Molecular Mechanisms Of The Receptorlike Cytoplasmic Kinase CECK3 In Regulating Plant Immunity And Cell Death

In the process of long-term interaction and co-evolution with pathogens,plants have developed the PAMP-triggered Immunity（PTI）and Effector-triggered Immunity（ETI）to prevent the invasion of pathogens.ETI,which is regulated by plants’R proteins,can sometimes lead to hypersensitive cell death（HCD）.Although HCD plays an important role in plant immunty,its regulatory mechanism is poorly understood.This thesis used the Arabidopsis（Arabidopsis thaliana）and tomato（Solanum lycopersicum）as research materials and studied the mechanism of Arabidopsis CRCK3 and MEKK2 in regulating plant cell death as well as the molecular mechanism of tomato Sl CRCK3and Sl CDPK10 in modulating plant immunity.The research results were obtained as follows:1. Silencing of MEKK1 by VIGS（VIGS-MEKK1）resulted in autoimmune phenotypes resembling the mekk1 mutant.While the mekk2,summ2-8 and crck3 mutants inhibited the cell death caused by silencing of MEKK1,indicating that MEKK2,SUMM2 and CRCK3positively regulate MEKK1-mediated plant cell death.2. Overexpression of MEKK2 and kinase dead MEKK2^KM in Arabidopsis wild-type caused plant cell death phenotype.Interestingly,overexpression of CRCK3 leaded to similar plant cell death,while overexpression of kinase mutant CRCK3^KM could not cause cell death.The results indicate that the kinase activity of CRCK3 is essential,whereas the kinase activity of MEKK2 is dispensable,for triggering cell death.3. The results of co-immunoprecipitation and pull-down assays show that MEKK2interacts with CRCK3.Interestingly,CRCK3 protein was unstable in mekk2 mutant,and transiently co-expression of MEKK2 and CRCK3 in tobacco significantly increased the accumulation of CRCK3 protein,indicating that MEKK2 positively regulates the stability of CRCK3.4. Tomato Solyc07g00780.2.1 shares 66.8%similarity with Arabidopsis CRCK3 in amino acid sequence,thus was named as Sl CRCK3.Sl CRCK3 contains a signal peptide and an intracellular kinase domain.Subcellular location results revealed that Sl CRCK3 mainly located on plasma membrane.5. Quantitative RT-PCR（q RT-PCR）results revealed that the transcription of Sl CRCK3increased significantly in response to Pst DC3000 infection.Besides,the tomato plants silencing of Sl CRCK3 were more susceptible to Pst DC3000 and fungal pathogen Botrytis cinerea,while Sl CRCK3 overexpressed tomato plants were more resistant to Pst DC3000.Those results indicate that Sl CRCK3 positively regulates the disease resistance of tomato.6. Heterologous overexpression of Sl CRCK3 elevated the expression of flg22-inducedgenes and early PTI response in Arabidopsis,and enhanced the resistance to Pst DC3000,indicating that Sl CRCK3 positively regulates plants’immune responses.7. The results of yeast two-hybrid and co-immunoprecipitation assays demonstrate that Sl CRCK3 interacts with Sl CDPK10.The disease inoculation test results showed that there was no significant difference in resistant to Pst DC3000 between tomato plants co-silencing of Sl CRCK3/Sl CDPK10 and tomato plants silencing of Sl CRCK3 or Sl CDPK10,indicating that Sl CRCK3 and Sl CDPK10 function synergistically in regulating disease resistance of tomato.In summary,Arabidopsis MEKK2 stabilizes CRCK3 and positively regulates plant cell death,and tomato Sl CRCK3 interacts with Sl CDPK10 and positively regulates plant immunity.