Analysis Of Fusarium Oxysporum Based On The Agrobacterium Tumefaciens-mediated T-DNA Insertional Mutagenesis

The Fusarium spp.is widely distributed and has various morphology and species.It can infect humans,animals and plants,causing Fusarium disease.In recent years,due to an increase in population with low immunity,invasive and disseminated Fusarium infections have been increasing year by year.It has becoming the most common pathogen causing fungal keratitis and is second only to Aspergillus spp.in the deep infection of filamentous fungi.The increasing incidence of Fusarium is a serious threat to human.In particular,the mortality of disseminated Fusarium is extremely high.Fusarium is a naturally occurring multidrug-resistant fungus.Clinically,Fusarium is resistant to almost all antifungal drugs,including azoles,echinocandins,and polyenes.At present,the researches on the resistance mechanisms of pathogenic fungi mostly focus on the Candida spp.and Aspergillus spp.,which cause more clinical infections.The formation of drug resistance is mostly caused by variation of the target gene,for example,resistance to azole drugs is mostly due to lanosterol 14?-demethylase(CYP51)mutations.In addition there are biofilm formation,drug efflux,drug degradationand and other mechanisms.However,there are few reports on the mechanism of drug resistance of Fusarium.Therefore,it is extremely urgent to study the mechanism of drug resistance of Fusarium.In this study,plasmid pXEN containing the G418 resistance gene(neo)screening tag was successfully constructed.Agrobacterium tumefaciens-mediated transformation(ATMT)was successfully performed on F.oxysporum by using this plasmid,and the main factors affecting the transformation efficiency were optimized,and an efficient and stable ATMT was established.After majorization the transformation efficiency was250 transformants per 10~4 spores.Using this transformation system,1,450 strains of T-DNA insertion mutants of F.oxysporum were obtained,and a mutant library of F.oxysporum was initially established.Biological traits and nucleic acid analysis showed that the obtained mutant was genetically stable,and the insertion site flanking sequence was obtained by TAIL-PCR amplification technique,and the insertion site was determined by bioinformatics analysis.This provide resources for the analysis of related mechanisms such as resistance and pathogenicity.In this study,we screened the F.oxysporum mutants above-mentioned by drug sensitivity test,and azoles,polyenes,and echinocandins was analyzed,found out a mutant FOM1123 with altered susceptibility to azole drugs.The mutant showed a significant increase in susceptibility to azoles other than fluconazole(FCZ),while there was no change in the minimum inhibitory concentration(MIC)for amphotericin B(AmB)and caspofungin(CAS).TAIL-PCR was used to obtain the T-DNA insertion site flanking sequence of FOM1123.Compared with the NCBI database,it was found that the hypothetical protein gene(HPG)and NADPH-cytochrome P450 reductase gene(CPR1)were interrupted.In order to determine which gene affected the drug sensitivity of the mutant,a single gene knockout was performed on these two genes,so we obtain the gene deletion strain?CPR1and?HPG.The drug sensitivity test showed that the drug sensitivity of?CPR1 was enhanced in keeping with FOM1123,there was no change in?HPG,indicating that the change in drug sensitivity of the mutant was associated with the gen of CPR1 only.Studies have shown that CPR and cytochrome b5 are electron transport proteins in organisms,also they provide electrons in fungi for a key enzyme in sterol synthesis-CYP51.Bioinformatics analysis revealed that there are four homologous genes of CPR in F.oxysporum,CPR1,CPR2,CPR3,and CPR4.Simultaneously,there are three homologous genes of CYP51,CYP51A,CYP51B and CYP51C.In order to investigate the relationship of CPR,cytochrome b5 and CYP51 in resistance of F.oxysporum,their transcriptional changes were detected in wild-type and?CPR1.The analysis of ergosterol content showed that compared with the wild type,the ergosterol content of?CPR1 was reduced,which was about 70%of the wild type content.When exposed to voriconazole,the?CPR1 and wild type ergosterol content were significantly reduced.The above results indicate that CPR1 is a key gene in the ergosterol synthesis pathway of Fusarium and it is the only CPR homologous genes associated with azole resistance.The lack of CPR1 can affect the synthesis of ergosterol and lead to sensitivity to azole drugs.In summary,this study successfully established a genetic transformation system of F.oxysporum mediated by A.tumefaciens,and constructed a T-DNA insertion mutant library of F.oxysporum,and analyzed CPR gene mutant drug resistance relationship deeply.This study found that the gene is associated with the synthesis of ergosterol,and the deletion results in a decrease in the synthesis of ergosterol and sensitivity to azole drugs.The above results lay a foundation for finding novel antifungal targets and revealing the resistance mechanism of pathogenic fungi.It also provides reference for other pathogenic fungi related researches.