Screening And Functional Analysis Of Genes Related To Growth And Drug Resistance Of Fusarium Oxysporum

Fusarium spp.exists widely in nature and is important plant pathogen and human opportunistic pathogen.In agriculture,it can infect corn,wheat,banana and other crops,causing economic losses.Clinically,it can mainly cause superficial infection.In patients with immune deficiency,Fusarium spp.can also cause invasive infection with a high mortality rate of 43-67%.On the other hand,Fusarium spp.has natural multiple drug resistance and low sensitivity to most commonly used antifungal drugs,limiting its therapeutic efficacy.(1)Hypothetical protein FoDbp40 regulates growth and virulence of Fusarium oxysporumThere is a close relationship between the growth and virulence of fungi.In many fungi such as Candida albicans and Aspergillus fumigatus,the virulence could also be affected after the growth restriction caused by gene mutation or defect.Therefore,observing the growth characteristics of Fusarium spp.,screening the key genes that regulate growth,and analyzing their functions will help to provide new targets for the development of antifungal drugs.Energy metabolism has regulatory effect on growth and virulence by affecting PKA,AMPK and other pathways.Among them,the role of PKA pathway in Candida albicans,Aspergillus fumigatus,Fusarium species and other fungi has been widely reported,while the effect of energy metabolism on growth and virulence through AMPK pathway has only been preliminarily studied in Saccharomyces cerevisiae.In addition to glucose glycolysis and TCA cycle,the energy metabolism of fungi also relies on glyoxylic acid cycle pathway.The glyoxylic acid cycle pathway can generate a certain amount of ATP while saving carbon source.Its regulatory effect on growth and virulence has been preliminarily studied in yeast fungi,but less reported in Fusarium spp.In the previous work of our research group,a mutant of Fusarium oxysporum FOM312 with significantly slower growth was obtained through Agrobacterium-mediated mutation(ATMT)technology.The T-DNA inserted gene in the mutant was verified as FOXG＿12762 by TAIL-PCR,which encodes a hypothetical protein.Sequence alignment analyzing showed that this hypothetical protein contains a CCCH zinc finger domain and three part of nuclear localization signal and was noted as CCCH containing DNA-binding protein in several fungi.Considering that various zinc finger proteins were reported previously to play a role in transcriptional regulation in the nucleus,we speculated that this putative protein may be a DNA-binding protein with transcriptional regulation function,temporarily named FoDbp40.In FOM312,the expression of genes related to the glyoxylate cycle pathway and the level of ATP all decreased,the expression of the rate-limiting enzyme isocitrate lyase(ICL)decreased the most significantly,which may play a role in the slowed growth.The gene expression and product level of ICL were detected in FOXG＿12762knockout/complementation strain.The results showed that the expression level of ICL was decreased after deficiency of FOXG＿12762.Meanwhile,the succinic acid and ATP were both decreased after deficiency of FOXG＿12762,indicating that the expression and activity of ICL might be affected by FoDbp40.The fluorescence localization of FoDbp40-EGFP showed that FoDbp40 was located in the nucleus.The interaction between FoDbp40 and ICL promoter region was analyzed by Dual luciferase reporter system.The results showed that FoDbp40 can promote the transcription and expression of genes downstream of ICL promoter,this indicated that FoDbp40 may play a role in regulating the transcription of ICL.After FoDbp40 deficiency,the ATP level and AMPK/m TOR pathway were affected.Human corneal epithelial cells(HCEC)was treated with the condia of Fusarium oxysporum,then the cell viability and expression of inflammatory factors were observed.The results showed that the deficiency of FoDbp40 resulted in the reduction of virulence to HCEC cells and the ability to induce inflammation of Fusarium oxysporum.The survival rate of zebrafish was observed after co-culture with the condia of Fusarium oxysporum.The results showed that the deficiency of FoDbp40 resulted in the decreased virulence to zebrafish.(2)Riboflavin metabolic pathway regulates drug resistance of Fusarium oxysporumThere are many kinds of azoles with a wide range of applications,which were commonly used in the treatment of fungal infections in clinical.The action mechanism of azoles is mainly through the inhibition of sterol 14α-Demethylase(CYP51)in ergosterol synthesis pathway and interfering with the synthesis of ergosterol,resulting in the decrease of cell membrane stability and killing of cells.The products of riboflavin metabolism,FMN(flavin mononucleotide)coenzyme and FAD(flavin adenine dinucleotide)coenzyme,are related to a variety of cell functions,including oxidation-reduction reactions,energy metabolism,etc.Both of them are involved in regulating the activity of CYP51,but the relationship between them and drug resistance is less reported at present.The key genes in the riboflavin metabolism pathway of Fusarium oxysporum were knockout,and the drug sensitivity test was proceeded in the knockout strains.The results showed that the the deficiency of FOXG＿03172 encoding riboflavin kinase could lead to increased sensitivity to voriconazole.The complementation strain of FOXG＿03172 was established,and its drug sensitivity was recovered to the same level as that of the wild type Fusarium oxysporum.The level of riboflavin kinase products,electron transfer protein and CYP51 expression were detected.The results showed that the deficiency of FOXG＿03172resulted in the decrease of FMN coenzyme,CPR2 protein and CYP51 A expression,and the ergosterol content.To sum up,ICL has a regulatory effect on energy level in Fusarium oxysporum.The hypothetical protein FoDbp40 can regulate the expression and activity of ICL at transcriptional level,affect energy metabolism and AMPK/m TOR pathway,and regulate growth and virulence.FoDbp40 and ICL are expected to become new antifungal drug targets;Riboflavin kinase in the riboflavin metabolic pathway can regulate the resistance of Fusarium oxysporum to azole drugs by regulating the synthesis of FMN,affecting the expression of CPR2 and CYP51 A,and the synthesis of ergosterol.Riboflavin kinase and CPR2 have the potential to become drug targets.