Molecular Mechanisms Of The Casein Kinase FgYck1 In Regulating Polarized Growth And Cell Wall Integrity In Fusarium Graminearum

Wheat is one of the most important food crops in China.Its stable and high yield is necessary for the national food security.Fusarium head blight（FHB）caused by Fusarium graminearum is one of the most serious fungal diseases on wheat.This disease not only significantly reduces the yield of wheat,but also produces mycotoxins in wheat kernels that are harmful to livestock and human health.At present,the management of FHB still depends mainly on the chemical fungicide due to the lack of FHB resistant wheat cultivars.However,the problems of fungicide resistance and environmental contamination caused by chemical pesticides are getting worse.Intensive study on the development and pathogenic molecular mechanism of F.graminearum can provide specific targets for novel environment-friendly fungicides and breeding of disease-resistant cultivars based on HIGS technology.Casein kinase I（CK1）family members are well-conserved serine/threonine protein kinases in eukaryotic organisms,which are involved in many essential life processes.At present,the functional researches of fungal casein kinase I have focused on budding yeast,whereas its functions are not well characterized in plant pathogenic fungi.In our previous study,we found that the Fg Yck1 plays an important role in polarized growth,asexual/sexual reproduction,and pathogenesis,while the underlying molecular mechanism is not clear.Interestingly,the Fgyck1 mutants was unstable and fast-growing suppressors emerged frequently at the edge of its colony.Suppressor phenomena are usually caused by spontaneous mutations in other genes which usually have physical or genetic interaction with the knocked-out gene.Therefore,suppressor mutations can be used as an important tool to investigate the gene functions and the underlying mechanism.In this study,we collected 25 suppressors with increased growth rates by different degrees,and found that the defect of Fgyck1 mutant in conidiation were partially restored in 15suppressors.However,the losses of sexual reproduction and pathogenicity in Fgyck1 mutant were not suppressed in all 25 suppressor strains.The sequencing analyses revealed that,6 of25suppressors had mutations in the FGRRES＿07218（ortholog of yeast BNI4,named Fg BNI4）,including five non-sense or frameshift mutations(R490*,S297fs^IR,R539fs,P684fs,and L712fs)and one missense mutation R699C.These six mutations could lead to the complete or partial truncations of the conserved CCT region（682-741 aa）of Fg Bni4,or the change of the conserved residue in the CCT region.Subsequently,we investigated the function of Fg BNI4gene in detail and found that the Fg Bni4 is indispensable for cell wall integrity,and its CCT region is essential for the function of Fg BNI4 gene.In addition,we also confirmed that the deletion of Fg BNI4 gene,as well as the CCT region truncation and the R699C mutation,can partially restore the defects of Fgyck1 mutant in vegetative growth and conidiation,indicating that there is a strong genetic interaction between Fg Yck1 and Fg Bni4.Furthermore,we found that the deletion of Fg BNI4 partially suppressed the defects of Fgyck1 mutant in polarized growth and cell wall integrity,indicating the Fg Yck1 and Fg Bni4 coordinately regulate the polarized growth and cell wall integrity in F.graminearum.Finally,we revealed that the Fgyck1 mutant defects of Fg Rho1 internalization,vacuolar fragmentation,abnormal accumulation of PI（4,5）P₂,and phosphatidylserine in plasma membrane,increased endocytosis,and metabolic disorder of lipid droplet,which were all partially suppressed by the deletion of Fg BNI4 gene.In summary,this study not only confirmed the genetic interaction relationship between Fg Yck1 and Fg Bni4,but also revealed that the Fg Yck1 and Fg Bni4 coordinately regulate the polarized growth,cell wall integrity,phospholipid synthesis,vacuolar fusion,endocytosis,and lipid droplet metabolism by the Fg Rho1 signaling pathway in F.graminearum.