假禾谷镰孢动力蛋白GTPase FpSey1的鉴定及功能研究

Identification and functional analysis of dynamin-like GTPase protein FpSey1 in Fusarium pseudograminearum

假禾谷镰孢(Fusarium pseudograminearum)引起的小麦茎基腐病在我国发生日益严重,已对我国小麦的产量和品质造成了严重影响。Sey1属于RHD3(Root hair defective 3)家族,其编码的类动力蛋白GTPase参与内质网的融合,而内质网参与多种病原真菌脱氧雪腐镰刀菌醇(DON)的合成,但在假禾谷镰孢中还没有相关报道。本研究对假禾谷镰孢Sey1蛋白(FpSey1)进行了亚细胞定位,发现FpSey1定位于内质网上。通过PEG介导原生质体转化和Southern杂交筛选获得了FpSEY1基因敲除突变体ΔFpSey1及其回补菌株。同野生型菌株相比,敲除突变体ΔFpSey1的菌丝生长速率、分生孢子产量以及DON合成相关基因(TRI1、TRI5、TRI10)的表达量均显著下降,在小麦胚芽鞘和大麦叶片上形成的病斑明显变小,且ΔFpSey1突变体对盐胁迫和过氧化氢(H2O2)敏感,但对二硫苏糖醇(DTT)耐受。上述结果表明,FpSey1参与菌丝生长、产孢和致病力形成,在假禾谷镰孢生长和侵染阶段发挥着重要作用。

The occurrence of Fusarium crown rot(FCR)caused by Fusarium pseudograminearum has been becoming increasingly serious in China,which has posed a severe threat to wheat yield and quality.The SEY1 belongs to the RHD3(Root Hair Defective 3)family and encodes a dynamin-like GTPase protein participating in endoplasmic reticulum(ER)fusion.The ER is involved in the synthesis of deoxynivalenol(DON)in different pathogenic fungi,while its function in F.pseudograminearum has not been reported.In this study,subcellular localization of GFP-tagged Sey1(FpSey1)protein in F.pseudograminearum was observed,and the results showed that FpSey1 was localized in the ER.The FpSEY1 deletion mutant(ΔFpSey1)was generated through PEG-mediated protoplast transformation and verified by Southern blot analysis,and complemented strains were obtained as well.Compared with the wild-type strain,theΔFpSey1 mutant exhibited significant reduction in vegetative growth,conidiation,relative expression of DON biosynthesis related genes(TRI1,TRI5,TRI10),and the virulence on wheat coleoptiles and barley leaves.In addition,theΔFpSey1 mutant is more sensitive to salt stress,hydrogen peroxide(H2O2),but more tolerant to dithiothreitol(DTT)than the wild-type and complemented strains.These results indicate that FpSey1 localized in the ER plays important roles in the growth and infection of F.pseudograminearum.