Functional Analysis And In Vitro Activity Assay Of Wheat Cysteine Protease TaCP During The Interaction Of Wheat And Puccinia Striiformis F.sp.tritici

The wheat stripe rust caused by Puccinia striiformis f.sp.tritici（Pst）seriously threatens the food security.The constantly emerging of new Pst races due to synergistic evolution between host and pathogen usually result in the loss of wheat resistance in Agriculture.Hence,it is greatly necessary to explore the mechanisms of Pst interaction and searchmore disease-resistant resources for breeding durable resistant cultivars,which is a huge challenge for global rust researchers.In the process of infecting wheat,Pst induced PTI（PAMP-triggered immunity）and ETI（Effectors-triggered immunity）.Cysteine protease（CP）is a kind of proteolytic enzyme involved in plant growth,immunity and a series of defense reactions.Many researches have reported that PLCPs（Papain-like cysteine Proteases）can be used as the central hubs to regulate the occurrence of immune response.Based on screening the c DNA library of wheat-Pst incompatible interaction,we obtained a cysteine protease gene of wheat-TaCP;which is highly induced in wheat changed by the avirulence.Thereby,in this study we futher investiagted its role during wheat and Pst interaction,which will lay foundation for the in-depth analysis of the molecular mechanism of wheat resistance to stripe rust and provid genetic resources for the genetic improvement of resistance to Pst in wheat.The main research results are as follows:1.Identify the characterization of cysteine protease family in wheat,TaCP gene was cloned from the c DNA library of wheat leaves infected with avirulence Pst,which encoded a protein of 377 amino acids with molecular weight 41.05KDa,the isoelectric point 5.60.SMART analysis showed that the wheat TaCP belongs to the PLCPs family of cysteine proteases,which contains signal peptide,Cathepsin propeptide inhibitor domain,Peptidase C1A and transmembrane structure at the N-terminus.The Phylogenetic relationship revealed that in the Arabidopsis thaliana PLCP subfamily classification,TaCP belongs to the RD19-Like subfamily.2.Clarify the function of TaCP in disease resistance to Pst:Quantitative Real-time PCR（Q-RT-PCR）analysis showed that the expression of TaCP reached the peak at 12 hpi in the incompatible interaction between wheat"Swon 11"and avirulence race CYR23.In the compatible interaction between"Suwon 11"and virulence race CYR31,the expression of TaCP was slightly up-regulated at 12 hpi.To futher analysis the function of TaCP in wheat"Swon 11"and CYR23,virus-induced gene silencing（VIGS）technology was used to silenced TaCP in wheat.The results showed that the TaCP knock-down plants,which changed by the avirulence race CYR23,obvious sporulation was observed.Q-RT-PCR analysises revealed that Pst CYR23 biomass was increased significantly,so it was speculated that TaCP may be reduced the resistance of wheat interact with avirulence Pst.3.Due to TaCP as a kind of cysteine protease,we analysis the protease activity in vitro by Z-Phe-Arg-MCA and the secretion function by Yeast signal sequence trap（YSST）and transient expression of TaCP-SP-eGFP in Nicotiana benthamiana.The research results indicated that the signal peptide of TaCP has asecretory function.The TaCP-GST,TaCP^△inhibitor-GST and TaCP^△SP-GST fusion protein by prokaryotic expressionto analyze the protease activity in vitro;We found that the fluorescence intensity of TaCP^△inhibitorwas higher than TaCP^△SP,which showed that the activity of protease was inhibited by the Cathepsin propeptide inhibitor domain.This experiment also proved that TaCP is a specific cysteine protease,which can degrade cysteine protease cathepsin-specific substrate（Z-Phe-Arg-MCA）,and the highest protease activity could be detected at 4℃.It is revealed that TaCP perform a crucial role to participate in the process of resistance to Pst.