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.

SET1/KMT2-governed histone H3K4 methylation coordinates the lifecycle in vivo and in vitro of the fungal insect pathogen Beauveria bassiana.

Biological control potential of insect-pathogenic fungi against pests is an overall output of various cellular processes regulated by signalling and epigenetic networks. In Beauveria bassiana, mono/di/trimethylation of histone H3 Lys 4 (H3K4me1/me2/m3) was abolished by inactivation of the histone lysine methyltransferase SET1/KMT2, leading to marked virulence loss, reductions in conidial hydrophobicity and adherence to insect cuticle, impeded proliferation in vivo, severe defects in growth and conidiation, and increased sensitivities to cell wall perturbation, H2 O2 and heat shock. Such compromised phenotypes correlated well with transcriptional abolishment or repression of carbon catabolite-repressing transcription factor Cre1, classes I and II hydrophobins Hyd1 and Hyd2 required for cell hydrophobicity, key developmental regulators, and stress-responsive enzymes/proteins. Particularly, expression of cre1, which upregulates hyd4 upon activation by KMT2-mediated H3K4me3 in Metarhizium robertsii, was nearly abolished in the Δset1 mutant, leading to abolished expression of hyd1 and hyd2 as homologues of hyd4. These data suggest that the SET1-Cre1-Hyd1/2 pathway function in B. bassiana like the KMT2-Cre1-Hyd4 pathway elucidated to mediate pathogenicity in M. robertsii. Our findings unveil not only a regulatory role for the SET1-cored pathway in fungal virulence but also its novel role in mediating asexual cycle in vitro and stress responses in B. bassiana.

Two white collar proteins protect fungal cells from solar UV damage by their interactions with two photolyases in Metarhizium robertsii.

The photolyases PHR1 and PHR2 enable photorepair of fungal DNA lesions in the forms of UV-induced cyclobutane pyrimidine dimer (CPD) and (6-4)-pyrimidine-pyrimidone (6-4PP) photoproducts, but their regulation remains mechanistically elusive. Here, we report that the white collar proteins WC1 and WC2 mutually interacting to form a light-responsive transcription factor regulate photolyase expression required for fungal UV resistance in the insect-pathogenic fungus Metharhizum robertsii. Conidial UVB resistance decreased by 54% in Δwc1 and 67% in Δwc2. Five-hour exposure of UVB-inactivated conidia to visible light resulted in photoreactivation rates of 30% and 9% for the Δwc1 and Δwc2 mutants, contrasting to 79%-82% for wild-type and complemented strains. Importantly, abolished transcription of phr1 in Δwc-2 and of phr2 in Δwc1 resulted in incapable photorepair of CDP and 6-4PP DNA lesions in UVB-impaired Δwc2 and Δwc1 cells respectively. Yeast two-hybrid assays revealed interactions of either WC protein with both PHR1 and PHR2. Therefore, the essential roles for WC1 and WC2 in both photorepair of UVB-induced DNA lesions and photoreactivation of UVB-inactivated conidia rely upon their interactions with, and hence transcriptional activation of, PHR1 and PHR2. These findings uncover a novel WC-cored pathway that mediates filamentous fungal response and adaptation to solar UV irradiation.

Essential Roles of Two FRQ Proteins (Frq1 and Frq2) in Beauveria bassiana's Virulence, Infection Cycle, and Calcofluor-Specific Signaling.

Two FRQ proteins (Frq1 and Frq2) distinct in molecular mass and structure coexist in Beauveria bassiana, an asexual insect-pathogenic fungus. Frq1 and Frq2 have been proven to have opposite nuclear rhythms that can persistently activate developmental activator genes and hence orchestrate nonrhythmic conidiation in vitro under light or in darkness. Here, we report the essentiality of either FRQ, but Frq2 being more important than Frq1, for the fungal virulence and infection cycle. The fungal virulence was attenuated significantly more in the absence of frq2 than in the absence of frq1 through either normal cuticle infection or cuticle-bypassing infection by intrahemocoel injection, accompanied by differentially reduced secretion of Pr1 proteases required for the cuticle infection and delayed development of hyphal bodies in vivo, which usually propagate by yeast-like budding in the host hemocoel to accelerate insect death from mycosis. Despite insignificant changes in radial growth under normal, oxidative, and hyperosmotic culture conditions, conidial yields of the Δfrq1 and Δfrq2 mutants on insect cadavers were sharply reduced, and the reduction increased with shortening daylight length on day 9 or 12 after death, indicating that both Frq1 and Frq2 are required for the fungal infection cycle in host habitats. Intriguingly, the Δfrq1 and Δfrq2 mutants showed hypersensitivity and high resistance to cell wall-perturbing calcofluor white, coinciding respectively with the calcofluor-triggered cells' hypo- and hyperphosphorylated signals of Slt2, a mitogen-activated protein kinase (MAPK) required for mediation of cell wall integrity. This finding offers a novel insight into opposite roles of Frq1 and Frq2 in calcofluor-specific signal transduction via the fungal Slt2 cascade.IMPORTANCE Opposite nuclear rhythms of two distinct FRQ proteins (Frq1 and Frq2) coexisting in an asexual fungal insect pathogen have been shown to orchestrate the fungal nonrhythmic conidiation in vitro in a circadian day independent of photoperiod change. This paper reports essential roles of both Frq1 and Frq2, but a greater role for Frq2, in sustaining the fungal virulence and infection cycle since either frq1 or frq2 deletion led to marked delay of lethal action against a model insect and drastic reduction of conidial yield on insect cadavers. Moreover, the frq1 and frq2 mutants display hypersensitivity and high resistance to cell wall perturbation and have hypo- and hyperphosphorylated MAPK/Slt2 in calcofluor white-triggered cells, respectively. These findings uncover a requirement of Frq1 and Frq2 for the fungal infection cycle in host habitats and provide a novel insight into their opposite roles in calcofluor-specific signal transduction through the MAPK/Slt2 cascade.

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+ , H2 O2 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.

Pleiotropic effects of Ubi4, a polyubiquitin precursor required for ubiquitin accumulation, conidiation and pathogenicity of a fungal insect pathogen.

Ubi4 is a polyubiquitin precursor well characterized in yeasts but unexplored in insect mycopathogens. Here, we report that orthologous Ubi4 plays a core role in ubiquitin- and asexual lifestyle-required cellular events in Beauveria bassiana. Deletion of ubi4 led to abolished ubiquitin accumulation, blocked autophagic process, severe defects in conidiation and conidial quality, reduced cell tolerance to oxidative, osmotic, cell wall perturbing and heat-shock stresses, decreased transcript levels of development-activating and antioxidant genes, but light effect on radial growth under normal conditions. The deletion mutant lost insect pathogenicity via normal cuticle infection and was severely compromised in virulence via cuticle-bypassing infection due to a block of dimorphic transition critical for acceleration of host mummification. Proteomic and ubiquitylomic analyses revealed 1081 proteins differentially expressed and 639 lysine residues significantly hyper- or hypo-ubiquitylated in the deletion mutant, including dozens of ubiquitin-activating, conjugating and ligating enzymes, core histones, and many more involved in proteasomes, autophagy-lysosome process and protein degradation. Singular deletions of seven ubiquitin-conjugating enzyme genes exerted differential Ubi4-like effects on conidiation level and conidial traits. These findings uncover an essential role of Ubi4 in ubiquitin transfer cascade and its pleiotropic effects on the in vitro and in vivo asexual cycle of B. bassiana.

Photoprotective Role of Photolyase-Interacting RAD23 and Its Pleiotropic Effect on the Insect-Pathogenic Fungus Beauveria bassiana.

RAD23 can repair yeast DNA lesions through nucleotide excision repair (NER), a mechanism that is dependent on proteasome activity and ubiquitin chains but different from photolyase-depending photorepair of UV-induced DNA damages. However, this accessory NER protein remains functionally unknown in filamentous fungi. In this study, orthologous RAD23 in Beauveria bassiana, an insect-pathogenic fungus that is a main source of fungal insecticides, was found to interact with the photolyase PHR2, enabling repair of DNA lesions by degradation of UVB-induced cytotoxic (6-4)-pyrimidine-pyrimidine photoproducts under visible light, and it hence plays an essential role in the photoreactivation of UVB-inactivated conidia but no role in reactivation of such conidia through NER in dark conditions. Fluorescence-labeled RAD23 was shown to normally localize in the cytoplasm, to migrate to vacuoles in the absence of carbon, nitrogen, or both, and to enter nuclei under various stresses, which include UVB, a harmful wavelength of sunlight. Deletion of the rad23 gene resulted in an 84% decrease in conidial UVB resistance, a 95% reduction in photoreactivation rate of UVB-inactivated conidia, and a drastic repression of phr2 A yeast two-hybrid assay revealed a positive RAD23-PHR2 interaction. Overexpression of phr2 in the Δrad23 mutant largely mitigated the severe defect of the Δrad23 mutant in photoreactivation. Also, the deletion mutant was severely compromised in radial growth, conidiation, conidial quality, virulence, multiple stress tolerance, and transcriptional expression of many phenotype-related genes. These findings unveil not only the pleiotropic effects of RAD23 in B. bassiana but also a novel RAD23-PHR2 interaction that is essential for the photoprotection of filamentous fungal cells from UVB damage.IMPORTANCE RAD23 is able to repair yeast DNA lesions through nucleotide excision in full darkness, a mechanism distinct from photolyase-dependent photorepair of UV-induced DNA damage but functionally unknown in filamentous fungi. Our study unveils that the RAD23 ortholog in a filamentous fungal insect pathogen varies in subcellular localization according to external cues, interacts with a photolyase required for photorepair of cytotoxic (6-4)-pyrimidine-pyrimidine photoproducts in UV-induced DNA lesions, and plays an essential role in conidial UVB resistance and reactivation of UVB-inactivated conidia under visible light rather than in the dark, as required for nucleotide excision repair. Loss-of-function mutations of RAD23 exert pleiotropic effects on radial growth, aerial conidiation, multiple stress responses, virulence, virulence-related cellular events, and phenotype-related gene expression. These findings highlight a novel mechanism underlying the photoreactivation of UVB-impaired fungal cells by RAD23 interacting with the photolyase, as well as its essentiality for filamentous fungal life.

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.

Phenotypic and molecular insights into heat tolerance of formulated cells as active ingredients of fungal insecticides.

Formulated conidia of insect-pathogenic fungi, such as Beauveria and Metarhizium, serve as the active ingredients of fungal insecticides but are highly sensitive to persistent high temperatures (32-35 °C) that can be beyond their upper thermal limits especially in tropical areas and during summer months. Fungal heat tolerance and inter- or intra-specific variability are critical factors and limitations to field applications of fungal pesticides during seasons favoring outbreaks of pest populations. The past decades have witnessed tremendous advances in improving fungal pesticides through selection of heat-tolerant strains from natural isolates, improvements and innovations in terms of solid-state fermentation technologies for the production of more heat-tolerant conidia, and the use of genetic engineering of candidate strains for enhancing heat tolerance. More recently, with the entry into a post-genomic era, a large number of signaling and effector genes have been characterized as important sustainers of heat tolerance in both Beauveria and Metarhizium, which represent the main species used as fungal pesticides worldwide. This review focuses on recent advances and provides an overview into the broad molecular basis of fungal heat tolerance and its multiple regulatory pathways. Emphases are placed on approaches for screening of heat-tolerant strains, methods for optimizing conidial quality linked to virulence and heat tolerance particularly involving cell wall architecture and optimized trehalose/mannitol contents, and how molecular determinants can be exploited for genetic improvement of heat tolerance and pest-control potential. Examples of fungal pesticides with different host spectra and their appropriateness for use in apiculture are given. KEY POINTS: • Heat tolerance is critical for field stability and efficacy of fungal insecticides. • Inter- and intra-specific variability exists in insect-pathogenic fungi. • Optimized production technology and biotechnology can improve heat tolerance. • Fungal heat tolerance is orchestrated by multiple molecular pathways.

Rei1-like protein regulates nutritional metabolism and transport required for the asexual cycle in vitro and in vivo of a fungal insect pathogen.

Rei1 is a cytoplasm-specific pre-60S subunit export factor that functions exclusively in cold-sensitive yeast growth but remains unexplored in filamentous fungi. Here, we report that Rei1-like BbRei1 is localized in both cytoplasm and nucleus and acts as a vital regulator in Beauveria bassiana. Deletion of BbRei1 resulted in delayed conidial germination, abnormally polarized germlings, severe growth defects on various carbon/nitrogen sources and reduced conidiation capacity as well as low temperature-sensitive growth. In ΔBbrei1, greatly attenuated virulence correlated with reduced activities of enzymes secreted for cuticular penetration and blocked formation of hyphal bodies in vivo essential for facilitation of host mummification. Revealed by transcriptomic analysis, 560 and 840 genes were significantly up- and down-regulated in ΔBbrei1 versus wild-type respectively, representing 13.5% of the fungal genome. Many repressed genes were involved in metabolism and transport of carbohydrates and amino acids. However, electrophoretic mobility shift assays presented no interactions of purified BbRei1 with 14 promoter DNA fragments. Conclusively, BbRei1 plays a pivotal role in gene expression and metabolism of nutrients and energy essential for the asexual cycle in vitro and in vivo of B. bassiana and functions much beyond the role for the yeast Rei1 in cold-sensitive cell growth.

The velvet protein VeA functions in asexual cycle, stress tolerance and transcriptional regulation of Beauveria bassiana.

VeA is a key velvet protein that regulates sexual/asexual development and secondary metabolism in filamentous fungi, particularly Aspergilli, but has not been explored yet in asexual insect mycopathogens, such as Beauveria bassiana. Here, we report a localization of B. bassiana VeA in the cytoplasm of hyphal cells exposed to either light or dark cue and its migration to the nucleus only in darkness. Deletion of veA resulted in facilitated hyphal growth and decreased cell length on rich media, light growth defects on scant media, and increased sensitivities to oxidation, high osmolarity and prolonged heat shock during colony growth. Compared to wild-type, the deletion mutant was much more triggered in conidiation at optimal 25 °C in darkness than in a light/dark (L:D) cycle of 12:12, indicating the role of VeA acting as a negative regulator of conidiation in a light-dependent manner. The mutant conidia produced at L:D 12:12 showed defects in germination, thermotolerance and UVB resistance but no change in virulence, contrasting to attenuated virulence for the mutant conidia produced in darkness. Intriguingly, fungal outgrowth and conidiation were markedly suppressed on the surfaces of the mutant-mummified insect cadavers, suggesting a significant role of VeA in fungal survival, dispersal and prevalence in host habitats. Transcriptomic analysis revealed 1248 and 1183 differentially expressed genes in the deletion mutant versus wild-type grown at L:D 0:24 and 12:12 respectively, including those involved in central developmental pathway and secondary metabolism. Altogether, VeA is functionally involved in asexual cycle, stress tolerance and transcriptional regulation of B. bassiana.

Two Photolyases Repair Distinct DNA Lesions and Reactivate UVB-Inactivated Conidia of an Insect Mycopathogen under Visible Light.

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.

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.

Insights into regulatory roles of MAPK-cascaded pathways in multiple stress responses and life cycles of insect and nematode mycopathogens.

Fungal entomopathogenicity may have evolved at least 200 million years later than carnivorism of nematophagous fungi on Earth. This mini-review focuses on the composition and regulatory roles of mitogen-activated protein kinase (MAPK) cascades, which act as stress-responsive signaling pathways. Unveiled by genomic comparison, three MAPK cascades of these mycopathogens consist of singular MAPKs (Fus3/Hog1/Slt2), MAPK kinases (Ste7/Pbs2/Mkk1), and MAPK kinase kinases (Ste11/Ssk2/Bck1). All cascaded components characterized in fungal entomopathogens play conserved and special roles in regulating multiple stress responses and phenotypes associated with biological control potential. Fus3-cascaded components are indispensable for fungal growth on oligotrophic substrata and virulence, and mediate cell tolerance to Na+/K+ toxicity, which is often misinterpreted as hyperosmotic effect but readily clarified by transcriptional changes of Na+/K+ ATPase genes and/or cell responses to osmotic polyols. Hog1-cascaded components regulate osmotolerance positively and phenylpyrrole-type fungicide resistance negatively, and also play differential roles in cell growth, conidiation, virulence, and responses to other stress cues. Ste11 has no stress-responsive role in the Beauveria Hog1 cascade despite an essential role in branched yeast Hog1 cascade. Slt2-cascaded components are required for mediation of cell wall integrity and repair of cell wall damage. A crosstalk between Hog1 and Slt2 cascades ensures fungal osmotolerance inside or outside insect. In nematode-trapping fungi, Slt2 is indispensable for cell wall integrity, conidiation, and mycelial trap formation, suggesting that the Slt2 cascade could have evolved along a distinct trajectory required for fungal carnivorism and dispersal/survival in nematode habitats. Altogether, the MAPK cascades are major parts of signaling network that regulate fungal adaptation to insects and nematodes and their habitats.

Daylight length-dependent translocation of VIVID photoreceptor in cells and its essential role in conidiation and virulence of Beauveria bassiana.

The fungal insect pathogen Beauveria bassiana has the blue-light photoreceptor VIVID (VVD) but lacks a pigmentation pattern to trace its light responses. Here, we show that the fungal vvd is transcriptionally expressed, and linked to other blue/red photoreceptor genes, in a daylight length-dependent manner. GFP-tagged VVD fusion protein was localized to periphery, cytoplasm and vacuoles of hyphal cells in light/dark (L:D) cycles of 24:0 and 16:8 and aggregated in cytoplasm with shortening daylight until transfer into nuclei in full darkness. Deletion of vvd caused more reduced (91%) conidiation capacity in L:D 12:12 cycle of blue light (450/480 nm) than of yellow-to-red (540-760 nm) and white lights (∼70%). The conidiation defect worsened with shortened daylight in different L:D cycles of white light, coinciding well with drastic repression of key activator genes in central development pathway. Intriguingly, the deletion mutant displayed blocked secretion of cuticle-degrading Pr1 proteases, retarded dimorphic transition in insect haemocoel, and hence a lethal action twice longer than those for control strains against Galleria mellonella regardless of the infection passing or bypassing insect cuticle. Conclusively, VVD sustains normal conidiation in a daylight length-dependent manner and acts as a vital virulence factor in B. bassiana.

C-terminal Ser/Thr residues are vital for the regulatory role of Ste7 in the asexual cycle and virulence of Beauveria bassiana.

The mitogen-activated protein kinase (MAPK) kinase Ste7 has a conserved Ser/Thr loop (S/T-X4(6)-S/T) that can activate the MAPK Fus3 or Kss1 for the regulation of pheromone response and filamentous growth in model yeast. Here, we show that not only the loop but also four C-terminal Ser/Thr residues are essential for Ste7 to function in the Fus3 cascade of Beauveria bassiana, a filamentous fungal insect pathogen. Mutagenesis of either looped S216/T220 or C-terminal S362 resulted in the same severe defects in conidial germination, hyphal growth, aerial conidiation, and submerged blastospore production as the ste7 deletion, followed by a complete loss of virulence and similarly increased cell sensitivities to osmotic salts, oxidants, heat shock and UV-B irradiation. Mutagenesis of three other Ser/Thr residues (S391, S440, and T485) also caused severe defects in most of the mentioned phenotypes. These defects correlated well with dramatically reduced transcript levels of some phenotype-related genes. These genes encode a transcription factor (CreA) essential for carbon/nitrogen assimilation, developmental activators (BrlA, AbaA, and WetA) and upstream transcription factor (FluG) required for conidiation, P-type N+/K+ ATPases (Ena1-5) required for intracellular N+/K+ homeostasis, and antioxidant enzymes involved in multiple stress responses. Our study unveils that the loop and four C-terminal Ser/Thr residues are all vital for the regulatory role of Ste7 in the growth, conidiation, virulence, and/or stress tolerance of B. bassiana and perhaps other filamentous fungi.

Antioxidant activities of four superoxide dismutases in Metarhizium robertsii and their contributions to pest control potential.

The superoxide dismutase (SOD) family of Metarhizium robertsii, a fungal insect pathogen, comprises six members functionally unknown yet, including Cu/ZnSODs (Sod1/5/6), MnSODs (Sod2/3), and FeSOD (Sod4). Here, we show a mitochondrial localization of Sod3 and Sod4 and a requirement of either sod4 or sod6 for the fungal life as suggested by an inability to be deleted. We found remarked roles of Sod1, Sod2, and Sod3 in sustaining antioxidant activity and the fungal potential against insect pests but no role of Sod5 in all examined phenotypes. Intracellular SOD activity decreased by 49% in Δsod1 and 22% in either Δsod2 or Δsod3. The decreased SOD activities concurred with altered enzymographs, in which one of two SOD-active bands in wild-type and rescued strains disappeared in Δsod1 rather than in Δsod2 and another band disappeared in Δsod3. Consequently, maximal cell sensitivity to superoxide anions generated by oxidant menadione occurred in Δsod1, followed sequentially by Δsod3 and Δsod2. The latter two mutants were more sensitive than Δsod1 to oxidant H2O2. Transcriptional analysis revealed partial compensation of one or two partner genes upregulated for the absence of sod1, sod2, or sod3 and full compensation of three partners largely upregulated for the absence of sod5, as well as differential expression of most catalase genes in each Δsod mutant. The three mutants also suffered defects in conidial thermotolerance, UVB resistance, and virulence. These findings unveil that, to adapt to different host spectra and habitats, some major SODs in M. roberstii are functionally differentiated from those known previously in Beauveria bassiana, a classic insect mycopathogen lacking Sod6.

Two histidine kinases can sense different stress cues for activation of the MAPK Hog1 in a fungal insect pathogen.

Filamentous fungi possess a large family of histidine kinases (HKs) classified to 11 distinct groups. Of those, only Group III (HK3) homologues in some fungi can sense osmotic, fungicidal and/or oxidative cues upstream of the signalling MAPK Hog1 cascade that usually governs two hallmark phenotypes, i.e., osmotolerance and dicarboximide/phenylpyrrole fungicide resistance. Here we examined functions of all eight HK groups with each possessing a single member in Beauveria bassiana. HK3 and HK8 (Group VIII) were evidently localized at hyphal cell periphery and had more functions in aerial conidiation and multiple stress responses than six other HKs. Increased osmosensitivity occurred uniquely in Δhk8 while high resistance to fludioxonil (phenylpyrrole) or iprodione (dicarboximide) fungicide appeared only in Δhk3. Phosphorylation of Hog1 was inhibited only in the Δhk3 cells triggered with fludioxonil cue and reduced only in the Δhk8 cells triggered with osmotic cue. However, an oxidative cue resulted in no signal change of phosphorylated Hog1 in either Δhk3 or Δhk8. Other Δhk mutants showed minor changes in some non-hallmark phenotypes. Altogether, HK3 and HK8 act as unique sensors of fungicidal and hyperosmotic cues respectively, upstream of the Hog1 cascade that regulates fungicidal resistance negatively and osmotolerance positively in B. bassiana.

Emulsifiable oil-formulated Beauveria bassiana competes with imidacloprid for seasonal control of cereal aphids in Zhejiang, China.

BACKGROUND: Alternatives to neonicotinoids against cereal aphids are needed to mitigate aphid resistance and non-target effects. The emulsifiable oil formulations of two Beauveria bassiana strains, namely Bb registered as a mycoinsecticide and TBb overexpressing an endogenous virulence factor, were tested for seasonal control of cereal aphids at the elongating (April 7) to milk ripening (May 12) stages of winter wheat crop in Yuhang, Zhejiang. Each of three field trials consisted of blank control and the treatments (three randomized 100-m2 plots per capita) of each fungal strain sprayed biweekly at rates of 1.0 × 1013 and 1.5 × 1013 conidia ha-1 and 10% imidacloprid WP sprayed biweekly at a label rate. RESULTS: Tiller infestation percentage and aphid density in the 5-week field trials after the first spray were reduced to 18.7-22.4% and 9.1-12.4 aphids per tiller in the fungal treatments, and 12.8-25.3% and 2.8-20.9 aphids per tiller in the chemical treatment, contrasting with 49.2-60.3% and 37.1-108.5 aphids per tiller in the control. Percent control efficacies (±SD) computed with weekly aphid densities over the period averaged 84.0 ± 1.6 and 85.3 ± 1.8 versus 78.0 ± 4.0 and 79.9 ± 3.2 in the high-rate versus low-rate treatments of Bb and TBb, respectively, and 84.5 ± 7.8 in the chemical treatment. Imidacloprid showed faster kill action but more variable efficacy than the fungal treatments throughout the trials. CONCLUSION: Either Bb or TBb formulation competes with imidacloprid in reducing percent infestation and aphid density. The overall efficacy was significantly higher in the treatments of TBb than of Bb. © 2024 Society of Chemical Industry.

Timing of Fungal Insecticide Application to Avoid Solar Ultraviolet Irradiation Enhances Field Control of Rice Planthoppers.

Thechemical control of rice planthoppers (RPH)is prohibited in annual rice-shrimp rotation paddy fields. Here, the fungal insecticides Beauveria bassiana ZJU435 and Metarizhium anisoplae CQ421 were tested for control of RPH populations dominated by Nilaparvata lugens in three field trials. During four-week field trials initiated from the harsh weather of high temperatures and strong sunlight, the rice crop at the stages from tillering to flowering was effectively protected by fungal sprays applied at 14-day intervals. The sprays of either fungal insecticide after 5:00 p.m. (solar UV avoidance) suppressed the RPH population better than those before 10 a.m. The ZJU435 and CQ421 sprays for UV avoidance versus UV exposure resulted in mean control efficacies of 60% and 56% versus 41% and 45% on day 7, 77% and 78% versus 63% and 67% on day 14, 84% and 82% versus 80% and 79% on day 21, and 84% and 81% versus 79% and 75 on day 28, respectively. These results indicate that fungal insecticides can control RPH in the rice-shrimp rotation fields and offer a novel insight into the significance of solar-UV-avoiding fungal application for improved pest control during sunny summers.