Functional Analysis Of The RxLR Effector PcSEP48 Of Phytophthora Capsici

Crop blight caused by Phytophthora species is one class of typical soil-borne diseases,which is widely distributed around the world.It often endangers the crop production and causes huge economic losses.During the infection processes,Phytophthora pathogens secrete a variety of effectors into plants to interfere with the normal cellular activities and manipulate the plant defense responses in order to promote their infection and colonization.Analyzing the biological functions of Phytophthora effectors is of great significance for elucidating the infection mechanisms of Phytophthora pathogens and formulating new management strategies for crop blight.In our previous study,5 proteins with host nuclear localization signals that were up-regulated during the P.capsici infection stage were obtained.In order to clarify their biological functions in the pathogenesis,one of them,namely the RxLR effector PcSEP48 was studied in this thesis,and the following analyses performed:(1)In order to determine whether the protein is secreted,the predicted signal peptide was analyzed by the yeast invertase secretion assay.The results showed that the signal peptide composed of the first 19 amino acids is functional in guiding the protein's secretion;(2)In order to analyze the localization of the protein in plants,the transient expression of the protein in Nicotiana benthamiana was observed under a laser confocal microscope.The results showed that PcSEP48 is localized in both the host cytoplasm and nucleus;(3)To analyze the role of PcSEP48 in the process of P.capsici infection,the Pcsep48 gene was knocked out using CRISPR/Cas9 technology.The results showed that the gene knockout significantly compromised the virulence of P.capsici on host plants,indicating that PcSEP48 plays an important virulence role in the infection process of P.capsici;(4)In order to analyze the effect of the protein on plant immunity,Agrobacterium-mediated overexpression of the protein in N.benthamiana was conducted.It was found that the transient overexpression of PcSEP48 induced N.benthamiana resistance to P.capsici infection.(5)In order to analyze the action mechanism of PcSEP48 contributing to pathogenicity,the yeast two-hybrid and bimolecular fluorescence complementation technology were utilized to identify and characterize its interacting proteins in N.benthamiana.The results showed that PcSEP48 interacts with a plant kinesin light chain-related 3-like protein(NbKLCR3).This thesis clarified that the RxLR effector PcSEP48 of P.capsici can be secreted into the plant cell nucleus,probably interacts with the plant KLCR3,and plays a virulent role in the infecting process of P.capsici.However,its overexpression can trigger the plant immunity.The results of this thesis not only lay the foundation for further analyzing the functions of PcSEP48,but also provide valuable experimental data for elucidating pathogenicity mechanism of Phytophthora pathogens.