Functional And Regulational Mechanism Of The Rust Induced Wheat Protein Kinase Gene TaRIPK1 During The Interaction Of Wheat And Stripe Rust Fungus

Wheat is one of the world’s most widely grown cereal crops and is the staple food grain of more than 2.5 billion people in the world.Wheat stripe rust,leaf rust and stem rust are the main diseases that threaten wheat production and even cause more than 90%yield loss in severe cases.Moreover,the resistant wheat varieties are constantly overcome by rust fungi due to the virulence variation of the rust pathogens,which results in these resistant wheat varieties cannot be used for the durable control of wheat rust.In addition,most rust resistance genes are race-specific,which makes it difficult to achieve broad-spectrum resistance in the case of complex and polymorphic rust fungi population in the field.Susceptible genes（S genes）or negative regulators are required for successful colonization and reproduction of plant pathogens and are essential for the establishment of compatible interactions between plants and pathogens.Blocking the evolved compatible interaction can achieve durable and broad-spectrum disease resistance of crops.Researches on susceptible genes and they mediated susceptible mechanisms can open up a new way for improvement of crop disease resistance.Up to data,a lot of wheat negative regulatory factors in the interaction between wheat and wheat stripe rust has been uncovered.However,little is known about the wheat susceptible genes in the interaction between wheat and wheat stripe rust and the susceptible mechanisms mediated by theses genes.Previously,virus induced transient silencing system was used to screen the up-regulated genes in the compatible c DNA library constructed in the early stage of interaction between stripe rust and wheat to identify the wheat susceptible genes.A gene named as Triticum aestivum Rust Induced Protein Kinase 1（TaRIPK1）was identified as a candidate wheat susceptible gene during in wheat-stipe rust interaction.In this study,molecular biology,genetics,biochemistry,histological assays were used to uncover the susceptible function of TaRIPK1 and it mediated susceptible mechanism.This study lays the foundation for effective control of wheat stripe rust and provide the new wheat stripe rust resistance wheat material.This study also deepens the understanding on the susceptible gene research and their action mechanisms.The main results are as follows:1.A rust induced S gene was identified,TaRIPK1,which belongs to RLCK receptor kinase gene.By analyzing the expression of TaRIPK1 in the compatible and incompatible of different wheat varieties,it was identified that the gene could only be induced in the early expression of the compatible interaction.The expression analysis of TaRIPK1 in different rusts（stripe rust,leaf rust and stem rust）showed that this gene could be induced by three species of rust.In contrast,TaRIPK1 could not be induced in transcriptome analysis of other wheat pathogens（Fusarium graminearum,Blumeria graminis and Septoria tritici）.This indicated that TaRIPK1 was specifically induced by rust and was most significantly up-regulated at the early stage of infection.Sequence alignment analysis revealed that TaRIPK1 contained typical ATP binding sites,activation rings,and conserved Ser/Thr protein kinase conserved domains.In addition,TaRIPK1 and PBL13 belong to RLCK VII kinases according to the evolutionary tree analysis of imported kinases in Arabidopsis thaliana.TaRIPK1 RNAi plants with different genetic backgrounds were created by particle bombardment-mediated wheat transformation,and TaRIPK1 RNAi plants phenotype changed from wild type（WT）high susceptibility to near-immunity or high resistance when inoculated with Pst race CYR32.Histological observation found that a large areas of H₂O₂TaRIPK1-RNAi Pst infection site than the WT.And H₂O₂ concentration were increased in TaRIPK1-RNAi plants by enzyme-linked immunoassay.Unlike WT plants,in which usually one SV（substomatal vesicle）forms per stoma,in the silenced plant lines multiple SVs formed at 33%-50%of stomata in TaRIPK1-RNAi plants.These results indicated that TaRIPK1-RNAi plants showed significant resistance to wheat stripe rust,mainly due to the abnormal development of Pst caused by the accumulation of H₂O₂.Thus,silencing of TaRIPK1,which plays a role in the interaction between wheat and stripe rust as an susceptible gene,may limit the expansion of Pst and block the susceptibility process of Pst.TaRIPK1 overexpressed plants were obtained by Agrobacterium-mediated genetic transformation,and TaRIPK1 overexpressed plants render the resistant wheat cultivar susceptible to Pst.Therefore,the overexpression of TaRIPK1 promoted Pst development in wheat,which further confirmed that TaRIPK1 was involved in the interaction between wheat and Pst as a susceptible gene.2.A septum-promoting GTP-binding protein 1,Ps Spg1,a key pathogenic factor secreted by Pst,enhances phosphorylation of TaRIPK1,resulting in its re-localization to the nucleus.A yeast two-hybrid system was used to screen the compatible yeast library between wheat and Pst,and Pst septum-promoting GTP-binding protein 1（Ps Spg1）was screened out and identified as Ps Spg1.The protein of Pt Spg1（P.triticina）and Pg Spg1（P.graminis）were obtained by sequence homologous cloning.TaRIPK1 could interact with Ps Spg1 and Pt Spg1,but not with Pt Spg1 were indicated by Y2H,co-immunoprecipitation and firefly luciferase complementation imaging assay（split-LUC assay）.Through sequence analysis,it was found that Ps Spg1 and Pt Spg1 had a high similarity in the variable region at the N-terminal of the protein sequence,leading to the interaction between Ps Spg1 or Pt Spg1 with TaRIPK1.LC-MS analysis indicated that TaRIPK1 is phosphorylated at five sites,and We wondered whether the interaction between Ps Spg1 and TaRIPK1 might affect the phosphorylation of TaRIPK1.In vitro and in vivo phosphorylation verification demonstrated that Ps Spg1 enhanced the phosphorylation of TaRIPK1.The interaction of Ps Spg1 with TaRIPK1 promotes translocation of TaRIPK1 from the plasma membrane into the nucleus,suggesting that Ps Spg1 promoted the translocation of TaRIPK1 from the plasma membrane to the nucleus by increasing the phosphorylation of TaRIPK1.The transient expression mediated by Agrobacterium invasion preliminarily confirmed that wheat plants with overexpression of Ps Spg1 were more susceptible to disease than the control plants,indicating that Ps Spg1 may promote the pathogenicity of Pst.The Ps Spg1silencing transgenic plants were obtained by RNAi mediated method,and it was found that the Ps Spg1-RNAi plants showed broad-spectrum resistance to the major predominant isolates of Pst.Thus,Ps Spg1 plays vital role in the important role in promoting the pathogenicity of Pst.3.TaRIPK1 phosphorylates the CCAAT box-binding transcription factor Ta CBF1,which promotes transcription of TaRIPK1.Among the selected candidate targets of TaRIPK1,CCAAT-box binding transcription factor Ta CBF1 was identified.There are three copies of Ta CBF1 in the wheat genome,located on chromosome 6A（Ta CBF1a）,6B（Ta CBF1b）and 6D（Ta CBF1d）.The proteins encoded by all three Ta CBF1 genes were found to interact with TaRIPK1 by using the Y2H assay,split-LUC assay and co-immunoprecipitation assay,and Ta CBF1d interacted more strongly with TaRIPK1 than did the other two homologues based on the split-LUC assay.In addition,due to phosphorylation assay,indicating that TaRIPK1 enters the nucleus and phosphorylates the transcription factor Ta CBF1d.Then,the candidate regulatory genes of Ta CBF1d were obtained by using the chromatin immunoprecipitation sequencing（Ch IP-seq）,among which the TaRIPK1 gene was enriched 13.5 fold compared with the control.Electrophoretic mobility shift assays（EMSAs）demonstrated the binding of Ta CBF1d to the promoter of TaRIPK1 containing these CCAAT-box elements.Dual luciferase reporter assays in wheat protoplasts showed that expression of Ta CBF1d-GFP increased expression from the TaRIPK1 promoter when compared to the GFP control and co-expression of Ta CBF1d-GFP and TaRIPK1significantly increased expression when compared to Ta CBF1d-GFP alone.Thus,phosphorylation of Ta CBF1d by TaRIPK1 increases expression of TaRIPK1,so providing a positive feedback loop,and the function of TaRIPK1 in susceptibility was further amplified.4.Phosphorylated Ta CBF1 binds to and represses the expression of resistance-associated gene Ta VPE4.The overexpressed transgenic plants of Ta CBF1d also showed resistance to the major predominant isolates of Pst,and the overexpressed transgenic plants of Ta CBF1d produced a large number of HRs on the leaf surface and a large amount of H₂O₂ burst during the infection stage,indicating that Ta CBF1d is essential for wheat resistance to Pst.Among the target genes regulated by Ta CBF1d,there are two significantly enriched vacuolar processing enzyme involved in ROS and cell necrosis,Ta VPEs.EMSAs demonstrated the binding of Ta CBF1d to the promoter of Ta VPE4 containing CCAAT-box elements,and phosphorylated Ta CBF1 can inhibit its transcription of this gene,thus TaRIPK1 helped to incite the resistant gene Ta CBF1 to function as a susceptible process.5.Broad-spectrum resistance wheat plant was obtained by CRISPR-Cas9-mediated TaRIPK1 editing.The three alleles of TaRIPK1 in wheat were simultaneously edited by CRISPR-Cas9editing technology.After the detection of the putative off-target genes predicted by Cas-OFFinder,it was found that TaRIPK1 gene editing plants to off-target other genes.Therefore,the TaRIPK1 gene knockout mutant plants were successfully obtained.It was found that major predominant isolates of Pst and P.triticina were inoculated against the TaRIPK1 gene knockout plant,which showed broad-spectrum resistance to the Pst and Pt,and the evaluation results of the main agronomic traits confirmed that the TaRIPK1 gene knockout plants retains the key agronomic traits.The TaRIPK1 gene knockout plants and the WT（Fielder）showed resistance to the Pst population in the field where heavy stripe rust infection occurred,and the key agronomic traits were not different from those of the Fielder.Therefore,TaRIPK1-knockout plants have the potential to be used for control the stripe rust in the field.