| Potato late blight caused by Phytophthora infestans is one of the devastating diseases in agricultural production.Potato late blight has led to the death of millions of people in parts of Europe in the 19th century,which has had a profound impact on the politics and economy of human society.At present,late blight still causes nearly 10 billion US dollars of crop losses every year,and it remains one of the major hidden dangers to human food security.Promoting potato staple foodization is a national strategy to ensure food security.However,late blight is seriously endangered in most potato producing areas in China,and it is one of the most important factors restricting potato production in China.The secreted protein of P.infestans plays an important role in regulating host physiology and immune response,and is the key to analyzing the mechanism of disease occurrence and plant disease resistance.It is one of the important pathogenic mechanisms for Phytophthora to transport large number of proteins to different parts of host cells to interfere with the normal immune response of plants.The whole genome and transcriptome sequencing of several Phytophthora showed that Phytophthora specifically expressed 800-1000 genes encoding proteins during the infection stage.According to the action site of these proteins in the host cells,the proteins could be classified into the cytoplasmic protein or the apoplast protein.The Phytophthora cytoplasmic protein mainly includes RxLR(N-terminal contains a conserved RxLR-dEER motif),CRN(crinkling-and necrosis-inducing proteins)and other types of proteins.These proteins enter plant cells and interact with plant immune-related proteins to regulate plant defense responses through different biochemical activities.A few domestic and foreign research groups have made rapid progress in the study of such protein.However,the biological functions and immune mechanisms of these Phytophthora apoplastic proteins are rarely reported.In this thesis,the functional identification of a type of intercellular protein-Small Cysteine Rich(SCR)protein,which is ubiquitous in eukaryotic pathogens,was carried out and the molecular mechanism of its regulation of plant resistance was analyzed.Screen the SCR proteins in P.infestans.The previous research team identified 28 genes encoding novel SCR proteins in the predicted secretome of P.infestans.These genes are significantly up-regulated during the infection and don’t have any similarity to known genes in the NCBI database.These 28 SCR proteins were heterologously expressed in N.benthamiana using the potato virus X(PVX)-mediated plant transient expression system,and the recombinant protein was transported to the N.benthamiana cell intercellular region through the signal peptide of the PR1 protein in the plant.PC2,one of these SCR proteins,was screened to trigger a hypersensitive response in plant cells.The mature protein encoded by the PC2 gene has 190 amino acids and is rich in 18 cysteine residues.The transient expression of PC2 in N.benthamiana induces significant up-regulation of plant defenserelated genes PR1 and RbohB and leads to a high level of ROS accumulation.And PC2 can trigger an immune response on a variety of Solanaceae plants such as potato and tomato.During the treatment with chemical inhibitors,the protease inhibitor Cocktail,metal ion chelating agent EDTA,reactive oxygen species inhibitor DPI,and calcium channel inhibitor LaC13 were found to inhibit PC2-induced hypersensitive response.All these experiments demonstrate that PC2-induced responses are an active,plant-dependent process.Since PC2 is conserved among all the Phytophthora species we examined so far and its homologous genes are up-regulated significantly in the early stages of infection by multiple Phytophthoraplant interaction systems,it is speculated that it has a universal and important regulatory role.Plant serine protease is involved in PC2-induced immune response.In the chemical reagent treatment experiment,the serine protease inhibitor,one component of the plant protease inhibitor cocktail,can specifically inhibit the PC2-induced immune response,suggesting that the plant serine protease participates in the PC2-induced plant immune response.Further studies have found that the serine protease inhibitor EPI1 secreted by P.infestans can specifically inhibit PC2-induced cell death.Previous reports have shown that the plant P69 gene family encodes a class of serine proteases,of which P69B is a pathogenesis-related gene that is strongly up-regulated by pathogens,and its protease activity can be inhibited by the serine protease inhibitor EPI1.So,we speculate that P69B may be involved in the cleavage of PC2.In vitro,we fund the plant serine protease P69B is capable of cleaving PC2 protein,and this cleavage can be inhibited by the serine protease inhibitors.Silencing P69B and its homologous genes in N.benthamiana and tomato can interfere with PC2-induced immune responses.It was demonstrated that the presence of serine proteases in plants is capable of cleaving PC2,and this cleavage is critical for PC2-induced immunity.PC2-induced immune responses are dependent on the conduction of plant immunerelated signaling pathways.Plants recognize pathogen-associated molecule patterns secreted by pathogen through surface receptors,and then conduct signal transduction to produce defense responses.This defense process consists of very complex network signals.In order to study the signaling pathway of PC2 immune response,we used TRV-mediated silencing system to silence important receptor kinases,MAPK cascade-related genes,and heterotrimeric G proteins.It was found that receptor-like kinase BAK1,receptor-like cytoplasmic kinase BIK1,MAPK related gene MAPKKKξ,and heterotrimeric G proteins are involved in the PC2 immune signal pathways.The P.infestans secreted effectors synergistically inhibit the PC2-induced immune responses.During the interaction between plants and Phytophthora,plants can recognize various effectors including PC2 through the immune system.However,in order to infect plants,Phytophthora can also secrete effectors to inhibit PC2-induced immune responses from different aspects.This study previously found that PC2-induced immune responses lead to accumulation of reactive oxygen species,up-regulation of plant serine protease P69B,and activation of the kinase MAPKKKξ in the mitogen-activated protein kinase cascade.Correspondingly,this study further found that the P.infestans secreted Nudix effectors inhibited PC2-induced immune responses by inhibiting the accumulation of reactive oxygen species;The pathogen secreted serine protease inhibitors EPI1 and EPI10 suppress PC2induced immune response by inhibiting plant serine protease activity.The RxLR effector PexRD2 blocks the MAPKKKξ signaling pathway,thereby inhibiting the PC2-induced immune response.The above results indicate that P.infestans can secrete a variety of effectors to synergistically inhibit PC2-induced plant immune responses from different levels,thereby achieving smooth infection.In this paper,a novel intercellular SCR protein PC2 was identified from P.infestans.We use the PC2 as a material to study the molecular mechanism of PC2-induced plant immune response and P.infestans inhibiting PC2 immune response.The results of the study initially revealed the biochemical function and mechanism of a broad-spectrum effector,and it is expected to provide a scientific basis for the design of disease-resistant resources screening and crop disease-resistant breeding for late blight. |
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