The ascomycete fungus Beauveria bassiana is a pathogen of hundreds of insect species and is commercially produced as an environmentally friendly mycoinsecticide. We sequenced the genome of B. bassiana and a phylogenomic analysis confirmed that ascomycete entomopathogenicity is polyphyletic, but also revealed convergent evolution to insect pathogenicity. We also found many species-specific virulence genes and gene family expansions and contractions that correlate with host ranges and pathogenic strategies. These include B. bassiana having many more bacterial-like toxins (suggesting an unsuspected potential for oral toxicity) and effector-type proteins. The genome also revealed that B. bassiana resembles the closely related Cordyceps militaris in being heterothallic, although its sexual stage is rarely observed. A high throughput RNA-seq transcriptomic analysis revealed that B. bassiana could sense and adapt to different environmental niches by activating well-defined gene sets. The information from this study will facilitate further development of B. bassiana as a cost-effective mycoinsecticide.

The biocontrol potential of entomopathogenic fungi against arthropod pests depends on not only their virulence to target pests but tolerance to outdoor high temperature and solar UV irradiation. Two Beauveria bassiana superoxide dismutases (SODs), BbSod2 and BbSod3, were characterized as cytosolic and mitochondrial manganese-cored isoenzymes (MnSODs) dominating the total SOD activity of the fungal entomopathogen under normal growth conditions. To probe their effects on the biocontrol potential of B. bassiana, ΔBbSod2, ΔBbSod3, and three hairpin RNA-interfered (RNAi) mutants with the transcripts of both BbSod2 and BbSod3 being suppressed by 91–97% were constructed and assayed for various phenotypic parameters in conjunction with ΔBbSod2/BbSod2, ΔBbSod3/BbSod3 and wild-type (control strains). In normal cultures, the knockout and RNAi mutants showed significant phenotypic alterations, including delayed sporulation, reduced conidial yields, and impaired conidial quality, but little change in colony morphology. Their mycelia or conidia became much more sensitive to menadione or H2O2-induced oxidative stress but had little change in sensitivity to the hyperosmolarity of NaCl and the high temperature of 45°C. Accompanied with the decreased antioxidative capability, conidial tolerances to UV-A and UV-B irradiations were reduced by 16.8% and 45.4% for ΔBbSod2, 18.7% and 44.7% for ΔBbSod3, and ∼33.7% and ∼63.8% for the RNAi mutants, respectively. Their median lethal times (LT50s) against Myzus persicae apterae, which were topically inoculated under a standardized spray, were delayed by 18.8%, 14.5% and 37.1%, respectively. Remarkably, the effects of cytosolic BbSod2 and mitochondrial BbSod3 on the phenotypic parameters important for the fungal bioncontrol potential were additive, well in accordance with the decreased SOD activities and the increased superoxide levels in the knockout and RNAi mutants. Our findings highlight for the first time that the two MnSODs co-contribute to the biocontrol potential of B. bassiana by mediating cellular antioxidative response.

Summary The catalase family of Beauveria bassiana (fungal entomopathogen) consists of catA (spore‐specific), catB (secreted), catP (peroxisomal), catC (cytoplasmic) and catD (secreted peroxidase/catalase), which were distinguished in phylogeny and structure and functionally characterized by constructing single‐gene disrupted and rescued mutants for enzymatic and multi‐phenotypic analyses. Total catalase activity decreased 89% and 56% in ΔcatB and ΔcatP, corresponding to the losses of upper and lower active bands gel‐profiled for all catalases respectively, but only 9−12% in other knockout mutants. Compared with wild type and complement mutants sharing similar enzymatic and phenotypic parameters, all knockout mutants showed significant (9−56%) decreases in the antioxidant capability of their conidia (active ingredients of mycoinsecticides), followed by remarkable phenotypic defects associated with the fungal biocontrol potential. These defects included mainly the losses of 40% thermotolerance (45°C) in ΔcatA, 46−48% UV‐B resistance in ΔcatA and ΔcatD, and 33−47% virulence to Spodoptera litura larvae in ΔcatA, ΔcatP and ΔcatD respectively. Moreover, the drastic transcript upregulation of some other catalase genes observed in the normal culture of each knockout mutant revealed functionally complimentary effects among some of the catalase genes, particularly between catB and catC whose knockout mutants displayed little or minor phenotypic changes. However, the five catalase genes functioned redundantly in mediating the fungal tolerance to either hyperosmotic or fungicidal stress. The differentiated roles of five catalases in regulating the B. bassiana virulence and tolerances to oxidative stress, high temperature and UV‐B irradiation provide new insights into fungal adaptation to stressful environment and host invasion.

Dimorphic plant and human mycopathogens require a switch from the usual yeast growth to filamentous growth for host tissue penetration, and the switch is controlled by multiple signaling systems other than the central developmental pathway. Unlike these fungi, dimorphic insect mycopathogens usually grow by hyphal extension, infect the host by hyphal penetration through the insect cuticle, and switch to unicellular blastospores from the penetrating hyphae only after entry into the host hemocoel, where blastospore propagation by yeast-like budding accelerates host mummification. Here, we report a dependence of the virulence-required dimorphic transition on the central pathway activators BrlA and AbaA in Beauveria bassiana. Deletion of brlA or abaA abolished both aerial conidiation and submerged blastospore formation in vitro despite no negative impact on hyphal growth in various media, including a broth mimic of insect hemolymph. The hyphae of either deletion mutant lost insect pathogenicity through normal cuticle penetration, contrasting with a high infectivity of wild-type hyphae. The mutant hyphae injected into the host hemocoel failed to form blastospores, resulting in slower lethal action. Uncovered by transcriptomic analysis, several genes involved in host adhesion and cuticle degradation were sharply repressed in both deletion mutants versus wild type. However, almost all signaling genes homologous to those acting in the dimorphic switch of other fungi were not differentially expressed at a significant level and hence unlikely to be involved in shutting down the dimorphic switch of each deletion mutant. Therefore, like aerial conidiation, the submerged dimorphic switch in vitro and in vivo is a process of asexual development governed by the two central pathway activators in B. bassiana. IMPORTANCE Dimorphic insect mycopathogens infect the host by hyphal penetration through the host cuticle and switch from the penetrating hyphae to unicellular blastospores after entry into the host hemocoel, where blastospore propagation by yeast-like budding accelerates host mummification to death. The fungal virulence-required dimorphic switch is confirmed as a process of asexual development directly regulated by BrlA and AbaA, two key activators of the central developmental pathway in an insect mycopathogen. This finding unveils a novel mechanism distinct from the control of the dimorphic switch by multiple signaling systems other than the central developmental pathway in dimorphic plant and human mycopathogens, which switch from the usual yeast growth to filamentous growth required for pathogenicity through host tissue penetration.

The flexible peptides (GGGGS)n (n < or = 3), the alpha-helical peptides (EAAAK)n (n < or = 3) and two other peptides were used as linkers to construct bifunctional fusions of beta-glucanase (Glu) and xylanase (Xyl) for improved catalytic efficiencies of both moieties. Eight Glu-Xyl fusion enzymes constructed with different linkers were all expressed as the proteins of ca. 46 kDa in Escherichia coli BL21 and displayed the activities of both beta-glucanase and xylanase. Compared to all the characterized fusions with the parental enzymes, the catalytic efficiencies of the Glu and Xyl moieties were equivalent to 304-426% and 82-143% of the parental ones, respectively. The peptide linker (GGGGS)(2) resulted in the best fusion, whose catalytic efficiency had a net increase of 326% for the Glu and of 43% for the Xyl. The two moieties of a fusion with the linker (EAAAK)(3) also showed net increases of 262 and 31% in catalytic efficiency. Our results highlight, for the first time, the enhanced bifunctional activities of the Glu-Xyl fusion enzyme by optimizing the peptide linkers to separate the two moieties at a reasonable distance for beneficial interaction.

Filamentous fungal insect pathogens represent a source of biological insecticides and acaricides formulated using intact cells, such as conidia or other spores. These mycoinsecticides infect arthropod pests through cuticular penetration. In field application, formulated fungal cells are exposed to environmental stresses, including solar UV irradiation, high temperature, and applied chemical herbicides and fungicides, as well as stress from host immune defenses. These stresses often result in accumulation of toxic reactive oxygen species (ROS), generating oxidative stress to the fungal cells and hence affecting the efficacy and persistency of fungi formulated for pest control. In response, fungi have evolved effective antioxidant mechanisms that include enzyme families that act as ROS scavengers, e.g., superoxide dismutases, catalases, peroxidases, thioredoxins /thioredoxin reductases, and glutaredoxins/glutathione reductases. Over two dozen antioxidant enzymes dispersed in different families have been characterized in Beauveria bassiana in recent years. This mini-review focuses on the progress detailed in the studies of these enzymes and provides an overview of their antioxidant activities and contributions to conidial thermotolerance, UV resistance and virulence. These activities are crucial for the biological control potential of mycoinsecticide formulation and have significantly advanced our understanding of how these organisms work. Several potent antioxidant genes have been exploited for successful genetic engineering of entomopathogenic fungi aimed at enhancing their potential against arthropod pests.

Fungal conidia serve as active ingredients of fungal insecticides but are sensitive to solar UV irradiation, which impairs double-stranded DNA (dsDNA) by inducing the production of cytotoxic cyclobutane pyrimidine dimers (CPDs) and (6-4)-pyrimidine-pyrimidine photoproducts (6-4PPs). This study aims to elucidate how CPD photolyase (Phr1) and 6-4PP photolyase (Phr2) repair DNA damage and photoreactivate UVB-inactivated cells in Beauveria bassiana, a main source of fungal insecticides. Both Phr1 and Phr2 are proven to exclusively localize in the fungal nuclei. Despite little influence on growth, conidiation, and virulence, singular deletions of phr1 and phr2 resulted in respective reductions of 38% and 19% in conidial tolerance to UVB irradiation, a sunlight component most harmful to formulated conidia. CPDs and 6-4PPs accumulated significantly more in the cells of Δphr1 and Δphr2 mutants than in those of a wild-type strain under lethal UVB irradiation and were largely or completely repaired by Phr1 in the Δphr2 mutant and Phr2 in the Δphr1 mutant after optimal 5-h exposure to visible light. Consequently, UVB-inactivated conidia of the Δphr1 and Δphr2 mutants were much less efficiently photoreactivated than were the wild-type counterparts. In contrast, overexpression of either phr1 or phr2 in the wild-type strain resulted in marked increases in both conidial UVB resistance and photoreactivation efficiency. These findings indicate essential roles of Phr1 and Phr2 in photoprotection of B. bassiana from UVB damage and unveil exploitable values of both photolyase genes for improved UVB resistance and application strategy of fungal insecticides. IMPORTANCE Protecting fungal cells from damage from solar UVB irradiation is critical for development and application of fungal insecticides but is mechanistically not understood in Beauveria bassiana, a classic insect pathogen. We unveil that two intranuclear photolyases, Phr1 and Phr2, play essential roles in repairing UVB-induced dsDNA lesions through respective decomposition of cytotoxic cyclobutane pyrimidine dimers and (6-4)-pyrimidine-pyrimidine photoproducts, hence reactivating UVB-inactivated cells effectively under visible light. Our findings shed light on the high potential of both photolyase genes for use in improving UVB resistance and application strategy of fungal insecticides.

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