Involvement Of Annexin C7 And Sexual Differentiation Process Proteins In Growth And Development In Two Thermophilic Fungi

Thermophilic fungi are a kind of eukaryotes which inhabit in extreme high temperature environment,including soils,composts,wood chip piles,nesting materials of birds and other animals,and municipal refuse.The thermophilic fungi Thermomyces lanuginosus and Chaetomium thermophilum inhabit in the same environments,reproduce in distinct patterns and different in lifespan.Based on the genomic and transcriptome biological information of these two thermophilic fungi,ANXC7(Annexin C7)and SDP(Sexual differentiation process protein)gene were found relative to filamentous fungi growth and development.Using deletion and replacement mutant construct,the characters from the perspectives of biology,physiology,biochemistry and molecular biology of these two gene has been revealed.Annexins(ANXs)are widely expressed and structurally related proteins which play multiple biological roles in animals,plants,and fungi.In this study,a single ANX homologue(ANXC7)was identified and characterized in T.lanuginosus.ANXC7 was 1401 bp,including one intron,two exons,and a 1341 bp coding region,which encoded a putative 466 amino acid protein.The deduced 3D structure of the ANXC7 protein indicated that the four annexin repeats were packed into one ?-helical disk with two Ca2+ binding sites.The ANXC7 protein accumulated in the mitochondria and membrane.The deletion of ANXC7(?ANXC7)resulted in no obvious phenotype related to colony growth.However,the dry weight of the mutant mycelia was significantly increased.Based on a surface hydrophobicity test,the ?ANXC7 strain was clearly less hydrophobic.The growth of ?ANXC7 was inhibited when grown under selected stress conditions,particularly with regard to salt stress;however,the oxidative resistance to exogenous H2O2 in ?ANXC7 was increased,and endogenous H2O2 levels within the ?ANXC7 were lower than in the wild-type,thereby suggesting that the ANXC7 specifically controls oxidative resistance.ANXC7 associated with precocious conidial maturation and shedding from the conidiophore stalk in this strain.Taken together,our data indicate that ANXC7 localizes to the mitochondria and is involved in controlling conidium development and oxidative resistance in T.lanuginosus.Fungal genes encoding sexual differentiation process proteins(SDPs),isp,have been known for decades.isp4/SDP and its homologues function as oligopeptide transporters(OPTs),yet their roles in reproduction are unknown.Here,we genetically analyzed all four isp4/SDP homologues in the sexual species C.thermophilum and asexual species T.lanuginosus.Using single gene deletion mutants,we found that T.lanuginosus SDP(Tl SDP)participated in asexual sporulation,whereas the other homologues participated in sexual morphogenesis.In complementary tests,C.thermophilum SDPs(Ct SDP1-3)restored sporulation defects in Tl SDP deletion strains(?Tl SDP),and their translated proteins,which were localized onto the cytomembrane,possessed OPT activity.Interestingly,Ct SDP2 accumulated at the top of the hyphae played a distinct role in determining the sexual cycle,glutathione transport,and lifespan shortening.A unique 72nt-insertion fragment(72INS)was discovered in Ct SDP2.Biological analysis of the 72 INS deletion and Ds RED-tagged fusion strains implied the involvement of 72 INS in fungal growth and development.In contrast to Tl SDP,which only contributes to conidial production,the three Ct SDPs play important roles in sexual and asexual reproduction,and Ct SDP2 harbors a unique functional 72 INS that initiates sexual morphogenesis.In summary,the molecular genetics technology was used to identify two types of growth and development related genes of the thermophilic fungi ANXC7 and SDP.Both ANXC7 and Tl SDP are involved in regulating the growth and development of T.lanuginosus and their response to stress,especially in regulating the formation,germination and shedding of conidia.In addition,this study comprehensively analyzed the biological functions of the four sexual differentiation process proteins genes.It was confirmed that the Ct SDPs gene of C.thermophilus with sexual reproduction as the main development mode not only has the characteristics of Tl SDP,but also participates in the defective phenotype of T.lanuginosus and also generates a sexual reproductive structure,revealing that Ct SDPs participate in the regulation sexual/asexual reproduction of thermophiles.In addition,it was found that the inserted fragment 72 INS of Ct SDP2 is involved in the regulation of sexual reproduction structure formation.This study explored the biological functions of ANXC7 and SDPs genes from the perspective of growth,development,and adaptation.The results of the study laid the foundation for further revealing the development,survival,and evolution of thermophilic fungi,and also provided theoretical evidence for the efficient use of thermophilic fungi.