Functional Analyses Of MAPK And APSES Family Proteins In Nematode-trapping Fungus Arthrobotrys Oligospora

Arthrobotrys oligospora is a typical nematode-trapping(NT)fungi,which can produce three-dimensional networks(traps)to capture nematodes.Traps are a tool for NT fungi to capture nematodes,and are also an important indicator of their switch from saprophytic to parasitic lifestyles.In this study,we identified the functions of MAPK and APSES family proteins in A.oligospora by gene knockout.The mechanisms of MAPK and APSES family proteins regulating phenotypes,such as conidiation and cell wall synthesis,were analyzed through real-time fluorescence quantitative PCR(RT-PCR)technology.Moreover,metabolomic and transcriptomic analyses were used to compare the metabolites and differential expression genes(DEGs)between wild-type(WT)and?Ao Swi6 mutant strains.Our results furtherly expand understanding of the functions and regulatory mechanisms of G protein signaling,especially MAPK family proteins and APSES transcription factors in the growth,sporulation,and trap formation of A.oligospora,which also provide a good foundation for further elucidating the mechanism of trap formation and lifestyle switch of NTFs.The main findings are described as follows:1.Functional analysis of MAPK family proteins in A.oligospora11 homologous MAPK family proteins were retrieved from the genome of A.oligospora,And a MAPK cascade model was proposed in A.oligospora by protein sequence alignment,conserved domain analysis,and cluster analysis.Similar to other filamentous fungi,four MAPK cascade regulatory pathways were predicted in A.oligospora,namely Fus3,Slt2,Hog1 and Ime2.1)Function of Slt2-MAPK proteinsSlt2-MAPK cascade is composed of Bck1,Mkk1,and Slt2.The?Ao Bck1 and?Ao Mkk1 mutants were obtained by gene knockout(The?Ao Slt2 mutant strains have been obtained in our previous study).Compared with WT strain,?Ao Bck1,?Ao Mkk1,and?Ao Slt2 mutants had slower growth,significantly reduced aerial hyphae and the number of nucleus in mycelial cells.The sensitivities of three mutant strains to several chemical stressors are increased,and their cell wall became deformed.In addition,the mutants were unable to produce conidia and traps,and can not kill nematodes.RT-PCR analysis showed that the transcription levels of several genes related to sporulation,cell wall synthesis,and H₂O₂metabolism in mutant strains were significantly down-regulated compared with WT strain.2)Function of Hog1-MAPK proteinsHog1-MAPK cascade is composed of Ssk2,Pbs2,and Hog1.The mutants of three protein-encoding genes(Ao Ssk2,Ao Pbs2,and Ao Hog1)were obtained through gene knockout.Phenotypic analysis revealed that the mycelium growth of mutant strains was slower and the aerial mycelium was significantly reduced than WT strain,but the hyphal septa were more than WT strain.Meanwhile,conidial germination rates of the mutants were also significantly lower than that of WT strain.The sensitivities of mutant strains to hyperosmotic,oxidative,and cell wall disrupting agents were increased significantly;in addition,the number of traps produced by mutant strains was increased compared with WT strain.The number of electron dense bodies(EDB)in traps of?Ao Hog1 mutant strains was increased,and a few of lipid droplets were observed in?Ao Hog1 mutants.3)Function of Fus3-MAPK proteinsFus3-MAPK cascade is composed of Ste11,Ste7,and Fus3.The?Ao Ste7 and?Ao Fus3 mutants were obtained by homologous recombination technology.Phenotypic analysis revealed that?Ao Ste7 and?Ao Fus3 mutant strains had reduced aerial hyphae but hyphal septa increased.The growth of?Ao Fus3 mutant strains slowed down,and the mycelium was significantly enlarged.The conidial yields of mutant strains were significantly reduced,and the conidiophores of?Ao Fus3 mutant strains changed significantly.Meanwhile,the mutants showed a significantly increased sensitivity to hypertonic,oxidative,and cell wall disrupting agents.The?Ao Ste7 mutant strains can produce few of traps,while?Ao Fus3 mutants can not produce traps instead of producing hyphal coil,and no EDB was observed in the hyphal coil.4)Function of Ime2-MAPK proteinDisruption of the gene Ao Ime2 caused defective growth,with slower mycelial growth in?Ao Ime2 mutants than WT strain,and the number of hyphal septa in mycelia was higher and cell nuclei in mycelia or conidia was considerably lower than in WT strain.The conidial yields of?Ao Ime2 mutants were decreased,and the transcription of several sporulation-related genes was markedly downregulated during the conidiation stage.The?Ao Ime2 mutants were highly sensitive to the osmotic stressors Na Cl and sorbitol,and the cell wall of partial hyphae in the mutants was deformed,and the cell wall of the traps produced by?Ao Ime2 mutants became loose,and the EDB in trap cells were also noticeably fewer than in WT strain.Moreover,Ao Ime2 disruption caused a reduction in trap formation and serine-protease production,and most hyphal traps produced by?Ao Ime2 mutants did not form an intact hyphal loop.2.Functional analysis of APSES family proteinsFive APSES homologous proteins(Ao Stu A,Ao Mbp1,Ao Swi6,Ao Bfp4,and Ao Xbp1)were retrieved from A.oligospora by genomic analysis.Among them,the?Ao Stu A mutant strains have been obtained in our previous study,we focus on the properties of the remaining four APSES proteins in this study.1)Function of four APSES proteinsFour APSES protein encoding genes(Ao Mbp1,Ao Swi6,Ao Bfp4,and Ao Xbp1)were disrupted by homologous recombination.Phenotypic analysis revealed that the growth rate of the?Ao Mbp1 and?Ao Swi6 mutants was slower than WT strain,and the aerial mycelium was weaker than that of WT strain,and the number of hyphal septa in mycelia was higher and the number of cell nuclei in mycelia and conidia was considerably lower than in WT strain,and their partial mycelia became swelling and deformation.The?Ao Mbp1 and?Ao Swi6 mutants showed increased sensitivities to cell wall perturbing,hyperosmotic and oxidative,and the cell wall of?Ao Swi6 mutant was deformed,and the number of Woronin body was decreased.Moreover,the traps produced by?Ao Mbp1 and?Ao Swi6 mutants were significantly reduced,and the number of EDB in traps was also reduced;interestingly,traps produced by?AoMbp1mutants are not three-dimensional nets but parallel with 3-5 hyphal coils,while traps of the?Ao Swi6 mutant was mainly produced near the nematode body.Furthermore,compared to WT strain,the protease hydrolysis activities of?Ao Mbp1 and?Ao Swi6mutants were decreased.RT-PCR analysis showed that the transcription levels of genes related to conidiation,stress-resisting and extracellular protease were significantly reduced in?Ao Swi6 mutant.In contrast,the phenotypes of?Ao Bfp4 and?Ao Xbp1 mutants were not significantly different from those of WT strain.2)Metabolomic and transcriptomic analysis between WT and?Ao Swi6 mutant strainsLC-MS analysis was used to compare the metabolites of the WT strain and the?Ao Swi6 mutant,their metabolic profiles and metabolites were significantly different.RNA-seq was used to compare DEGs between WT and?Ao Swi6 mutant strains at different time points induced by C.elegans.Compared with WT strain,the transcriptomic profile was significantly different in?Ao Swi6 mutant in the vegetative growth stage and the pathogenic stage,among them,the genes involved in DNA replication,mismatch repair,base excision repair,nucleotide excision repair,homologous recombination and non-homologous end joining pathways were significantly up-regulated.Combining of KEGG and GO enrichment analyses,we identified several genes might involve in trap formation and pathogenicity.3)Prediction and analysis of downstream target genes and interacting proteins of Ao Swi6Based on transcriptomic data,we also predicted the target downstream genes of Ao Swi6.A total of 401 target genes were predicted,of which 83 genes were involved in the regulation of cellular processes.Meanwhile,the interacting proteins of Ao Swi6were further predicted,and the yeast two-hybrid technology(Y2H)was used to verify the interaction of Ao Swi6 and related proteins.Our results showed that Ao Swi6 can interact with Ao Slt2 and Ao Pkc,which suggested that Ao Pkc may directly regulate Ao Swi6 in A.oligospora,thereby regulating the fungal growth,sporulation,and trap formation.Innovations in this paper:1.The functions of MAPK family proteins were identified in A.oligospora,and the mechanism of MAPK family proteins regulating phenotypes such as sporulation and stress resistance was analyzed by RT-PCR.2.The functions of four APSES family proteins were identified in A.oligospora,and DEGs between WT and?Ao Swi6 mutant strains at different time points in the nematode induction were compared using transcriptomic analysis.Moreover,the downstream target genes and interacting proteins of Ao Swi6 were predicted,and the interaction between Ao Swi6 and Ao Slt2 or Ao Pkc was verified by Y2H.