Isolation And Functional Analysis Of Three Kinase Genes In Wheat Response To Rhizoctonia Cerealis

Wheat sharp eyespot, mainly caused by Rhizoctonia cerealis, is one of the most devasting diseases ofwheat in the world. Because the genetics of wheat resistance to R. cerealis is not fully understood andthe applied wheat cultivars are susceptible to sharp eyespot, it is urgent to unravel the mechanism ofwheat defense response to R. cerealis and to identify the genes involved in wheat defense response to R.cerealis. Protein kinase played important roles in perceiving pathogen infection, subsequent signalingpathways and ultimately inducing plant defense response. However, no kinase gene involved in wheatresistance to R. cerealis had yet been identified. In this study, using microarray, RNA-Sequencing(RNA-Seq), and semi-RT-PCR analysis, three protein kinase genes, including TaCRK1, TaWAK5, andTaPK-R1, were identified to be associated with the resistance degrees of wheat lines to R. cerealisinfection.The TaCRK1gene encoded a cystein-rich receptor protein kinase with680amino acid residues.Transcription of TaCRK1was induced in CI12633after infection with R. cerealis and exogenousabscisic acid (ABA) treatment. The TaWAK5gene encoded a wall-associated kinase of703amino acidresidues. The expression of TaWAK5was induced by infection with R. cerealis and treatments withexogenous salicylic acid (SA), abscisic acid, and methyl jasmonate (MeJA). Subcellular localizationanalysis in onion epidermal cells or wheat protoplasts indicated that the TaCRK1protein and TaWAK5protein were localized to the plasma membranes. Characterization of TaCRK1silencing and TaWAK5induced by virus-mediated method in CI12633, respcetively showed that the down-regulation ofTaCRK1or TaWAK5transcript did not obviously impair resistance to R. cerealis, suggesting that thesetwo genes were not the major genes involved in the wheat defense response to R. cerealis.The TaPK-R1gene, including13exons and12introns, encoded a wheat protein A/proteinG/protein C with557amino acid residues. The expression of the TaPK-R1transcript was induced by R.cerealis and by exogenous ethylene and hydrogen peroxide. Moreover, transcript abundance ofTaPK-R1were higher in the R. cerealis-resistant lines (CI12633, Shanhongmai, Navit14, Shannong0431, and Xifeng) than that in the R. cerealis-susceptible lines (Yangmai158, Wenma6, and Zhoumai18). Transient expression of TaPK-R1in onion epidermal cells or wheat protoplast indicated that theTaPK-R1protein was localized to the cytoplasm and nucleus. The protein kinase domain ofrecombinant GST-TaPK-R1showed autophosphorylated activity. Characterization of TaPK-R1silencinginduced by virus-mediated method in CI12633showed that down-regulation of TaPK-R1gene inCI12633significantly compromise wheat resisitance to R. cerealis. Over-expression of TaPK-R1gene intransgenic wheat plants confers increased resistance to wheat sharp eyespot accompanied withdecreased R. cerealis abundance. These results indicated that the TaPK-R1gene played an importantrole in wheat defense response to R. cerealis. This study indicated that the AGC protein kinase wasinvolved in plant defense response to necrotrophic pathogen in wheat for the first time. The TaPK-R1transgenic plants improved wheat resistance to R. cerealis and lay a foundation for unraveling the mechanis mof wheat defense response to sharp eyespot.