Functional Analysis Of Casein Kinase TaCK2α Involved In Wheat Resistance To Puccinia Triticina

Casein kinase 2(CK2),a ubiquitous and highly conserved serine/threonine kinase,which is belong to polygene family.It is involved in plant response to various stresses and regulates the expression of downstream target genes.The role of CK2 in the interaction between wheat and Puccinia triticina(Pt)is not yet clear,so it is of great significance to explore whether CK2 gene in wheat is involved in the defense reaction against Pt.In our previous study,transcriptome analysis was carried out on TcLr19 wheat inoculated with Pt and a CK2 was screened.In this study,a CK2 gene named TaCK2α was cloned from resistant wheat variety TcLr19,and its genetic characteristics,kinase activity,antifungal ability and function involved in wheat resistance to Pt was analyzed.The main results of this study are as followings:1.In order to clarify the genetic relationship between TaCK2α and CK2 in different plant species,sequence alignment was carried out and phylogenetic trees were constructed.The results showed that TaCK2α was classified into a single branch of CK2α,which is closely related to TaCK2α of Brachypodium distachyon,T.aestivum and Lolium perenne.Quantitative real-time PCR(qPCR)was used to detect the expression pattern of TaCK2α.The expression level of TaCK2α peaked at 48 hours post inoculation(hpi)in the incompatible combination between wheat and Pt,which was about 6.32 times higher than that at 0 hpi,indicating that the expression of TaCK2α was induced by Pt.TaCK2αexpression peaked at 2 hours after treated with salicylic acid(SA),which was about 3 times higher than that at 0 hours,indicating that TaCK2α respond to SA signaling pathway.Kinase activity experiments show that TaCK2α has kinase activity.2.In order to verify whether TaCK2α can inhibit the germination and growth of Pt,antifungal ability tests in vitro and in vivo were carried out.The results in vitro showed that the average urediospore germination rate and mycelium length of TaCK2α protein decreased by 23%and 56%compared with the control group,respectively,which suggest that the germination of urediospore and the growth of mycelium could be inhibited by TaCK2α protein.The results in vivo showed that the disease severity of wheat treated with TaCK2α protein decreased,with fewer urediospore and accumulation of H2O2,indicating that wheat resistant ability to Pt was enhanced.In addition,the function of TaCK2α was verified by virus-induced gene silencing(VIGS)technology.The mycelium length and infection area in silenced plants increase significantly compared with wild type,and the accumulation of H2O2 decreased significantly,which suggested that TaCK2α can be involved in wheat resistance to Pt.3.In order to screen the interacting targets of TaCK2α,the recombinant vector pGBKT7-TaCK2α was constructed successfully.34 candidate targets that may interact with TaCK2α were obtained by yeast two-hybrid(Y2H).Among them,TaCK2β is the regulatory subunit of CK2,the interaction between TaCK2α and TaCK2β was preliminarily verified by Y2H.TaCK2β gene was cloned from TcLr19 wheat,and its expression pattern was detected by qPCR.It is clear that the TaCK2β was induced by Pt.In order to explore the significance of the interaction between TaCK2α and TaCK2β,the expression patterns of TaCK2α and TaCK2β in wheat after inoculation with Pt were analyzed.The expression pattern of TaCK2β in wheat was similar to TaCK2α at different time points after Pt infection,they were highly expressed at 48 hpi,suggesting that TaCK2α and TaCK2β have synergistic effects in wheat resistance to Pt.Taken together,TaCK2α encoding casein kinase was cloned in wheat.It was proved that the TaCK2α protein has kinase activity,and might be involved in wheat defense response to Pt.The interacting target of TaCK2α was obtained,TaCK2β and TaCK2α play a synergistic role in wheat disease resistance to Pt,which will provide a theoretical basis for further analysis of the molecular mechanism of TaCK2α involved in wheat Pt resistance.