豆科植物能与根瘤菌形成共生体－根瘤，根瘤的形成是根瘤菌侵染和根瘤器官发生协同作用的结果。遗传学分析表明，根瘤菌侵染和根瘤器官发生可以是两个相对独立的过程，而CCaMK在协调这两个过程中起关键调控作用。CYCLOPS是CCaMK的一个磷酸化底物，CCaMK和CYCLOPS形成复合体是根瘤菌侵染所必需的而独立于根瘤器官发生。申请者以CCaMK的蛋白激酶结构域为诱饵，在百脉根中鉴定到CCaMK的另一个磷酸化底物CIP73。本申请项目拟在此基础上，进一步研究不同组成型激酶活性的CCaMK（缺失自我抑制结构域或自磷酸化位点突变）磷酸化CIP73和CYCLOPS之间的异同，揭示CIP73被CCaMK磷酸化的生物学意义。另外，研究CCaMK、CIP73和CYCLOPS能否形成三元复合体，以及筛选与CIP73相互作用的新蛋白，以揭示CIP73的生化和生物学功能，阐明CIP73在结瘤信号通路中的调控机制。

The formation of nitrogen-fixing nodules between legume plants and rhizobia requires the coordinated development of rhizobial infection and nodule organogenisis, which can be separated genetically. Calcium/calmodulin-dependent protein kinase (CCaMK) is a central regulator for both rhizobial infection and nodule organogenesis, and CYCLOPS is a phosphorylation substrate of CCaMK. CYCLOPS forms an ancient signal transduction complex with CCaMK that is specifically required for infection, but is dispensable for nodule organogensis. Using the constitutively active CCaMK as bait in yeast two-hybrid screening, we identified a novel ubiquitin-like protein named CIP73 which is another phosphorylaton target of CCaMK. The CCaMK autophosphorylation defect mutant (T265I) led to spontaneous nodule development. This mutant can also phosphorylate myelin basic protein in a Ca2+/CaM dependent manner, although the activity was lower as compared to the wild-type. The deletion mutant, CCaMK (1-311) and CCaMK (1-326), which also led to spontaneous nodulation, phosphorylated GS peptide constitutively independent of Ca2+ and CaM. Although both of the T265I and the deletion mutants can activate nodule morphogenesis, only the T265I mutant is fully functional to allow bacterial entry, but not the deletion mutants. Based on these, we would like to test whether different CCaMK mutants were functionally different in the phosphorylation of CYCLOPS or CIP73. Determining the differences in the biochemical mechanisms will help to illustrate the functional significance of phosphorylation of CIP73 and CYCLOPS by CCaMK. On the other hand, studying whether CCaMK, CYCLOPS and CIP73 could form ternary complex and screening new CIP73-interacting proteins will help to understand the biochemical and biological functions of CIP73 in the nodulation signaling pathway.