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.     

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.     

Pleiotropic effects of the histone deacetylase Hos2 linked to H4‐K16 deacetylation,H3‐K56 acetylation,and H2A‐S129 phosphorylation in Beauveria bassiana

Histone acetyltransferases and deacetylases maintain dynamics of lysine acetylation/deacetylation on histones and nonhistone substrates involved in gene regulation and cellular events. Hos2 is a Class I histone deacetylases that deacetylates unique histone H4‐K16 site in yeasts. Here, we report that orthologous Hos2 deacetylates H4‐K16 and is also involved in the acetylation of histone H3‐K56 and the phosphorylation of histone H2A‐S129 and cyclin‐dependent kinase 1 CDK1‐Y15 in Beauveria bassiana, a filamentous fungal insect pathogen. These site‐specific modifications are evidenced with hyperacetylated H4‐K16, hypoacetylated H3‐K56, and both hypophosphorylated H2A‐S129 and CDK1‐Y15 in absence of hos2. Consequently, the Δhos2 mutant suffered increased sensitivities to DNA‐damaging and oxidative stresses, disturbed cell cycle, impeded cytokinesis, increased cell size or length, reduced conidiation capacity, altered conidial properties, and attenuated virulence. These phenotypic changes correlated well with dramatic repression of many genes that are essential for DNA damage repair, G₁/S transition and DNA synthesis, hyphal septation, and asexual development. The uncovered ability for Hos2 to directly deacetylate H4‐K16 and to indirectly modify H3‐K56, H2A‐S129, and CDK1‐Y15 provides novel insight into more subtle regulatory role for Hos2 in genomic stability and diverse cellular events in the fungal insect pathogen than those revealed previously in nonentomophathogenic fungi.     

Use of uridine auxotrophy (ura3) for markerless transformation of the mycoinsecticide Beauveria bassiana

Genetic engineering offers a practical route for enhancing the insect biological control potential of entomopathogenic fungi such as Beauveria bassiana. To date, however, such efforts have relied upon transformation protocols that utilize antibiotic or herbicidal resistance markers as selection agents for the introduction of genes into the fungus. In order to avoid the use of such markers for the development of field-usable fungal strains, a markerless transformation system based upon complementation of uridine auxotrophy was developed. A targeted gene deletion knockout of orotidine 5′-phosphate decarboxylase (ura3) was isolated using a positive screening protocol with 5′-fluoro-orotate. Although growth was restored when the mutant, ΔBbura3, was grown in the presence of exogenous uridine, conidiation remained impaired and conidial yield was reduced. Insect bioassays revealed that the ΔBbura3 strain was essentially avirulent using both topical and intrahemocoel injection assays, indicating that the deletion mutant was unable to scavenge uridine from the host during infection. A series of plasmid constructs were developed for complementation of the ura3 mutant, and complemented strains were restored to wild-type growth and virulence. These data indicate that the ura3 mutant and corresponding complementation vectors can be used to construct markerless strains for the bioengineering of desired traits in B. bassiana.     

Allee Effect in the Infection Dynamics of the Entomopathogenic Fungus Beauveria bassiana (Bals) Vuill. on the Beetle, Mylabris pustulata

Successful infection by Beauveria bassiana as with all other entomopathogenic fungi, is accomplished only at a high conidial dose while, theoretically, a single conidium should be sufficient. Indeed, this is a major deterrent in its use as a biocontrol agent. High pathogen load for infection is required by organisms which display ‘Allee’ effect. In such organisms, a threshold exists for pathogen dose, below which no infection can be caused. B. bassiana has a semelparous life cycle and, therefore, its infection dynamics are expected to conform to the mass action principle with a linear relationship between dose and successful infection observable as mortality of the insect. Whether the need for a high conidial dose to induce insect mortality by B. bassiana is due to the operation of Allee effect was examined. A sample of 34 isolates was bioassayed on Mylabris pustulata (Coleoptera: Meloidae) at four conidial concentrations. With more than half of the isolates in the sample, the lowest dose tested (10⁴ conidia/insect) did not cause insect mortality. Thus, a threshold pathogen load is required to cause successful infection. In these isolates, the dose–mortality relationship was sigmoid. Allee effect is thus identified in the infection dynamics of B. bassiana–M. pustulata system. The isolates that induced mortality at the lowest dose tested are concluded to be highly virulent with a lower threshold dose required for successful infection. With some isolates, at high conidial dose, the infection rate decreased either due to a decrease in the proportion of insects showing mycosis, to the speed of death, or both. Such a response could result from intra scramble competition arising from overload of pathogen at very high dose.     

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.     

Characterization of the velvet regulators in Aspergillus fumigatus

Fungal development and secondary metabolism is intimately associated via activities of the fungi‐specific velvet family proteins. Here we characterize the four velvet regulators in the opportunistic human pathogen Aspergillus fumigatus. The deletion of AfuvosA, AfuveA and AfuvelB causes hyperactive asexual development (conidiation) and precocious and elevated accumulation of AfubrlA during developmental progression. Moreover, the absence of AfuvosA, AfuveA or AfuvelB results in the abundant formation of conidiophores and highly increased AfubrlA mRNA accumulation in liquid submerged culture, suggesting that they act as repressors of conidiation. The deletion of AfuvosA or AfuvelB causes a reduction in conidial trehalose amount, long‐term spore viability, conidial tolerance to oxidative and UV stresses, and accelerated and elevated conidial germination regardless of the presence or absence of an external carbon source, suggesting an interdependent role of them in many aspects of fungal biology. Genetic studies suggest that AfuAbaA activates AfuvosA and AfuvelB expression during the mid to late phase of conidiation. Finally, the AfuveA null mutation can be fully complemented by Aspergillus nidulans VeA, which can physically interact with AfuVelB and AfuLaeA in vivo. A model depicting the similar yet different roles of the velvet regulators governing conidiation and sporogenesis in A. fumigatus is presented.     

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.