Regulatory Rolesof Asexual Developmental Activators And Hydrophobins In Conidiation And Conidial Maturation Of Metarhizium Robertsii

Metarhizium robertsii and Beauveria bassiana act as main sources of environmentally friendly wide-spectrum fungal insecticidies and are classified to Clavicipitaceae and Cordycipitaceae in Hypocreales,respectively.Metarhizium is considered to have evolved insect pathogenicity from phytopathogenic or phytoendophytic fungi 130 million years later than that Beauveria.Due to this big difference,the two lineages have differential or even different gene networks involved in asexual development and adapatation to host and environemtn.The yield and quality of aerial conidia serving as active ingredients of fungal insecticides to large extent determine the potential of biological control and the field efficacy of formulated conidia.In Aspergillus nidulans,aerial conidiation and conidial maturation is asexual developmental processes under the control of central developmental pathway（CDP）,and is regulated by the CDP activators BrlA,AbaA and WetA and the downstream velvety protein VosA.Similar regulatory roles of their orthologs have also been elucidated in B.bassiana.Hydrophobicity,one of important indices for mature conidia,is determinant to condial adhesion required for initiation of fungal infection to insect and largely dependent on the assembly of synthesized hydrophobins into rodlet bundles on conidial coat.The difference in evolutionary histories of insect pathogenicity between the two fungal lineages implicates that regulatory mechanisms underlying asexual development could be subtly different between M.robertsii and B.bassiana.Fully understanding the mechanistic difference will help to improve mass production and quality control technology of conidia and,hence,is crucial for biological control of insect pests.This study seeks ot characterize biological fungctions of brlA,abaA,wet A,vos A and three hydorphobin genes（hyd1–3）in M.robertsii and elucidate regulatory roles of those CDP genes in conidiation and conidial maturation and possible roles of hyd1–3 in conidal maturation.The main results are summarized as follows.Regulatory roles of BrlA and AbaA in asexual and insect-pathogenic lifecycles of M.robertsii.BrlA and AbaA are key CDP activators that control asexual development in Aspergillus but their roles remain understood insufficiently in hypocerealean insect pathogens.Here,regulatory roles of BrlA and AbaA orthologs in M.robertsii are characterized for comparison to those elucidated previously in B.bassiana at phenotypic and transcriptomic levels.Time-course transcription profiles of brlA,abaA and wet A revealed that they were not so sequentially activated in M.robertsii as elucidated in A.nidulans.Aerial conidiation essential for fungal infection and dispersal,submerged blastospore production mimicking yeast-like budding proliferation in insect hemocoel,and insect pathogenicity via cuticular penetration were all abolished as a consequence of brlA or abaA disruption,which had little impact on normal hyphal growth.The disruptants were severely compromised in virulence via cuticle-bypassing infection（hemocoel injection）and differentially impaired in cellular tolerance to oxidative and cell wall-perturbing stresses.In the brlA and abaA disruptants,255 and 233 genes were dysregulated（up/down ratios:52:203 and 101:122）respectively,including 108 genes co-dysregulated.These counts were very small in comparison to 1513 and 2869 dysregulated genes（up/down ratios:707:806 and 1513:1356）identified in the previous brlA and abaA knockout mutants of B.bassiana.These results reveal not only conserved roles for BrlA and AbaA in asexual developmental control but also their indispensable roles in fungal adaptation to insect-pathogenic lifecycle and host habitats.Intriguingly,gene expression networks controlled by BrlA or AbaA are largely different between the two insect pathogens,in which similar phenotypes were compromised in the absence of either brlA or abaA.Essential role of WetA,but no role of VosA,in conidiation,conidial maturation and insect pathogenicity of M.robertsii.Conidial maturation vital for conidial quality is controlled by the asexual development activator WetA and the downstream velvety protein VosA in Aspergillus.Their orthologs have proved functional in conidial quality control of B.bassiana as seen in Aspergillus but remain functionally unclear in M.robertsii,the other hypocrealean insect pathogen.Here,WetA and VosA proved essential and nonessential for M.robertsii’s lifecycle,respectively.Disruption of wet A increased hyphal sensitivity to oxidative stress and Congo red-induced cell wall stress despite little effect on radial growth on various media.The wet A disruptant was severely compromised in conidiation capacity and conidial quality,which was featured by slower germination,decreased UV resistance,reduced hydrophobicity and deformed hydrophobin rodlet bundles assembled onto conidial coat.The disruptant’s virulence was greatly attenuated via normal cuticle infection due to a blockage of the infection-required cellular processes.All phenotypes examined were not affected at all for the vos A disruptant.Intriguingly,mannitol was much less accumulated in the 7-and 15-day-old cultures of the wet A and vos A disruptants than of their control strains while accumulated trehalose was undetectable,revealing little link of intracellular polyol accumulation to conidial maturation.Identified from the transcriptome of the wet A disruptant versus the wild-type strain were 160 dysregulated genes（up/down ratio:72:88）mostly involved in cellular component,biological process and molecular function but very few associated with asexual development.Conclusively,WetA plays relatively conserved role in M.robertsii’s spore wall assembly but has functionally differentiated in some other conidial maturation-related cellular processes between the two insect pathogens.The functional redundancy of VosA in M.robertsii is distinct from a requirement of its ortholog for asexual development of B.bassiana.The present study unveils that M.robertsii WetA or VosA is functionally differential or different from the orthologs of either characterized in B.bassiana and other ascomycetes.Functional characterization of three hydrophobins（Hyd1–3）in M.robertsii.Fungal hydrophobin family consists of small amphiphilic proteins that are secreted to take part in aerial growth,development and conidial maturation.Three hydrophobins（Hyd1–3）exist in M.robertsii.Based on conserved domain analysis,Hyd1 vectoring an N-terminal signal peptide（NSP）and NSP-free Hyd2 are Class I hydrophobins while NSP-containing Hyd3 falls into Class II hydrophobins.In M.robertsii,threeΔhyd1 mutants were consistently compromised in aerial conidiation during the first-week incubation due to its hydrophilic or wettable hyphae although its conidial yield was restored to a wild-type level at the end of 15-day incubation.Compared to the wild-type strain,theΔhyd1 mutants produced defective conidia,which featured differential reductions of hydrophobicity（58%）,adhesion to insect cuticle（36%）,tolerances to UVB irradiation（20%）and 45℃ wet-heat stress（10%）,and virulence via normal cuticle infection(LT₅₀prolonged by 37%).In contrast,deletion of hyd2 or hyd3 had little impact on all examined phenotypes.Shown in scanning electron microscopic images,hydrophobin rodlet bundles assembled on conidial coat were disordered in the absence of hyd1 but not affected in the absence of either hyd2 or hyd3.In conclusion,only Hyd1 was proven to act a core regulator of hydrophobicity and play an important role in conidial quality control and insect pathogenicity of M.robertsii.Hyd2 and Hyd3 were proved functionally redundant in the insect mycopathogen.