Functional Analysis Of Rab GTPases In Fusarium Graminearum

Fusarium raminearum is a filamentous fungus that causes head blight in wheat and other cereals,and has emerged as one of the most important wheat diseases worldwide.It produces mycotoxins such as deoxynivalenol(DON)in the infected cereals which poses health threat to both humans and animals.So it is essential to better understand the pathogenesis mechanism and develop a novel strategy for controlling the Fusarium head blight.Rab GTPases,which constitute the largest subfamily of Ras-related small GTPases that are evolutionarily conserved and regulate membrane trafficking in all eukaryotic organisms,but their roles in plant pathogens are not well understood.So we systematically characterized the sub-cellular localization and functions of the F.graminearum Rabs(FgRabs)by live cell imaging and genetic analysis in this study.FgRab4,FgRab51,FgRab52,FgRab7 and FgYptA are found on punctate endosomal structures and on the vacuolar membrane,respectively,while FgRabl,FgRab2 and FgRab6 are localized to exocytic organelles such as the ER and Golgi complex.Remarkably,the two recycling Rabs,FgRab8 and FgRabl1,are highly concentrated at the growth tip of vegetative hyphae and conidia.Gene knock out experiments show that most FgRabs are important for fungal growth,with FgRab1 and FgRab11 essential for survival,instead we generated FgrabllDN(dominant negative)and FgrabllCA(constitutively active)mutants.However,we were unable to obtain a viable mutant of FgrablDN.We found FgRab51,FgRab52,FgRab6,FgRab7 and FgRab8 are important to the vegetative growth,conidiogenesis,sexual reproduction as well as pathogenesis and DON metabolism.However,FgYPTA,FgRABX,FgRAB2 and FgRAB4 are not very crucial for the growth and development.In addition,we found FgRab51 and FgRab52 played an important role in the regulation of early endocytosis in F.graminearum.FgRab52 is involved in the control of cell membrane and cell wall stresses.FgRab7 controls the homotypic fusion of vacuoles and the formation of autophagosomes.FgRab8 is involved in the formation of polarisome and recruitment of FgSpa2 for polarized growth and secretion in F.graminearum,deletion of FgRAB8 resulted in the production of aberrant conidia with collapsed ends.Furthermore,in our pursuit to investigate the regulatory networks of FgRab,we subsequently selected known network regulators and effectors of FgRab8 which includes,the guanine nucleotide exchange factors(GEFs),GTPase-activating proteins(GAPs)and corresponding effectors.In yeast,Sec2p or Sro7p act as a GEF or effector for Sec4p/Rab8 respectively,Gyplp and Msb3/4p showed the GAP activity for Sec4p.We therefore decided to knock out these genes and upon successful deletion,we noticed FgSec2A,FgGypl and FgMsb3 are important for growth and development of F.graminearum.We further proceeded to explore the interactions between these genes using yeast two-hybrid and results that emanated from this investigation showed that,FgSec2A can interact with FgRab8DN(GDP-bound),but could not interact with FgRab8CA(GTP-bound),suggesting that FgSec2A is a GEF of FgRab8.However,FgGyp1,FgMsb3 and FgSro7 neither interacted with FgRab8CA nor FgRab8DN,for this observation we deduced that there be a possible weak interaction that could not be detected by the yeast two-hybrid assay and hence,reasoned that it would be prudent to other methods such as pull-down,Co-IP or BIFC to further examine their interactions.In summary,both endocytic and exocytic FgRabs significantly affect conidiogenesis,sexual reproduction as well as pathogenesis and DON metabolism,suggesting that FgRabs and membrane trafficking play an important role in the regulation of critical physiological functions in F.graminearum.Our work is the first study on the whole family of Rab GTPases in a pathogenic filamentous fungus and thus providing a theoretical basis for clarifying the toxin producing mechanism in F.graminearum and control strategy for wheat scab disease.