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Deregulation of a Ca2+/calmodulin-dependent kinase leads to spontaneous nodule development


Induced development of a new plant organ in response to rhizobia is the most prominent manifestation of legume root-nodule symbiosis with nitrogen-fixing bacteria. Here we show that the complex root-nodule organogenic programme can be genetically deregulated to trigger de novo nodule formation in the absence of rhizobia or exogenous rhizobial signals. In an ethylmethane sulphonate-induced snf1 (spontaneous nodule formation) mutant of Lotus japonicus, a single amino-acid replacement in a Ca2+/calmodulin-dependent protein kinase (CCaMK) is sufficient to turn fully differentiated root cortical cells into meristematic founder cells of root nodule primordia. These spontaneous nodules are genuine nodules with an ontogeny similar to that of rhizobial-induced root nodules, corroborating previous physiological studies1. Using two receptor-deficient genetic backgrounds we provide evidence for a developmentally integrated spontaneous nodulation process that is independent of lipochitin–oligosaccharide signal perception and oscillations in Ca2+ second messenger levels. Our results reveal a key regulatory position of CCaMK upstream of all components required for cell-cycle activation, and a phenotypically divergent series of mutant alleles demonstrates positive and negative regulation of the process.

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Figure 1: Phenotype of snf1-1 mutants and ontogeny of spontaneous nodules.
Figure 2: Nod-factor receptor-independent development of spontaneous nodules and complementation of the ccamk-2 loss of function mutation.
Figure 3: The CCaMK gene and expression of CCaMK and Nin in wild-type and hypernodulation har1-5 mutants.
Figure 4: Domains and in vitro kinase activity of CCaMK protein.

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We thank Finn Pedersen for plant care and crossing.

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Correspondence to Jens Stougaard.

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The sequences for the L. japonicus ecotype GIFU CCaMK gene and mRNA are deposited in the EMBL nucleotide database under accession numbers AM230792 and AM230793. Reprints and permissions information is available at The authors declare no competing financial interests.

Supplementary information

Supplementary Figure 1

Calcium oscillations in root hairs in response to purified Nod factor. (PPT 294 kb)

Supplementary Figure 2

In vitro culture of wild type and snf1-1 mutant tissues. (PPT 33607 kb)

Supplementary Figure 3

Alignment of chimeric Ca2+/calmodulin dependent protein kinases from Lotus, Medicago, pea, tobacco, lily, rice and Physcomitrella. (RTF 165 kb)

Supplementary Figure 4

The log-log plot corresponding to figure 4e. (PPT 47 kb)

Supplementary Methods

This file contains a detailed Methods section. (DOC 30 kb)

Supplementary Table 1

Summary of loss of function ccamk mutations and gain of function snf1 mutations. (DOC 24 kb)

Supplementary Table 2

This file shows complementation of ccamk-2 in the presence of Mesorhizobium loti; transformation of ccamk-2 in the absence of Mesorhizobium loti; and transformation of snf1-1 in the absence of Mesorhizobium loti. (DOC 41 kb)

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Tirichine, L., Imaizumi-Anraku, H., Yoshida, S. et al. Deregulation of a Ca2+/calmodulin-dependent kinase leads to spontaneous nodule development. Nature 441, 1153–1156 (2006).

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