Molecular Mechanism Of The GTPase-activating Protein FgMsb3 Mediated Pathogenesis Of Fusarium Graminearum

Wheat scab is the main disease in wheat production in the world which is caused by Fusarium graminearum.Previous studies have shown that vesicle transport mediated by the Rab proteins plays important roles in the development and pathogenesis of F.graminearum.However,the regulatory mechanisms of Rab proteins are still unclear,so it is of great significance to study the regulatory mechanism of Rab proteins.GTPaseactivating proteins(GAPs)are negative regulators of Rab proteins.In mammals and yeasts,the identified GAPs of Rab proteins contain conserved TBC(Tre2/Bub2/Cdc16)domains.In order to study the regulatory network of Fg Rab proteins in this fungus,all 12 hypothetical GAPs containing the TBC domain were identified from the SMART database,and further knocked out by replacing each gene with the hygromycin phosphotransferase gene as a selection marker.Preliminary analysis showed that the deletion of the gene Fg MSB3(FGSG＿04033)significantly reduced the growth,germination rate,and pathogenicity of the fungus.Since Fg Msb3 contains the conserved TBC domain and Arg681 residue,we deleted the TBC domain and mutated Arg681 of Fg Msb3 to know the role of TBC domain and Arg681 residue to the function of Fg Msb3,and found both TBC domain and Arg681 residue are important to the vegetative growth,conidiation,pathogenicity and DON production.To further confirm that if Fg Msb3 could act as the GAP for Fg Rab,Fg Msb3 and several Fg Rabs proteins were induced and purified by prokaryotic expression in vitro with several Fg Rabs successfully induced and purified,but failure in induction of Fg Msb3.Furthermore,we identified many different secreted proteins between the mutant and wild type PH-1 through proteomic analysis.To identify the up-regulator and down-regulator of Fg Msb3,GFP-Fg Msb3 pull-down and affinity capture mass spectrometry were then used to identify interacting proteins.We found that one of the candidate protein Fg Ndpk(FGSG＿05972)played an important role in regulating the vegetative growth,conidiation and pathogenicity of F.graminearum.However,yeast two hybrid assay showed that Fg Ndpk could not directly interact with Fg Msb3,and their relationship needs to be further investigated.In summary,our study here revealed how Fg Msb3 mediates the pathogenesis of Fusarium graminearum from the aspects of domain function,activity of GTPase-activating protein,secreted proteome,and interaction proteins.