Minisymposium 11: Plant-symbiont interactions

Abs # 24004: Identification and characterization of novel calcium binding proteins in the symbiosome space of M. truncatula nodules

Presenter:	Liu, Junqi , liuxx162@tc.umn.edu
Authors	Liu, Junqi  (A)   Miller, Sue  (D)   Bucciarelli, Bruna  (D)   Fedorova, Maria  (A)   Matsumoto, Peter  (A)   Graham, Michelle  (B)   Sherrier, Janine  (E)   Samac, Deborah  (C) (D)  Gannt, Steve  (B)   Vance, Carroll P (A) (D)
Affiliations:	(A): Department of Agronomy and Plant Genetics, University of Minnesota (B): Plant Biology, University of Minnesota (C): Plant Pathology, University of Minnesota (D): USDA-ARS, Plant Science Research Unit (E): Department of Plant and Soil Sciences, University of Delaware

Calcium is a key messenger and has been implicated in the mediation of a variety of signaling processes including the legume-rhizobium symbiosis. Calmodulins (CaMs) are the prevalent calcium sensors which respond to transient changes in the cytosolic free [Ca²⁺]. Upon Ca²⁺-CaM binding, the subsequent Ca²⁺/CaM complex can affect the function of diverse proteins, such as transcription factors, metabolic enzymes, and ion channels. The specificity and subcellular nature of calcium signaling remains largely unclear. Analysis of the Medicago truncatula root nodule transcriptome revealed a group of 6 nodule specific transcripts encoding calmodulin-like proteins, designated as NodCaMs (for nodule specific calmodulin-like proteins). Unlike typical CaMs with four EF hands, NodCaMs only have two to three EF hands. All 6 NodCaMs are predicted to possess an N-terminal signal peptide. By using NodCaM fusion proteins overexpressed in E.coli, we have proven that NodCaMs bind Ca²⁺ in vitro. Moreover, we found that two of the NodCaM genes are located within a 7 kb region of the Medicago genome, in tandem orientation. A NodCaM promoter was fused to GUS and EGFP and used in the transformation of Medicago sativa. Expression of the NodCaM::GUS reporter gene was highly specific being detected only during nodule development. Semi-quantitative RT-PCR analysis further showed a striking increase in mRNA abundance in nodules between 6 to 8 days after inoculation. Both in situ hybridization and GUS staining showed NodCaM expression was very strong in infected cells whereas little to no expression was observed in uninfected cells in mature nodules. In addition, purified symbiosome space protein contained a highly enriched polypeptide that is recognized by NodCaM antibody. The subcellular localization of NodCAMs in purified symbiosome space protein was confirmed by MS/MS sequencing of antibody reactive protein. Phylogenetic analysis of NodCAMs and all full-length plant calmodulins in the GenBank nonredundant database showed that NodCaMs consistently grouped outside a clade of all typical calmodulins, suggesting diversification of NodCaMs within M. truncatula. Taken together the genomic, biochemical, histological and molecular data indicate that NodCaMs represent a novel class of calcium sensors located in the symbiosome space with root nodule specific function.