Identification And Functional Analysis Of FgKAR9 And Its Interaction Protein In Fusarium Graminearum

Fusarium graminearum is one of the dominant species of wheat scab,which not only seriously affects the yield and quality of wheat,but also produces deoxynivalenol(DON)mycotoxins in the process of infection,which poses a great threat to human and livestock health.Microtubule is an important component of the cytoskeleton and play an important role in many biological processes such as the growth of the fungus,transportation of matter,mitosis,and maintenance of cell morphology.At the same time,microtubules as a target of benzimidazole fungicides have great significance in disease prevention and control.Microtubule function is dependent on microtubule-associated proteins that bind to it.At present,studies on microtubules and microtubule-associated proteins have developed into an important branch of cell biology.In Fusarium graminearum there are two β-tubulins,FgTub1 and FgTub2.It is known that there is a significant functional differentiation between the two β-tubulins,FgTUB1 is required for sexual reproduction,FgTUB2 is closely related to vegetative growth,knocking out FgTUB2 will result in slow vegetative growth and small colony.In our study,we found the Fgtub2 deletion mutant is unstable and produced spontaneous suppressors on V8 agar plates.38 sectors were collected and classified by different growth rate and colony morphology,and we chose 14 sectors to make the genomic sequencing to identify FgTUB2-related genes.Combining with bioinformatics software,we found that suppressors b-9-1s and b-10-6 had different termination mutations in the conserved domain of FgKAR9 gene.To verify that FgKAR9 deletion suppresses Fgtub2 deletion,we used the Split-PCR method to knock out FgKAR9 in the Fgtub2 mutant and knock out FgTUB2 in the Fgkar9 mutant to obtain the Fgtub2kar9 and Fgkar9tub2 double mutants.The phenotypes of b-9-1s,b-10-6,Fgtub2kar9 and Fgkar9tub2 were observed.The growth rate and virulence of b-9-1s and b-10-6 were partially restored compared with that of Fgtub2,conidiation and sexual reproduction are normal.While the mutant Fgtub2kar9,Fgkar9tub2 and b-9-1s,b-10-6 in the growth rate,virulence and other aspects of the same phenotype,indicating FgKAR9 deletion can suppress Fgtub2 deletion.Two Fgkar9 deletion mutant strains were obtained by knockout,PCR and southern validation.Phenotypic observations revealed that both vegetative growth and sexual reproduction were normal.By fusion of FgKAR9 into the pFL2 plasmid with strong RP27 promoter,the fusion expression vector was transferred to PH-1 to obtain the FgKAR9 overexpression strain,and there was no abnormality in its phenotype.Subcellular localization of FgKAR9 was observed.It was found that FgKar9 protein moves at a high speed along the FgTub2 tubulin filament in the cell,showing a discontinuous line shape and a small amount of enrichment at the tip of the germ tube.BiFC experiments have shown that FgTub2 interacts with FgKar9.This shows that FgKAR9 plays an important role in microtubule function.The yeast two-hybrid experiment showed that FgKar9 interacts with FgBim1,and it is speculated that FgKAR9 may regulate microtubule function with FgBIM1.In summary,starting from the spontaneous suppressors generated by Fgtub2 mutants,we found that the deletion of the microtubule-associated protein FgKAR9 could suppress Fgtub2 deletion.Through the identification and functional analysis of FgKAR9 and its interaction proteins,the mechanism of the suppressors of Fgtub2 mutants caused by FgKAR9 in Fusarium graminearum was initially explored.