| Fusarium head blight(FHB)is one of the most destructive diseases of wheat in the world.As the causal agent of this disease,Fusarium graminearum is a major threat to global wheat production.In addition,it produces trichothecene mycotoxin deoxynivalenol(DON)during plant infection which is harmful to human and animal health.As a hemibiotrophic fungus,F.graminearum has a short biotrophic stage during wheat infection.In other pathogenic fungi,such as Ustilago maydis and Magnaporthe oryzae,a lot of effector proteins expressed in this stage were functionally characterized.However,up to now,little is known about the function of effector proteins in F.graminearum.Orphan secretory proteins of F.graminearum,considered as potential effectors,were systematically identified.Deletion of Osp24(Orphan Secretory Protein 24)resulted significant reduction in pathogneicity.In this study,we further investgated the exact function of Osp24.Ta Sn RK1 was identified as the target of Osp24 in wheat.Regulation of Ta Sn RK1 by Osp24 was also revealed.Our results will not only improve our understanding of effectors function,but also lead to the development of novel strategies for Fusarium head blight control.The results are as follows:In order to ensure the main phase of Osp24’s function in infection stage,we observed the infection structure and the ability of development invasive hyphae in the wheat lemma and rachis tissues.Compared with wild type,fewer hyphae were observed in rachis tissues infected spikelet inoculated with the osp24 mutant.Similar to many other fungal effectors,Osp24 is a cysteine-rich protein,containing 8 cysteine residues.Cys94 and Cys105 were important for the function of Osp24.However,the results of yeast two-hybrid showed that Cys94 and Cys105 are for intramolecular but not intermolecular disulfide bond.They were more likely to regulate the function of Osp24 by mediating the formation of intramolecular disulfide bonds in infection stage.Interestingly,a disulfide bridge between Cys94 and Cys105 was predicted by EDBCP tool with highest probability.It is possible that the role of Osp24 in infection was dependent of intramolecular disulfide bond.Ta Sn RK1,a target protein of Osp24 in wheat,was identified by yeast two-hybrid screen library technology.Furthermore,interaction between Osp24 and Ta Sn RK1 was confirmed by Y2 H,Bi FC and in vitro pull down assays.To determine the region requirement of Osp24 for its association with Ta Sn RK1,we divided Osp24 into three segments and found the C-terminal region of Osp24 interact with Ta Sn RK1 in Y2 H.Therefore,the C-terminal region of Osp24 was responsible for the interaction with Ta Sn RK1.Sn RK1 is a conservative protein kinase in plants.To determine Ta Sn RK1 function in resistance against F.graminearum,Ta SNRK1 gene was overexpressed and silenced in wheat plant KN199.The results showed that Ta SNRK1 over expressed wheat plants showed increased resistance to F.graminearum,while the Ta Sn RK1 silencing plants were more sensitive to F.graminearum infection.These results indicate that Ta Sn RK1 regulates plant defense against F.graminearum infection.Then in vitro cell-free degradation experiments were carried out to reveal the molecular regulation of Osp24 on Ta Sn RK1.The result showed that Osp24 mediated the degradation of Ta Sn RK1,while MG132 inhibited the degradation of Ta Sn RK.These suggested that the degradation of Ta Sn RK1 depends on 26 S proteasome.Further,in vitro pull down experiments showed that the interaction between Ta Sn RK1 and 26 S proteasomes was significantly enhanced in the presence of Osp24.This suggests that Osp24 may stimulate the degradation of Ta Sn RK1 through the 26 s proteasome pathway during the infection.In this paper,the mechanism of action of Osp24,a potential effector of F.graminearum was analyzed,the molecular mechanism of interaction between F.graminearum and wheat was preliminarily revealed,therefore it is helpful for the prevention and control of FHB. |
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