Functional Analysis Of Effectors ChEp011 And ChEp113 Of Colletotrichum Higginsianum

Colletotrichum higginsianum is an important fungal pathogen which employs a two-stage lifestyle refers to as hemibiotrophy.It has a wide range of hosts and can damage a variety of cruciferous plants,causing the famous anthracnose.Effector is one of the main pathogenic factors of pathogenic fungi,which can regulate the immune defense response of plants and promote the infection and colonization of pathogenic fungi.However,there are still few studies on the effectors of C.higginsianum.Based on the prediction and screening of the C.higginsianum effectors conducted previously in my laboratory,this study intend to analyze the functions of some key effectors,so as to better understand the pathogenic mechanism of C.higginsianum and provide a theoretical basis for the control of anthracnose of cruciferous plants caused by C.higginsianum.In this study,the effectors ChEP011 and ChEP113 of C.higginsianum were selected for functional analysis.First,the genes of these two effectors were cloned,and the bioinformatics analysis and expression pattern analysis were carried out,then the PVX expression vector was constructed,and Phytophthora infestans elicitor INF1 was used as a positive control to analyze its functions in inducing tobacco cell death or inhibiting the cell necrosis response induced by INF1;the signal peptide and functional domain of ChEP113 was predicted,then the vectors lacking signal peptide or functional domain were constructed,and the functions of ChEP113 to inhibit necrosis or induce necrosis after deletion of signal peptide and functional domain were analyzed.At the same time,the fluorescent expression vector was constructed to analyze the effect of signal peptides on subcellular localization of effectors.And Botrytis cinerea Pers.was inoculated after transient expression of ChEP011 and ChEP113 to research the effects of ChEP011 andChEP113 on the disease resistance of Nicotiana benthamiana L..Finally,the genes ChEP011 and ChEP113 were knocked out by using ATMT technique,then the biological traits of the knockout mutants were observed and their pathogenicity to Arabidopsis thaliana(L.)Heynh.were determined.The main results are as follows.1.The ChEP011 gene of C.higginsianum was cloned,its full length sequence was 369 bp with two introns,65 bp and 70 bp in length,respectively;CDS sequence length was 234 bp,encoded a total of 77 amino acids(aa)with a 19 aa signal peptide and no domain.The full length sequence of ChEP113 gene was 306 bp with no intron,and encoded a total of101 aa,including a 18 aa signal peptide and a CFEM domain at 64-216 nt.The gene expression pattern analysis showed that both effector genes ChEP011 and ChEP113 were induced to be up-regulation in the early stage of infection,that is,they were highly expressed in the stage of biotrophy.2.Induction of necrosis and inhibition of necrosis and subcellular localization analysis showed that the effectors ChEP011 and ChEP113 of C.higginsianum could not induce tobacco leaf necrosis,but both the effectors could inhibit the necrosis caused by INF1.However,ChEP113 lacked the signal peptide could still inhibit the necrosis reaction induced by INF1,but ChEP113 lacked the CFEM domain could not inhibit the necrosis.It was suggested that ChEP011 and ChEP113 might inhibit the PTI by inhibiting necrosis caused by pathogen-associated molecular patterns,and the signal peptide was not essential for the activity of ChEP113,it might only play a secretory role;while the deletion of CFEM domain affected the activity of ChEP113,indicating that it was essential for the activity of ChEP113.Subcellular localization showed that ChEP113 was localized on the cell membrane of tobacco cells,and the loss of signal peptide affected its precise localization.3.Using Agrobacterium-mediated plant transient expression system,B.cinerea was inoculated in tobacco leaves after the transient expression of effectors ChEP011 and ChEP113,respectively.It was found that the expression of ChEP011 and ChEP113 stimulated the defensive response of N.benthamiana and improved the disease resistance of N.benthamiana to B.cinerea.4.The ChEP011 and ChEP113 knockout mutants were obtained by using ATMT technique.It was found that compared with the wild-type strain,the aerial hyphae werevigorous,hyphae melanin was reduced,growth rate was slowed down,the amount of sporulation and the dry weight of the hyphae were increased,and the hyphal tip became straight with few branches,but the mechanical penetration of hyphae was not affected in ChEP011 and ChEP113 knockout mutants.The conidia abnormally germinated to produce two germ tubes and two blackened appressoria,and the germ tubes became longer,but did not affect the pathogenicity to A.thaliana in the ChEP011 knockout mutants.Most of the conidia of ChEP113 knockout mutants only produced germ tubes on one side,and no blackened appressorium.However,a small number of conidia could germinate normally to produce blackened appressorium,and the pathogenicity to A.thaliana was weakened.It indicated that ChEP011 gene was involved in the regulation of colony morphology,mycelial growth,sporulation and spore germination of C.higginsianum.ChEP113 gene was involved in the regulation of colony morphology,mycelial growth,sporulation,spore germination and pathogenicity of C.higginsianum.