Identification And Functional Verification Of The Effector Protein Of Colletotrichum Fructicola,the Pathogen Of Camellia Oleifera Anthracnose

Camellia oleifera,as the leading industry for the prosperity of the forest and the people in rural areas and the production industry for poverty alleviation,has always attracted attention.Camellia anthracnose is a common and important disease in my country’s Camellia oleifera planting areas.It causes severe fruit drop,bud drop,and even the whole plant decline.Effector proteins are pathogenic factors secreted by pathogenic fungi in the process of infecting plant hosts,and play a vital role in the pathogenic process.In this thesis,the main pathogen of Camellia anthracnose(Colletotrichum fructicola)secreted effector proteins during the dead vegetative period was studied to predict,screen and verify their functions,and to understand the functions and rules of action of these effector proteins for the prevention of Camellia oleifera The spread of anthracnose and revealing the mechanism of the interaction between Camellia oleifera and anthracnose have positive significance.This paper has obtained the following research results:1.Predictive screening of candidate effector proteins of C.fructicola dead body vegetative stage.The anthracnose-Camellia oleifera lesion tissue that was in the dead body vegetative stage for 96 hours was selected for transcriptome sequencing.The transcriptome result data showed that there were 7850 differentially expressed genes during the infection period compared with the conidia period,and the coding proteins of the differentially expressed genes were predicted and analyzed 345 of them were found to be classical secreted proteins.With amino acid length<300 and cysteine residue number≥ 4 as the screening conditions,the proteins with annotated functions were filtered,and finally 17 candidate effector proteins were screened out.According to the homology comparison of the 17 candidate effector proteins obtained from the screening,it is found that the candidate effector protein also has high homology proteins in fungal pathogens related to plant diseases.2.Functional verification of C.fructicola candidate effector protein.Agrobacterium-mediated tobacco transient expression system was used to verify the function of candidate effector proteins.CfEP1,CfEP2,CfEP3,CfEP4,CfEP6,CfEP15 were identified to cause shrinkage,thinning,and dry necrosis of tobacco leaves to varying degrees;the results of 1:1 mixed co-injection of candidate effector protein and Bax protein showed that 17 candidates The effector protein did not exhibit the function of inhibiting cell necrosis;the subcellular localization results showed that the candidate effector protein with the function of inducing necrosis could not capture the fluorescence signal.The same was true in the result of co-injection with Bax.It is speculated that leaf cell death would affect the fluorescence of the fusion protein.expression.3.qRT-PCR quantitative analysis of C.fructicola candidate effector protein.The seven time periods of sorting conidia,vegetative growth 48h,72h,96h,and 48h,72h,96h infecting Camellia oleifera leaves,quantitatively analyzed the genes of 17 candidate effector proteins,and found that among them,the most effective in inducing cell death The expression level of CfEP15 was the highest at 48h,and continued to be expressed in the subsequent infection stage.It is speculated that this protein played a key role in the process of C.fructicola infecting Camellia oleifera.In summary,17 candidate effector proteins of C.fructicola in dead body vegetative phase were screened in this article;the construction of transient expression vector successfully verified that CfEP1,CfEP2,CfEP3,CfEP4,CfEP6,CfEP14,CfEP15 candidate effector proteins have the function of causing tobacco leaf necrosis;Through multi-time quantitative analysis,the expression rule of candidate effector protein is obtained.The data of C.fructicola effector protein is enriched,and it provides a favorable reference for other host plants to resist anthracnose.At the same time,it provides a basis for the study of the molecular mechanism of the fruiting Echinochloa spp.infecting Camellia oleifera and the breeding of disease-resistant varieties of Camellia oleifera.