| Lignocellulose is the most abundant biomass resource on Earth.Cellulases and hemicellulases,which can degrade lignocellulose,have been widely used in food,feed and textile industries,and show great application potential in the production of fine chemicals and alternative energy.Filamentous fungi represented by Trichoderma reesei,Penicillium oxalicum,Aspergillus niger,and Myceliophthora thermophila have become the main sources of cellulase in industrial production due to their ability to efficiently synthesize and secrete cellulases.The synthesis of cellulase is mainly controlled at the transcriptional level.Many sequence specific transcription factors are directly or indirectly involved in the activation or repression of cellulase gene transcription.The activity,stability,subcellular localization,and DNA-affinity of transcription factors are affected by various post-translational modification processes,including phosphorylation,ubiquitination,acetylation,etc.After transcription factors bind with DNA,cofactors are recruited to co-regulate transcription.Therefore,finding and identifying proteins that interact with transcription factors and confirming whether they contain enzymes/proteins that perform post-translational modifications and whether they contain cofactors are important research contents to explore the mechanism of transcription factors regulating gene expression.The study of complex patterns of relations among genes,sequence-specific transcription factors,and interacting proteins is of great theoretical significance for revealing the gene transcription regulation mechanism in eukaryotes at a deeper level.Meanwhile,the identified proteins that interact with transcription factors may also become targets for genetic engineering of cellulase-producing strains.Research has profound theoretical and practical significance.In this paper,the mechanisms of transcription factor Ace1,which directly regulates cellulase gene expression,and CrzA,which indirectly regulates cellulase gene expression,are studied using Penicillium oxalicum,an important cellulase-producing fungus,as the research object.The F-box protein Fbx23,which has a direct interaction with Acel,was captured,identified,and characterized.We propose a possible model by which Acel is controlled by the ubiquitin ligase SCF complex(Skpl,Cullin,F-box complex)containing the F-box protein Fbx23,which may regulate the expression of cellulase genes.Proteins that interact with the transcription factor CrzA were captured,and the calcineurin-transcription factor CrzA pathway in P.oxalicum was drafted.The main research contents and results of the paper are as follows:1.Capture and identification of interacting proteins with P.oxalicum transcription factor PoAce1The transcription factor Ace1 was originally considered as a transcriptional activator of cellulase gene expression,and its name(activator of cellulase expression)was derived from it.Subsequent studies have found that Ace1 is essentially a transcriptional repressor of cellulase gene expression.Ace1 deletion in T.reesei and P.oxalicum can increases cellulase synthesis.However,the mechanism of Ace1 regulating gene expression has not yet been elucidated.P.oxalicum Ace1(PoAce1)was used as the bait protein to capture and identify proteins interacting with PoAce1 using tandem affinity purification(TAP)combined with mass spectrometry(TAP-MS).Among the 8 captured proteins,the abundance of the two components of the ubiquitin ligase SCF complex,Skp1 and F-box protein Fbx23,are the top two positions.The other two components of the SCF complex,Rbx1 and Cull,are also discovered.The results suggest that the transcription factor Ace1 is related to the SCF complex which is composed of Rbxl-Cull-Skp1-Fbx23.The results of yeast two hybrid further support the direct interaction of PoAce1 with the F-box protein PoFbx23.Results from bimolecular fluorescence complementation(BiFC)confirm that the PoAce-PoFbx23 interaction occurs within the nucleus.The PoFbx23 protein in the SCFFbx23 complex(superscript representing the F-box subunit of ubiquitin ligase)plays the role in recognizing and binding the transcription factor Ace1 as a substrate.2.Characterization of P.oxalicum F-box protein PoFbx23The functions of P.oxalicum Fbx23(PoFbx23)in hyphae growth,asexual development,cellulase gene transcription and cellulase synthesis were studied.PoFbx23 is localized in the nucleus.When PoFbx23 is absent(Δfbx23),the biomass of the Δfbx23 is no different from that of the wild type strain,but the conidiation is impaired and the number of conidia decreases.In the Δfbx23 mutant strain,the transcription of the gene encoding the key transcription factor BrlA located in the central regulatory pathway controlling conidiation is significantly downregulated,suggesting that PoFbx23 indirectly regulated conidiation by affecting the central regulatory pathway.At the same time,the transcription levels of genes in the dihydroxynaphthyene(DHN)-melanin synthesis pathway(abr2→abr1→ayg1→arp1→arp2→alb1)related to spore pigment synthesis were also downregulated.Compared with that of the wild type strain,the secretion of cellulase,hemicellulase and extracellular protein of Δfbx23 mutant is significantly increased.The qPCR results show that the transcription level of the main cellobiohydrolase gene cbh1 and endoglucanase gene eg1 were significantly upregulated,indicating that PoFbx23 affects cellulase synthesis by regulating the expression of cellulase-encoding genes.Transcriptome data also supports that the expression of key cellulase and hemicellulase genes in the Δfbx23 mutant is upregulated.The two most important cellobiohydrolase genes cbh1 and cbh2,two endoglucanase genes eg1/cel7B and eg2/cel5B,β-glucosidase gene bgl1,two xylanase genes xyn10A and xyn11A,lysaccharide monooxygenase(LPMO)gene AA9 that exhibits synergistic effects on cellulose degradation,are significantly upregulated.In addition,PoFbx23 is also associated with the expression of secondary metabolic gene clusters.Of the 28 gene clusters predicted in the P.oxalicum genome,4 clusters were downregulated in theΔfbx23 mutant.Compared with the starting strain,the protein abundance of the transcription factor PoAcel in the Δfbx23 mutant strain increased,but the transcription level of ace1 gene does not increase.The results indicate that the deletion of Pofbx23 gene mainly affected the abundance of PoAce1 at the level of protein synthesis or degradation.The possible mechanism by which transcription factor Acel controlled by ubiquitin ligase SCFFbx23 to regulate cellulase gene expression is proposed:the inactive precursor of transcriptional repressor Acel was degraded and cleaved into an active state by,cFFbx23,playing its roles in repressing cellulase gene expression.3.Capture and identification of interacting proteins with transcription factor CrzAThe calcium-calmodulin-calcineurin signaling pathway is a conserved cascade pathway in fungi that senses environmental signals and coordinates fungal growth,development,and metabolism.The transcription factor Crz1/CrzA(calcineurin-responsive zinc finger)is one of the most important targets downstream of this pathway,involved in various fungal life processes,such as mycelial growth,cell wall integrity,sexual/asexual development,calcium homeostasis,stress resistance,virulence,and secondary metabolite synthesis,etc.The study of the function of transcription factor CrzA has always been a hot topic in the research field of filamentous fungi.The biological functions of P.oxalicum CrzA(PoCrzA)has been characterized in our laboratory.In the paper,proteins interacting with PoCrzA were captured and identified by tandem affinity purification and mass spectrometry(TAP-MS).Fifty proteins with potential interactions with PoCrzA were discovered,including:the catalytic subunit of calcineurin(Cnal,calcineurin A),the regulatory subunit of calcineurin(Cnb1,calcineurin B),and the 14-3-3 protein Bmh1.Two proteins associated with nuclear protein transport,the exportin Los1 and the importin Srp1,two phosphokinases Fus3 and Slt2,and subunit Cyc8 of the transcriptional cofactor complex Tupl-Cyc8 were also discovered.Based on the results of TAP-MS,the calcineurin-transcription factor CrzA pathway in P.oxalicum is drafted.The conjecture that Tupl-Cyc8 may be involved in transcriptional regulation as a cofactor of PoCrzA was proposed. |
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