Functional Characterization Of ChODC,ChCDC25,and ChPhb1/2 In Colletotrichum Higginsianum

Anthracnose of cruciferous plants,including Brassica and Raphanus,that caused by Colletotrichum higginsianum affects quality and yield of the cruciferous vegetable production.In this study,functional studies were conducted on pathogenicity related genes,Ch ODC,Ch CDC25,and Ch Phb1/Ch Phb2 in C.higginsianum,which elucidated the molecular mechanism of pathogenicity with polyamine metabolism,Ras protein signaling pathway,mitochondrial function,and autophagy.The main results of the study are described as follows:(1)In C.higginsianum,ornithine decarboxylase(ODC)orthologous gene,Ch ODC,contains three exons and two introns,and codes a protein with 456 aa.Knockout and complementation of Ch ODC showed that Ch ODC deletion mutants were auxotrophic of putrescine and restored by exogenous putrescine.Deletion of Ch ODC resulted in defects in vegetative growth,melanin pigmentation,conidiation,cell wall integrity,appressorial turgor pressure,and pathogenicity.Subcellular localization of Ch ODC showed it was localized to the cytoplasm.Metabonomic and transcriptome analysis showed that Ch ODC was involved in regulating amino acid metabolism,lipid metabolism,and carbon metabolism and thus affected the turgor pressure of appressorium,penetrability,and pathogenicity.Besides,comprehensive analysis of transcriptome analysis of autophagyrelated genes,microscopic observations of autophagy,and western blot indicated that Ch ODC was involved in autophagy by enhancing acetylation level of histone H3.Deletion of autophagy-related gene Ch ATG8 indicated that autophagy plays important roles in conidiation,appressorium formation,and pathogenicity in C.higginsianum.Furthermore,Ch ODC deletion mutants showed increased sensitivity to rapamycin,and the function of appressorium was partially restored by both exogenous c AMP and rapamycin in Ch ODC deletion mutants.Overall,Ch ODC was involved in c AMP and m TOR signaling pathways to affect appressorial function and autophagy in C.higginsianum.(2)In C.higginsianum,Ras nucleotide exchange factor Ch CDC25 gene contains five exons and four introns,codes a 1199 aa protein with three conserved domains including SH3,Ras GEFN,and Ras GEF.Functional analysis of Ch CDC25 in C.higginsianum indicated that Ch CDC25 was involved in vegetative growth,conidiation,appressorial formation,pathogenicity,and enhanced tolerance to salt stress,cell wall integrity,and oxidative stresses.Subcellular localization observation indicated that Ch CDC25 was localized in the cytoplasm.Intracellular c AMP level detection showed that it was lower in Ch CDC25 deletion mutant than that was in the wild-type.Exogenous c AMP and IBMX treatments were able to induce the formation of appressorium in Ch CDC25 deletion mutants.Yeast two-hybrid and western blot analysis showed that Ch CDC25 interacts with Ras2 and affects the abundance of Ras2 protein.This study suggested that Ch CDC25 interacts with Ras2 to integrate c AMP signal pathway,thus affects development conidiation,and pathogenicity in C.higginsianum.(3)Homologous analysis showed that there are two prohibitin genes in C.higginsianum,named Ch Phb1 and Ch Phb2.Ch Phb1 contains two exons and one intron,codes a protein with 276 aa.Ch Phb2 contains four exons and three introns,codes a protein with 312 aa.Functional analysis of Ch Phb1 and Ch Phb2 indicated that Ch Phb1 deletion mutants had serious defects in mitochondrial ridge morphology,mitochondrial membrane potential,and pathogenicity,while Ch Phb2 deletion mutants showed defects in vegetative growth,conidiation,mitochondrial ridge morphology,mitochondrial membrane potential,and pathogenicity.Subcellular localization observation indicated that both Ch Phb1 and Ch Phb2 were localized to mitochondria.Yeast two-hybrid,co-immunoprecipitation,and bimolecular fluorescence complementary experiments indicated Ch Phb1 and Ch Phb2 interacted in mitochondria.The phenotype of Ch Phb1/Ch Phb2 double-knock mutant was similar with Ch Phb2 deletion mutant.More importantly,mitophagy observation suggested that deletion of Ch Phb1 or Ch Phb2 led to defects in mitophagy.Yeast two-hybrid,Coimmunoprecipitation and bimolecular fluorescence complementary experiments revealed that Ch ATG24 interacted with Ch Phb1 and Ch Phb2 in mitochondria respectively.In order to understand the relationship between Ch ATG24 and Ch Phb1/Ch Phb2,Ch ATG24 was knockout in C.higginsianum.Ch ATG24 deletion mutants were also defected in vegetable growth,conidiation,mitophagy and pathogenicity.Subcellular localization observation indicated that Ch ATG24 was localized to cytoplasm including mitochondria and lipid droplets under nitrogen starvation conditions.Further study showed that PX and BAR domains of Ch ATG24 protein were indispensable for its vegetative growth,conidiation,and pathogenicity.And the PX domain of Ch ATG24 was found to be essential for its localization.Taken together,our results indicated that Ch Phb1 and Ch Phb2 were involved in fungal development,mitochondrial function,and pathogenicity in C.higginsianum,and may interact with Ch ATG24 to mediate mitophagy.