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PNAS 101 (13): 4701-4705

Copyright © 2004 by the National Academy of Sciences.

From The Cover

Plant Biology

A Ca2+/calmodulin-dependent protein kinase required for symbiotic nodule development: Gene identification by transcript-based cloning

Raka M. Mitra *, Cynthia A. Gleason {dagger}, Anne Edwards {dagger}, James Hadfield {dagger}, J. Allan Downie {dagger}, Giles E. D. Oldroyd {dagger}, and Sharon R. Long * {ddagger}

*Department of Biological Sciences, 371 Serra Mall, Stanford University, Stanford, CA 94305-5020; and {dagger}John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, United Kingdom

Contributed by Sharon R. Long, January 27, 2004

Abstract: In the establishment of the legume-rhizobial symbiosis, bacterial lipochitooligosaccharide signaling molecules termed Nod factors activate the formation of a novel root organ, the nodule. Nod factors elicit several responses in plant root hair cells, including oscillations in cytoplasmic calcium levels (termed calcium spiking) and alterations in root hair growth. A number of plant mutants with defects in the Nod factor signaling pathway have been identified. One such Medicago truncatula mutant, dmi3, exhibits calcium spiking and root hair swelling in response to Nod factor, but fails to initiate symbiotic gene expression or cell divisions for nodule formation. On the basis of these data, it is thought that the dmi3 mutant perceives Nod factor but fails to transduce the signal downstream of calcium spiking. Additionally, the dmi3 mutant is defective in the symbiosis with mycorrhizal fungi, indicating the importance of the encoded protein in multiple symbioses. We report the identification of the DMI3 gene, using a gene cloning method based on transcript abundance. We show that transcript-based cloning is a valid approach for cloning genes in barley, indicating the value of this technology in crop plants. DMI3 encodes a calcium/calmodulin-dependent protein kinase. Mutants in pea sym9 have phenotypes similar to dmi3 and have alterations in this gene. The DMI3 class of proteins is well conserved among plants that interact with mycorrhizal fungi, but it is less conserved in Arabidopsis thaliana, which does not participate in the mycorrhizal symbiosis.

Abbreviations: CaM, calmodulin; CaMKII, calcium/CaM-dependent kinase II; CCaMK, calcium/CaM-dependent protein kinase; TCs, tentative consensus sequences.

Data deposition: The sequences reported in this paper have been deposited in the GenBank database (accession no. AY496049 for M. truncatula DMI3) and in the European Molecular Biology Laboratory database (accession no. AJ621916 for P. sativum SYM9).

See Commentary on page 4339.

{ddagger} To whom correspondence should be addressed. E-mail: srl{at}stanford.edu.

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