A nuclear-targeted cameleon demonstrates intranuclear Ca2+ spiking in Medicago truncatula root hairs in response to rhizobial nodulation factors.
Lipochitooligosaccharide nodulation factors (NFs) secreted by endosymbiotic nitrogen-fixing rhizobia trigger Ca(2+) spiking in the cytoplasmic perinuclear region of host legume root hairs. To determine whether NFs also elicit Ca(2+) responses within the plant cell nucleus we have made use of a nucleoplasmin-tagged cameleon (NupYC2.1). Confocal microscopy using this nuclear-specific calcium reporter has revealed sustained and regular Ca(2+) spiking within the nuclear compartment of Medicago truncatula root hairs treated with Sinorhizobium meliloti NFs. Since the activation of Ca(2+) oscillations is blocked in M. truncatula nfp, dmi1, and dmi2 mutants, and unaltered in a dmi3 background, it is likely that intranuclear spiking lies on the established NF-dependent signal transduction pathway, leading to cytoplasmic calcium spiking. A semiautomated mathematical procedure has been developed to identify and analyze nuclear Ca(2+) spiking profiles, and has revealed high cell-to-cell variability in terms of both periodicity and spike duration. Time-lapse imaging of the cameleon Förster resonance energy transfer-based ratio has allowed us to visualize the nuclear spiking variability in situ and to demonstrate the absence of spiking synchrony between adjacent growing root hairs. Finally, spatio-temporal analysis of the asymmetric nuclear spike suggests that the initial rapid increase in Ca(2+) concentration occurs principally in the vicinity of the nuclear envelope. The discovery that rhizobial NF perception leads to the activation of cell-autonomous Ca(2+) oscillations on both sides of the nuclear envelope raises major questions about the respective roles of the cytoplasmic and nuclear compartments in transducing this key endosymbiotic signal.
Sieberer
Björn J
BJ
Laboratory of Plant-Microbe Interactions, UMR CNRS-INRA 2594/441, F-31320 Castanet-Tolosan, France.
Chabaud
Mireille
M
Timmers
Antonius C
AC
Monin
André
A
Fournier
Joëlle
J
Barker
David G
DG
D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D031541:Fluorescence Resonance Energy Transfer; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D046913:Medicago truncatula / Q000235:genetics / Q000378:metabolism*; D018613:Microscopy, Confocal; D056489:Nucleoplasmins / Q000378:metabolism; D055170:Plant Root Nodulation; D018517:Plant Roots / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D015398:Signal Transduction; D016962:Sinorhizobium meliloti / Q000502:physiology; D000071857:Tumor Suppressor p53-Binding Protein 1
D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D056489:Nucleoplasmins; D011994:Recombinant Proteins; C498302:Trp53bp1 protein, mouse; D000071857:Tumor Suppressor p53-Binding Protein 1; C507867:yellow cameleon 2.1; D002118:Calcium
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