Functional Characterization Of Arabidopsis Calmodulin-binding Protein 60b In Immunity

Plants are sessile.To cope with various pathogens they encounter,plants have developed two strategies to sense and respond to pathogens.Firstly,Pathogen-or microbe-associated molecular patterns(PAMPs/MAMPs)are detected as nonself by host pattern recognition receptors(PRRs),composed of receptor-like kinase(RLKs)and receptor-like protein(RLPs),and activate pattern-triggered immunity(PTI).To counter PTI,pathogens release effectors that attack and destroy the plant immunity.However,the intracellular receptor resistant protein(R protein)of plants can sense these effectors in a direct or indirect way and trigger the effector-triggered immunity(ETI).When the immunity is activated,a number of key signaling modules are triggered,including rapid phosphorylation of receptor-like cytoplasmic kinases(RLCKs),an influx of calcium across PM,a burst of ROS,and activation of mitogen-activated protein kinase(MAPK)cascades,etc.Among them,transcriptional reprogramming induced by immune responses is an important process.A large number of immune response genes are activated to resist pathogen invasion.CBP60g and SARD1,members of the CBP60 family,are well characterized transcription factors that play major transcriptional functions in the immune response,and they redundantly activate the expression of immune genes in ETI,PTI,salicylic acid(SA)and systemic acquired resistance(SAR)pathways.Another member of the family,CBP60a,has been reported to negatively regulate immunity.However,the function of the remaining five highly conserved members of the CBP60 family,known as CBP60b-f,has not yet been elucidated.In this study,we examined the expression patterns of these five genes and determined that CBP60b is highly and constitutively expressed.Subsequently,by combining RT-q PCR,Ch IP-PCRs,luciferase(LUC)reporter assays,as well as genetic experiments,we demonstrated that CBP60b plays a central role in plant immunity.The main results and conclusions presented in this thesis are as follows:(1)CBP60b is constitutively expressedIn this study,we first analyzed the expression patterns of the remaining five members of the CBP60 family.Histochemical GUS staining of CBP60Bb-f genomic-GUS transgenic plants showed that CBP60b was highly and constitutively expressed,especially in the shoot tip and root tip.However,CBP60c-f was only slightly expressed in few tissues.The expression patterns of the five CBP60 family members suggested that CBP60b may play a role in plant development.(2)CBP60b loss-of-function compromises plant growth and results in autoimmunityNext,we analyzed their mutants and found that the growth of cbp60b was compromised,showing leaf malformation,defective SAM,abnormal distribution and accumulation of auxin,and so on.In comparison,the quadruple mutant cbp60c;d;e;f developed normally.Further analysis indicated that cbp60b was an autoimmune mutant,reflected by its reduced size and dwarfism,high levels of SA,enhanced immune genes expression,increased hydrogen peroxide(H₂O₂)and programmed cell death(PCD),and hyposensitivity to pathogens.(3)CBP60b acts as a transcriptional activator to positively regulate immunityWe determined that CBP60b is localized in the nucleus.CBP60b was induced by DC3000at both transcriptional and post-translational levels.By combining Ch IP-PCRs and luciferase(LUC)reporter assays,we determined that CBP60b bound to the consensus motif G(A/T)AATT(T/G)to activate the expression of immune genes,which are also target genes of CBP60g and SARD1.We also characterized the transcriptional activation motif of CBP60b.These results indicate that CBP60b acts as a transcriptional activator to positively regulate immunity.Further,we determined that overexpressing CBP60b also led to the constitutive activation of plant immunity,indicating that CBP60b positively regulates immunity.In addition,we demonstrated that the DNA binding domain and the CAM binding domain of CBP60b are necessary for its function.(4)R proteins monitor CBP60b or its pathwayThat CBP60b being a transcriptional activator for immune genes did not reconcile with the fact that cbp60b is an autoimmune mutant.We thus considered the possibility that CBP60b is a guardee monitored by R proteins.According to guard model,functionally impaired guardees activate cognate R proteins to induce ETI,leading to autoimmunity.To test whether autoimmunity of the cbp60b was due to guard-activation-induced ETI,we introduced eds1,pad4 and ndr1 into cbp60b,and found that functional loss of EDS1 or PAD4 completely suppressed the defective growth of cbp60b and functional loss of NDR1 significantly suppressed the defective growth of cbp60b,suggesting that CBP60b or its signaling pathway is monitored by both TNL and CNL type R proteins.In addition,TNL SNC1 mutation can partially restore the cbp60b phenotype,suggesting that SNC1 is one of the R proteins that monitor CBP60b.(5)CBP60b plays redundant yet distinct roles from CBP60g and SARD1 in immunitySince CBP60b,CBP60g and SARD1 regulate the same downstream genes,it is suggested that they have functional redundancy.Moreover,after cbp60g;sard1 was introduced into cbp60b,the cbp60b;cbp60g;sard1 was extremely weak,which also indicated this point.However,cbp60g;sard1 did not show autoimmune phenotype.By detecting their expression levels,we found that the expression level of CBP60b was much higher than that of CBP60g and SARD1 under normal growth condition,which explained that cbp60b showed developmental defects while cbp60g;sard1 did not.However,CBP60g or SARD1 driven by the promoter of CBP60b did not restore the autoimmunity of cbp60b,indicating their functional distinction.Surprisingly,we found that CBP60b can directly bind to the promoters of CBP60g and SARD1 to activate their expression.CBP60b is the only transcription factor that has been found to bind both CBP60g and SARD1 promoters to regulate their expression.In conclusion,CBP60b,as the core transcription factor in immunity,not only maintains the expression of immune genes during plant development,but also activates the expression of immune genes upon pathogen invasion,supporting plant defense to pathogens.