Studies On Molecular Mechanisms Of TaCBL4/TaCIPK5 And TaCIPK10 In Wheat Resistance Against Stripe Rust

Plants have evolved complex mechanisms in response to invasion of pathogens,which are involved in recognition of the pathogens,signals transduction and defense genes expression.The calcium ions,as secondary messenger,play an important role in signal transduction of plant development and response to environmental stimuli.Intracellular increase in Ca²⁺consentration is perceived by various Ca²⁺sensors to initiate various cellular signal transduction events.Calcineurin B-like proteins（CBLs）are one of calcium sensors in plant cell,which regulate the activity of CBL interacting protein kinase（CIPK）.It has been confirmed that salicylic acid（SA）signal is critical for plant resistant to biotrophic fungus.In addition,reactive oxygen species（ROS）has been shown to be invovled in plant resistance.Wheat stripe rust,caused by an obligate biotrophic fungus Puccinia striiformis f.sp.tritici（Pst）poses a tremendous threat to the production of wheat worldwide.However,the roles of CIPKs in immune signaling of wheat and the relationship of CBL-CIPK system and SA or ROS signals in wheat resistane to Pst are under debate.Therefore,we characterized the roles of TaCBL/TaCIP5 and TaCIPK10 in the interaction between wheat and Pst.1.Based on wheat genome database,a genome-wide search using protein sequences from Arabidopsis and rice CBLs and CIPKs as query revealed a total of 22 putative CBL（7,6,and 9 loci in sub-genomes A,B,and D,respectively）and 74 putative CIPK（25,24,and25 loci in sub-genomes A,B,and D,respectively）loci in wheat.We tentatively designated them as TaCBL1.1 to TaCBL9 and TaCIPK2 to TaCIPK32,following the relationship with homologs in rice.As for AtCBLs and AtCIPKs,four EF hands were found in all TaCBLs and all TaCIPKs consist of a conserved N-terminal kinase domain,followed by a variable junction domain and a C-terminal regulatory domain.In the rather divergent regulatory domain,the conserved NAF/FISL and protein-phosphatase interaction（PPI）motifs were identified.An active loop motif was also identified in the kinase domain.The resulting dendrogram showed that CBLs from various plants cluster in three major groups:type I,type II,and type III.TaCBLs are distributed among all three clusters.Proteins belonging to the type I group begin with a lipid modification motif（i.e.,MGCXXS/T）,and CBLs in the type II group contain the consensus motif MSQCXDGXKHXCXSXXXCF,which also exists in tonoplast targeting sequences（TTSs）of members in the type III group possesses an extended N-terminus.In addition,all CIPKs were classified into two distinct clusters based on the number of introns:the intron-less group and the intron-rich group.Subcellular localization assays indicated that TaCBL1.1,TaCBL2,TaCBL6 and TaCBL9 were targeted to plasma membrane of wheat cells.TaCBL3 was located in vacuolar membrane,whereas the TaCBL4 was detected throughout wheat cells,including the cytoplasm and the nucleus.For wheat CIPK proteins,except for TaCIPK2 and TaCIPK31,which appear exclusively in the plasma membrane,all other selected TaCIPKs displayed a localization pattern similar to that of the GFP control,indicating a cytoplasmic and nuclear localization.2.In our previous study,the transcript level of TaCBL4 had been shown to be consistence with that of TaCIPK5 under Pst infection.Additionally,both TaCBL4 and TaCIPK5 were rapidly induced under the flg22 treatment or in the Pst322（a putative effector of Pst）overexpressed leaves,with similar expression patterns.Thus,we supposed that TaCBL4 and TaCIPK5 co-regulated wheat resistance to Pst.To further verify our speculation,the interaction between TaCIPK5 and TaCBL4 in planta was confirmed by yeast two-hybrid system,BiFC and Co-IP assay.Transient silencing of TaCBL4 or TaCIPK5 respectively expressed weakened resistance characterized by decreased necrotic area,increased fungal infection structures and uredia production in response to virulent Pst race CYR31.Co-silencing of TaCBL4 and TaCIPK5 significantly weakened wheat resistance against Pst infection.Moreover,the disease resistance phenotype of TaCBL4 and TaCIPK5 co-silenced plants（TaCBL4/TaCIPK5）was consistent with that of TaCBL4-or TaCIPK5-knockdown plants.In addition,the accumulation of ROS was significantly altered in all silencing plants during Pst infection.Ten RBOH-type genes were identified from Triticum aestivum by a genome-wide search.One of them,designed as TaRBOHi,was confirmed to interact with TaCIPK5 in Y2H system.Moreover,the relative expression of TaRBOHiwassignificantlyreducedinTaCBL4-,TaCIPK5-and TaCBL4/TaCIPK5-knockdown plants.Together these findings demonstrate that the TaCBL4-TaCIPK5 complex positively modulates wheat resistance in an ROS-dependent manner.3.The result of qRT-PCR assay showed that TaCIPK10 is significantly induced by Pst infection and SA treatment,suggesting that TaCIPK10 participates in wheat resistance against Pst in a SA-dependent manner.The in vitro phosphorylation assay indicated that the kinase activity of TaCIPK10 is regulated by Ca²⁺and TaCBL4.Knockdown TaCIPK10significantly reduced wheat resistance to Pst,whereas TaCIPK10 overexpression resulted in enhanced wheat resistance to Pst by the induction of defense response in different aspects,including hypersensitive cell death,ROS accumulation and pathogenesis-relative genes expression.Moreover,TaCIPK10 physically interacted with and phosphorylated TaNH2,which is the homolog of AtNPR3/4.In addition,Ser-518 of TaNH2 is the principal amino acid for phosphorylation of TaNH2 in vitro.Transient silencing of TaNH2 was significantly reduced wheat resistance indicating that TaNH2 plays a positive role in wheat resistance against Pst.Together these findings indicate that TaCIPK10 positively regulate wheat resistance to Pst as molecular links between of Ca²⁺and downstream components of defense response and TaCIPK10 interacts with and phosphorylates TaNH2 to regulate wheat resistance to Pst.Our results imply a new mechanism of crosstalk between the SA signals and CBL-CIPK pathways in regulating wheat disease resistance against Pst.