Identification And Mechanistic Elucidation Of High Virulence Factors In Entomopathogenic Fungi

Insect-pathogenic fungi in the genera Beauveria and Metarhizium serve as main sources of fungal pesticides and play increasing roles in biological control programs of agricultural and forestry arthropod pests.Their biological control potential is an overall output of multipe cellular processes and events involved in normal cuticle infection and subsequent hemocoel localization,particularly a capability of their adaptation to host hemocoel environmen where fungal cells are exposed to the stresses of hemolymphic pH,high osmolarity and reactive superoxide species(ROS)generated from host immune defense response.Therefore,fungal insecticidal activity to large extent relies upon an ability to collapse host immune defense response during cuticular penetration and hemocoel colonization,which determines a speed of mycosis development and host death from mummification.This study sought to discover novel virulence factors by characherization of functional genes involved in different cellular processes and events,elucidate possible mechanisms underlying their roles,and explore an biotechnological approach to better utilization of those genes for the development of more efficatious fungal pesticides through genetic improvement.The characterized genes encode P-type Na+/K+ATPases involved in cellular homeostasis and responses to cation-induced stresses,COP9 signalosome subunit 5(Csn5)involved in transcriptional regulation,DNA repair,cell cycle and differentation and asexual development,and small secreted proteins(SSPs)presumably involved in fungus-insect interactions.A markerless transformation system was also developed and applied for non-risky engineering of an endogenous gene to effectively enhance insecticidal activity of Beauveria bassiana.The main results are summarized as follows:P-type Na+/K+ ATPases essential and nonessential for cellular homeostasis and insect pathogenicity of B.bassiana.ENA1 and ENA2 are P-type IID/ENA subfamily of Na+/K+ ATPases required for cellular homeostasis in model yeast but remain poorly understood in filamentous fungal insect pathogens.In B.bassiana,seven genes encode five ENA1/2 homologs(ENA1a-c and ENA2a/b)and two P-type IIC/NK subfamily of Na+/K+ATPases(NK1/2).Most of these genes were highly responsive to alkaline pH and Na+/K+cues at transcription level.Cellular Na+,K+ and H+homeostasis was disturbed only in the absence of enala or ena2b.The disturbed homeostasis featured acceleration of vacuolar acidification,elevation of cytosolic Na+/K+level at pH 5.0 to 9.0,and stabilization of extracellular H+level to initial pH 7.5 during a 5-d period of submerged incubation.Despite little defect on hyphal growth and asexual development,the Δena1a and Δena2b mutants were less tolerant to metal cations(Na+,K+,Li+,Zn2+,Mn2+ and Fe3+),cell wall perturbation,oxidation,noncation hyperosmolarity and UVB irradiation,severely compromised in insect pathogenicity via normal cuticle infection,and markedly attenuated in virulence via hemocoel injection.The deletion mutants of five other ENA and NK genes showed little change in vacuolar pH and all examined phenotypes.Therefore,only ENA1a and ENA2b evidently involved in both transmembrane and vacuolar activities are essential for cellular cation homeostasis,insect pathogenicity and multiple stress tolerance in B.bassiana.These findings provide a novel insight into ENAla-and ENA2b-dependent vacuolar pH stability,cation-homeostatic process and fungal fitness to host insect and environment.Essential role of COP9 signalosome subunit 5(Csn5)in insect pathogenicity and asexual development of B.bassiana.Csn5 is subunit 5 of COP9 signalosome complex in model fungi.In B.bassiana,orthologous Csn5 was proven to accumulate more heavily in nucleus than in cytoplasm and also proved indispensable for insect pathogenicity and virulence-related cellular events.Deletion of csn5 led to 68%increase of intracellular ubiquitin accumulation and dysregulation of 18 genes encoding ubiquitin-activating(E1),conjugating(E2),and ligating(E3)enzymes and ubiquitin-specific proteases,suggesting a crucial role for Csn5 in balancing ubiquitination and deubiquitination.Consequently,the deletion mutant displayed abolished insect pathogenicity,marked reductions in conidial hydrophobicity and adherence to insect cuticle,abolished secretion of cuticle penetration required enzymes,blocked haemocoel colonization,and reduced conidiation capacity despite unaffected biomass accumulation.These phenotypes correlated well with sharply repressed or abolished expressions of key hydrophobin genes required for hydrophobin biosynthesis/assembly and of developmental activator genes essential for aerial conidiation and submerged blastospore production.In the mutant,increased sensitivities to heat shock and oxidative stress also correlated with reduced expression levels of several heatresponsive genes and decreased activities of antioxidant enzymes.Altogether,Csn5-reliant ubiquitination/deubiquitination balance coordinates expression levels of those genes and quality control of functionally important enzymes,which are collectively essential for fungal pathogenicity,virulence-related cellular events,and asexual development.A small cysteine-free protein acts as a novel regulator of insect-pathogenic life cycle and genomic expression in B.bassiana.Small secreted proteins(SSPs),particularly cysteine-rich proteins secreted during fungal infection,comprise virulence effectors in plant-pathogenic fungi but remain unknown in insect-pathogenic fungi.In B.bassiana,only a small cysteine-free protein(CFP)is indispensable for insect pathogenicity among 10 studied SSPs(99 to 274 amino acids[aa]),including seven hypothetical proteins containing 0 to 12 Cys residues.CFP(120 aa)features an N-terminal signal peptide(residues 1 to 17)and a nuclear localization signal motif(residues 24 to 57)but no predictable domain.Its homologs exist exclusively in asexual species of insect pathogens in Cordycipitaceae and Clavicipitaceae.Green fluorescence-tagged CFP fusion protein was localized in nucleus but extracellularly undetectable,suggesting an inability for CFP to be secreted out.Disruption of cfp resulted in abolished pathogenicity via normal cuticle infection,attenuated virulence via hemocoel injection,compromised conidiation capacity versus little growth defect,impaired conidial coat,blocked secretion of cuticle-degrading enzymes,impeded proliferation in vivo,disturbed cell cycle,reduced stress tolerance,and 1,818 dysregulated genes(genomic 17.54%).Hundreds of those genes correlated with phenotypic changes observed in the disruption mutant.Intriguingly,nearly 40%of those dysregulated genes encode hypothetical or unknown proteins,and another 13%encode transcription factors and enzymes or proteins involved in genome-wide gene regulation.However,purified CFP showed no DNA-binding activity in an electrophoretic mobility shift assay.These findings unveil that CFP is a novel regulator of fungal insect-pathogenic life cycle and genomic expression and that cysteine richness is dispensable for distinguishing virulence effectors from putative SSPs in B.bassiana.Three small cysteine-free proteins(Cfpl-3)act as essential virulence factors of Metarhizium robertsii.One to three homologs of the unique CFP characterized as an extraordinary virulence factor in B.bassiana exist exclusively in asexual species of Cordycipitaceae(Beauveria/Cordyceps spp.)and Clavicipitaceae(Metarhizium spp.),suggesting that such homologs fuction in the fungi as does CFP in B.bassiana.Thus,three CFP homologs(Cfpl-3,128-145 aa)in M.robertsii were functionally characterized to clarify the hypothesis.Despite limited effects on asexual cycle in vitro,knockout mutations of cfpl,cfp2 and cfp3 led to so impaired a capability of normal cuticle infection that most G.mellonella larvae survived topical application of a 2 × 107 conidia/mL suspension.The blocked cuticle infection concurred with reduced activities of cuticle-degrading extracellular enzymes and Prl proteases secreted into the supernatants of the mutants’ liquid cultures.Intrahemocoel injection of～250 conidia per larva for cuticle-bypassing infection resulted in more attenuated virulence in the absence of cfp1/(82%)than of cfp2(21%)or cfp3(25%).Microscopic examination of hemolymph samples taken from surviving larvae revealed a delayed release of fungal cells by aggregated insect hemocytes for yeast-like budding proliferation in vivo.Transcriptomic analysis resulted in identification of 604 genes(up/down ratio:251:353)dysregulated in the Δcfp1 mutant.Many of them were involved in virulence-related cellular processes and events aside from 154 functionally unknown genes(up/down ratio:56:98).These results reinforce essential roles of small CFP homologs in hypocrealean fungal adaptation to insect pathogenic lifestyle and unravel their exploitability in genetic improvement of fungal insecticides.Fungal insecticidal activity is elevated by non-risky markerless overexpression of an endogenous cysteine-free protein gene in B.bassiana.Fungal insecticides are notorious for slow kill action,an intrinsic trait that can be readily improved by genetic engineering of an exogenous or endogenous virulence factor.However,transgenic insecticides expressing exogenous toxin and herbicide-resistant marker genes may cause unexpected ecological risks and hence suffer strict regulatory hurdles to be hardly stridden for field release.It is necessary to improve biotechnology that enables to not only speed up fungal insect-killing action but exclude ecological risk source.A markerless transformation system of B.bassiana was hence reconstructed based on the fungal uridine auxotrophy(Δura3)and applied for overexpression of cfp characterized as an extraordinary virulence factor.Three selected strains with cfp expression being greatly upregulated showed much faster kill action to Galleria mellonella larvae than the parental wild-type via normal cuticle infection but no change in vegetative growth and aerial conidiation.The faster kill action was achieved due to an increase of conidial adherence to insect cuticle,more secretion of cuticle-degrading Prl proteases,elevation of antioxidant enzyme activity crucial for collapse of insect immune defense,and acceleration of hemocoel localization,proliferation in vivo and host death from mummification.The markerless system is free of any foreign DNA fragment as a source of ecologic risk and provides a novel biotechnological approach to enhancing fungal insecticidal activity with non-risky endogenous genes and striding over the regulatory hurdles.