Rab GTPase And Their GEF Regulate DON Biosynthesis In Fusarium Graminearum

Fusarium graminearum is a homozygous ascomycete fungus that can infect a variety of cereal crops such as wheat,barley,and corn,causing Fusarium head blight（FHB）,stem rot,root rot and ear rot.And moreover,this fungus can produce toxic secondary metabolites including Deoxynivalenol（DON）in the infected crops,which results in severe yield and quality loss and threatens the health of humans and animals.Therefore,an in-depth understanding of the pathogenesis and DON biosynthesis mechanism of F.graminearum will be conducive to developing a new strategy for better control of FHB.Rab GTPases act as a molecular switch in vesicle trafficking through the conformational transition between the inactive GDP-bound state and the active GTP-bound state.Previous studies have shown that the deletion of Fg RAB51,Fg RAB52,Fg RAB7 or Fg RAB8 significantly reduced the DON content in infected grains,but the involved mechanism is still unclear.In this study,we measured the intracellular and extracellular DON in these RAB deletion mutants under trichothecene biosynthesis induction condition（TBI）,and the results showed that the intracellular and extracellular DON content,the expression level of trichothecene biosynthesis-related genes（TRIs）were both significantly declined in these mutants,and the expressions of toxisome marker protein Fg Tri1and Fg Tri4 could not be detected either.Previous study has shown that the absence of Fg TRI12 reduced DON content in infected wheat.Here we also found that both the intracellular and extracellular DON content and the expression levels of TRI genes were remarkably reduced in Fgtri12mutant,indicating that Fg Tri12 is also involved in DON biosynthesis.Further studies demonstrated that,compared with the wild-type strain,the expression level of TRI12 and the fluorescence signal of Fg Tri12-GFP in the above RAB mutants were dramatically reduced.Taken together,we speculated that Rab GTPases regulate DON biosynthesis by affecting the expression levels of TRI genes.Guanine nucleotide exchange factor（GEF）can convert the Rab protein from the inactive GDP-bound state to the active GTP-bound state.Previous studies have shown that Fg Mon1 and Fg Sec2A,acting as GEFs of Fg Rab7 and Fg Rab8 respectively,are involved in the growth,development and pathogenicity of F.graminearum,besides,the extracellular DON content is reduced in their deletion mutants.In this study,we found that Fg Vps9,a yeast Vps9 homolog,could specifically interact with the GDP-bound state Fg Rab51DN and Fg Rab52DN by yeast two hybrid,indicating that Fg Vps9 may function as GEF of Fg Rab51 and Fg Rab52.In order to clarify whether the absence of GEFs could also affect the biosynthesis of DON,we measured the DON content in the deletion mutants of the above three GEFs,and the results showed that similar to Fg RAB deletion mutants,the expression levels of TRI genes and the intracellular and extracellular DON content were also significantly reduced in these GEF deletion mutants,indicating that GEFs of F.graminearum could also function in DON biosynthesis by regulating the expressions of TRI genes.Besides,functional analysis of three Vps9 domain-containing proteins also showed that Fg Vps9 can co-localize with Fg Rab51 and Fg Rab52 on the early endosomes,and participate in the endocytosis and autophagy of F.graminearum.The deletion of Fg VPS9 severely weakened the vegetative growth,asexual reproduction,and the pathogenicity on wheat ear and coleoptile.In addition,the deletion of the Vps9 domain and the simultaneous mutation of two GEF activity sites(Asp₅₂₅and Glu₅₆₂)in Fg Vps9 both led to similar phenotypic defects with the entire gene deletion mutants,suggesting that the Vps9 domain and the GEF activity sites are necessary for the biological functions of Fg Vps9.Although Fg Muk1 and Fg Vrl1 have been found to co-localize with Fg Rab51 and Fg Rab52 on the early endosomes,they are dispensable for all the analyzed phenotypes in F.graminearum.In conclusion,our results have demonstrated that Fg Rabs and their GEFs control DON biosynthesis by regulating the expression levels of DON biosynthesis-related genes.Furthermore,Fg Vps9,a Rab5 GEF,is essential for the growth,development and pathogenicity of F.graminearum.These results will provide a theoretical basis for controlling DON biosynthesis and prevention of FHB.