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 Ca 2+ /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.

The Small GTPase ROP6 Interacts with NFR5 and Is Involved in Nodule Formation inLotus japonicus

“…The ROP6 RNAi hairy roots were inoculated with a lacZlabeled strain of M. loti, which provided a marker for identification of ITs after staining with 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (X-gal) solution (Kang et al, 2011;Chen et al, 2012). Nine days after Rhizobium inoculation, the cellular locations and the numbers of ITs in the RNAi hairy roots were recorded.…”

Recent Advances in Calcium/Calmodulin-Mediated Signaling with an Emphasis on Plant-Microbe Interactions

“…Ca 2+ binding to the three EF hands in the VLD induces a change in the tertiary structure of CCaMK, activates its kinase activity, and also makes the Thr-271 accessible for autophosphorylation (state 2, transient and active, may phosphorylate substrates; Sathyanarayanan et al, 2000Sathyanarayanan et al, , 2001Gleason et al, 2006;Swainsbury et al, 2012). Thr-271 was reported to be the preferred and likely the first autophosphorylated site of CCaMK (Sathyanarayanan et al, 2001;Routray et al, 2013) Messinese et al, 2007;Yano et al, 2008;Kang et al, 2011). In addition, CaM binding could protect Ser-343 and Ser-344 from being phosphorylated by CCaMK, which is supported by the in vitro phosphorylation assays that demonstrated that CaM binding decreases the autophosphorylation level of CCaMK .…”

A MAP Kinase Kinase Interacts with SymRK and Regulates Nodule Organogenesis in Lotus japonicus

“…An M. loti strain labeled with lacZ (Tansengco et al, 2003;Kumagai et al, 2006;Kang et al, 2011) was used to infect hairy roots. In SIP2 RNAi hairy roots, the initiation of ITs and IT growth could still be observed ( Figure 5E).…”