Investigating The Roles Of MilRNAs And Their Target Genes In Arthrobotrys Oligospora

Arthrobotrys oligospora is a nematode-trapping fungus widely distributed in the environment.It can kill nematodes by producing adhesive three-dimensional nets and completes the lifestyle transition from saprophytes into pathogenesis.Even though it is a model organism for studying the interaction between fungi and nematodes,the mechanism of its trap formation is still unclear.As an important regulator of gene expression in organisms,the regulation of the traps formation by milRNAs needs to be further explored in A.oligospora.Previous data in our laboratory showed that the expression of mil-289 increased during the formation of traps induced by the extract of nematodes,while the expression of mil-799 appeared from nothing.These two milRNAs may be involved in the formation of three-dimensional network.From this point of view,the mechanism of interaction between milRNAs and target genes in A.oligosporus to regulate the formation of traps was studied.After knocking out genes ao?289 and ao?799 respectively,traps formation was delayed under urea induction in strain_?799,while the traps in strain_?289 were reduced compared with wild type(WT)strains.In addition,the capacity of traps production in the knockout strains_?799 and_?289 was also weaker than that of WT strain when induced by Caenorhabditis elegans extract.Which indicated that milR-289 and milR-799 were indeed involved in the traps formation in A.oligosporus.Endonuclease Dicer is a key enzyme in the synthesis of mature micro RNAs in animals and plants.RT-PCR analysis showed that in the Semi-knockout strain_?Dicer,the transcriptional levels of the five mature milRNAs(milR-19,milR-36,milR-352,milR-799,milR-837),as well as Dicer gene,decreased significantly while milR-764and milR-289 increased.Therefore,there may be different synthetic pathways of mature milRNAs in A.oligospora in addition to Dicer.Further phenotypic experiments showed that strain_?Dicer grew weaker in every medium than WT strain,as well as the hyphae erupted by the semi-knockout strain_?Dicer spores showed a curling phenomenon,which was different from WT,suggesting that Dicer may be an essential gene in A.oligospora.In addition,strain_?Dicer had a weaker ability to produce traps than WT under the urea induction,which suggested that endonucleases in the synthesis of milRNAs from A.oligosporus can also regulate the formation of traps to some extent.Bioinformatic analysis showed that there were incomplete complementary pairing sites between the 3'UTR region of genes ao0300281(encoding glycosyltransferase)and ao0304365(encoding thioredoxin)and the seed sequences of milR-289 and milR-799.Two knockout strains were obtained by homologous recombination as_?0281 and_?4365.Compared with WT,knockout strain_?0281produced more traps in the presence of urea,nematode extract or live nematodes,and with shorter induction times.In contrast,knockout strain_?4365 lost its ability to produce traps.Further dual-luciferase reporter experiments showed that milR-289 and milR-799 could regulate the gene ao0300281 simultaneously through complementation in the 3'UTR region,as the interaction disappeared after the addition of the milRNAs inhibitor.But,there is no interaction between the two milRNAs and genes ao0304365 3'UTR.So these two milRNAs regulate nematode trapping by regulating the expression of a target gene in A.oligospora.Further RT-PCR experiments of knockout strain_?0281 showed that the transcription levels of genes GS1,GDH2 related to ammonia production and amet related to ammonia secretion were all significantly increased.Therefore,the effect of gene ao0300281 on traps formation may be regulated through ammonia metabolic pathway.In the medium containing SDS,a cell wall synthesis inhibitor,the growth of_?0281 mutant strains was inhibited,indicating that cell wall synthesis may also affect traps formation.In addition,phenotypic assays showed that the conidia yield of ao?289 knockout strain increased significantly,while that of_?4365 mutant strain decreased.Therefore,both genes may be involved in the conidia formation process.In stress tolerance experiments,_?4365 could not grow normally in medium containing H₂O₂and grew worse in medium containing SDS.Therefore,gene ao0305365 may also participate in antioxidant and cell wall synthesis pathways.Knockout strain_?0281 grew weaker than wild type in medium containing H₂O₂or Na Cl,suggesting that gene ao0300281may be involved in maintaining osmotic pressure and oxidoreduction balance.Therefore,in addition to the formation of traps,these genes also participate in other biological pathways in A.oligospora.The originality and novelty of this thesis are as follows:1.Proposed the likely regulatory pathways of milR-289 and milR-799 involved in trap formation in A.oligospora.2.For the first time,glycosyltransferases and thioredoxins were found to be involved in some biological processes such as formation of nematode traps in A.oligospora.