Phenotypic Screening Of Fusarium Oxysporum T-DNA Insertional Mutants And Functional Analysis Of Genes Related To Drug Resistance

Fusarium spp.is widely distributed in soil and plants,and it has strong ecological adaptability.It can not only attack plants but also infect humans and animals.Fusarium spp.infecting plants results in fusarius and verticillium wilt and it has caused huge economic losses to agriculture.Fusarium spp.causes superficial infections and it is common to cause fungal keratitis clinically.In recent years,the incidence of systemic infection has increased with the increase of people with low immunity.Systemic infections caused by Fusarium spp.has risen to the second place and the first place is Aspergillus spp.At present,the research on the resistance mechanism of pathogenic fungi mostly focuses on Candida spp.and Aspergillus spp.And the mechanism has been identified in the following aspects: changes in target enzymes of drug,overexpression of drug efflux,changes in cell membrane or cell wall composition and biofilm formation.As a new,global epidemic fungal infection,Fusarium spp.has a significant multidrug resistance to clinical antifungal drugs such as azoles,echinocandins and polyenes.Multi-drug resistance has become the key to its prevention.However,there are few reports on the resistance mechanism of Fusarium spp.This study aims to find resistance-related genes and analyze its resistance mechanisms.In this study,T-DNA insertional mutant library has been established by Agrobacterimu tumefaciens-mediated transformation based on multidrug-resistant Fusarium oxysporum.Previous study has successfully established and optimized the method of ATMT transformation of Fusarium oxysporum.A total of 1120 mutants were obtained through ATMT and T-DNA insertion is successfully determined and confirmed to be stable through PCR.It has provided sufficient resources for studying the functions of genes related to growth,pathogenicity and drug resistance in Fusarium oxysporum.The phenotypes of 1120 mutants were screened and a total of 6 mutants with abnormal growth were screened as FOM159,FOM312,FOM364,FOM589,FOM977 and FOM1012.The T-DNA insertion site is located by TAIL-PCR and the interrupted genes are identified as TIF31 protein and 5 hypothetical protein.Drug susceptibility tests with four clinically used antifungal drugs,itraconazole,voriconazole,amphotericin B and caspofungin revealed no significant changes in the drug sensitivity of the six mutants compared with wild strain.700 randomly selected mutants have been screened for drug sensitivity usin g the above 4 drugs and one mutant FOM263 whose sensitivity to amphotericin B increased has been selected.FOM263's T-DNA insertional site is obtained b y TAIL-PCR and it has been found that FOXG₁8528?925bp?,FOXG₁8529?539bp?and FOXG₀3305?1174bp?are interrupted.All of the three genes encode h ypothetical protein.To identify drug resistance-related genes,single gene knocko ut strains of FOXG₀3305 and FOXG₀18528 were constructed respectively.Dr ug sensitivity tests were performed on the knockout strains ?03305 and ?018528.The results showed that drug sensitivity of ?03305 was consistent with FOM263.The deletion of FOXG₀3305 resulted in an increase in FOM263's sensitiv ity to AmB,indicating that FOXG₀3305 is related to drug resistance of Fusari um oxysporum.Compared with wild strain,?03305 produces more pigments.The colony,morphology,and growth rate of ?03305 are not significantly different from thos e of wild strain,indicating that FOXG₀3305 does not affect the vegetative gr owth.FOXG₀3305 encodes hypothetical protein and its function is unknown.By NCBI Blast alignment,the protein was predicted to contain two conserved d omains: a Zn?II?2Cys6 binucleus cluster domain similar to GAL4 p and a dimeri zed leucine zipper domain.The protein was speculated to be Zn?II?2Cys6 zinc cluster protein transcription factor.A large number of transcription factors relate d to drug resistance have been found in yeast fungi but there are few reports o n the transcriptional regulation of filamentous fungi in response to drug stress.Wild strain and ?03305 were cultivated on PDA medium containing SDS and congo red.The results showed that the growth of ?03305 has no significant change compared with wild strain,indicating that FOXG₀3305 is not related to cell wall integrity and ?03305‘s increase sensitivity to AmB has nothing to do with cell wall integrity.Then the ergosterol content of wild-type and ?03305 was detected.The ergosterol content of ?03305 increased significantly,indicating that FOXG₀3305 is involved in regulating the ergosterol biosynthesis.Next qPCR was used to verify mRNA expression levels of genes related to ergosterol biosynthetic pathway.The mRNA expression levels of ERG7,ERG6,ERG3,ERG4 in the knockout strain ?03305 have significantly increased compared with wild strain,while the mRNA expression levels of CYP51 and ERG9 were significantly decreased.The expression levels of other genes related to ergosterol biosynthetic pathway were not significantly changed.And qPCR was used to test mRNA expression levels of 14 ABC transporters related to pleiotropic drug resistance in the Fusarium oxysporum‘s genome.Compared with wild strain,the expression levels of two genes?FOXG₁2868,FOXG₀5904?in the knockout strain ?03305 have significantly decreased,while the expression levels of the other 12 genes remained unchanged.Above all,deletion of FOXG₀3305 not only causes changes in the expression of genes related to ergosterol biosynthetic pathway,but also affects the expression of the drug efflux pump.In summary,the deletion of FOXG₀3305 leads to changes in the expression of genes related to ergosterol biosynthetic pathway,an increase in the content of ergosterol and a low expression of drug efflux pump in Fusarium oxysporum,which in turn leads to an increase in the sensitivity of the strain to AmB.FOXG₀3305regulates AmB resistance and it is related to ergosterol biosynthetic pathway and drug efflux pump.