title: Structural implications of mutations in the pea SYM8 symbiosis gene, the DMI1 ortholog, encoding a predicted ion channel.
abstract: The Pisum sativum SYM8 gene plays an essential part in both rhizobial and mycorrhizal symbioses. Mutation of sym8 in the original type line R25 blocks nodulation, mycorrhization, and Nod-factor-induced calcium spiking, an early component of the nodulation signaling pathway. We describe four new sym8 alleles of pea, which fall into the same complementation group as R25. The sym8 mutants are phenotypically similar to Medicago truncatula dmi1 mutants and map to a syntenic location. We used sequence homology to isolate the pea ortholog of M. truncatula DMI1 and have shown that the cloned pea ortholog can complement a M. truncatula dmi1 mutant for nodulation. Each of the five pea sym8 mutants carries a mutation in the DMI1 ortholog, confirming that the pea SYM8 is the DMI1 ortholog. Based on predicted structural similarities with an archaebacterial ion channel, we propose that SYM8 forms a tetrameric calcium-gated channel of a predicted structure similar to the archaebacterial potassium channel but containing a filter region that is different. The predicted structure identifies four aspartate residues (one from each subunit) forming the channel opening. We made a mutation changing the aspartate to valine and identified a missense mutation (changing alanine to valine adjacent to the aspartate residues) in this predicted filter region; both mutations caused a loss of function. We also identified a loss-of-function missense mutation (changing arginine to isoleucine) in a domain proposed to link the predicted channel and the gating ring domains, indicating that this mutation may block function by preventing a protein conformational change being transmitted from the gating-ring domain to the pore domain.
authors.affiliation: Department of Molecular Microbiology, John Innes Centre, Colney Lane, NR4 7UH, Norwich, UK. anne.edwards@bbsrc.ac.uk
authors.forename: Anne
authors.initials: A
authors.lastname: Edwards
authors.forename: Anne B
authors.initials: AB
authors.lastname: Heckmann
authors.forename: Faridoon
authors.initials: F
authors.lastname: Yousafzai
authors.forename: Gerard
authors.initials: G
authors.lastname: Duc
authors.forename: J Allan
authors.initials: JA
authors.lastname: Downie
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018506:Gene Expression Regulation, Plant; D007473:Ion Channels / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D046913:Medicago truncatula / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D018532:Pisum sativum / Q000235:genetics* / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D013559:Symbiosis / Q000235:genetics*
chemical_list: D007473:Ion Channels; D010940:Plant Proteins