Cucumber Translationally Controlled Tumor Protein Interacts With Rab11A And Promotes Activation Of Target Of Rapamycin In Response To Sphaerotheca Fulignea

Cucumber（Cucumis sativus L.）is an important protected vegetable in China.Cucumber powdery mildew[Sphaerotheca fuliginea（Schlecht）poll.]is a widespread disease,which seriously affects the yield and quality of cucumber fruit.TOR（target of rapamycin）kinase is a highly conserved regulatory center in eukaryotic growth,development and immune response,and plays an important role in plant stress response.At present,little is known about the function and dynamic regulation mechanism of TOR in photosynthetic plants.The previous research of our research group made it clear that CsTCTPs,an important upstream component of TOR,negatively regulates the disease resistance of cucumber to S.fuliginea,but the molecular mechanism in the process of disease resistance is unclear.In animals,TCTP can regulate and control disease occurrence and immune response by activating Rheb（Ras type small GTPase）,the upstream regulator of TOR signalling pathway.Whether TCTP in cucumber can regulate the activation of TOR signalling by combining with some Ras type small GTPases,and then regulate the defense response of plants,there is still a lack of direct and powerful evidence.Based on this,our study intends to verify the interaction between CsTCTPs and CsRabs by using yeast two hybrid（Y2H）,firefly luciferase complementation image assay（LCI）and bimolecular fluorescence complementation assay（BiFC）;The role of CsRab11A in response to S.fuliginea was studied by using cucumber cotyledon transient transformation technology;Y2H,LCI and in vivo kinase assay were used to explore the interaction between CsRab11A and TOR and the regulation of TOR signalling pathway;ITRAQ phosphorylated proteomics was used to analyze the differentially expressed phosphorylated proteins of S.fuliginea inoculated under the condition of inhibited TOR activity by AZD-8055,and to analyze the TOR signalling function in coordinating the growth and stress response of cucumber seedlings.The purpose is to clarify the mechanism of CsTCTPs/Rabs/TOR pathway regulating cucumber defense response,and to lay a foundation for exploring the balanced regulation of cucumber"stress stimulation and development".The main results are as follows:（1）24 Rab GTPases were searched in Cucumber database,among which CsRab11A,CsRab F1 and CsRab F2b had high similarity with Rabs interacting with At TCTP in Arabidopsis.Through the interaction methods of Y2H,LCI and BiFC,the interaction between CsTCTPs and CsRabs is explored.The results show that CsTCTPs can interact with CsRabs,and the interaction between CsTCTP2 and CsRab11A is the most stable.Subcellular localization showed that both CsTCTPs and CsRab11A were widely expressed in the cytoplasm,while the co-expression of CsTCTPs and CsRab11A showed that the overexpression of CsTCTPs would promote the transfer of CsRab11A to the plasma membrane.（2）Through the transient transformation experiment of cucumber mediated by Agrobacterium tumefaciens,CsRab11A overexpression and silencing cucumber cotyledons were obtained respectively.The disease index of transformed cotyledons and the expression of defense related genes were investigated to clarify the role of CsRab11A in response to S.fuliginea.The results showed that CsRab11A silencing cotyledons increased the resistance of cucumber to S.fuliginea.At the same time,the expression of defense related genes was significantly up-regulated,while CsRab11A overexpression increased the accumulation of S.fuliginea on cucumber cotyledons,which showed that it was susceptible,and the expression of defense related genes did not change much.The results showed that CsRab11A negatively regulated the resistance of cucumber to S.fuliginea.（3）The interaction between CsRab11A and TOR was studied by Y2H and LCI assay,and the activation of TOR kinase by CsTCTPs and CsRab11A was studied by in vivo kinase assay.The results showed that CsRab11A could interact with TOR in yeast and tobacco,mainly with c TOR（4315～7314 bp）.Kinase assay showed that CsRab11A could promote the phosphorylation level of TOR.In addition,CsTCTPs could also promote the activation of TOR.The co-expression of CsTCTPs and CsRab11A showed that CsTCTP1 played a promoting role in the process of CsRab11A promoting TOR activation.At the same time,the resistance of CsTCTPs and CsRab11A to S.fuliginea in the process of activating TOR activity was studied.The results showed that the transient expression of CsTCTPs and CsRab11A decreased the resistance of cucumber to S.fuliginea,while the cotyledons co-expressed with CsTCTPs and CsRab11A were the most susceptible.In conclusion,CsRab11A can interact with TOR to activate TOR activity and reduce resistance to S.fuliginea.CsTCTP1 plays a promoting role in this process.（4）ITRAQ phosphorylation proteomics was used to analyze the regulatory network of TOR signalling response to S.fuliginea.The results showed that AZD-8055 pretreatment significantly inhibited the growth and development of cucumber seedlings,5μmol·L^-1AZD-8055 significantly improved the disease resistance of cucumber seedlings.GO enrichment and KEGG pathway enrichment of proteins corresponding to differentially expressed phosphorylated peptides were analyzed.The results showed that after TOR activity was inhibited or inoculation of S.fuliginea,the phosphorylation level of proteins controlling cell process and cell localization,participating in biological regulation and stress response,and molecular functions such as nucleotide binding,catalytic activity and signalling transduction were significantly induced;The signalling pathway related to spliceosome,photosynthesis,endocytosis,RNA transport and phosphatidylinositol signaling system also changed significantly.The differentially expressed proteins in the two comparison groups AZD vs AZDP and DMSOP vs AZDP,which represent the response of S.fuliginea regulated by TOR,were analyzed.There were 233 differential proteins in the intersection of the two groups.Some candidate proteins that may be related to the response to S.fuliginea infection were screened,mainly including plant hormone signallings,transcription factors,G protein related and defense related proteins.The function and phosphorylation site of the candidate differentially expressed phosphorylated protein bZIP4150 transcription factor were analyzed and verified by Western blot.The results of in vivo kinase assay showed that the activation of bZIP4150 at Ser110 site played an important role in TOR regulating plant resistance to S.fuliginea.Through the above experiments,the interaction between CsTCTPs and CsRabs in cucumber was confirmed,and the negative regulation of CsRabs in cucumber response to S.fuliginea was clarified.In addition,the interaction between CsRab and TOR and its activation on the TOR signalling pathway were revealed.Finally,iTRAQ phosphorylation proteomics analyzed the function of TOR signalling in regulating cucumber seedling growth and development and stress response,and identified several downstream components regulated by TOR kinase.