Screening And Preliminary Exploration Of Effector Proteins In Rhizocatonia Solani

Rice sheath blight is one of the three major diseases of rice,which seriously affects rice yield and quality.The pathogenic mechanism of rice sheath blight and its interaction mechanism with host plants are very complex,and the role of effector proteins is particularly important in the interaction process between plants and microorganisms.In this paper,the main study is the Rhizocatonia solani in the pathogenic bacteria of rice sheath blight.At present,the identification and functional analysis of the effector protein of rice sheath blight have gradually deepened,so it is of great significance to build a secretion protein library of rice sheath blight to improve the research of effector proteins.In this study,the predictor proteins were screened and identified by yeast double hybridization technology and bimolecular fluorescence complementary technology,and the functions of the presumed effector proteins screened were preliminarily explored by subcellular localization and agrobacterium-mediated expression of tobacco,and this paper provides a theoretical basis for studying the molecular mechanism of rice sheath blight.The main research results achieved are as follows:In this study,Signal P and TMHMM software were used to predict the effector proteins of rice sheath blight,and finally 420 proteins were secreted by Rhizocatonia solani.Protein cloning and vector construction of the predicted secreted proteins: the p GBKT7 vector was digested to obtain a linearized carrier,and the DNA fragments that matched the size of the predicted proteins were cloned by PCR technology,a total of 285 secreted proteins were cloned,and then the linear vector was connected with the dna fragments obtained by the clones by transforming the competent cells of E.coli,and the positive clones were randomly selected for colony PCR verification,digestion verification and sequencing identification.A total of 226rice-secreted proteins were obtained in the BD vector library.In this study,31 rice sheath blight disease resistance proteins related to chloroplast synthesis and development,including Os ACS2,Os OXO4,Os PGIP1,etc.,21 rice sheath blight resistance protein vectors were successfully constructed.Their interaction with the BD vector library of rice secreted protein was screened by using Yeast two-hybrid system(Y2H).Firstly,the self-activating detection of the BD vector library of rice secretory proteins was carried out,and it was found that there were 44 secreted proteins such as Rs6,Rs26,Rs40 and p GADT7no-load co-rotating yeast colonies that could still grow normally and turn blue on SD/-Lue-TrpHis-Ade medium containing Ab A,X-α-gal,so it was concluded that a total of 44 secreted proteins had self-activation.One-on-one screening of 182 secretory proteins without selfactivation and candidate rice blight disease resistance proteins: first two-by-two cotransformation with SD/-Lue-Trp medium,and then the positive clones are transferred to SD/-Lue-Trp-His-Ade medium containing Ab A and X-α-gal,if they can still grow normally and turn blue,the two proteins are considered to interact.The results showed that os1 and Rs333,Os2 and Rs145,Os2 and Rs155,Os3 and Rs117,os4 and Rs161 could interact with each other.Since in yeast double hybridization technology,some kind of stable protein complex may form when the two proteins are co-rotated,which causes the expression of the reporter gene,producing false positives.Therefore,this study further verified the Y2 H results by Bimolecular fluorescence complementarity technology(Bi FC).Rice Nippon Qing was cultivated in artificial climate incubators to extract rice protoplasts,and the five sets of interactions screened by Y2 H were co-rotated into protoplast cells in good condition,and after incubation,they were placed under a confocal microscope for observation,and it was observed that after the co-transformation of Os1 and Rs333 in rice protoplasts,the fluorescence of ECFP and m Cherry was localized in the nucleus and membrane,indicating that both proteins were successfully transfected and expressed in this cell,and m Vneus showed that fluorescence and chloroplast autofluorescence coincided in cell chloroplasts.From this,it can be concluded that Os1 interacts with Rs333 in chloroplasts.Os2 and Rs145,Os2 and Rs155,Os3 and Rs117,Os4 and Rs161 co-rotated as above.The experimental results of Y2 H and Bi FC were comprehensively analyzed,and the functions of the five presumed blight effector proteins screened were preliminarily explored.The hypothetical blight effector protein was constructed on the GFP-PUC19 vector,and the rice protoplast transformation system and GFP fluorescence observation were used to demonstrate that the five hypothetical effector proteins were localized in the cell chloroplast.The agrobacterium-mediated transient expression system of tobacco was used to study the ability of hypothetical effector proteins to induce cell death.PTA7001 vector was constructed and transformed in Agrobacterium EHA105,Agrobacterium carrying the assumed effector protein gene was inoculated on age-appropriate and healthy tobacco leaves,and after DEX induction expression for 12-13 days,Agrobacterium carrying Rs117,Rs161,Rs333 genes induced tobacco leaf cell death,while Rs145 and Rs155 did not trigger the HR response of tobacco leaves.From this,it can be preliminarily determined that Rs117,Rs161 and Rs333 can interact with the host and exert toxic effects to affect plant immunity.