Functional Analysis Of The Secreted Protein PcSEP24 Of Phytophthora Capsici And Anti-oomycete Trial Of A Peptide Aptamer

Crop blight caused by oomycete pathogen Phytophthora capsici is one class of soil-borne destructive diseases,which seriously affect the production of hundreds of crops around the world and cause huge economic losses annualy.There has been lack of effective and safe management methods.The disease management mainly depends on the chemicals.It has been know that like many other oomycetes,P.capsici secretes a large number of effectors into the host plants during the infection process,which manipulate the physiological processes of the plants,deactivate the immune system,to foster their own infection and colonization.Deep understanding of pathogenicity mechanisms of P.capsici and other oomycete pathogens,and developing of novel disease management measures are compelling need to secure safety of crop productions.Our previous study identified from P.capsici five secreted proteins whose expression were highly up-regulated during the P.capsici infection of host plants.In order to clarify the functions of the proteins in the pathogenesis,this thesis focused on one of them,namely PcSEP24,and the following analyses were performed:(1)It was predicted that this protein possesses a signal peptide(SP).Therefore,the yeast invertase secretion assay was performed.It was proved that SP of the protein is functional;(2)It was predicted that the protein contains a nuclear localization signal.To clarify its subcellular localization,the leaves expressing the protein fused with GFP of Nicotiana benthamiana were observed under a laser confocal microscopy.It was found that PcSEP24 resided in the cytoplasm and nucleus of the plant cells;(3)To analyze if the protein contributes to the virulence of the pathogen,the N.benthamiana leaves transiently expressing the protein were inoculated with P.capsici.The results showed that the transient expression of PcSEP24 facilitates the host infection of P.capsici;(4)To directly ensure if the protein is involved in the pathogenesis,the Pcsep24 gene was knocked out in P.capsici using CRISPR/Cas9 technology.The inoculation results showed that the gene knockout significantly compromised the pathogenicity of P.capsici on the host plant;(5)In order to determine the effect of the protein on the host defenses,the Pcsep24-transgenic N.benthamiana plants were constructed.It was found that the reactive oxygen burst,callose deposition,the resistance of the plants to two pathogens(P.capsici and TMV)were suppressed,indicating that the protein could suppress the host immunity resistance during the infection;(6)To reveal the action mechanism of the protein during the infection,by yeast two-hybrid screening of an in-planta infection library and through analyses using bimolecular fluorescence complementation and GST pull-down technologies,an unknown interaction protein Pc511533 was identified from P.capsici;(7)Based on a previous report,this thesis analyzed the inhibition effect on phytopathogenic oomycetes by a peptide aptamer targeting the cellulose synthase of Plasmopara viticola and its application potential used as an oomycete disease control agent.On-plate inhibition experiments showed that the peptide aptamer can significantly suppress the vegetative growth of Peronophythora litchi,Phytophthora parasitica,P.melonis,Phytopythium helicoides,P.cactorum,P.palmivora,P.capsici,and P.cinnamomi.After spraying peptide aptamer on litchi fruits,it was found that the occurrence of litchi downy blight was significantly suppressed.The results together indicated that the peptide aptamer can be used as a novel biological control agent for oomycete diseases.