Unveiling Functions And Regulatory Roles Of Putative Regulators Upstream Of Central Developmental Pathway In Beauveria Bassiana

The insect-pathogenic fungus Beauveria bassiana serves as a main source of environment-friendly wide-spectrum fungal insecticides comprising formulated conidia as active ingredients.The yield and quality of aerial conidia produced by a fermentation technology determines the cost and field performace of a product.In nature,fungal infection cycle and survival/dispersal in host habitats rely upon aerial conidia produced on the surfaces of mycotized insect cadavers.Filamentous fungal conidiation and conidial maturation essential for conidial yield and quality is the process of asexual development under the control of central developmental pathway（CDP）,which comprise three sequentially activated genes brl A,aba A and wet A and is aided by the downstream gene vos A for condial maturation.The regulatory role of CDP elucidated previously in the asexual development of Aspergillus nidulans has been fully evidenced in B.bassiana.In A.nidulans,the key CDP gene brl A is transcriptionally activated by upstream developmental activation pathway（UDAP）consisting of flu G known as a core UDAP regulator and five fluffy genes（flb A-flb E）,which are orchestrated by flu G to form three flu G-cored cascades for activation of brl A.However,increasing evidences of fungal genome divergence in the postgenomic era make it necessary to clarify whether the genetic control principles on asexual developmental of A.nidulans are applicable to Pezizomycotina.Understanding molecular mechanisms of CDP activation required for asexual development is of great importance for design and improvement of fermentation technology for maximization of conidial yield and quality.Therefore,this study sought to characterize functions and regulatory roles of Flu G homolog,Flu G-like regulator Flr A and Flb A-Flb E orthologs in B.bassiana and unviel whether the principles on the CDP activation of A.nidulans fit the fungal insect pathogen.The main results are summarized below:Functions and regulatory role of Flu G.In A.nidulans,UDAP comprises the flu G-cored cascades flu G-flb A,flu G-flb E/B/D and flu G-flb C to activate expression of the key CDP gene brl A required for initiation of conidiation.In B.bassiana,homologous Flu G tagged by green fluorenscence was localized mainly in the nuclei of hyphae,conidia and blastospores from plate or submerged cultures in a fashion independent of photoperiod change.Disruption of flu G resulted in limited conidiation defect,which was mitigated with incubation time and associated with time-course upregulation and downregulation of all flb and CDP genes and the other flu G-like gene（BBA＿06309）.In the（35）flu G mutant,increased sensitivities to oxidative,osmotic,cell wall perturbing and heat-shocking stresses correlated well with repressed expression 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（35）flu G cultures mimicking insect hemolymph,greatly increased blastospore production concurred with drastic upregulation of the key CDP genes brl A and aba A,which was associated with earlier upregulation of most flb genes in the cultures.These results unveil an essentiality of flu G for fungal adaptation to insect-pathogenic lifecycle and suggest the other flu G-like gene to likely act as an alternative player in the UDAP of B.bassiana.Functions and regulatory roles of flb A-flb E.Five fluffy genes,namely flb A-flb E,are well-known UDAP regulators in activating the key CDP gene brl A in A.nidulans.In B.bassiana,their orthologs were found playing insignificant roles in radial growth under normal culture conditions and different stresses although flb A and flb D were evidently involved in cellular responses to stresses induced by heat shock and H₂O₂respectively.Aerial conidiation level was lowered in the deletion mutants of flb B and flb E（～15%）less than of flb A and flb C（～30%）,in which the key CDP genes brl A and aba A were repressed consistently during normal incubation.The CDP-controlled blastospore production in submerged cultures mimicking insect hemolymph was abolished in the（35）flb A mutant with brl A and aba A being sharply repressed,and decreased by 55%in the（35）flb C mutant with only aba A being downregulated.The fungal virulence against a model insect was attenuated in the absence of flb A more than of flb C irrespective of normal cuticle infection or cuticle-bypassing infection（intrahemocoel injection）.These findings unravel more important role of flb A than of flb C,but null roles of flb B/D/E,in the insect-pathogenic lifecycle of B.bassiana and hence a scenario distinctive from what has been characterized in A.nidulans.Functions of Flu G-like regulator Flr A and comparative roles of and Flr A and Flu G in genome-wide gene regulation.Flu G-like regulators（FLRs）are present in many filamentous fungi genomes and are noted as glutamine synthetase or putative proteins,but their functions in ascomycetes have not been reported.In this study,single-disruption（SD）mutants of flr A and double-disruption（DD）mutants of flr A and flu G were analyzed to clarify whether Flr A and Flu G are UDAP regulators required for CDP activation and elucidate their distinct or similar functions in B.bassiana.Frl A fusion protein tagged by fluorescence protein accumulated more heavily in the nuclei than in the cytoplasm of hyphae like the preivous Flu G counterpart,but no signal of protein-protein interaction was detected between Flr A and Flu G in a yeast two-hybrid assay,suggesting functional independence of each other.Three SD mutants exhibited phenotypes similar to those by the previous（35）flu G mutant,including limited conidiation defects,facilitated blastospore production,impaired spore quality,blocked host infection,delayed proliferation in vivo,greatly attenuated virulence,and increased cell sensitivities to oxidative,osmotic,cell wall perturbing and thermal stresses.Three DD mutants were similar to the SD mutants in all phenotypes except more compromised insect pathogenicity and cell tolerance to heat shock or calcofluor white（cell wall stressor）.Surprisingly,all CDP and fluffy genes remained active at transcription level in the SD and DD mutants.Transcriptomic analyses revealed dysregulation of 1,622 and 2,234 genes（up/down ratios:635:987 and 780:1454）in the respective（35）flr A and（35）flu G mutants,not including CDP genes.More surprisingly,the majority（up/down ratio:540:875;genomic 13.65%）of those dysregulated genes were individually co-upregulated or co-downregulated at similar levels in the（35）flr A and（35）flu G mutants.These results unravel largely overlapping roles for flr A and flu G in transcriptional regualtion of massive genes vital for B.bassiana’s adaptation to insect-pathogenic lifestyle and environment but no role of either flr A or flu G in the fungal UDAP.This finding highlights that the principles on the CDP activation of A.nidulans are not applicable to B.bassiana in Cordycepitaceae of Hypocreales.