Study On The Function And Mechanism Of The Translationally Controlled Tumor Protein In Wheat-Puccinia Triticina Interaction

Wheat leaf rust disease that caused by Puccinia triticina is one of the most wide-spread and severe phyto-diseases.Study on the molecular mechanism of host disease resistance can be conductive to fundamentally reduce the adverse impact of such diseases on food production.Our laboratory has long been engaged in the research of the host resistance mechanism in the wheat-P.triticina interaction,and found that intracellular signals such as calcium(Ca2+),hydrogen peroxide(H2O2),and nitric oxide(NO)and the phytohormone salicylic acid(SA)are involved in the regulatory process of hypersensitive responseprogrammed cell death(HR-PCD)in the wheat-P.triticina interaction.Translationally controlled tumor protein(TCTP)exists widely in eukaryotic cells and participates in the regulation of various biological processes including development,cell expansion,and programmed cell death.Through high-throughput sequencing analysis,we found that the expression of TaTCTP was induced in the incompatible combination of wheat near-isogenic line TcLr26 and P.triticina race 260,and the induction was regulated by Ca2+.In this study,the function and mechanism of TaTCTP in wheat resistance to P.triticina infection were investigated using virus-induced gene silencing(VIGS),tandem affinity purification-mass spectrometry(TAP-MS),and bimolecular fluorescence complementation(BiFC).The main results obtained in this study are as follows:1.The expression of TaTC.TP was detected by RT-qPCR in the incompatible and compatible combinations of TcLr26 and its recurrent parent Thatcher with P.triticina race 260.In the incompatible combination,the transcription level of TaTCTP was induced by P.triticina infection,and reached its peaks at 12 and 96 h post-inoculation,whereas almost unaffected in the compatible combination.Meanwhile,the increased transcription level of TaTCTP was compromised by the pre-injection of extracellular Ca2+ chelator EGTA,and the result was consistent with what was obtained in the transcriptome analysis.The protein level of TaTCTP was analyzed by Western blotting,and it was found that the accumulation of TaTCTP was induced by P.triticina infection in the incompatible combination,and reached its peaks at 48 and 120 h after inoculation,while the protein product of TaTCTP was not detected in the compatible combination.It is speculated that TaTCTP may be positively involved in the defense response against P.triticina infection.2.The function of TaTCTP in the wheat-P.triticina interaction process was investigated by reverse genetics approaches VIGS and RNAi.It was found that P.triticina produces a greater number of haustorial mother cells(HMC)on the TaTCTP-silenced wheat leaves compared with that on the control plants,and the area of dying cells was also larger.It is speculated that the silence of TaTCTP reduces the resistance of wheat to P.triticina.3.The interactants of TaTCTP were identified by TAP-MS.The interaction between TaTCTP and wheat calcineurin B-like protein-interacting protein kinase 23(CIPK23)was examined by yeast two-hybrid,co-immunoprecipitation(Co-IP)and BiFC analysis,and it was found that the interaction between TaTCTP and TaCIPK23 occurs in the endoplasmic reticulum in plant cells.By introducing truncated mutants,it was found that TaTCTP binds to the N-and C-terminals of TaCIPK23.4.Phosphorylation analysis revealed that TaCIPK23 phosphorylates TaTCTP in its Ser17 and Thr73 residues,and the phosphorylation effect weakens their interaction.In addition,phosphorylated TaTCTP induces HR-type cell death in plant.5.The expression of TaCIPK23 was continuously up-regulated and regulated by P.triticina infection in a Ca2+-dependent manner in the incompatible combination,whereas it was not significantly changed in the compatible combination.VIGS was used to silence TaCIPK23 in the incompatible combination,and it was found that P.triticina produced more HMCs that resulted in a larger area of cells occurring HR-PCD in the silenced plants compared with that in the control plants.In addition,P.triticina produced similar numbers of HMCs and induced similar areas of cells undergoing HR-PCD in plants that silencing both TaTCTP and TaCIPK23 compared with the plants that TaTCTP or TaCIPK23 was silenced along,and the expression of disease-related genes TaPRl,TaPR2,and TaPR5 were also similar,indicating that TaTCTP and TaCIPK23 may participate in the same signaling pathway conferring disease resistance.In conclusion,our findings suggest that TaTCTP and TaCIPK23 are involved in the defense response against P.triticina infection in wheat,and the possible mechanisms are as follows:the expression of TaTCTP and TaCIPK23 are induced by P.triticina infection in the incompatible combination;TaCIPK23 phosphorylates TaTCTP.leading to an increased level of phosphorylated TaTCTP that promote HR-PCD,thereby limiting the nutrients accessible for P.triticina.Our findings uncovered a disease-resistance pathway downstream of Ca2+signal in the wheat-P.triticina interaction,and that TaTCTP acts as a target protein that was regulated by TaCIPK23.These results not only improved the mechanism of HR-PCD in the resistance against P.triticina in wheat,but also provided candidate valuable genetic resources for breeding disease-resistant wheat cultivars.