It is almost certain that Ca2+ fluxes play a crucial role in the activation of defense responses. Induction of ROS and activation of mitogen-activated protein kinase cascades are two examples of many responses that are facilitated by Ca2+ influxes.
- However, to date, little is known about the molecular identity of the channels that generate this Ca2+ flux after pathogen recognition. Based on genomic sequence data, Arabidopsis contains over 150 cation transport proteins. Among them, candidate ion channels that may be involved in pathogen-inducible Ca2+ influx include the two-pore channel, TPC1, the ionotropic glutamate receptors (GLRs), and cyclic nucleotide-gated ion channels (CNGCs).
- So far, no strong evidence for a role of GLRs in plant immunity has been presented and the role of TPC1 in defense in Arabidopsis is disputed. However, in rice and tobacco, TPC1 plays an important role in elicitor-induced defense responses.
- Thus, CNGCs have emerged as one of the strongest candidates for Ca2+ -conducting channels involved in plant immune responses. Their overall physiological functions, structural aspects as well as ion selectivity have already been summarized elsewhere. Therefore, in this review, we concentrated on the role of CNGCs in plant immunity, focusing mostly on the well studied Arabidopsis CNGCs. Arabidopsis CNGC family comprises 20 members.
- Through Arabidopsis mutant screens, four CNGC mutants have been identified that are involved in immune responses. The first one was discovered through a screen that aimed to identify mutants that lacked HR upon infection with the avirulent bacterial pathogen, Pseudomonas syringae pv. glycinea expressing avrRpt2.
- The mutant identified was named dnd1 (defense, no death1) to reflect its phenotype, which maintains R gene-mediated resistance with greatly reduced HR cell death. This mutant was later revealed to be a null mutant of AtCNGC2 due to a creation of a premature stop codon. dnd1 mutant plants not only exhibit a lack of HR phenotype upon infection with an avirulent pathogen, but also display constitutively activated defense responses, such as elevated levels of SA, constitutive expression of PR genes, and increased resistance against virulent strains of P. syringae and H. arabidopsidis (Yu et al., 1998; Genger et al., 2008). Further analysis also revealed that this mutant is a conditional lesion mimic mutant in that it displays spontaneous lesion formation without induction by the presence of an avirulent pathogen (Clough et al., 2000). In addition, these lesions appear to be dependent on environmental conditions, as the degree of lesion formation increases with increased light intensity and lower humidity (Jirage et al., 2001). The second and the third mutants reported to be involved in defense responses are hlm1/dnd2 (HR-like lesion mimic1/defense no-death2) that are null mutants for AtCNGC4. hlm1/dnd2 exhibit remarkably similar phenotypes to dnd1, such as impaired HR responses, while maintaining resistance against avirulent pathogens, exhibiting enhanced broad-spectrum resistance against virulent pathogens, elevated levels of SA, and constitutive expression of PR genes (Balague´ et al., 2003; Jurkowski et al., 2004). The barley nec1 mutant, which also displays elevated levels of PR genes and a lesion mimic phenotype, was also found to have a mutation in the barley homolog of AtCNGC4. AtCNGC2 and AtCNGC4 are phylogenetically closest to each other and comprise one sub-group in the CNGC family tree and double knockout mutants exhibited additive. The fourth mutant identified was a rare gain-of-function mutant, cpr22.This mutant also shares similarity in its phenotypes with dnd1 and dnd2/hlm1, such as constitutive expression of PR genes, elevated levels of SA, and broadspectrum resistance against virulent pathogens. However, it does induce HR upon infection with Pseudomonas syringae pv. tomato carrying avrRpt2, which distinguishes cpr22 from dnd1 and hlm1/dnd2 (Yoshioka et al., 2006). Genetic and molecular analysis revealed the cpr22 mutation as a 3-kb deletion that fuses two tandemly repeated CNGCs (AtCNGC11 and AtCNGC12) to generate a novel chimeric gene AtCNGC11/12 and that phenotypes conferred by cpr22 are attributable to the expression and function of the chimeric AtCNGC11/12 and not the loss of AtCNGC11 or 12 (Yoshioka et al., 2006; Baxter et al., 2008). cpr22 also shows spontaneous lesion formation that is environmentally sensitive (conditional). In addition, T-DNA insertion knockout mutants for AtCNGC11 and 12 exhibit a partial breakdown of resistance against avirulent, but not virulent H. arabidopsidis, indicating that both AtCNGC11 and 12 act as positive regulators of R gene-mediated resistance responses (Yoshioka et al., 2006). Additional support for this notion stems from experiments using the bacterial pathogen P. syringae. Infection with avirulent P. syringae (avrRpt2) led to 5–10 times greater pathogen growth in null mutants for either of genes cngc11-1 and cngc12-1 compared to wild-type plants, indicating a partial breakdown of R gene-mediated resistance against different pathogens (Figure 1A). On the other hand, after infection with virulent P. syringae, or after treatment with a MAMP, the bacterial flagellin peptide, flg22 (Felix et al., 1999), no significant difference in bacterial growth or callose deposition, respectively, was observed in both knockout lines compared to wild-type plants (Figure 1B and data not shown). These observations further confirmed the involvement of AtCNGC11 and 12 specifically in R gene-mediated resistance and not MAMP-triggered basal resistance. Since cpr22 has a mutation that affects both genes, double silencing lines for AtCNGC11 and AtCNGC12 have been created (Urquhart, 2010 and Supplementary Data). As shown in Figure 1A, two silencing lines (11.12-RNAi-1 and –2) exhibited a similar degree of breakdown in R gene-mediated resistance compared to the corresponding single knockout mutants cngc11-1 and cngc12-1. This suggested that, although AtCNGC11 and AtCNGC12 are both involved in R genemediated resistance, there is no clear synergism. AtCNGC11 and 12 are not only tandemly repeated, but also are phylogenetically the closest related CNGC family members (Ma¨ser et al., 2001). These data, together with the data obtained from single knockout mutants, further support the notion that AtCNGC11 and 12 may be subunits in the same heterotetrameric channel, as previously suggested (Yoshioka et al., 2006).