Screening Of Candidate Effectors And Function Analysis Of Effector ChEP085 Of Colletotrichum Higginsianum

Colletotrichum higginsianum Sacc.is a typical hemibiotrophic ascomycetous fungus of Colletotrichum,which can damage cruciferous plants including the model plant Arabidopsis thaliana worldwide,cause anthracnose diseases and serious economic losses.Effector is a kind of proteins secreted by pathogens that act in the apoplast or inside of plant cells to regulate host-pathogen interactions,so it is also called effector protein.Fungal effetors can facilitate the infection of pathogenic fungi by manipulating host physiological process or inhibit host immunity.However,little is known about the function of these effectors of Colletotrichum higginsianum at present.In this study,on the basis of previous prediction and screening of effetors conducted in author’s laboratory,10 candidate effectors of C.higginsianum were screened again,and an effector ChEP085,which could stably cause cell death in tobacco leaves and specifically expressed in the infection stage,was deeply analyzed to clarify its function so as to better understand the pathogenic mechanism of C.higginsianum and provide a theoretical basis for the study of host-pathogen interactions.Firstly,the gene expression patterns of 10 candidate effectors were analyzed after Arabidopsis thaliana was infected by C.higginsianum.At the same time,the genes of 10 candidate effectors were cloned and constructed into PVX expression vector,using Phytophthora infestans elicitor INF1,which can induce cell death in tobacco leaves,as positive control,to analyze whether these candidate effectors could cause leaf necrosis of Nicotiana benthamiana.The effector ChEP085 which can stably induce cell death in tobacco was obtained through above experiments.The bioinformatics analysis of ChEP085 was carried out to predict it’s signal peptide,then the deletion of signal peptide sequence of ChEP085 gene was constructed into PVX vector and enhanced green fluorescent protein vector p Bince GFP respectively to analyze the function of signal peptide,to find out if the signal peptide influences the function of inducing tobacco cell death,inhibiting the necrosis induced by INF1 and sbucellular localization of ChEP085.Then,after transient expression of ChEP085 in tobacco leaves,Botrytis cinerea Pers.was inoculated to study its effect on tobacco disease resistance.Finally,the ChEP085 gene was knocked out and then complemented by using ATMT technology,the biological characteristics and pathogenicity of the knockout mutants were determined,and the pathogenicity of the complement was determined to clarify the function of the effector ChEP085.The results are as follows:1.The feature of effector gene ChEP085.The ChEP085 gene is 675 bp in length,without intron,encodes 224 amino acids,the translation product of this gene is a hypothetical protein.From 1 to 20 amino acid is signal peptide and without known functional domain.Gene expression pattern analysis showed that ChEP085 gene was specifically highly expressed at 24 hours after inoculation.Furthermore,after injecting tobacco leaf with Agrobacterium containing the ChEP085 gene the results showed that ChEP085 was the only one of the 10 candidate effectors that could stably cause tobacco leaf necrosis.2.The function of signal peptide of effector ChEP085.Agrobacterium-mediated transient expression of ChEP085 gene in tabacco leaf showed that tobacco leaf necrosis could be induced by ChEP085 protein with or without signal peptide,which indicated that signal peptide did not affect the function of ChEP085.ChEP085 could not inhibit leaf necrosis induced by INF1 either in full length or without signal peptide sequence.Subcellular localization experiment showed that ChEP085 was located in the cell membrane,and the deletion of signal peptide affected its accurate localization.3.Effector ChEP085 influence the infection of Botrytis cinerea on tobacco.Transient expression of effector ChEP085 in tobacco leaf could promote the infection of Botrytis cinerea,which suggests that ChEP085 might facilitate the infection of pathogens by its own virulence function.4.The effect of ChEP085 on biological traits and pathogenicity of Colletotrichum higginsianum.After knockout of ChEP085 gene,there was no significant difference between knockout mutants and wild type(WT)isolate in colony morphology,hyphae growth rate,hyphae biomass,hyphae tip morphology and hyphae mechanical penetration.However,the spore production of knockout mutants varied with culture time.After 5 days and 6 days culture,the spore production of knockout mutants was slightly higher or equal with WT isolate,after 7 days and 8 days culture,the spore production of WT isolate was significantly higher than those of knockout mutants.Secondly,the spore germination and appressoria formation are also different,the conidia of WT isolate produce germ tubes and form a melanized appressoria,while the conidia of knockout mutants germinate to produce longer germ tube but not appressoria,except one or two conidia might form a melanized appressoria.The spore germination rate of (?)ChEP085-2 was significantly lower than that of WT isolate,but there was no significant difference between (?) ChEP085-8 and WT isolate.In terms of appressorium formation rate,two knockout mutants were significantly lower than WT isolate,indicating that ChEP085 gene affected the formation of appressoria.The pathogenicity of (?)ChEP085-2 and (?) ChEP085-8 to Arabidopsis thaliana and Chinese flowering cabbage was lower than that of WT isolate.After the ChEP085 gene was supplemented,the inoculation experiments showed that three of the four complement could recover the pathogenicity.It is suggested that ChEP085 gene is involved in the regulation of spore germination and appressorium formation of C.higginsianum,and it is also necessary for the pathogenicity of C.higginsianum.