A Novel Interaction between CCaMK and a Protein Containing the Scythe_N Ubiquitin-Like Domain in Lotus japonicus
A Novel Interaction between CCaMK and a Protein Containing the Scythe_N Ubiquitin-Like Domain in Lotus japonicus
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DOI：
10.1104/pp.110.167965
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发表时间：
2011-03-01
期刊：
影响因子：
7.4
通讯作者：
Zhang, Zhongming
中科院分区：
文献类型：
--
作者：
Kang, Heng;Zhu, Hui;Zhang, Zhongming
在根瘤菌-豆科植物共生系统中，钙/钙调素依赖性蛋白激酶（CCaMK）是根瘤菌感染和根瘤器官形成的关键调节因子。通过自磷酸化位点的点突变或羧基末端调节结构域的缺失，CCaMK的失调导致在没有根瘤菌的情况下自发形成根瘤。然而，对潜在的生化机制知之甚少。在这里，使用的激酶结构域的CCaMK作为诱饵在酵母双杂交筛选，我们确定了一种新的蛋白质，CIP 73（CCaMK相互作用的蛋白质约73 kD），与CCaMK相互作用。CIP 73含有一个Scythe_N泛素样结构域，属于泛素超家族。缺失和突变分析表明，CIP 73只能与CCaMK相互作用时，钙调素结合结构域和三个EF-手基序从激酶结构域被删除。CCaMK的氨基末端80个氨基酸残基（80-160）是在酵母细胞中与CIP 73相互作用所必需的。本氏烟草（Nicotiana benthamiana）的蛋白质下拉实验和双分子荧光互补实验表明，全长CCaMK在体外和植物体内均能与CIP 73相互作用。重要的是，CCaMK在体外以Ca 2 +/钙调蛋白依赖性方式磷酸化CIP 73的氨基末端。CIP 73转录物优先在根中表达，并且在叶、茎和根瘤中检测到非常低的表达。接种中慢生根瘤菌后，根中的表达量显著降低。RNA干扰敲低CIP 73表达的毛状根转化在百脉根导致根瘤形成减少，表明CIP 73在结瘤中发挥重要作用。
In the Rhizobium-legume symbiosis, calcium/calmodulin-dependent protein kinase (CCaMK) is a key regulator for both rhizobial infection and nodule organogenesis. Deregulation of CCaMK by either a point mutation in the autophosphorylation site or the deletion of the carboxyl-terminal regulatory domain results in spontaneous nodule formation without rhizobia. However, the underlying biochemical mechanisms are poorly understood. Here, using the kinase domain of CCaMK as a bait in yeast two-hybrid screening, we identify a novel protein, CIP73 (for CCaMK-interacting protein of approximately 73 kD), that interacts with CCaMK. CIP73 contains a Scythe_N ubiquitin-like domain and belongs to the large ubiquitin superfamily. Deletion and mutagenesis analysis demonstrate that CIP73 could only interact with CCaMK when the calmodulin-binding domain and three EF-hand motifs are removed from the kinase domain. The amino-terminal 80 amino acid residues (80-160) of CCaMK are required for interacting with CIP73 in yeast cells. On the other hand, protein pull-down assay and bimolecular fluorescence complementation assay in Nicotiana benthamiana show that the full-length CCaMK could interact with CIP73 in vitro and in planta. Importantly, CCaMK phosphorylates the amino terminus of CIP73 in a Ca2+/calmodulin-dependent manner in vitro. CIP73 transcripts are preferentially expressed in roots, and very low expression is detected in leaves, stems, and nodules. The expression in roots is significantly decreased after inoculation of Mesorhizobium loti. RNA interference knockdown of CIP73 expression by hairy root transformation in Lotus japonicus led to decreased nodule formation, suggesting that CIP73 performed an essential role in nodulation.