Light stimulates conidiation of the entomopathogenic fungus Beauveria bassiana

Beauveria bassiana is a commercially important entomopathogenic fungus. Like other insect fungal pathogens, B. bassiana usually produces asexual reproductive bodies, conidia, for dispersal, transmission and infection of insects. Adequate mass-production of high quality conidia is crucial to development of an efficient B. bassiana insecticide. However, little is known about details of conidiation in this fungus in response to environmental signals, which limits understanding of the mechanism of conidiation and improvement in conidia production. Here, morphologenetic changes of B. bassiana under different light conditions are reported. When cultured in total darkness, B. bassiana hyphae can grow continuously with few reproductive structures differentiated, while illumination with white light resulted in prolific formation of conidiophores bearing abundant conidia, indicating that light could stimulate conidiation of B. bassiana. Among the single colour lights tested, blue light was the most effective to stimulating sporulation. Colonies became adapted for blue light stimulus only after hyphae had grown in total darkness for at least 96 h, whereas the photoadaptation obviously declined after 144 h. For the exposure time, 3 min of blue light pulse was enough to stimulate conidiation in the photoadapted mycelia, while prolonged light exposure over 3 min resulted in a decrease in conidia yield. Our results provided useful clues for understanding the mechanism of conidiation mediated by light in B. bassiana.     

A novel Botrytis cinerea-specific gene BcHBF1 enhances virulence of the grey mould fungus via promoting host penetration and invasive hyphal development

Botrytis cinerea is the causative agent of grey mould on over 1000 plant species and annually causes enormous economic losses worldwide. However, the fungal factors that mediate pathogenesis of the pathogen remain largely unknown. Here, we demonstrate that a novel B. cinerea-specific pathogenicity-associated factor BcHBF1 (h yphal b ranching-related f actor 1), identified from virulence-attenuated mutant M8008 from a B. cinerea T-DNA insertion mutant library, plays an important role in hyphal branching, infection structure formation, sclerotial formation and full virulence of the pathogen. Deletion of BcHBF1 in B. cinerea did not impair radial growth of mycelia, conidiation, conidial germination, osmotic- and oxidative-stress adaptation, as well as cell wall integrity of the ∆Bchbf1 mutant strains. However, loss of BcHBF1 impaired the capability of hyphal branching, appressorium and infection cushion formation, appressorium host penetration and virulence of the pathogen. Moreover, disruption of BcHBF1 altered conidial morphology and dramatically impaired sclerotial formation of the mutant strains. Complementation of BcHBF1 completely rescued all the phenotypic defects of the ∆Bchbf1 mutants. During young hyphal branching, host penetration and early invasive growth of the pathogen, BcHBF1 expression was up-regulated, suggesting that BcHBF1 is required for these processes. Our findings provide novel insights into the fungal factor mediating pathogenesis of the grey mould fungus via regulation of its infection structure formation, host penetration and invasive hyphal branching and growth.     

Distinctive role of fluG in the adaptation of Beauveria bassiana to insect-pathogenic lifecycle and environmental stresses

The upstream developmental activation (UDA) pathway comprises three fluG-cored cascades (fluG-flbA, fluG-flbE/B/D and fluG-flbC) that activate the key gene brlA of central developmental pathway (CDP) to initiate conidiation in aspergilli. However, the core role of fluG remains poorly understood in other fungi. Here, we report distinctive role of fluG in the insect-pathogenic lifecycle of Beauveria bassiana. Disruption of fluG resulted in limited conidiation defect, which was mitigated with incubation time and associated with time-course up-regulation/down-regulation of all flb and CDP genes and another fluG-like gene (BBA_06309). In ΔfluG, increased sensitivities to various stresses correlated with repression of corresponding stress-responsive genes. Its virulence through normal cuticle infection was attenuated greatly due to blocked secretion of cuticle-degrading enzymes and delayed formation of hyphal bodies (blastospores) to accelerate proliferation in vivo and host death. In submerged ΔfluG cultures mimicking insect haemolymph, largely increased blastospore production concurred with drastic up-regulation of the CDP genes brlA and abaA, which was associated with earlier up-regulation of most flb genes in the cultures. Our results unveil an essentiality of fluG for fungal adaptation to insect-pathogenic lifecycle and suggest the other fluG-like gene to act as an alternative player in the UDA pathway of B. bassiana.     

Semi-defined Medium for in vitro Cultivation of the Fastidious Insect Pathogenic Fungus Cordyceps unilateralis

Cordyceps unilateralis is a fastidious fungal pathogen affecting ants. Up to now, only the complex and expensive Grace’s insect cell culture medium has been used for in vitro cultivation (as blastospores and mycelium) of this fungus. To obtain an inexpensive and less complicated medium, the effects of carbon and nitrogen sources, salt solution and carbon-to-nitrogen (C:N) ratio on the growth of this fungus were examined. Glucose was the most important factor for blastospore formation, and yeast extract could be used as a nitrogen source for blastospore formation and mycelial growth. A suitable C:N ratio (glucose: yeast extract) was 33.3:1. As a result, a new semi-defined medium was achieved, composed of 26.68 g L⁻¹ glucose, 3.3 g L⁻¹ yeast extract and salt solution. This medium supported blastospore formation and mycelial growth of all tested C. unilateralis isolates.     

Ras subfamily GTPases regulate development,aflatoxin biosynthesis and pathogenicity in the fungus Aspergillus flavus

Ras subfamily proteins are molecular switches in signal transduction pathways of many eukaryotes that regulate a variety of cellular processes. Here, the Ras subfamily, encoded by six genes, was identified in Aspergillus flavus: rasA, rasB, rasC, rab-33, rheb and rsr1. The rsr1 deletion mutant (∆rsr1), rheb deletion mutant (∆rheb) and double deletion mutant (∆rheb/rsr1) displayed significantly decreased growth and sporulation. Sclerotia formation was significantly decreased for ∆rheb or ∆rheb/rsr1 but increased for ∆rsr1. Aflatoxin production was significantly increased in ∆rheb but decreased in ∆rsr1 and ∆rheb/rsr1. We found that rsr1 and rheb are crucial for the pathogenicity of A. flavus. Quantitative proteomics identified 520 differentially expressed proteins (DEPs) for the ∆rsr1 mutant and 133 DEPs for the ∆rheb mutant. These DEPs were annotated in multiple biological processes and KEGG pathways in A. flavus. Importantly, we identified the cytokinesis protein SepA in the protein–protein interaction network of rsr1, and deletion mutants showed that SepA has pleiotropic effects on growth and AF biosynthesis, which may depend on Rsr1 for regulation in A. flavus. Our results indicated that these Ras subfamily proteins exhibited functional redundancy with each other but there were also differences in A. flavus.     

A vacuolar glucoamylase,Sga1, participates in glycogen autophagy for proper asexual differentiation in Magnaporthe oryzae

Nutrient limitation acts as a trigger for the synthesis of glycogen, which serves as a carbon and energy reserve during starvation. Recently, we reported that an autophagy-deficient mutant (atg8Δ) shows severe reduction in aerial hyphal growth and conidiation in the rice-blast fungus Magnaporthe oryzae, and proposed that autophagy plays an important role in facilitating glycogen homeostasis to ensure proper asexual differentiation in Magnaporthe. Here, we identify and characterize a vacuolar glucoamylase function (Sga1) that hydrolyses glycogen to meet the energy requirements during asexual development in Magnaporthe. Loss of SGA1 resulted in significant reduction in conidiation compared to the wild-type Magnaporthe strain. More importantly, an sga1Δ atg8Δ double deletion mutant showed further reduction in conidiation compared to the atg8Δ mutant in Magnaporthe. Forced localization of GFP-Sga1 to the cytoplasm (through removal of the predicted signal peptide) led to increased conidiation in wild type and the sga1Δ, but more interestingly, significantly restored conidiation in the atg8Δ mutant. Our results indicate that autophagy and Sga1 act cooperatively in vacuolar glycogen breakdown, which is essential for conidia formation but dispensable for pathogenicity in Magnaporthe.     

球孢白僵菌响应氧化胁迫的分子机制研究进展

球孢白僵菌作为模式丝状真菌,以分生孢子、菌丝体、虫菌体等多种形态存在,在真菌孢子发育、寄主与宿主互作的研究中具有重要意义。同时,球孢白僵菌又是一类广泛应用的真菌杀虫剂,对森林防护和农业生产具有实际应用价值。球孢白僵菌的相关基因被敲除后,突变体响应氧化胁迫,孢子发育和毒力会发生改变。本文综述了近年来球孢白僵菌在响应氧化胁迫方面的研究进展,为丝状真菌氧化胁迫信号途径的研究提供参考。     

The DUF1996 and WSC domain‐containing protein Wsc1I acts as a novel sensor of multiple stress cues in Beauveria bassiana

Wsc1I homologues featuring both an N‐terminal DUF1996 (domain of unknown function 1996) and a C‐terminal WSC (cell wall stress‐responsive component) domain exist in filamentous fungi but have never been functionally characterized. Here, Wsc1I is shown to localize in the vacuoles and cell wall/membrane of the insect mycopathogen Beauveria bassiana and hence linked to cell membrane‐ and vacuole‐related cellular events. In B. bassiana, deletion of Wsc1I resulted in marked increases of hyphal and conidial sensitivities to hyperosmotic agents, oxidants, cell wall perturbing chemicals, and metal cations (Cu²⁺, Zn²⁺, Fe²⁺, and Mg²⁺) despite slight impact on normal growth and conidiation. Conidia produced by the deletion mutant showed not only reduced tolerance to both 45°C heat and UVB irradiation but also attenuated virulence to a susceptible insect through normal cuticle infection or cuticle‐bypassing infection. Importantly, phosphorylation of the mitogen‐activated protein kinase Hog1 was largely attenuated or nearly abolished in the Wsc1I‐free cells triggered with hyperosmotic, oxidative, or cell wall perturbing stress. All changes were well restored by targeted gene complementation. Our findings highlight a novel role of Wsc1I in sensing multiple stress cues upstream of the Hog1 signalling pathway and its pleiotropic effects in B. bassiana.     

Three proline rotamases involved in calcium homeostasis play differential roles in stress tolerance,virulence and calcineurin regulation of Beauveria bassiana

FK506‐sensitive proline rotamases (FPRs), also known as FK506‐binding proteins (FKBPs), can mediate immunosuppressive drug resistance in budding yeast but their physiological roles in filamentous fungi remain opaque. Here, we report that three FPRs (cytosolic/nuclear 12.15‐kD Fpr1, membrane‐associated 14.78‐kD Fpr2 and nuclear 50.43‐kD Fpr3) are all equally essential for cellular Ca²⁺ homeostasis and contribute significantly to calcineurin activity at different levels in the insect‐pathogenic fungus Beauveria bassiana although the deletion of fpr1 alone conferred resistance to FK506. Radial growth, conidiation, conidial viability and virulence were less compromised in the absence of fpr1 or fpr2 than in the absence of fpr3, which abolished almost all growth on scant media and reduced growth moderately on rich media. The Δfpr3 mutant was more sensitive to Na⁺, K⁺, Mn²⁺, Ca²⁺, Cu²⁺, metal chelate, heat shock and UVB irradiation than was Δfpr2 while both mutants were equally sensitive to Zn²⁺, Mg²⁺, Fe²⁺, H₂O₂ and cell wall‐perturbing agents. In contrast, the Δfpr1 mutant was less sensitive to fewer stress cues. Most of 32 examined genes involved in DNA damage repair, Na⁺/K⁺ detoxification or osmotolerance and Ca²⁺ homeostasis were downregulated sharply in Δfpr2 and Δfpr3 but rarely so affected in Δfpr1, coinciding well with their phenotypic changes. These findings uncover important, but differential, roles of three FPRs in the fungal adaptation to insect host and environment and provide novel insight into their essential roles in calcium signalling pathway.     

Development of the entomopathogenic fungus Beauveria bassiana in liquid cultures

Growth and development of B. bassiana was followed in four liquid media: peptone, peptone-glucose, glucose and glucose-peptone-yeast extract. Six developmental stages were defined: (I) the unswollen conidium, (II) the swollen conidium, (III) emergence of the germ tube, (IV) elongation of the germ tube and formation of the first septum, (V) polar and bipolar elongation (growth) of the resulting mycelium and initiation of a blastospore and, (VI) seccession of that blastospore. Conidia of B. bassiana produced germ tubes in all liquid media. Blastospores were produced in all liquid media except glucose. In peptone-glucose, the yield of blastospores was four-fold higher than in glucose-peptone-yeast extract. However, biomass production was highest in peptone-glucose-yeast extract.