Functional Analyses Of Ras And Rho GTPases And Ar-GAPs In Arthrobotrys Oligospora

Nematode-trapping fungi(NTF)are widely distributed in terrestrial and aquatic ecosystems.Their broad adaptability and flexible lifestyles make them ideal agents for controlling pathogenic nematodes.Arthrobotrys oligospora is a model species employed for understanding the interaction between fungi and nematodes.It captures nematodes using adhesive networks.Its extracellular proteases play an important role in the infection of nematodes.Small GTPases are highly conserved in fungi and are involved in morphogenesis,cell polarity,development and host infection.To further understand the effects of small GTPases on the growth,development and pathogenicity of A.oligospora.In this study,eight homologous genes(Aoras2,Aoras3,Aorheb,Aorho2,Aocdc42,Aorac,Aoagel and Aoage2)of the Ras and Rho GTPases,and the Arf-GAP family were studied in A.oligospora.It were further characterized by gene knockout and phenotypic analysis.In addition,transcriptome and untargeted metabolomics analyses were combined to explore the regulatory role of small GTPases in growth and development and trap formation.The results of this study provide insight into the role of small GTPases in NTF,which is of great significance for the exploration of genetic strategies of fungal candidate strains and the development of more efficient and durable nematocides.The main results as follows:Ⅰ.Functional study of Rho GTPases.In this study,RNA-Seq technique was used to detect the transcriptome of A.oligospora induced by live nematodes at different time points(0-48 h).The transcriptome of A.oligospora during nematode-induced trap formation and infection of nematodes was studied.Differentially expressed genes(DEGs)accounted for about half of the genome(5752 genes),and rac genes were significantly upregulated.Alternative splicing(AS)events were identified in 2012 genes(about 17.5%of the genome).Analysis of genes in which AS occurred and DEGs during these processes showed that trap formation is an energy-consuming process involving a series of complex cellular processes.Meanwhile,rac and rho2 are also affected by AS.In addition,the identification of three Rho proteins(AoRho2,AoCdc42,and AoRac)by knockout and comparison of multiple phenotypes showed that the three Rho GTPases play different roles in mycelial growth and morphology,lipid accumulation,DNA damage and apoptosis,spore production and morphology,trap formation,nematocidal ability and ROS production.In this paper,we describe gene expression and AS events of A.oligospora during trap development and nematode predation,and emphasize the important role of Rho family in fungal growth,development,trap formation and lifestyle transformation.More importantly,Rho family proteins,especially AoRac and AoCdc42,play an indispensable role in the growth,development and pathogenicity of A.oligospora.This discovery helps to clarify the mechanism of trap morphogenesis and provide more effective strategies for biological control of pathogenic nematodes.Ⅱ.Functional study of Ras GTPases.In this study,three Ras GTPases(Ras2,Ras3,and Rheb)from A.oligospora were functially analyzed by gene knockout and multiple phenotypic analyses.In addition,comparative transcriptome analysis was performed to explore the regulatory role of AoRas2 in vegetative growth and trap formation.Our transcriptome analysis(12 h)showed that nearly one-fifth of the genes are regulated directly or indirectly by AoRas2,allowing us to understand the pleiotropic effects of Ras GTPases.Our results show that they play a variety of roles in regulating mycelial growth,conidia,stress resistance and pathogenicity of A.oligospora.In addition,the loss of Aoras2 and Aorheb significantly affected mitochondrial activity,reactive oxygen species,lipid storage and autophagy.Transcriptome analysis of the AAoras2 mutants showed that many of the repressed genes were involved in signal transduction,energy production,and carbohydrate transport and metabolism.In addition,metabolic profiling showed that AoRas2 and AoRheb affected the biosynthesis of secondary metabolites of A.oligospora.Taken together,these findings provide insight into the important role of Ras small GTPases in vegetative growth,development,and pathogenicity,and broaden our understanding of the biological functions of Ras GTPases in A.oligospora.These results lay a foundation for revealing the lifestyle transformation mechanism of NTF and exploring the potential application of oligospora arthroplexus in biological control of pathogenic nematodes.Ⅲ.Functional study of Arf-GAPs.In this study,two Arf-GAPs homolog in S.cerevisiae,Agel(AoAge1)and Age2(AoAge2),were knocked out in A.oligospora.Then,phenotypic traits of wild-type(WT)strains and mutant strains were systematically studied.AoAge2,in particular,plays an important role in vegetative growth,conidia,trap formation,DNA damage,endoplasmic reticulum stress,mitochondrial activity,endophagy,heat shock stress,ROS level,and autophagy of A.oligospora.Importantly,transcriptome analysis(12 h)showed that nearly 62.7%of A.oligospora genes were directly or indirectly regulated by AoAge2,which enabled us to understand the pleiotropic effect of Arf-GAP.At the same time,untargeted metabolome analysis showed that AoAge2 regulates secondary metabolism of A.oligospora.These findings provide insights into the important role of Arf-GAP in growth,development and pathogenicity,and broaden our understanding of the biological functions of Arf-GAP in fungi.The main innovation as follows:Ⅰ.In this study,RNA-Seq technique was used to detect the transcriptome of A.oligospora induced by live nematodes at different time points(0-48 h).The transcriptome of A.oligospora during nematode-induced trap formation and infection was studied.These results suggest that AS events play an important role in trap formation and lifestyle transformation of A.oligospora.The function of three Rho GTPases(AoRho2,AoCdc42,and AoRac)was identified by analyzing DEGs and AS events.They play different roles in mycelial growth and development,lipid accumulation,DNA damage,sporulation,trap formation,nematocidal ability and ROS production of A.oligospora.This discovery helps to clarify the mechanism of trap formation and pathogenicity of NTF.Ⅱ.In this study,the functions of Ras GTPases(AoRas2,AoRas3,and AoRheb)in A.oligospora were identified.Combined with the analysis of multiple omics,phenotypic and biochemical experiments,these Ras GTPases play an important role in the regulation of mycelial growth,sporulation,stress resistance and pathogenicity.These findings provide insight into the important role of Ras GTPases in growth,development and pathogenicity,thus laying the foundation for revealing the mechanism of lifestyle transformation of NTF.Ⅲ.In this study,we found that two Arf-GAPs(AoAgel and AoAge2)play an important role in the vegetative growth,sporulation,trap formation,DNA damage,endoplasmic reticulum stress,mitochondrial activity,endophagy,heat shock stress,ROS levels,autophagy and secondary metabolism of A.oligospora.These findings provide insights into the important role of Arf-GAPs in growth,development and pathogenicity,and broaden our understanding of the biological functions of Arf-GAPs in fungi.