The Function Of TaSnRK2.1 In The Interaction Between Wheat And Puccinia Triticina

Wheat(Triticum aestivum L.)leaf rust is caused by the infection of the highly specialized Puccinia triticina,which is a kind of parasitic fungi lived on wheat.The outbreak of P.triticina had led to a significant reduction in wheat production,which is a serious threat to food security.Our laboratory has long been engaged in the molecular mechanism of wheat resistance to P.triticina infection.In the incompatible combination,wheat inhibits the development of P.triticina by hypersensitive reaction-PCD(HR-PCD).Previous studies in the laboratory found that wheat translationally controlled tumour proteinl(TaTCTP)can respond to P.triticina infection and participates in the formation of HR-PCD,which has a positive regulatory effect on the cell death of HR-PCD.In order to further investigate the mechanism of TaTCTP in the process of wheat resistance to P.triticina,our group used TAP(tandem affinity purification)and mass spectrometry to identify a Ser/Thr protein kinase,which might be a potential interaction protein of TaTCTP.Phylogenetic tree analysis revealed that the protein kinase belongs to the sucrose non-fermentative protein kinase 2(SnRK2)family and was higher homology with the SnRK2.1 subfamily protein,so we named it TaSnRK2.1.In the transcriptome data,it was found that the transcription level of TaSnRK2.1 was up-regulated and higher in the affinity combination(Tc×260)than that in the incompatibility combination(TcLr26×260).Therefore,we take TaSnRK2.1 as the gene of interest.The full-length coding sequence of TaSnRK2.1 was obtained by gene cloning technology,and then its function and mechanism were further studied.In terms of function,we first analyzed the expression changes of TaSnRK2.1 in different affinity combinations,and then further to study the effect of TaSnRK2.1 on wheat resistance on leaf rust by virusinduced gene silencing technique.We also analyzed the subcellular localization of TaSnRK2.1 by GFP fusion expression technology.In terms of mechanism,we first studied the interaction state of TaSnRK2.1 and TaTCTP by protein interaction technology,and further analyzed whether TaTCTP can be served as a phosphorylation substrate of TaSnRK2.1 by in vitro phosphorylation assay.The main results obtained in this assay were as follows:1 The transcriptional level expression of TaSnRK2.1 gene in Tc and TcLr26 was detected by qRT-PCR after inoculated with P.triticina races 260.The results showed that TaSnRK2.1 was up-regulated after inoculation with P.triticina,and the relative expression in Tc was higher than that inTcLr26.Thus,it was proved that TaSnRK2.1 gene was involved in wheat resistance against P.triticina.2 The distribution of TaSnRK2.1 in tobacco epidermal cell was analyzed by GFP fluorescent protein fusion expression technique.The results showed that TaSnRK2.1 was not only present in the cytoplasm but also in the nucleus.3 The gene silence of TaSnRK2.1 was conducted by VIGS technology,The results showed that after silencing TaSnRK2.1 in the affinity combination(Tc×260),the area of hyphae decreased compared with the control,indicating that the development of P.triticina was blocked and the plant resistance was enhanced.After silencing TaSnRK2.1 in the unaffinity combination(TcLr26×260),the HR area and the number of haustorial mother cells(HMC)were reduced compared with the control,which also demonstrated the plant resistance was enhanced.4 It was verified TaSnRK2.1 and TaTCTP could physically interact with each other by yeast two-hybrid(Y2H).Subsequent bi-molecular fluorescence complementation(BiFC)and co-immunoprecipitation(Co-IP)demonstrated that TaSnRK2.1 can interact with TaTCTP under physiological conditions in plant.The results of bimolecular fluorescence complementation assay showed that the interaction between TaSnRK2.1 and TaTCTP not only existed in the nucleus but also in cytoplasm,which is consistent with the subcellular localization of TaSnRK2.1.5 In vitro phosphorylation assay demonstrated that TaSnRK2.1 has significant kinase activity,whereas TaSnRK2.1 could not phosphorylate TaTCTP.Further studies showed that TaTCTP inhibited the kinase activity of TaSnRK2.1.