A novel yeast two-hybrid approach to identify CDPK substrates: characterization of the interaction between AtCPK11 and AtDi19, a nuclear zinc finger protein

Calcium-dependent protein kinases (CDPKs) are sensor-transducer proteins capable of decoding calcium signals in diverse phosphorylation-dependent calcium signaling networks in plants and some protists. Using a novel yeast two-hybrid (YTH) approach with constitutively active and/or catalytically inactive forms of AtCPK11 as bait, we identified AtDi19 as an AtCPK11-interacting protein. AtDi19 is a member of a small family of stress-induced genes. The interaction was confirmed using pull-down assays with in vitro translated AtCPK11 and GST-AtDi19 and localization studies in Arabidopsis protoplasts cotransfected with AtCPK11:GFP and AtDi19:DsRed2 protein fusions. We further showed that the interaction of AtDi19 is specific to both AtCPK4 and AtCPK11, whereas other closely related CPKs from Arabidopsis interacted weakly (e.g., AtCPK12) or did not interact (e.g., AtCPK26, AtCPK5 and AtCPK1) with AtDi19. Deletion analyses showed that a region containing two predicted nuclear localization signals (NLS) and a nuclear export signal (NES) of AtDi19 is essential for interaction with AtCPK11. We further demonstrated that AtDi19 is phosphorylated by AtCPK11 in a Ca(2+)-dependent manner at Thr105 and Ser107 within the AtDi19 bipartite NLS using in vitro kinase assays. Our data suggest that disruption of the autoinhibitor domain leading to the formation of a constitutively active CDPK may stabilize kinase-substrate interactions without affecting specificity.     

番茄AT-hook基因家族的鉴定及胁迫条件下的表达分析

AT-hook蛋白家族在植物生长发育、器官构建及逆境胁迫和激素信号应答中发挥重要作用。本研究在番茄基因组范围内,利用生物信息学方法对番茄AT-hook基因家族的成员、分布、结构和功能进行分析。结果表明,番茄AT-hook家族包含32个成员,分为3种类型,其中类型Ⅰ含有13个成员;遗传进化分析表明番茄AT-hook基因成员与拟南芥家族基因具有相似分类。利用实时荧光定量PCR对番茄32个基因开展组织表达分析,结果表明AT-hook基因具有表达差异,主要在根和花中表达较高。氧化胁迫分析结果表明,32个基因受ABA、SA、盐、高温和低温诱导表达,其中部分基因显著上调或下调表达,很可能参与了番茄逆境胁迫条件下的防御应答反应。本研究结果将为番茄AT-hook家族基因的深入研究提供依据,为进一步解析番茄AT-hook基因的功能奠定基础。     

Expression Profiles of Arabidopsis thaliana VQ Gene Family in Defense Related Hormones Treatments

Several VQ proteins are recently identified as WRKY factors interacting partners in Arabidopsis and involved in regulating physiological processes. Searching of genomic databases found that VQ gene family is specific to land plants and these VQ genes encode proteins characteristic of a conserved VQ motif. It consists of 34 representatives in Arabidopsis and can be divided into two groups based on the similarity of the amino acid sequences. To understand the functions of Arabidopsis VQ proteins, we examined the expression profiles of AtVQ genes in various defense related hormones treatments. qRT PCR analysis revealed that a majority of them were differently regulated in response to salicylic acid (SA), methyl jasmonate (MeJA), or 1 aminocyclopropane 1 carboxylate (ACC). And some members are induced by two of these three hormones. Moreover, four members (AtVQ3, AtVQ18, AtVQ23 and AtVQ24) are induced by SA, MeJA and ACC simultaneity. However, there is only one AtVQ gene (AtVQ27) is up regulated after spraying of abscisic acid (ABA). These results suggest that Arabidopsis VQ genes may be involved in plant defense responses.     

拟南芥VQ基因家族响应抗性相关激素表达谱分析

近年来与WRKY转录因子相互作用的VQ-motif蛋白逐渐引起广泛关注.它是一类植物特异性蛋白,目前在拟南芥(Arabidopsis thaliana)中已鉴定到34个成员,据其所编码氨基酸的序列相似性构建系统进化树,这34个成员聚集成两大簇.实时定量荧光PCR分析表明在SA(水杨酸),MeJA(茉莉酸甲酯),ACC(乙烯前体),ABA(脱落酸)处理下AtVQ基因家族中有多个成员分别受SA,JA,ET高倍诱导,部分成员受SA,JA,ET中两种激素诱导,其中AtVQ3,AtVQ18,AtVQ23和AtVQ24同时受SA,JA,ET诱导,仅AtVQ27受ABA诱导,推测拟南芥VQ家族成员在抗病方面起作用.