Identification of a functionally essential amino acid for Arabidopsis cyclic nucleotide gated ion channels using the chimeric AtCNGC11/12 gene

Abstract

We used the chimeric Arabidopsis cyclic nucleotide-gated ion channel AtCNGC11/12 to conduct a structurefunction study of plant cyclic nucleotide-gated ion channels (CNGCs). AtCNGC11/12 induces multiple pathogen resistance responses in the Arabidopsis mutant constitutive expresser of PR genes 22 (cpr22). A genetic screen for mutants that suppress cpr22-conferred phenotypes identified an intragenic mutant, #73, which has a glutamate to lysine substitution (E519K) at the beginning of the eighth b-sheet of the cyclic nucleotide-binding domain in AtCNGC11/12. The #73 mutant is morphologically identical to wild-type plants and has lost cpr22-related phenotypes including spontaneous cell death and enhanced pathogen resistance. Heterologous expression analysis using a K + -uptake-deficient yeast mutant revealed that this Glu519 is important for AtCNGC11/12 channel function, proving that the occurrence of cpr22 phenotypes requires active channel function of AtCNGC11/12. Additionally, Glu519 was also found to be important for the function of the wild-type channel AtCNGC12. Computational structural modeling and in vitro cAMP-binding assays suggest that Glu519 is a key residue for the structural stability of AtCNGCs and contributes to the interaction of the cyclic nucleotide-binding domain and the C-linker domain, rather than the binding of cAMP. Furthermore, a mutation in the a-subunit of the human cone receptor CNGA3 that causes total color blindness aligned well to the position of Glu519 in AtCNGC11/12. This suggests that AtCNGC11/12 suppressors could be a useful tool for discovering important residues not only for plant CNGCs but also for CNGCs in general.

Multiple Calmodulin-binding Sites Positively and Negatively Regulate Arabidopsis CYCLIC NUCLEOTIDE-GATED CHANNEL12

“…Transient heterologous expression of chimeric At-CNGC11/12 in N. benthamiana triggers Ca 2+ -dependent spontaneous HR-like PCD, suggesting that CNGC11/12 is a constitutively active and/or misregulated form of CNGC12, which is a positive regulator of immunity (Baxter et al, 2008;Urquhart et al, 2007). Low temperature is known to enhance immune responses in plants (Hua, 2013), and shifting to lower temperature has been previously shown to enhance the PCD phenotype induced by CNGC11/12 mutants (Mosher et al, 2010;Chin et al, 2010).…”

“…CNGC12 is a positive regulator of immunity (Moeder et al, 2011), and CNGC11/12 was hypothesized to represent a misregulated form of CNGC12 that constitutively induces autoimmune phenotypes including hypersensitive response (HR)-like programmed cell death (PCD) in a constitutive manner (Yoshioka et al, 2006;Urquhart et al, 2007;Baxter et al, 2008). Activation of such autoimmunity can be assessed via Agrobacterium tumefaciens-mediated transient expression of At-CNGC11/12 in Nicotiana benthamiana, where CNGC11/12-GFP, but not CNGC12-GFP, can induce PCD (Yoshioka et al, 2006).…”

“…Because the chimeric CNGC11/12 is hypothesized to comprise a misregulated form of CNGC12 and constitutively activates PCD, it has proven to be an excellent tool to study channel structurefunction (Baxter et al, 2008;Chin et al, 2010;Abdel-Hamid et al, 2013). CNGC11/12 possesses the C-terminal half of CNGC12 and therefore shares the CNGC12 IQ and CT motifs.…”

“…CNGC12 fragments were expressed using either the pET28 or pGEX4T3 vectors (for 6xHis-or GST-tagged fusion proteins, respectively, as indicated in the figures) in BL21 (DE3) codon plus E. coli cells. Constructs for in planta expression as GFP fusions were cloned into the pJP (pMBP3) binary plasmid (Baxter et al, 2008) in frame with a C-terminal GFP tag. Site-directed mutagenesis was performed in vitro using overlapping PCR reactions.…”

Calmodulin binding to Arabidopsis cyclic nucleotide gated ion channels

“…15,16 It has been reported that the expression of AtCNGC11/12 and its channel activity is attributable for the cpr22 phenotype. 17,18 A genetic screen for mutants that suppress cpr22-conferred phenotypes identified over 20 novel mutant alleles in AtCNGC11/12. 1,18 These intragenic mutants are excellent tools to study the structure-function relationship of plant CNGCs.…”