Study On Trap Formation Mechanism In Orbilia Oligospora

Nematophagous fungi are a large group of filamentous fungi that can capture and kill nematode pests using their infection weapon of specified predacious devices(traps).However,much remains unknown about the mechanism of trap formation in these fungi.Our prior work has shown that ammonia signals and mediates the production of fungal trap structures in some nematophagous fungi,but the associated pathway underlying how this small molecule initiates and controls these fungi to develop predacious hyphae is unclear.As a species with full genome information and clearly described life history,Orbilia oligospora has been used as a model for dissecting mechanisms of traps formation.In this study,I used ammonia as the inducing factor to identify the genes involved in trap formation in the fungus O.oligospora.This fungus can capture and consume nematodes by producing adhesive hyphal nets.My analyses revealed that traps formation requires a novel cascade “Torc1-Npr1-Art-Rsp5”,the ESCRT machinery,and DUBs.The main findings are as follows:1.Cell membrane endocytosis and collapse-reconstruction play important roles in trap formation.Transcriptome sequencing for the four time points after ammonia treatment revealed the involvements of pathways related to Endocytosis,Ubquitination-mediated proteosome hydrolysis,and Phosphatidylinositol signaling,all of which were enriched throughout the processes of trap formation.Of obvious note is the endocytosis pathway in the first three time points.In addition,using FM4-64 staining and TEM,we observed that the added ammonia was directed to a highly visible endocytosis appearance within the cells of O.oligpspora.Furthermore,we noted an obvious damage-remodeling of cellular membranes triggered by the condition of ammonia addition.2.The TORC1-Npr1 cascade is related to the endocytosis pathway.Quantitative Real-time PCR screening revealed that the expression levels of AOL＿s00043g12,an ortholog of the yeast Torc1(Target of rapamycin),was increased by over1.5 folds.In yeast and animals,Torc1 is a protein kinase and acts as a key in the regulation of endocytosis.While ammonia could induce trap formation;rapamycin supplement inhibited cellular endocytosis,and completely blocked trap formation.Npr1(Nitrogen regulation reactivator 1)functions downstream in Torc1-mediated endocytosis and can be phosphorylated by the Torc1 kinase and is subsequently inhibited in the presence of the favored nitrogen source.We therefore hypothesized that,in A.oligospora,the deleting of Npr1 homologous gene AOL＿s00006g439 would enhance trap production,even forming traps spontaneously without the signaling of ammonia and others.As expected,the mutant strain OoNpr1 showed a significant ability to form traps even in the absence of ammonia.Further,this mutant possessed more remarkable phenomenon of endocytosis.These analyses suggested that Npr1 kinase is a crucial effector that plays roles in the lifestyle switch of nematode-trapping fungi from vegetative growth to predacious status.3.OoArt mediated OoRsp5 to regulate endocytosis of OoAmts and traps production.Yeast 2 hybrid(Y2H)assays revealed O.oligospora E3 ligase Rsp5 could interact with the adaptor protein OoArt,which was bound to the ammonium transporter OoAmts.The number of traps of strain ?OoRsp5 was decreased by 83%,80.3% and83% with the ammonia induction at 36 h,48 h and 60 h respectively;and that in?OoArt mutant by 33.7%,35.5% and 56.9% respectively.Furthermore,in response to nematodes after 24 h,36 h and 48 h,?OoRsp5 resulted in trap number decreases by63.3%,90.7% and 88.5%;and that in the ?OoArt mutants the decreases were 9.6%,11.6% and 4.2% respectively.4.ESCRT-0 bound to deubiquitinases regulated endocytosis and trap formation.ESCRT-0 is the initial complex of ESCRT(endosomal sorting complex required for transport),which acts as a key mediator in endocytosis pathway.Y2 H experiments showed that both components of the ESCRT-0,OoHse and OoVps27,could bind to the proteins of deubiquitinases OoSst2 and OoDoa4.For O.oligospora,lacking of OoHse and OoVps27 removed the ability of fungal endocytosis and generated traps when exposed to ammonia and nematodes.Moreover,the loss of OoSst2 and OoDoa4 diminished the effect of endocytosis.Accordingly,the trap number induced by ammonia was significantly decreased by 100%,96.7%,and 89.5% in the ?OoSst2 strain and 16.1%,27.6% and 29.4% in the ?OoDoa4 strain at the time of 6 h,48 h and 60 h after ammonia addition.Further,upon the addition of nematodes,after 24 h,36 h,and48 h,the trap number of the mutant ?OoSst2 is declined by 77.6%,77% and 64.8%,respectively.5.Trap formation-related genes are not only involved in the regulation of endocytosis and traps formation,but also involved in the regulation of colony growth,mycelial morphology,sporulation and stress resistance,respectively.The ?OoNpr1,?OoRsp5,?OoHse,?OoVps27 and ?OoSst2 mutants showed lower growth rate and more hyphal septation.For the mutants ?OoNpr1,?OoRsp5 and ?OoSst2,the numbers of conidia were significantly decreased,and some spores changed in their morphology.Beyond that,?OoHse and ?OoVps2 even lost the capability to sporulate:?OoNpr1 showed enhanced resistance to osmotic and oxidatve stresses;?OoRsp5 presented weakened resistance to osmotic and oxidative stresses and reduced susceptibility to agents that interfere with the cell wall synthesis;?OoHse displayed enhanced osmotic pressure resistance;?OoSst2 showed reduced susceptibility to agents that interfere with the cell wall synthesis.Innovations in this thesis:For the first time,the endocytosis pathway regulated by "TORC1-NPR1-ART-RSP5",ESCRT and the deubiquitinating enzyme DUBs is demonstrated to participated in the formation of traps in nematophageous fungi,and the role of collapse-reconstruction of cell membranes in the formation of traps.