The Fungistatic Mechanism Of Ammoniaaganist Nematode-Trapping Fungi Arthrobotrys Oligospora And The Survival Mechanism Of Conidia Under Ammoniafungistasis

In this study,Arthrobotrys oligospora was used as the research object.Multi-omics of A.oligospora conidia under ammonia fungistasis combined with related gene function studies revealed the molecular mechanism of ammonia fungistasis,conidia survival,and nutrients-mediated relief of fungistasis in A.oligospora.Ubiquitination proteome data and Western Blot result showed the up-regulation of protein ubiquitination level in AO conidia under ammonia fungistasis,suggesting that ammonia induced integral protein misfolding,which was further confirmed by up-regulation of ubiquitin-proteasome pathway in proteome.In proteome,down-regulated proteins（especially protein synthesis related proteins）were significantly correlated with up-regulated ubiquitin proteins in pathway distribution.In conclude,the molecular mechanism of ammonia inhibiting conidial germination was speculated: ammonia inhibits conidial germination by inducing integral protein misfolding,inducing endoplasmic reticulum stress and inhibiting cell protein synthesis.According to the omics data,after endoplasmic reticulum stress induced by ammonia,in order to survive,conidia up-regulate ubiquitin-proteasome system through unfolded protein response pathway to facilitate the degradation of misfolded proteins,and correct protein misfolding by up-regulating molecular chaperones.These two strategies can reduce the toxicity caused by the accumulation of misfolded proteins.In addition,conidia could survive through up-regulating a range of carbon and amino acid metabolic pathways to solidify ammonia.Nutrients relieve ammonia fungistasis experiments could support this survival mechanism.Based on the above mechanism analysis,we chose lactoyl glutathione lyase,20 S proteasome subunit α7 and Bi P,which were up-regulated b the omics data,used homologous recombination method to konck out the genes encoding these proteins（AOL＿s00004g335,AOL＿s00054g963,AOL＿s00210g337）and do function studies to validate the above mechanism.（1）AOL＿s00004g335 encodes lactoyl glutathione lyase that is more than 2-fold up-regulated in response to ammonia fungistasis and benzaldehyde fungistasis,which is a detoxifying enzyme for methylglyoxal（MG）.AOL＿s00004g335 knockout mutant strains showed significantly higher conidial germination rates under ammonia fungistasis compared to the wild type.The exogenous addition of MG at low concentrations can relieve the ammonia fungistasis,but MG at high concentrations will inhibit the germination of conidia deversely.This suggests that AO conidia can regulate carbon metabolism under ammonia fungistasis,such as producing methylglyoxal to improve conidia tolerance to ammonia,while up-regulation of lactoyl glutathione lyase can limit the toxicity from excessive accumulation of methylglyoxal.（2）AOL＿s00054g963 encodes 20 S proteasome subunit α7,which is up-regulated in response to ammonia fungistasis.The proteasome plays a key role in the degradation of misfolded proteins.The mutant strain ΔAOL＿s00054g963 showed sensitivity to ammonia,which has a significantly lower germination rate than the wild type under ammonia fungistasis.At the same time,after removing the ammonia fungistasis,its conidial germination rate was significantly lower than that of the wild type.AOL＿s00210g337 encodes Bi P,which is a key protein in UPR.The mutant strainΔAOL＿s00210g337 showed significantly higher germination rates compared to the wild type under low concentration of ammonia fungistasis.This is because the deletion of Bi P protein,on the one hand,activates the PERK pathways of the UPR and reduces the synthesis of protein,on the other hand,activiates the IRE pathway and up-regulates the protein degradation system and chaperone system,thereby reducing ammonia-induced protein misfolding.After removing the ammonia fungistasis,the mutants’ conidial germination rate was significantly lower than that of the wild type.These results confirmed that ammonia could induce protein misfolding and conidia could survive under ammonia fungistasis by activating UPR reaction.In addition,the mutant strain under the fungistasis of benzaldehyde and soil also showed similar research results under ammonia fungistasis,which proved that the ammonia inhibition mechanism and conidia survival mechanism disclosed in this article are universal.