Studies On The Regulatory Network Of Autophagy-related Protein Atg1 And The Mechanisms Involved In Pexophagy Of Beauveria Bassiana

Beauveria bassiana is a well-investigated filamentous insect pathogenic fungus and has a great potential in biological control of insect pests.Autophagy is a conserved intracellular degradation mechanism in eukaryotes and has been implicated in a plethora of physiologies in filamentous fungi,including vegetative growth,cell differentiation,and virulence.This study aims to reveal the regulatory mechanisms and biochemical pathways involved in autophagic processes of B.bassiana.Analysis of phosphorylation regulation function of protein kinase BbAtg1Autophagy is a conserved intracellular degradation mechanism in eukaryotes and is initiated by the protein kinase autophagy-related protein 1（Atg1）.However,except for the autophosphorylation activity of Atg1,the target proteins phosphorylated by Atg1 are largely unknown in filamentous fungi.In Beauveria bassiana,Atg1 is indispensable for autophagy and is associated with fungal development.Comparative omic-based analyses revealed that B.bassiana Atg1（BbAtg1）has key influence on the proteome and phosphoproteome during conidiogenesis.In terms of its physiological functions,BbAtg1-mediated phosphoproteome is primarily associated with metabolism,signal transduction,cell cycle,and autophagy.At the proteomic level,BbAtg1 mainly regulates genes involved in protein synthesis,protein fate,and protein with binding function.Furthermore,integrative analyses of phosphoproteomic and proteomic data led to the identification of several potential targets regulated by BbAtg1 phosphorylation activity.Notably,we demonstrated that BbAtg1phosphorylated BbAtg3,an essential component of ubiquitin-like conjugation system in autophagic progress.Our findings indicate that in addition to being a critical component of the autophagy initiation,Atg1 orchestrates the autophagosome elongation via its phosphorylation activity.The data from our study will facilitate future studies on the noncanonical targets of Atg1 and decipher the Atg1-mediated phosphorylation networks.Functional analysis of BbATG7 and BbATG10 genes in the ubiquitin-like conjugation system In B.bassiana,Atg7 and Atg10 which involved in the ubiquitin-like conjugation system were identified and functionally analyzed.Ablation of BbATG7 and BbATG10 genes blocked the autophagic process under starvation stress,respectively.Both mutants ofΔBbatg7 andΔBbatg10 exhibited impaired growth on the media with chitin as single nitrogen source.On rich media,genes loss did not cause notable effect on vegetative growth,but resulted in a considerable reduction in conidiation（71.6%）and blastospore yield（61.1%）inΔBbatg7 mutant.At the same time,the conidiation ofΔBbatg10 decreased by 95.6%.In addition,theΔBbatg7 mutant displayed increased sensitivity to stress caused by menadione and Congo red,while theΔBbatg10 mutant was only senitive to menadione.The virulence ofΔBbatg7 andΔBbatg10 mutants was significantly attenuated as indicated in topical and intrahemocoel injection assays.Our study indicates that BbATG7 and BbATG10contribute to B.bassiana virulence and asexual development via regulating autophagy pathway and playing non-autophagic functions in the infection cycle.Functional analysis of BbPEX14 Peroxisomes are single-membrane-bound organelle which play indispensable roles in regulating cell metabolism,growth and development in fungi.This study identified and functionally characterized the biological functions of B.bassiana PEX14（BbPEX14）.Disruption of BbPEX14 gene led to severe functional impairment of B.bassiana in various physiological processes such as asexual development,stress resistance,and virulence.Compared with the wild-type strain,the blastospore yield of theΔBbpex14 mutant strain decreased by57.3%,the conidia yield decreased by 97.6%,and the sporulation structure could not form normally;The virulence of theΔBbpex14 mutant strain to infect host by injection and immersion was significantly weakened.Based on the data from mass spectrometry analysis,five ubiquitination sites were revealed at lysine of BbPex14.Point-mutation analyses demonstrated that two sites（K149,K155）in BbPex14 were significantly involved in conidiation.These results indicate that BbPex14 plays critical roles in physiologies of B.bassiana,in which its function in conidiation is regulated by post translation modulation.BbPex14 mediates pexophagy In this study,we found that BbPex14 mediated pexophagy in B.bassiana.Yeast two-hybrid assay was used to screen the potential proteins that interact with BbPex14,in which a coding gene of one protein was identified as next to BRCA gene 1（NBR1）.Bb Nbr1 is involved in the development and pathogenesis of B.bassiana.Compared to the wild strain,deletion of Bb NBR1resulted in a 55.2%decrease in fungal conidia yield,a 36.3%decrease in blastospore yield,and a 1.02 d prolongation of LT₅₀in the cuticle infection mode.Meanwhile,ablation of Bb NBR1 blocked pexophagy in B.bassiana.Bioinformation analysis indicated that Bb Nbr1 contained Atg8 interaction motif.Interaction between them was validated by the protein interaction assay.Therefore,we propose a model of pexophagy in B.bassiana,in which BbPex14 act as peroxisomal adaptor,Bb Nbr1 as selective autophagy receptor,and BbAtg8 as the acceptor on autophagosome.This is the first study to highlight a pathway involved in pexophagy in filamentous fungi.Functional analysis of a peroxisomal sterol carrier protein 2（Scp2）in B.bassiana Sterol carrier protein 2（Scp2）represents a family of proteins binding a variety of lipids and plays essential roles in cellular physiology.However,its physiological roles are largely unknown in filamentous fungi.In this study,we functionally characterized an orthologous SCP2 gene in the filamentous insect pathogenic fungus Beauveria bassiana（Bb SCP2）.Bb Scp2 was verified to be a peroxisomal protein and displayed different affinities to various lipids,with strong affinity to palmitic acid（PA）and ergosterol（ES）.No significant binding activity was detected between protein and oleic acid（OA）or linoleic acid（LA）.Ablation of Bb SCP2 did not cause significant effects on fungal growth on various carbon sources,but resulted in a modest reduction in conidial（49%）and blastospore yield（45%）.In addition,exogenous lipids could recover the defectives in conidiation ofΔBbscp2mutant strain.Bb Scp2 was required for the cytomembrane functionality in germlings,and its loss resulted in a more significant decrease in virulence indicated by cuticle infection assay than intrahemocoel injection assay.Our findings indicate that Scp2links the lipid trafficking to the asexual differentiation and virulence of B.bassiana.Summary,in addition to its function as the autophagy initiator,Atg1 mediates comprehensive proteome and phosphoproteome in B.bassiana and perform feedforward control on the ubiquitin-like conjugation pathway in autophagy.Peroxisomal proteins contribute to development,stress response,and virulence in B.bassiana.Notably,three proteins（peroxin 14,Nbrl and Atg8）form a 3-step pathway which mediates the selective degradation of peroxisomes.This study updates our understandings of mechanisms involved in physiological process and autophagy pathway of entomopathogenic fungi.