title: A novel interaction between CCaMK and a protein containing the Scythe_N ubiquitin-like domain in Lotus japonicus.
abstract: 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.
authors.affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Heng
authors.initials: H
authors.lastname: Kang
authors.forename: Hui
authors.initials: H
authors.lastname: Zhu
authors.forename: Xiaojie
authors.initials: X
authors.lastname: Chu
authors.forename: Zhenzhen
authors.initials: Z
authors.lastname: Yang
authors.forename: Songli
authors.initials: S
authors.lastname: Yuan
authors.forename: Dunqiang
authors.initials: D
authors.lastname: Yu
authors.forename: Chao
authors.initials: C
authors.lastname: Wang
authors.forename: Zonglie
authors.initials: Z
authors.lastname: Hong
authors.forename: Zhongming
authors.initials: Z
authors.lastname: Zhang
mesh_terms: D000595:Amino Acid Sequence; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D000070116:Lotus / Q000201:enzymology* / Q000382:microbiology; D008969:Molecular Sequence Data; D038821:Mycorrhizae / Q000502:physiology; D010641:Phenotype; D010766:Phosphorylation; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism*; D055170:Plant Root Nodulation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D034622:RNA Interference; D012231:Rhizobium / Q000502:physiology; D013347:Subcellular Fractions / Q000378:metabolism; D013379:Substrate Specificity; D020798:Two-Hybrid System Techniques
chemical_list: D010940:Plant Proteins; D017871:Calcium-Calmodulin-Dependent Protein Kinases