Essential roles of ferric reductase-like proteins in growth, development, stress response, and virulence of the filamentous entomopathogenic fungus Beauveria bassiana.

In yeasts, ferric reductase catalyzes reduction of ferric ion to ferrous form, which is essential for the reductive iron assimilation system. However, the physiological roles of ferric reductases remain largely unknown in the filamentous fungi. In this study, genome-wide annotation revealed thirteen ferric reductase-like (Fre) proteins in the filamentous insect pathogenic fungus Beauveria bassiana, and all their functions were genetically characterized. Ferric reductase family proteins exhibit different sub-cellular distributions (e.g., cell periphery and vacuole), which was due to divergent domain architectures. Fre proteins had a synergistic effect on fungal virulence, which was ascribed to their distinct functions in different physiologies. Ten Fre proteins were not involved in reduction of ferric ion in submerged mycelia, but most proteins contributed to blastospore development. Only two Fre proteins significantly contributed to B. bassiana vegetative growth under the chemical-induced iron starvation, but most Fre proteins were involved in resistance to osmotic and oxidative stresses. Notably, a bZIP-type transcription factor HapX bound to the promoter regions of all FRE genes in B. bassiana, and displayed varying roles in the transcription activation of these genes. This study reveals the important role of BbFre family proteins in development, stress response, and insect pathogenicity, as well as their distinctive role in the absorption of ferric iron from the environment.

Homologs of bacterial heat-labile enterotoxin subunit A contribute to development, stress response, and virulence in filamentous entomopathogenic fungus Beauveria bassiana.

Introduction: Enterotoxigenic bacteria commonly excrete heat-labile enterotoxins (LT) as virulence factors that consist of one subunit A (LTA) and five B subunits (LTB). In fungi, there are a large number of genes encoding the homologs of LTA, but their biological roles remain largely unknown. Methods: In this study, we identified 14 enterotoxin_A domain proteins in filamentous fungus B. bassiana in which five proteins were functionally characterized. Results: Five proteins displayed diverse sub-cellular localizations but perform convergent functions in stress response, development, and virulence. The loss of five LTA genes resulted in significant reduction in conidial production, blastospore formation, and the increased sensitivity to oxidative and cell wall -perturbing stresses. The virulence of five disruptants was notably weakened as indicated by topical and intrahemocoel injection assays. Notably, the loss of these five proteins led to the significant changes in the carbohydrate profiles of cellular surface, which induced the enhanced host immune reactions of encapsulation and melanization. Discussion: Thus, LTA proteins contribute to the fungus-host interaction via maintaining the carbohydrate profiles of cellular surface. This study expands our understanding of the enterotoxin_A domain proteins in fungal physiology and deepens mechanisms involved in the lifestyle of fungal insect pathogens.

Two aminopeptidase I homologs convergently contribute to pathobiology of fungal entomopathogen Beauveria bassiana via divergent physiology-dependent autophagy pathways for vacuolar targeting.

INTRODUCTION: In yeast, the cytoplasm-to-vacuole targeting (Cvt) pathway acts as a biosynthetic autophagy-related process, in which vacuolar targeting of hydrolase is mediated by the machineries involved in the selective autophagy. However, the mechanistic insights into vacuolar targeting of hydrolases through the selective autophagy pathway still remain enigmatic in filamentous fungi. OBJECTIVES: Our study aims to investigate the mechanisms involved in vacuolar targeting of hydrolases in filamentous fungi. METHODS: The filamentous entomopathogenic fungus Beauveria bassiana was used as a representative of filamentous fungi. We identified the homologs of yeast aminopeptidase I (Ape1) in B. bassiana by bioinformatic analyses and characterized their physiological roles by gene function analyses. Pathways for vacuolar targeting of hydrolases were investigated via molecular trafficking analyses. RESULTS: B. bassiana has two homologs of yeast aminopeptidase I (Ape1) which are designated as BbApe1A and BbApe1B. The two homologs of yeast Ape1 contribute to starvation tolerance, development, and virulence in B. bassiana. Significantly, BbNbr1 acts as a selective autophagy receptor to mediate the vacuolar targeting of the two Ape1 proteins, in which BbApe1B interacts with BbNbr1 also directly interacting with BbAtg8, and BbApe1A has an additional requirement of the scaffold protein BbAtg11 that interacts with BbNbr1 and BbAtg8. Protein processing occurs at both terminuses of BbApe1A and only at carboxyl terminus of BbApe1B, which is also dependent on the autophagy-related proteins. Together, the functions and translocation processes of the two Ape1 proteins are associated with autophagy in fungal lifecycle. CONCLUSION: This study reveals the functions and translocation processes for vacuolar hydrolases in the insect-pathogenic fungi and improves our understandings of the Nbr1-mediated vacuolar targeting pathway in the filamentous fungi.

Autophagy-Related Gene 4 Participates in the Asexual Development, Stress Response and Virulence of Filamentous Insect Pathogenic Fungus Beauveria bassiana.

Autophagy is a conserved mechanism for the turnover of intracellular components. Among the 'core' autophagy-related genes (ATGs), the cysteine protease Atg4 plays an important role in the activation of Atg8 by exposing the glycine residue at its extreme carboxyl terminus. In the insect fungal pathogen Beauveria bassiana, a yeast ortholog of Atg4 was identified and functionally analyzed. Ablation of the BbATG4 gene blocks the autophagic process during fungal growth under aerial and submerged conditions. Gene loss did not affect fungal radial growth on various nutrients, but ΔBbatg4 exhibited an impaired ability to accumulate biomass. The mutant displayed increased sensitivity to stress caused by menadione and hydrogen peroxide. ΔBbatg4 generated abnormal conidiophores with reduced production of conidia. Additionally, fungal dimorphism was significantly attenuated in gene disruption mutants. Disruption of BbATG4 resulted in significantly weakened virulence in topical and intrahemocoel injection assays. Our study indicates that BbAtg4 contributes to the lifecycle of B. bassiana via its autophagic roles.

Transcriptomic investigation reveals a physiological mechanism for Beauveria bassiana to survive under linoleic acid stress.

In integrated pest management program (IPM), the compatibility of mycoinsecticides with bioactive fungicides [e.g., unsaturated fatty acids (UFAs)] has attracted more and more attention; however, the mechanisms underlying fungal resistance to UFAs remain largely unknown. In this study, Beauveria bassiana, an entomopathogenic fungus, was used to explore fungal responses to linoleic acid (LA). Genome-wide expression revealed the transcriptomic responses of fungal cells to LA in a stress-intensity-dependent manner. Enrichment analyses indicated that the up-regulated differentially expressed genes (DEGs) are associated with the metabolism of lipid and fatty acids. Notably, a lipid-droplet protein (BbLar1) maintains the intracellular homeostasis of fatty acids and is crucial to fungal tolerance to LA stress, which significantly contributes to fungal compatibility with UFAs. Additionally, BbLar1 links the lipid droplets to global expression profiles in B. bassiana under LA stress. Our investigations provide an initial framework for improving the efficacy of insect pathogenic fungi in practical application.

Divergent Physiological Functions of Four Atg22-like Proteins in Conidial Germination, Development, and Virulence of the Entomopathogenic Fungus Beauveria bassiana.

In yeast, Atg22 functions as a vacuolar efflux transporter to release the nutrients from the vacuole to the cytosol after the degradation of autophagic bodies. There are more than one Atg22 domain-containing proteins in filamentous fungi, but their physiological roles are largely unknown. In this study, four Atg22-like proteins (BbAtg22A through D) were functionally characterized in the filamentous entomopathogenic fungus Beauveria bassiana. These Atg22-like proteins exhibit different sub-cellular distributions. BbAtg22A localizes in lipid droplets. BbAtg22B and BbAtg22C are completely distributed in the vacuole, and BbAtg22D has an additional association with the cytomembrane. The ablation of Atg22-like proteins did not block autophagy. Four Atg22-like proteins systematically contribute to the fungal response to starvation and virulence in B. bassiana. With the exception of ∆Bbatg22C, the other three proteins contribute to dimorphic transmission. Additionally, BbAtg22A and BbAtg22D are required for cytomembrane integrity. Meanwhile, four Atg22-like proteins contribute to conidiation. Therefore, Atg22-like proteins link distinct sub-cellular structures for the development and virulence in B. bassiana. Our findings provide a novel insight into the non-autophagic roles of autophagy-related genes in filamentous fungi.

The Entomopathogenic Fungus Beauveria bassiana Employs Autophagy as a Persistence and Recovery Mechanism during Conidial Dormancy.

Many filamentous fungi develop a conidiation process as an essential mechanism for their dispersal and survival in natural ecosystems. However, the mechanisms underlying conidial persistence in environments are still not fully understood. Here, we report that autophagy is crucial for conidial lifespans (i.e., viability) and vitality (e.g., stress responses and virulence) in the filamentous mycopathogen Beauveria bassiana. Specifically, Atg11-mediated selective autophagy played an important, but not dominant, role in the total autophagic flux. Furthermore, the aspartyl aminopeptidase Ape4 was found to be involved in conidial vitality during dormancy. Notably, the vacuolar translocation of Ape4 was dependent on its physical interaction with autophagy-related protein 8 (Atg8) and associated with the autophagic role of Atg8, as determined through a truncation assay of a critical carboxyl-tripeptide. These observations revealed that autophagy acted as a subcellular mechanism for conidial recovery during dormancy in environments. In addition, a novel Atg8-dependent targeting route for vacuolar hydrolase was identified, which is essential for conidial exit from a long-term dormancy. These new insights improved our understanding of the roles of autophagy in the physiological ecology of filamentous fungi as well as the molecular mechanisms involved in selective autophagy. IMPORTANCE Conidial environmental persistence is essential for fungal dispersal in ecosystems while also serving as a determinant for the biocontrol efficacy of entomopathogenic fungi during integrated pest management. This study identified autophagy as a mechanism to safeguard conidial lifespans and vitality postmaturation. In this mechanism, the aspartyl aminopeptidase Ape4 translocates into vacuoles via its physical interaction with autophagy-related protein 8 (Atg8) and is involved in conidial vitality during survival. The study revealed that autophagy acted as a subcellular mechanism for maintaining conidial persistence during dormancy, while also documenting an Atg8-dependent targeting route for vacuolar hydrolase during conidial recovery from dormancy. Thus, these observations provided new insight into the roles of autophagy in the physiological ecology of filamentous fungi and documented novel molecular mechanisms involved in selective autophagy.

Genome-wide identification of BCS1 domain-containing proteins reveals the mitochondrial bcs1 essential for growth, stress response, and virulence of the filamentous entomopathogenic fungus Beauveria bassiana.

In yeasts, bcs1 is a mitochondrial AAA protein (ATPase associated with diverse cellular activities) and required for biogenesis of the complex III in mitochondrial electron transfer chain. However, the presence and biological roles of bcs1 remain largely unknown in the filamentous fungi. In present study, genome-wide identification revealed that there were six BCS1-domain containing proteins (Bbbcs1a through f) in the filamentous insect pathogenic fungus Beauveria bassiana, five of which (except for Bbbcs1f) were functionally analyzed. Phenotypic evaluation revealed that only Bbbcs1b and Bbbcs1c contributed to fungal physiologies, and they localized to nuclei and mitochondria, respectively. Hence, Bbbcs1c is considered as the ortholog of yeast bcs1 in B. bassiana. Ablation of Bbbcs1c did not affect biogenesis of mitochondria, but its loss significantly attenuated mitochondrial functionality (e.g., ATP synthesis and mitochondrial targeting of proteins) significantly. ΔBbbcs1c mutant displayed the impaired phenotypes in vegetative growth, stress response, development, and virulence. Notably, ΔBbbcs1c mutant displayed the increased sensitivity to linoleic acid (LA) stress and lost the intracellular fatty acid homeostasis. The Bbbcs1c loss compromised the mitochondrial membrane potential, and LA stress exacerbated this damage. These findings indicate that Bbbcs1c is a functional homolog of yeast bcs1 in B. bassiana and links mitochondrial functionality to unique lifestyle in the entomopathogenic fungi.

Functional characterization of two homologs of yeast acetyl-coenzyme A synthetase in the entomopathogenic fungus Beauveria bassiana.

Acetyl-coenzyme A (CoA) synthetase (Acs) links cellular metabolism and physiology by catalyzing acetate and CoA into acetyl-CoA. However, the biological roles of Acs are not well studied in entomopathogenic fungi. In this study, two Acs proteins (BbAcs1 and BbAcs2) was functionally characterized in the filamentous insect pathogenic fungus Beauveria bassiana. BbAcs1 and BbAcs2 localize in cytoplasm and peroxisome, respectively. BbAcs1 contributes to vegetative growth on fatty acids as carbon source, and BbAcs2 did not. Both genes did not contribute to fungal response to stresses. The BbAcs1 loss conferred a slight influence on conidiation, and did not result in the defects in blastospore formation. On the contrary, BbAcs2 significantly contributes to lipid metabolism in germlings, blastospore formation, and virulence. The results indicated that Acs2 played a more predominant role than Acs1 in B. bassiana, which links the acetyl-CoA metabolism with the lifestyle of entomopathogenic fungi.

Succinate Dehydrogenase Subunit C Contributes to Mycelial Growth and Development, Stress Response, and Virulence in the Insect Parasitic Fungus Beauveria bassiana.

Succinate dehydrogenase (SDH), also known as respiratory chain complex II, plays a crucial role in energy production in which SdhC functions as an anchored subunit in the inner membrane of mitochondria. In this study, domain annotation analyses revealed that two SdhC domain-containing proteins were present in the filamentous insect-pathogenic fungus Beauveria bassiana, and they were named BbSdhC1 and BbSdhC2, respectively. Only BbSdhC1 localized to mitochondria; hence, this protein is considered the ortholog of SdhC in B. bassiana. Ablation of BbSdhC1 led to significantly reduced vegetative growth on various nutrients. The ΔBbsdhc1 mutant displayed the significantly reduced ATP synthesis and abnormal differentiation under aerial and submerged conditions. Notably, the BbSdhC1 loss resulted in enhanced intracellular levels of reactive oxygen species (ROS) and impaired growth of mycelia under oxidative stress. Finally, insect bioassays (via cuticle and intrahemocoel injection infection) revealed that disruption of BbSdhC1 significantly attenuated fungal virulence against the insect hosts. These findings indicate that BbSdhC1 contributes to vegetative growth, resistance to oxidative stress, differentiation, and virulence of B. bassiana due to its roles in energy generation and maintaining the homeostasis of the intracellular ROS levels. IMPORTANCE The electron transport chain (ETC) is critical for energy supply by mediating the electron flow along the mitochondrial membrane. Succinate dehydrogenase (SDH) is also known as complex II in the ETC, in which SdhC is a subunit anchored in mitochondrial membrane. However, the physiological roles of SdhC remain enigmatic in filamentous fungi. In filamentous insect-pathogenic fungus B. bassiana, SdhC is required for maintaining mitochondrial functionality, which is critical for fungal stress response, development, and pathogenicity. These findings improve our understanding of physiological mechanisms of ETC components involved in pathogenicity of the entomopathogenic fungi.

Systematic contributions of CFEM domain-containing proteins to iron acquisition are essential for interspecies interaction of the filamentous pathogenic fungus Beauveria bassiana.

Common in fungal extracellular membrane (CFEM) domain is unique in fungal proteins and some of which contribute to iron acquisition in yeast. However, their roles in iron acquisition remain largely unknown in filamentous fungi. In this study, 12 CFEM-containing proteins were bioinformatically identified in the filamentous entomopathogenic fungus Beauveria bassiana, and the roles of 11 genes were genetically characterized. Transmembrane helices were critical for their association with intracellular membranes, and their number varied among proteins. Eleven CFEM genes significantly contribute to vegetative growth under iron starvation and virulence. Notably, the virulence of most disruptants could be significantly weakened by a decrease in iron availability, in which the virulence of ΔBbcfem7 and 8 strains was partially recovered by exogenous hemin. ΔBbcfem7 and 8 mutants displayed defective competitiveness against the sister entomopathogenic fungus Beauveria brongniartii. All 11 disruptants displayed impaired growth in the antagonistic assay with the saprotrophic fungus Aspergillus niger, which could be repressed by exogenous ferric ions. These findings not only reveal the systematic contributions of CFEM proteins to acquire two forms of iron (i.e. heme and ferric ion) in the entire lifecycle of entomopathogenic fungi but also help to better understand the mechanisms of fungus-host and inter-fungus interactions.

Proteomic and Phosphoryproteomic Investigations Reveal that Autophagy-Related Protein 1, a Protein Kinase for Autophagy Initiation, Synchronously Deploys Phosphoregulation on the Ubiquitin-Like Conjugation System in the Mycopathogen Beauveria bassiana.

Autophagy is a conserved intracellular degradation mechanism in eukaryotes and is initiated by the protein kinase autophagy-related protein 1 (Atg1). However, except for the autophosphorylation activity of Atg1, the target proteins phosphorylated by Atg1 are largely unknown in filamentous fungi. In Beauveria bassiana (a filamentous insect-pathogenic fungus), Atg1 is indispensable for autophagy and is associated with fungal development. Comparative omics-based analyses revealed that B. bassiana Atg1 (BbAtg1) has key influence on the proteome and phosphoproteome during conidiogenesis. In terms of its physiological functions, the BbAtg1-mediated phosphoproteome is primarily associated with metabolism, signal transduction, cell cycle, and autophagy. At the proteomic level, BbAtg1 mainly regulates genes involved in protein synthesis, protein fate, and protein with binding function. Furthermore, integrative analyses of phosphoproteomic and proteomic data led to the identification of several potential targets regulated by BbAtg1 phosphorylation activity. Notably, we demonstrated that BbAtg1 phosphorylated BbAtg3, an essential component of the ubiquitin-like conjugation system in autophagic progress. Our findings indicate that in addition to being a critical component of the autophagy initiation, Atg1 orchestrates autophagosome elongation via its phosphorylation activity. The data from our study will facilitate future studies on the noncanonical targets of Atg1 and help decipher the Atg1-mediated phosphorylation networks. IMPORTANCE Autophagy-related protein 1 (Atg1) is a serine/threonine protein kinase for autophagy initiation. In contrast to the unicellular yeast, the target proteins phosphorylated by Atg1 are largely unknown in filamentous fungi. In this study, the entomopathogenic fungus Beauveria bassiana was used as a representative of filamentous fungi due to its importance in the applied and fundamental research. We revealed that Atg1 mediates the comprehensive proteome and phosphoproteome, which differ from those revealed in yeast. Further investigation revealed that Atg1 directly phosphorylates the E2-like enzyme Atg3 of the ubiquitin-like conjugation system (ULCS), and the phosphorylation of Atg3 is indispensable for ULCS functionality. Interestingly, the phosphorylation site of Atg3 is conserved among a set of insect- and plant-pathogenic fungi but not in human-pathogenic fungi. This study reveals new regulatory mechanisms of autophagy and provides new insights into the evolutionary diversity of the Atg1 kinase signaling pathways among different pathogenic fungi.

A homologue of yeast acyl-CoA synthetase Faa1 contributes to cytomembrane functionality involved in development and virulence in the insect pathogenic fungus Beauveria bassiana.

Acyl-CoA synthetase (ACS) functions as a hub linking lipid metabolism with in cellular physiologies by producing active intermediate of catalyzes acyl-CoA. However, the biological roles of ACS are largely unknown in filamentous fungi. In this study, an ortholog of yeast Faa1, named BbFaa1, was functionally characterized in the filamentous entomopathogenic fungus Beauveria bassiana. BbFaa1 was associated with vesicular membrane, and its loss resulted in the impaired cytomembrane integrity. Notably, in ΔBbfaa1 mutant strain, the translocation of hydrophobins across cell membrane was significantly hampered, which resulted in the reduced hydrophobicity of aerial mycelia and conidia. In addition, loss of BbFaa1 significantly weakened fungal virulence. Our findings indicate that the metabolism of acyl-CoA synthetase Faa1 contributes to the cytomembrane functionality which cascades hydrophobin translocation and differentiation, thus affecting virulence of B. bassiana.

A virulence-related lectin traffics into eisosome and contributes to functionality of cytomembrane and cell-wall in the insect-pathogenic fungus Beauveria bassiana.

Lectins are characterized of the carbohydrate-binding ability and play comprehensive roles in fungal physiology (e.g., defense response, development and host-pathogen interaction). Beauveria bassiana, a filamentous entomopathogenic fungus, has a lectin-like protein containing a Fruit Body_domain (BbLec1). BbLec1 could bind to chitobiose and chitin in fungal cell wall. BbLec1 proteins interacted with each other to form multimers, and translocated into eisosomes. Further, the interdependence between BbLec1 and the eisosome protein PliA was essential for stabilizing the eisosome architecture. To test the BbLec1 roles in B. bassiana, we constructed the gene disruption and complementation mutants. Notably, the BbLec1 loss resulted in the impaired cell wall in mycelia and conidia as well as conidial formation capacity. In addition, disruption of BbLec1 led to the reduced cytomembrane integrity and the enhanced sensitivity to osmotic stress. Finally, ΔBbLec1 mutant strain displayed the weakened virulence when compared with the wild-type strain. Taken together, BbLec1 traffics into eisosome and links the functionality of eisosome to development and virulence of B. bassiana.

Transcription Activator Swi6 Interacts with Mbp1 in MluI Cell Cycle Box-Binding Complex and Regulates Hyphal Differentiation and Virulence in Beauveria bassiana.

Mbp1 protein acts as a DNA-binding protein in MluI cell cycle box-binding complex (MBF) and plays an essential role in filamentous myco-pathogen Beauveria bassiana.In the current study, BbSwi6 (a homologue of yeast Swi6) was functionally characterized in B.bassiana. Both BbSwi6 and BbMbp1 localize in the nucleus and display a direct interaction relationship which is indicated by a yeast two-hybrid assay. BbSwi6 significantly contributes to hyphal growth, asexual sporulation and virulence. On the aerial surface, ΔBbSwi6 grew slower on various nutrients and displayed abnormal conidia-producing structures, which hardly produced conidia. In liquid media, BbSwi6 loss led to 90% reduction in blastospore yield. Finally, the virulence of the ΔBbSwi6 mutant was modestly weakened with a reduction of 20% in median lethal time. Comparative transcriptomics revealed that BbSwi6 mediated different transcriptomes during fungal development into conidia and blastospores. Notably, under the indicated condition, the BbSwi6-mediated transcriptome significantly differed to that mediated by BbMbp1. Our results demonstrate that, in addition to their roles as the interactive components in MBF, BbSwi6 and BbMbp1 mediate divergent genetic pathways during morphological transitions in B. bassiana.

HapX, an Indispensable bZIP Transcription Factor for Iron Acquisition, Regulates Infection Initiation by Orchestrating Conidial Oleic Acid Homeostasis and Cytomembrane Functionality in Mycopathogen Beauveria bassiana.

In pathogenic filamentous fungi, conidial germination not only is fundamental for propagation in the environment but is also a critical step of infection. In the insect mycopathogen Beauveria bassiana, we genetically characterized the role of the basic leucine zipper (bZIP) transcription factor HapX (BbHapX) in conidial nutrient reserves and pathogen-host interaction. Ablation of BbHapX resulted in an almost complete loss of virulence in the topical inoculation and intrahemocoel injection assays. Comparative transcriptomic analysis revealed that BbHapX is required for fatty acid (FA)/lipid metabolism, and biochemical analyses indicated that BbHapX loss caused a significant reduction in conidial FA contents. Exogenous oleic acid could partially or completely restore the impaired phenotypes of the ΔBbHapX mutant, including germination rate, membrane integrity, vegetative growth, and virulence. BbHapX mediates fungal iron acquisition which is not required for desaturation of stearic acid. Additionally, inactivation of the Δ9-fatty acid desaturase gene (BbOle1) generated defects similar to those of the ΔBbHapX mutant; oleic acid also had significant restorative effects on the defective phenotypes of the ΔBbOle1 mutant. A gel retarding assay revealed that BbHapX directly regulated the expression of BbOle1 Lipidomic analyses indicated that both BbHapX and BbOle1 contributed to the homeostasis of phospholipids with nonpolar tails derived from oleic acid; therefore, exogenous phospholipids could significantly restore membrane integrity. These data reveal that the HapX-Ole1 pathway contributes to conidial fatty acid/lipid reserves and that there are important links between the lipid biology and membrane functionality involved in the early stages of infection caused by B. bassiana IMPORTANCE Conidial maturation and germination are highly coupled physiological processes in filamentous fungi that are critical for the pathogenicity of mycopathogens. Compared to the mechanisms involved in conidial germination, those of conidial reserves during maturation are less understood. The insect-pathogenic fungus Beauveria bassiana, as a representative species of filamentous fungi, is important for applied and fundamental research. In addition to its conserved roles in fungal adaptation to iron status, the bZIP transcription factor HapX acts as a master regulator involved in conidial virulence and regulates fatty acid/lipid metabolism. Further investigation revealed that the Δ9-fatty acid desaturase gene (Ole1) is a direct downstream target of HapX. This study reveals the HapX-Ole1 pathway involved in the fatty acid/lipid accumulation associated with conidial maturation and provides new insights into the startup mechanism of infection caused by spores from pathogenic fungi.

Transcriptomic analyses reveal comprehensive responses of insect hemocytes to mycopathogen Beauveria bassiana, and fungal virulence-related cell wall protein assists pathogen to evade host cellular defense.

Entomopathogenic fungi naturally infect insect hosts in environment. Fungal invasion and host immune defense are still in the progress of co-evolution. In this study, entomopathogenic fungus Beauveria bassiana and lepidopteran insect Galleria mellonella were used to investigate host cellular immunity and fungal strategy to evade host defense. First of all, genome-wide expression revealed the transcriptomic responses of hemocytes to insect mycopathogen, which dynamically varied during infection process. Enrichment analysis indicated that differentially expressed genes were primarily involved in metabolism, cellular process and immune system. Notably, cellular response involved a series of hydrolytic enzyme and antimicrobial peptide genes which were sorted together in clustering analysis. In B. bassiana, a cell-wall protein gene (BbCwp) contributes to fungal development in host hemocoel and virulence. RT-qPCR analyses indicated that infection by ΔBbCwp mutant strain caused the up-regulated expression of a series of immunity-related genes, including ß-1, 3-glucan recognition protein, hydrolytic enzyme and antimicrobial peptide genes. Disruption of BbCwp resulted in a significant change in conidial lectin-binding feature and the enhanced encapsulation by the host hemocytes. After being treated with hydrolytic enzymes, ΔBbCwp mutant displayed a significantly enhanced sensitivity to osmotic and oxidative stresses. In conclusion, fungal invasion initiates comprehensive physiological responses in the host hemocytes. For mycopathogen, cell-wall protein plays an important role in fungal evasion of immunity defense and colonization in host. Our studies provide an initial framework for exploring more mechanistic details about the fungus-host interaction.

Mbp1, a component of the MluI cell cycle box-binding complex, contributes to morphological transition and virulence in the filamentous entomopathogenic fungus Beauveria bassiana.

The Mbp1 protein functions as a DNA-binding protein in the MluI cell cycle box-binding complex and plays significant roles in yeast development. In this study, an ortholog of yeast Mbp1, BbMbp1, was characterized in a filamentous insect mycopathogen, Beauveria bassiana. BbMbp1 plays an important role in morphological changes under aerial and liquid environments. On the aerial surface, BbMbp1 was indispensable for the biogenesis of conidiophores and conidiation. Under submerged conditions, the ∆BbMbp1 mutant displayed abnormal spore-producing structures, with a dramatic decrease in blastospore yield (~95%). The virulence of the ∆BbMbp1 mutant was notably weakened, which might be due to the defect in in vivo blastospore formation in the insect. Moreover, disruption of BbMbp1 resulted in a substantial reduction in hyphal growth on cadavers. Comparative transcriptomics revealed that BbMbp1 mediated different transcriptomes during the formation processes of conidia and blastospores. Yeast one-hybrid assays demonstrated that BbMbp1 was required for transcriptional control of a cell wall protein gene, BbCwp, and an integral membrane protein gene, BbImp that played significant roles in conidiation and blastospore formation respectively. Our results demonstrate that BbMbp1 contributes to the morphological transitions in the pathogenic and saprophytic growth of B. bassiana via different genetic pathways.

Upregulation of ADAMTS­7 and downregulation of COMP are associated with spontaneous abortion.

A disintegrin and metalloproteinase with thrombospondin motifs 7 (ADAMTS­7) has been revealed to serve an important role in inflammation­associated diseases. However, the role of ADAMTS­7 in spontaneous abortion (SA) remains unclear. In the present study, human and mouse decidual tissues were used to detect the expression of ADAMTS­7 and cartilage oligomeric matrix protein (COMP) in mice with lipopolysaccharide (LPS)­induced abortion (10 mice/group), and in SA humans and the corresponding control group (21 participants in the SA group and 15 participants in the control group). The results revealed that ADAMTS­7 expression was upregulated and that COMP expression was downregulated in the mouse decidual tissue of the LPS­induced abortion group, when compared with that of the normal control group. The results were further confirmed by western blot analysis and reverse transcription­quantitative polymerase chain reaction (RT­qPCR) analysis, which revealed increased ADAMTS­7 and decreased COMP expression at the protein and mRNA levels in mice treated with LPS. Additionally, the expression of ADAMTS­7 was negatively correlated with the expression of COMP in mice, with a correlation coefficient of ­0.936 (P<0.001). In addition, the expression of ADAMTS­7 and COMP exhibited was similar in the decidual tissue of SA patients when compared with the levels observed in the tissues of the normal control participants, as demonstrated by increased ADAMTS­7 expression and decreased COMP expression. Western blotting and RT­qPCR analysis revealed that ADAMTS­7 was increased and COMP was decreased in the decidual tissue of SA subjects. The correlation analysis of ADAMTS­7 and COMP in human decidual tissue also revealed a similar result, with a correlation coefficient of ­0.836 (P<0.001). The results of the present study demonstrated that ADAMTS­7 was upregulated and COMP was downregulated in the decidual tissues of humans and mice with SA, and a negative correlation was identified between the expression levels of ADAMTS­7 and COMP, thereby providing novel evidence for a better understanding of the pathogenesis of SA, which may lead to improvements in the clinical pregnancy outcomes of these individuals.

Glc8, a regulator of protein phosphatase type 1, mediates oxidation tolerance, asexual development and virulence in Beauveria bassiana, a filamentous entomopathogenic fungus.

Protein phosphatase type 1 (PP1) plays an important role in cellular metabolism and development in yeast. In PP1 enzyme complex, Glc8 protein is a global regulatory subunit and regulates many physiological processes. However, its biological roles are unexplored in filamentous fungi. In this study, we characterized a yeast ortholog of Glc8 in Beauveria bassiana, a filamentous entomopathogenic fungus. Gene disruption of BbGlc8 had no significant effect on vegetative growth, but resulted in a significant reduction in conidiation (51%) and blastospore yield (55%) in the mutant. The ΔBbGlc8 mutant displayed an enhanced sensitivity to oxidative stress and a weakened virulence as indicated by cuticle infection and intrahemocoel injection assays. Transcriptomic analysis indicated that the genes regulated by BbGlc8 during conidiation were primarily associated with metabolism, cell rescue and cell wall formation. Notably, as a down-regulated gene in ΔBbGlc8 mutant, BbOsmC2 (a member of OsmC protein family) contributes to fungal resistance to salt stress, spore differentiation and virulence. Thus, BbOsmC2 functions as a down-stream target of BbGlc8 during spore differentiation, but not in stress response. Our findings indicate that BbGlc8 contributes to the biocontrol potential of B. bassiana by mediating comprehensive genetic pathways.