Functional Characterization Of CaCDPK15 And Its Application On Screening Of Small Chemicals Inducing Plant Defense

Calcium-dependent protein kinases(CDPKs)are a protein family functioning as important sensors of Ca2+ flux in plants.Through sensing and transmitting Ca2+ signal,they play essential roles in regulating plant growth,development and adaptation to biotic and abiotic stresses.However,the present work on this protein family is limited to a few of model plants and crop.The function and molecular mechanism of most members has not been reported yet.In order to elucidate the disease resistance mechanism of pepper which is an important kind of solanaceae vegetable,we studied the function of CaCDPK15,an important member of CDPKs,in pepper immune system against disease.Using transgenic tobacco lines containing the inductive promoter of CaCDPK15 fused to the reporter gene GUS,we constructed a high throughput screening system for small chemicals inducing plant defense and have obtained some practically useful chemicals.The main results are as follows:(1)The expression of CaCDPK15 was obviously up-regulated in wild type pepper GZ03 treated with salicylic acid(SA),Methyl Jasmonate(MeJA),ethrel(ETH)and infected with the bacterial pathogen Ralstonia solanacearum.And the expression of CaCDPK15 responded faster to the treatment of ETH and Ralstonia solanacearum than to the application of SA and MeJA.Pepper plants overexpressing CaCDPK15 exhibited serious hypersensitive response(HR)and a up-regulated expression level of hypersensitive response marker gene CaHIR1,as well as several defense-associated genes CaPO2,CaDEF,CaACO1,CaNPR1,CaPR1,CaPR1b and CaABR1.Consistently,pepper plants with CaCDPK15 silenced by virus-induced gene silencing(VIGS)showed enhanced susceptibility to the highly virulent Ralstonia solanacearum strain FJC100301,and this was correlated with decreased transcripts of defense-related pepper genes.These CaCDPK15 silenced pepper plants dead one week post infection of Ralstonia solanacearum and the colony forming unit(CFU)was higher than the control.Together,the above data suggests that CaCDPK15 can induce hypersensitive response and is involved in pepper defense responses through SA-,JA-,ET-,ABA-mediated signal transduction pathways.(2)In order to find SA-,JA-,ET-and Ralstonia solanacearum-responsive regions in the CaCDPK15 promoter,we constructed transgenic tobacco lines containing promoter fragments ranging from-240 to-1091 bp fused to GUS reporter gene(lines CaCDPK15-240::GUS,CaCDPK15-463::GUS,CaCDPK15-558::GUS,CaCDPK15-1091::GUS)and performed the 5' mutants assay.The GUS activity quantitative assay of transgenic tobacco lines treated with SA,MeJA,ETH and Ralstonia solanacearum showed that lines containing CaCDPK15-46::GUS,CaCDPK15-558::GUS,CaCDPK15-1091::GUS displayed obvious GUS up-regulation expression in contrast to the control.Moreover,GUS expression in the CaCDPK15-558::GUS and CaCDPK15-1091::GUS lines can also be induced by ETH.While no induction was observed for the CaCDPK15-240::GUS line.These results suggest that SA-,JA-and Ralstonia solanacearum-responsive regions must be located between positions-463bp and-240bp,while ET-responsive regions must be located between positions-558bp and-463bp.(3)Using transgenic tobacco containing the CaCDPK15 promoter fragment fused to GUS,we constructed a high throughput screening system for small chemicals inducing plant defense and have obtained two useful chemicals:C8H6CINO4(Methyl 4-Chloro-3-nitrobenzoate)and C13H7Cl4NO(2,4-Dichloro-6-{(E)-[(2,4-dichlorophenyl)imino]methyl phenol).Real-time PCR assay indicated that the expression level of SA-related gene NPR1 increased in tobacco treated with these two small chemicals suggesting that they could be involved in the SA signaling process.