A mutation affecting symbiosis in the pea line Risnod27 changes the ion selectivity filter of the DMI1 homolog

After identifying regions of cDNA conserved between the symbiotic gene DMI1 of the model species Medicago truncatula and the homologous genomic region of Arabidopsis thaliana, universal primers were designed from 8 of 12 exons to allow the routine amplification of plant homologs. As an example, the complete homologous sequence from the pea (Pisum sativum L.) was amplified and sequenced, although the poorly conserved 5′-end and 5′-flanking region of the gene had to be amplified using a modified TAIL-PCR strategy. The identity of this amplified homolog with the SYM8 gene was independently confirmed by the presence of a single nucleotide change in the coding sequence of the mutant line Risnod27 (sym8) that cosegregated with the asymbiotic phenotype. Five insertions in pea introns responsible for increasing the total length of SYM8 by 1443 bp, compared to the M. truncatula homolog DMI1, belong to known transposon and retrotransposon families of pea and legumes in general. In view of the predicted function of SYM8 as an ion channel, the Risnod27 mutation (His309Tyr) appears to be localized in the selectivity filter domain. This finding confirms the essential role of histidine 309 in the symbiotic function of SYM8 and provides a guide to its ionic specificity. In view of the Risnod27 symbiotic phenotype, we hypothesize that SYM8 does not have identical functions in the transduction of rhizobial and mycorrhizal signals. The variability of the N-proximal region of the known legume homologs of DMI1 suggests an interaction with a variable ligand.