Molecular Mechanism Underlying The Transcription Of CfHac1 To Regulate Pathogenicity In Colletotrichum Fructicola

The tea-oil tree Camellia oleifera is native to China and is cultivated in many parts of southern China.This plant has been grown for over two thousand years,mainly for its high-quality cooking oil.Anthracnose is the main disease of tea-oil tree and results in a huge loss annually.Colletotrichum fructicola is a major pathogen causing anthracnose on Ca.oleifera,and results in a huge loss annually.But the detailed molecular mechanisms of its pathogenicity is still largely unclear.Understanding the molecular pathogenic mechanism of the pathogen will help us to explore new ways of controlling this disease.The main results of the research are as follows:(1)Functional analysis of BRLZ motif of the transcription factor Cfhac1 in C.fructicola.In previous study,we characterized that the Cfhac1 controls the development and pathogenicity of C.fructicola,and the structure prediction revealed that CfHac1 contains one basic region-leucine zipper motif(BRLZ).Here,we constructed the motif deletion vector and obtained the complementation strains,and analyzed the phenotypes by bioassay.The mycelial growth rate,conidiation and appressorium formation rate of the △Cfhac1△BRLZ were significantly reduced,and the△Cthac1△BRLZ strain was sensitive to dithiothreitol.The pathogenicity test showed that the △Cfhac1△BRLZ lost the ability to infect Ca.oleifera leaves.BRLZ motif of Cfhac1 is essential for growth,development,tolerance to ER stresses and full virulence of C.fructicola.(2)To identify the role of Cfhac1 in the endoplasmic reticulum(ER)stress in C.fructicola,the growth of a ΔCfhac1 mutant strain of C.fructicola was determined on the PDA medium containing dithiothreitol,an ER stress factor.The results revealed that the △Cfhac1 strain was sensitive to dithiothreitol.To uncover the potential mechanisms of Cfhac1 contributions to pathogenicity and response to ER stress,the transcriptome profiles of the wild type strain and mutant △Cfhac1 of C.fructicola were characterized with high-throughput sequencing technology.The data indicated that,relative to the wild type,there were 2 680 differentially expressed genes(DEGs)in mutant ΔCfhac1,of which 1 181 were up-regulated and 1 499 were down-regulated.The Gene Ontology classification showed that the DEGs were mainly related to catalytic activity,binding,metabolic process,cellular process,cellular component organization or biogenesis,regulation of biological process and response to stimulus pathway.The KEGG pathway analysis also revealed that the up-regulated expressed genes were mainly related to ribosome,ribosome biogenesis in eukaryotes,RNA transport and cyanoamino acid metabolism pathways;the down-regulated expressed genes were mainly related to protein processing in ER,N-glycan biosynthesis,steroid biosynthesis,and protein export pathways.Genes involved in ER stress response and virulence were down-regulated at transcriptional level in the ΔCfhac1 strain.This study provides useful data concerning the effect of CfHac1 in endoplasmic reticulum stress response at the genome level and a basis for illuminating pathogenic mechanism of C.fructicola.(3)We previously revealed that C.fructicola was a major pathogen causing anthracnose on tea-oil treeand that the bZIP transcription factor CfHacl controlled the development and pathogenicity of C.fructicola.Here,we identified and characterized the function of CfVAM7 gene,which showed significant down-regulated transcript abundance in the △Cfhac1 mutant strain under dithiothreitol stress.Targeted gene deletion revealed that CfVam7 is important in growth,pathogenicity and responses to endoplasmic reticulum related stresses.Further analysis revealed that CfVam7 is required for appressorium formation,homotypic vacuole fusion and autophagy,which is important for fungal pathogen invasion.Moreover,we demonstrated that CfVam7 is localized to vacuole membranes and that the localization is dependent on the conserved PX(Phox homology)and SNARE domains,both of which are also indispensable for normal CfVam7 functions.Taken together,our results suggested that CfVam7-mediated vacuole membrane fusion promotes growth,autophagy and pathogenicity of C.fructicola.(4)To investigate the role of CfVps39 which interacts with CfVam7 in the fungal plant anthracnose pathogen C.fructicola,the CfVPS39 gene was deleted.This resulted in reduced mycelial growth,conidial production and complete loss of pathogenicity on tea-oil leaves and fruits.In addition,△Cfvps39 showed increased sensitivity to cell wall stress,osmotic stress and endoplasmic reticulum stress.Further analysis revealed that CfVps39 is required for appressorium formation and homotypic vacuole fusion,both important for fungal pathogen invasion.Our study provides evidence that CfVps39 has a role in conidiation,stress response,appressorium formation,homotypic vacuole fusion and virulence against tea-oil.This research not only provide the theories for revealing the pathogenic molecular mechanism of C.fructicola,but also has important guiding significance to reveal new fungicide targets to control this devastating disease.