Abstract
To investigate the structure-function relationship of plant cyclic nucleotide-gated ion channels (CNGCs), we identified a total of 29 mutant alleles of the chimeric AtCNGC11/12 gene that induces multiple defense responses in the Arabidopsis (Arabidopsis thaliana) mutant, constitutive expresser of PR genes22 (cpr22). Based on computational modeling, two new alleles, S100 (AtCNGC11/12:G459R) and S137 (AtCNGC11/12:R381H), were identified as counterparts of human CNGA3 (a human CNGC) mutants. Both mutants lost all cpr22-mediated phenotypes. Transient expression in Nicotiana benthamiana as well as functional complementation in yeast (Saccharomyces cerevisiae) showed that both AtCNGC11/12:G459R and AtCNGC11/12:R381H have alterations in their channel function. Site-directed mutagenesis coupled with fast-protein liquid chromatography using recombinantly expressed C-terminal peptides indicated that both mutations significantly influence subunit stoichiometry to form multimeric channels. This observation was confirmed by bimolecular fluorescence complementation in planta. Taken together, we have identified two residues that are likely important for subunit interaction for plant CNGCs and likely for animal CNGCs as well.
Highlights
To investigate the structure-function relationship of plant cyclic nucleotide-gated ion channels (CNGCs), we identified a total of 29 mutant alleles of the chimeric AtCNGC11/12 gene that induces multiple defense responses in the Arabidopsis (Arabidopsis thaliana) mutant, constitutive expresser of PR genes22
We describe a total of 29 mutant alleles of AtCNGC11/12, including the three previously published alleles (Baxter et al, 2008; Chin at al., 2010), and compare their predicted three-dimensional structural positions with equivalent mutations of a human CNGC, CNGA3
Important Residues for CNGC Subunit Interaction findings that cpr22 (AtCNGC11/12)-mediated phenotypes are attributable to the expression of AtCNGC11/ 12 (Yoshioka et al, 2006; Baxter et al, 2008)
Summary
To investigate the structure-function relationship of plant cyclic nucleotide-gated ion channels (CNGCs), we identified a total of 29 mutant alleles of the chimeric AtCNGC11/12 gene that induces multiple defense responses in the Arabidopsis (Arabidopsis thaliana) mutant, constitutive expresser of PR genes (cpr). We have reported two functionally important residues in plant CNGCs (Baxter et al, 2008; Chin et al, 2010) These residues were discovered using a suppressor screen of the rare gain-of-function Arabidopsis mutant constitutive expresser of PR genes (cpr22; Yoshioka et al, 2006). It has been reported that the cpr phenotype is attributable to the expression of AtCNGC11/12 and its channel activity (Yoshioka et al, 2006; Baxter et al, 2008), thereby making the suppressor screen an invaluable tool for identifying intragenic mutants to further elucidate the structure-function relationship of plant CNGCs (Baxter et al, 2008; Chin et al, 2010)
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