred in two independent events, with one event likely facilitated by a type II DNA transposon. These horizontal transfer events are now in various processes of decay in each species due to the repeated insertion of new transposons and subsequent rounds of targeted mutation by a fungal genome defense mechanism known as repeat induced point mutation. This work highlights the role that HGT plays in the evolution of host adaptation in eukaryotic pathogens. It also increases the growing body of evidence indicating that transposons facilitate adaptive HGT events between fungi present in similar environments and hosts.

ABSTRACT The emergence of azole resistance in the pathogenic fungus Aspergillus fumigatus has continued to increase, with the dominant resistance mechanisms, consisting of a 34-nucleotide tandem repeat (TR34)/L98H and TR46/Y121F/T289A, now showing a structured global distribution. Using hierarchical clustering and multivariate analysis of 4,049 A. fumigatus isolates collected worldwide and genotyped at nine microsatellite loci using analysis of short tandem repeats of A. fumigatus (STRAf), we show that A. fumigatus can be subdivided into two broad clades and that cyp51A alleles TR34/L98H and TR46/Y121F/T289A are unevenly distributed across these two populations. Diversity indices show that azole-resistant isolates are genetically depauperate compared to their wild-type counterparts, compatible with selective sweeps accompanying the selection of beneficial mutations. Strikingly, we found that azole-resistant clones with identical microsatellite profiles were globally distributed and sourced from both clinical and environmental locations, confirming that azole resistance is an international public health concern. Our work provides a framework for the analysis of A. fumigatus isolates based on their microsatellite profile, which we have incorporated into a freely available, user-friendly R Shiny application (AfumID) that provides clinicians and researchers with a method for the fast, automated characterization of A. fumigatus genetic relatedness. Our study highlights the effect that azole drug resistance is having on the genetic diversity of A. fumigatus and emphasizes its global importance upon this medically important pathogenic fungus. IMPORTANCE Azole drug resistance in the human-pathogenic fungus Aspergillus fumigatus continues to emerge, potentially leading to untreatable aspergillosis in immunosuppressed hosts. Two dominant, environmentally associated resistance mechanisms, which are thought to have evolved through selection by the agricultural application of azole fungicides, are now distributed globally. Understanding the effect that azole resistance is having on the genetic diversity and global population of A. fumigatus will help mitigate drug-resistant aspergillosis and maintain the azole class of fungicides for future use in both medicine and crop protection.

More than 1.5 million fungal species are estimated to live in vastly different environmental niches. Despite each unique host environment, fungal cells sense certain fundamentally conserved elements, such as nutrients, pheromones and stress, for adaptation to their niches. Sensing these extracellular signals is critical for pathogens to adapt to the hostile host environment and cause disease. Hence, dissecting the complex extracellular signal-sensing mechanisms that aid in this is pivotal and may facilitate the development of new therapeutic approaches to control fungal infections. In this review, we summarize the current knowledge on how two important pathogenic yeasts, Candida albicans and Cryptococcus neoformans, sense nutrient availability, such as carbon sources, amino acids, and ammonium, and different stress signals to regulate their morphogenesis and pathogenicity in comparison with the non-pathogenic model yeast Saccharomyces cerevisiae. The molecular interactions between extracellular signals and their respective sensory systems are described in detail. The potential implication of analyzing nutrient and stress-sensing systems in antifungal drug development is also discussed.

In the past few years, fungal diseases caused estimated over 1.6 million deaths annually and over one billion people suffer from severe fungal diseases (Brown et al., 2012; Anonymous, 2017b). Public health surveillance of fungal diseases is generally not compulsory, suggesting that most estimates are conservative (Casadevall, 2017; Anonymous, 2017a). Fungal disease can also damage plants and crops, causing major losses in agricultural activities and food production (Savary et al., 2012). Animal pathogenic fungi are threatening bats, amphibians and reptiles with extinction (Casadevall, 2017). It is estimated that fungi are the highest threat for animal-host and plant-host species, representing the major cause (approximately 65%) of pathogen-driven host loss (Fisher et al., 2012). In this complex scenario, it is now clear that the global warming and accompanying climate changes have resulted in increased incidence of many fungal diseases (Garcia-Solache and Casadevall, 2010). On the basis of all these factors, concerns on the occurrence of a pandemic of fungal origin in a near future have been raised (Casadevall, 2017). In this context, to stop forgetting and underestimating fungal diseases is mandatory.

Short-wave ultraviolet (UV-C) treatment represents a potent, clean and safe substitute to chemical sanitizers for fresh fruit preservation. However, the dosage requirement for microbial disinfection may have negative effects on fruit quality. In this study, UV-C was found to be more efficient in killing spores of Botrytis cinerea in dark and red light conditions when compared to white and blue light. Loss of the blue light receptor gene Bcwcl1, a homolog of wc-1 in Neurospora crassa, led to hypersensitivity to UV-C in all light conditions tested. The expression of Bcuve1 and Bcphr1, which encode UV-damage endonuclease and photolyase, respectively, were strongly induced by white and blue light in a Bcwcl1-dependent manner. Gene mutation analyses of Bcuve1 and Bcphr1 indicated that they synergistically contribute to survival after UV-C treatment. In vivo assays showed that UV-C (1.0 kJ/m2) abolished decay in drop-inoculated fruit only if the UV-C treatment was followed by a dark period or red light, while in contrast, typical decay appeared on UV-C irradiated fruits exposed to white or blue light. In summary, blue light enhances UV-C resistance in B. cinerea by inducing expression of the UV damage repair-related enzymes, while the efficiency of UV-C application for fruit surface disinfection can be enhanced in dark or red light conditions; these principles seem to be well conserved among postharvest fungal pathogens.

ABSTRACT Delineating species and epidemic lineages in fungal plant pathogens is critical to our understanding of disease emergence and the structure of fungal biodiversity and also informs international regulatory decisions. Pyricularia oryzae (syn. Magnaporthe oryzae) is a multihost pathogen that infects multiple grasses and cereals, is responsible for the most damaging rice disease (rice blast), and is of growing concern due to the recent introduction of wheat blast to Bangladesh from South America. However, the genetic structure and evolutionary history of M. oryzae, including the possible existence of cryptic phylogenetic species, remain poorly defined. Here, we use whole-genome sequence information for 76 M. oryzae isolates sampled from 12 grass and cereal genera to infer the population structure of M. oryzae and to reassess the species status of wheat-infecting populations of the fungus. Species recognition based on genealogical concordance, using published data or extracting previously used loci from genome assemblies, failed to confirm a prior assignment of wheat blast isolates to a new species (Pyricularia graminis-tritici). Inference of population subdivisions revealed multiple divergent lineages within M. oryzae, each preferentially associated with one host genus, suggesting incipient speciation following host shift or host range expansion. Analyses of gene flow, taking into account the possibility of incomplete lineage sorting, revealed that genetic exchanges have contributed to the makeup of multiple lineages within M. oryzae. These findings provide greater understanding of the ecoevolutionary factors that underlie the diversification of M. oryzae and highlight the practicality of genomic data for epidemiological surveillance in this important multihost pathogen. IMPORTANCE Infection of novel hosts is a major route for disease emergence by pathogenic microorganisms. Understanding the evolutionary history of multihost pathogens is therefore important to better predict the likely spread and emergence of new diseases. Magnaporthe oryzae is a multihost fungus that causes serious cereal diseases, including the devastating rice blast disease and wheat blast, a cause of growing concern due to its recent spread from South America to Asia. Using whole-genome analysis of 76 fungal strains from different hosts, we have documented the divergence of M. oryzae into numerous lineages, each infecting a limited number of host species. Our analyses provide evidence that interlineage gene flow has contributed to the genetic makeup of multiple M. oryzae lineages within the same species. Plant health surveillance is therefore warranted to safeguard against disease emergence in regions where multiple lineages of the fungus are in contact with one another.