A Preliminary Study On The Functions Of Rab And Arf Small G Proteins Of Arthrobotrys Oligospora

Small GTPases are important signaling proteins,which are homologous to the G?subunit with a GTPase active site and a GDP/GTP binding site.It can also bind GTP alone and hydrolyze it to GDP to achieve regulation of downstream effector proteins.At present,the small GTPases reported in filamentous fungi mainly include Ras,Rho,Rab and Arf protein subfamilies.The nematode-trapping fungi capture nematodes through producing special trapping devices(traps)and acquire nutrients for growth while killing nematodes.Traps is not only served as a weapon to capture nematodes,but it is also an important signal for the fungal lifestyle translation from saprophytic to parasitic stages.However,the signal transduction mechanism of trap formation is poorly understood.Arthrobotrys oligospora is a typical species of nematode-trapping fungi,four genes of Rab and Arf small GTPases in A.oligospora was knocked out in this study,and their functions were preliminary analyzed.The main experimental results:1.Amino acid sequence and cluster analysis of AoRab-7A,AoRab-2,AoArf-GAP and AoArl proteinsAnalysis of the amino acid sequences and conserved domains of the four small GTPases in A.oligospora,revealed that except for AoArf-GAP,which has a relatively large molecular weight and contains 476 amino acid residues,the other three small G proteins have relatively small molecular weights,with only 200 amino acids,and the functional domains of AoArf-GAP protein differs from the other three small GTPases,the functional domains of AoArf-GAP mainly include zinc finger domains(IPR037278)and Arf GTPase activating protein domains(IPR001164),while another three small GTPases share a conserved P-loop containing nucleoside triphosphate hydrolases(IPR027417)and a small GTP-binding protein domain(IPR005225).Cluster analysis showed that AoRab-7A and AoRab-2 clustered in two different evolutionary branches,similarly,AoArf-GAP and AoArl also clustered in two different branches,suggested that two members of Rab and Arf small GTPases may play different functions.2.Knock-out of four small GTPase genes and comparison of their phenotypesUsing the method of homologous recombination,four mutants of the small GTPase genes were successfully obtained,and the phenotypic characteristics of the wild type strain(WT)and the corresponding mutants were compared,including colony morphology,growth rate,formation and the number of traps,conidiation and activity of extracellular protease.The results showed that the growth rate of the ?AoRab-7A mutant was slower than the WT and the aerial mycelium was weaker than that of the WT.Meanwhile,the conidia of the ?AoRab-7A mutants were significantly reduced,and it could not produce traps,and the activity of extracellular protease was also reduced.Similarly,the growth rate of ?AoArf-GAP mutants were slower than the WT and its colonies were irregular,especially,the number of conidia was also greatly reduced and produced malformed spores,and the nematocidal activity was significantly reduced.However,there were no significant changes in phenotypes between the ?AoRab-2 and?AoArl mutants and their corresponding WTs.3.Analysis of the transcriptional levels of several phenotype-related genes in?AoRab-7A and ?AoRab-2 mutantsThe mycelia of the WT,?AoRab-7A and ?AoRab-2 mutants were collected,the total RNAs were extracted and transcribed reversely into cDNA,respectively.Several genes related to conidiation,stress-resisting and extracellular proteases were selected and their transcriptional levels were compared between the WT and mutants using RT-PCR method.The transcription of these genes in the ?AoRab-7A mutants showed significantly change compared against the WT,suggested that the AoRab-7A gene can regulate the expression of these genes and plays important roles in the growth,development and pathogenicity in of A.oligospora.However,the expression of these genes in the ?AoRab-2 mutants showed no significantly change compared to the WT,which was consistent to the phenotypic differences.4.The AoRab-7A is involved in the formation of autophagosome in A.oligosporaUnder nitrogen deprivation,a fluorescent dye experiment on autophagosome was carried out to compare the variations between the WT and ?AoRab-7A strains.Ourresults showed that the deletion of the gene AoRab-7A heavily blocked the autophagy process and the formation of autophagosome,which suggested that the AoRab-7A involved in the occurrence of autophagy.Innovations in this paper:1.Four small GTPases genes were successfully deleted in A.oligospora,AoRab-7A and AoArf-GAP were found to be involved in the regulation of important biological processes,such as growth,sporulation,and trap formation.2.The transcription levels of several genes related to conidiation,stress resistance and serine proteases were compared between the WT and ?AoRab-7A mutants,and our results suggested that AoRab-7A may involve in the regulation of these genes in A.oligospora.3.Our results confirmed that the AoRab-7A involved in the formation of the autophagic bodies under nitrogen starvation in A.oligospora.