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

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.

Fig. 1.

Sequence of DMI3. Amino acid sequence of M. truncatula DMI3 (MtDmi3) aligned with the Lilium longiflorum CCaMK 1 (Llyck1) is shown. Amino acids that are identical in the two proteins are connected by vertical bars. Residues shown in red indicate the serine/threonine kinase domain. Residues shown in blue indicate the CaM-binding domain. Residues shown in green indicate EF-hand motifs that specify three different calcium-binding domains. The dmi3 mutation causes a frameshift resulting in a 9-aa tail before the protein is prematurely terminated (shown in orange above the DMI3 sequence). Mutations in pea result in translation termination marked by orange asterisks above the sequence. Mutation R72 results in termination at amino acid 216, mutations P1 and P2 result in termination at amino acid 222, and mutations P53 and P54 result in termination at amino acid 232.