Figure 6. SNI1 enhances binding of CBNAC to the E0-1-1 element. (A) Nucleotide sequence of the PR1 promoter indicating E0-1-1 element. The numbers indicate the position of the element relative to the PR1 translation start site. (B) EMSA analysis of the effect of SNI1. EMSA was performed using 32P-labeled E0-1-1 element (lanes 1 to 5), without (lane 1) or with the addition GST (lane 2; negative control), CBNAC (lanes 4 and 5) and SNI1 (lane 5). Equal amounts of CBNAC were used in the two lanes. the phenotypes of transgenic plants overexpressing the C-terminal truncated form of CBNAC/NTL9, but the cbnac1/ntl9-1 mutant and transgenic plants overexpressing the full-length CBNAC/NTL9 did not exhibit any pheno- types. It seems likely that the biological function of CBNAC/NTL9 could be misinterpreted from the pheno- types reported for transgenic plants overexpressing CBNAC/NTL9 devoid of its regulatory domain. In essence, the physiological function of CBNAC/NTL9 has not been elucidated to sufﬁcient detail. CBNAC is a CaM-binding transcriptional repressor that interacts with DNA through a GCTT core sequence ﬂanked on both sides by frequently repeating sequences (26). This study demonstrates CBNAC binds to the promoter of PR1, a marker gene for SA-induced defense responses and also inhibits pathogen-induced increase in PR1 expression. CBNAC alone, and in synergism with SNI1, negatively regulates basal resistance to PstDC3000. As CaM regulates the repressor function of CBNAC (26), these results imply that CBNAC is a point of crosstalk between calcium- and SA-mediated defense signaling. Linker-scanning (LS) mutagenesis of the PR1 promoter revealed at least three cis-elements (LS5, LS7 and LS10) displaying positive or negative characters (34). LS5 and LS7 both contain the binding sequence (TGACG) for Nucleic Acids Research, 2012, Vol. 40, No. 18 9189 TGA transcription factors and TGA2 independently binds the LS5 and LS7 promoter elements in vitro (10). The LS5 element seems to contribute to the negative regu- lation of PR1 expression both in the absence and presence of SA, whereas LS7 is required for SA-mediated PR1 in- duction (34). The GCTT cis-element for CBNAC binding to the PR1 promoter represents a novel PR1 regulatory module. Interestingly, CBNAC is recruited to the PR1 promoter in both non-treated and SA-treated tissues (Supplementary Figures S5C and S7). SA-independent binding of CBNAC to the PR1 promoter suggests that the DNA-binding activity of CBNAC is not altered by SA. Both PstDC3000 and SA induced CBNAC quickly in a transient way (Figure 1). The induction of the negative regulator during the early stages of infection might serve as a mechanism to prevent gratuitous or even harmful overactivation of pathogen-induced defense mechanisms when the population of the pathogen remains at relatively low levels (35). As pathogen growth increases, enhanced defense mechanisms would be necessary, and this could be achieved at least partially by suppressed expression and inactivation of negative regulators such as CBNAC and WRKY factors. Pathogen-induced expression of negative defense regu- lators could be explained by their involvement in the an- tagonistic crosstalk of distinct signaling pathways against various types of microbial pathogens. SA-mediated signal- ing activates defense mechanisms effective against biotrophic pathogens but can suppress ET/JA-mediated signaling in defense against necrotrophic pathogens (3). For example, overexpression of the transcription factor WRKY33 enhances susceptibility to the biotrophic pathogen PstDC3000 and increases resistance to necro- trophic fungal pathogens (36). cbnac1 plants showed enhanced resistance to biotrophic pathogen PstDC3000 but no difference to necrotrophic fungal pathogens. Consistently, the transcription levels of JA-responsive marker genes, PDF1.2 and LOX2, were similar in cbnac1 mutant and wild-type plants (Supplementary Figure S8). These results suggest that CBNAC is not involved in the antagonistic crosstalk of defense signaling pathways against biotrophic and necrotrophic pathogens. It is possible that CBNAC is involved in crosstalk between SA- and NPR1-mediated defense signaling and as yet un- identiﬁed environmental stress signaling. CBNAC acts synergistically with SNI1 as a transcriptional repressor of the PR1 gene SNI1, a transcriptional repressor of PR gene expression, has no apparent DNA-binding domain and is predicted to bind to DNA indirectly by interactions with DNA-binding proteins (23). CBNAC has been identiﬁed as one DNA-binding protein that interacts with SNI1 (Figure 5). SNI1 enhances the DNA-binding activity of CBNAC to a PR1 promoter element (Figure 6B). There are several reported instances where an interaction partner enhances or inhibits DNA-binding activity of a transcrip- tion factor. The human viral protein TAX stimulates the DNA binding of bZIP proteins (37), whereas Arabidopsis