Prediction And Screening Of Candidate Effectors And The Functional Analysis Of ChEP011 In Colletotrichum Higginsianum

Colletotrichum higginsianum is one of the important plant pathogenic fungi,which can infect a variety of cruciferous plants such as the vegetable crops in the genera Raphanus and Brassica and cause severe anthracnose.The climatic conditions of high temperature and high humidity in South C hina are very suitable for the occurrence of anthracnose in cruciferous vegetables such as C hinese flowering cabbage,which caused serious yield losses up to 30% to 40%.So far,little study on pathogenic mechanism of C.higginsianum at the cellular and molecular levels has been conducted.In this study,through the investigations of prediction,screening and functional analysis of candidate effectors in C.higginsianum,aims to better understand the functions of effectors during the pathogenesis,so as to provide important evidences for the study of molecular mechanism during the interaction between the pathogen and the host plant.The results are as follows.According to the whole genome sequence of C.higginsianum released recently,their candidate effectors were predicted by using bioinformatics technology.Through bioinformatics software and databases such as SignalP,TMHMM,Protcomp,big-PI Predictor and TargetP,the candidate secreted proteins were predicted and obtained.Then the candidate effectors were further screened through cysteine content and the size of sequence.Finally,the candidate effectors were analyzed by using Blastp tool and the database of non-redundant protein and those having no or lower protein homology sequences were obtained,and thus 135 candidate candidate molecules were obtained.Specific primers were designed according to the sequences of 135 candidate effectors of C.higginsianum.The genes of candidate effectors were cloned based on the cDNA of different growth stages of this pathogen and were used to construct the plant efficient expression vector for the transient expression in Nicotiana benthamiana.As a result,12 effectors of C.higginsianum,which could induce the cell necrosis in N.benthamiana,were successfully obtained.However,in the experiments of the co-injection of effectors and INF1,all effectors could not inhibit the necrosis in N.benthamiana and so that the effectors that inhibited the host cellular immune response could not been obtained.There was only one effector having a functional domain,and the others were putative proteins with unknown functions.Finally,12 effectors were validated to be expressed successfully in N.benthamiana by using Western blot technique.The above results also showed that it is feasible to screen the functional effectors by means of Agrobacterium-mediated transient expression in N.benthamiana leaves.Finally,the effector C hEP011,which is the effector to induce most tobacco necrosis,was selected as the object of study.First of all,the gene sequence,signal peptide sequence,the physical and chemical properties and the function of the translated protein of C hEP011 were analyzed by using bioinformatics.Then,the signal peptide of ChEP011 was predicted,and the position and length of signal peptide were analyzed,and the vector was constructed so as to study the function of signal peptide.The results showed that ChEP011,which was removed the signal peptide,could not induce the necrosis of the leaves of N.benthamiana.At the same time,the effects of signal peptide on the subcellular localization of effector molecules were studied.The results showed that ChEP011 was secreted on the host cell membrane during the interaction between pathogen and host plant.Taken together,the signal peptide sequence of C hEP011 plays an important role in its function.