Function Analysis Of CHIMPB,CHSCH9 And CHCOT1 In The Crucifer Anthracnose Fungus,Colletotrichum Higginsianum

The hemibiotrophic fungus Colletotrichum higginsianum is the causal agent of anthracnose disease of cruciferous plants,including many cultivated species of Brassica and Raphanus as well as the model plant Arabidopsis thaliana.It is necessary to improve our understanding of molecular fungal pathogenic mechanisms for developing novel control strategy of this important anthracnose disease.In this study,we explored gene functions of Ch Impb,Ch Sch9 and Ch Cot1 in C.higginsianum.The main results of the study were described as follows.(1)Homologous analysis revealed that two Importin-? genes,named Ch Impb1 and Ch Imb2 were present in the genome of C.higginsianum.Ch Impb1 codes a protein with 841 aa and contains three domains,IBN_N,Heat_EZ and ARM whereas Ch Imb2 codes a protein with 755 aa and only contains Heat_EZ domain.Functional analysis of Ch Impb1 and Ch Imb2 showed that the deletion mutants of Ch Impb1 or Ch Impb2 both cause serious defects in vegetative growth,conidiation,tolerance to hyperosmotic,cell wall and oxidative stresses and pathogenicity of C.higginsianum.Yeast two-hybrid experiments revealed that Ch Impb1 and Ch Imb2 both interacted with Ch Mig2,which contained two C2H2 Zinc finger domains.In order to further understand the relationship between Ch Impb and Ch Mig2,Ch Mig2 was deleted in C.higginsianum.Ch Mig2 deletion mutants were also defected in vegetative growth,conidiation,tolerance to hyperosmotic,cell wall and oxidative stresses and virulence of C.higginsianum.Taken together,our results indicated that Ch Impb1 and Ch Impb2 might interact with Ch Mig2 and were involved in fungal development and pathogenicity in C.higginsianum.(2)The kinase protein Ch Sch9 codes an 897 aa protein with three conserved domains including Protein kinase C conserved region 2(C2),Serine/Threonine protein kinase catalytic(S_TKc)and Extension to Ser/Thr-type protein kinases(S_TK_X).Phylogenetic analysis revealed that Ch Sch9 is closely related to AGC kinase family,which is conserved in fungi.Deletion of Ch Sch9 caused defects in hyphal morphology,growth and conidiation.Moreover,deletion of Ch Sch9 was associated with reduced tolerance to hyperosmotic,cell wall and oxidative stresses in C.higginsianum,and Ch Sch9 deletion mutants were more tolerant to heat.Furthermore,we found that Ch Sch9 might be involved in the pyruvate–acetaldehyde fermentation pathway.Importantly,deletion of Ch Sch9 led to a reduction of appressorial and infective hyphae formation resulting in defects in virulence.Overall,our data indicated that Ch Sch9 plays important roles in fungal infection-related morphogenesis and virulence in C.higginsianum.(3)In previous lab work,we obtained an ATMT mutant G323.Functional analysis showed that the mutant G323 was defective in appressorium production and pathogenicity on Arabidopsis plants.A single T-DNA insertion in ATMT mutant G323 targeted a gene coding serine/threonine-protein kinase Ch Cot1.Ch Cot1 protein contains two domains including Serine/Threonine protein kinase catalytic(S_TKc)and Extension to Ser/Thr-type protein kinases(S_TK_X).To further verify the functions of Ch Cot1,this gene was deleted in C.higginsianum.Deletion and complementation of Ch Cot1 revealed that Ch Cot1 deletion mutants were defected in vegetative growth and virulence.Overall,the results indicate that Ch Cot1 is involved in fungal morphogenesis and virulence in C.higginsianum.