Study On The Function Of FgStu2 Including Its Regulation To Carbendazim-sensitivity In Fusarium Graminearum

Resistance of Fusarium graminearum to carbendazim is a serious problem in wheat production.It has been established that a single amino acid mutation in β₂-tubulin of Fusarium graminearum,which causes FHB in wheat,leads to resistance to carbendazim.However,the mechanism of drug resistance induced by tubulin point mutation in plant pathogenic fungi has been stagnated due to the barrier of tubulin expression and purification technology.Purification of tubulin is a prerequisite to solve the mechanism of drug resistance.However,at present,purification of tubulin directly is difficult,so co-expression and purification of tubulin by the interaction protein of tubulin is one of the feasible methods.XMAP215 is a class of microtubule-associated proteins（MAPs）found in African claw toad.It is characterized by containing two TOG（tumor overexpress gene）domains that interacting with tubulins.Stu2,a member of the XMAP215 family of Saccharomyces cerevisiae,has been used successfully purified tubulin from several species through the interaction between its TOG domain and tubulin.However,Stu2 in Saccharomyces cerevisiae can not interact with β₂-tubulin of Fusarium graminearum,it can not be used forβ₂-tubulin purification of filamentous fungi.In this study,the highest homologous protein in Fusarium graminearum was first identified by comparison from the sequence of Saccharomyces cerevisiae Stu2 and the co-localization of FgStu2 and Fusarium graminearum β-tubulin in cells was found by fluorescence labeling technology.The interaction between this protein and β-tubulin was verified by Co-IP technology,which further proved the presence of this protein in cells through the formation of complexes with tubulin.This discovery provided a new technical idea for the co-expression and purification of β₂-tubulin with FgStu2 in Fusarium graminearum.It has known that Stu2 is also involved in some related processes of cell mitosis in other species,interacting with y-tubulin to participate in microtubule nucleation,and interacting with NDC80 complex to regulate the contact between microtubule and centromere to form spindle,and etc.In this study,the interaction between FgStu2 of Fgγ-tubulin and FgNDC80 in Fusarium graminearum was preliminarily explored,which laid a foundation for further research on the role of FgStu2 in cell mitosis in Fusarium graminearum and in the cell cycle such as tubulin functional conversion.It provides a preliminary reference basis and foreshadowing for further study of its function.It has been reported that Stu2 of Saccharomyces cerevisiae and Alp A of XMAP215 homologous protein in Aspergillus nidulans are involved in the regulation of sensitivity to benomyl.Furthermore,we found that the function loss of FgStu2 in the β-tubulin strain labeled with green fluorescent also affected the microtubule polymerization in Fusarium graminearum,resulting in sparse microtubule degradation.Therefore,we wondered that FgStu2 might was also involved in the sensitive regulation to carbendazim in Fusarium graminearum.Pzear is one of the induced promoters in Fusarium graminearum and zearalenone is the inducer.To explore whether FgStu2 in Fusarium graminearum regulates the sensitivity to carbendazim and other biological functions of this gene,in this study,we construct a silencing vector on the basis of Pzear.By means of PEG-mediated protoplast transformation,the silent vector was transformed into wild strain PH-1 and carbendazim moderate resistant strain Fgβ₂F167Y,and the transformants were named as FgStu2-Si and Fgβ₂F167Y-Stu2-Si respectively.And the revertants were obtained by adding the inducerβ-est（analogue of zearalenone）,which indicated the silencing system containing zearalenone inducer promoter was successfully applied in Fusarium graminearum.To reveal the regulation of FgStu2 in the sensitivity to carbendazim of Fusarium graminearum,carbendaziam was used to measure the EC50 values both of the PH-1 and FgStu2-Si,showing that compared with PH-1,EC50 values of FgStu2-Si decreased 18%and the sensitivity to carbendazim increased.EC50 values of Fgβ₂F167Y-Stu2-Si decreased 34%compared with Fgβ₂F167Y,which means a significant decrease in the level of drug resistance.In addition,the results meaused with respect to other biological phenotypes,such as growth rate,mycelium morphology,sporogenesis ability,morphology,sexual reproduction ability,pathogenicity ability,virulence ability and etc,showed that these biological phenotypes of FgStu2-Si were significant down regulated compared to PH-1.The above results indicated that the function of FgStu2 reduced in Fusarium graminearum not only affected the sensitivity to carbendaziam,but also accompanied by a series of biological phenotype changes related to growth,development and pathogenicity.The reduced function of this gene would reduce the biological fitness for the survival of Fusarium graminearum.The study not only deepened the toxicology of tubulin inhibitor signaling and the regulation mechanism of sensitivity or resistance to know,but also provided a possible co-expression tool for the study of microtubule purification and a new target point to development tubulin inhibitor synergistic agent.