9184 Nucleic Acids Research, 2012, Vol. 40, No. 18 of CBNAC was detected by RNA gel blot analysis. RNA was separated on 1.5% agarose–formaldehyde gels and transferred to nylon membranes. Membranes were incubated with an (a-32P)dATP-labeled gene-speciﬁc probe at 65 C overnight and washed under high strin- gency conditions as described (29). Electrophoretic mobility shift assays For mapping of the CBNAC-binding promoter region of the PR1 gene, DNA probes were generated by PCR amp- liﬁcation with a Klenow fragment polymerase, a-32P-ATP (6000 Ci/mmol; Amersham) and the following primers, for E0, E1, E2, E3, E4, E5, E6, E0-1, E0-2, E0-3, E0-4, E3-1, E3-2, E4-1, E4-2, E4-3, E5-1, E5-2, E6-1 and E6-2 and by end labeling with polynucleotide kinase, g-32P-ATP and the following primers, for E0-1-1, E0-1-2, E0-4-1, E0-4-2, E3-1-1, E3-1-2, E3-1-3, E4-1-1, E4-1-2, E5-3-1, E5-3-2, E6-1-1 and E6-1-2 (Supplementary Table S2). DNA-binding reactions were conducted at 25 C for 20 min in binding buffer [20 mM HEPES/KOH (pH 7.9), 0.5 mM DTT, 0.1 mM ethylenediaminetetraacetic acid (EDTA)], 50 mM KCl, 15% glycerol, 1 mg poly(dI-dC) and 0.5 mg bacterially produced fusion protein puriﬁed with glutathione-Sepharose. 32P-labeled DNA probes (40 000 cpm) were added and incubated with the mixture at 25 C for 30 min. The reactions were separated on an 8% polyacrylamide gel in 0.5 tris-borate-EDTA buffer at 80 V for 3 h. The gel was dried, mounted for autoradiography with intensifying screens and exposed at –70 C. Chromatin immunoprecipitation assay Chromatin samples were prepared as described previously (30). Wild-type and 35S:Flag-CBNAC overexpression lines were ﬁxed with 1% formaldehyde for 10 min. The eluted DNA was analyzed by PCR using the following speciﬁc primers: E0, E3, E4, E5 and E6 (Supplementary Table S2). The ampliﬁed bands were visualized on a 2% agarose gel. Yeast two-hybrid assay CBNAC cDNA was digested with EcoRI and XhoI and ligated into the pAS2-1 plasmid (bait vector), which contains the Trp1 selection marker. The SNI1 cDNA was cloned into the pGAD424 plasmid (prey vector), which harbors the Leu2 selection marker. For mapping, the interacting domain deletions of CBNAC were PCR ampliﬁed using gene-speciﬁc primers and cloned in the pAS2-1 plasmid. Prey and different bait plasmids were co-transformed in the pJ69-4A (31) strain of yeast. Two-hybrid assays were performed as described in CLONTECH’s Yeast Protocols. Positive interactions were veriﬁed by the b-galactosidase assay. Luciferase complementation imaging assay Luciferase (Luc) complementation imaging (LCI) assay was carried out as described previously (32). CBNAC was fused with the C-terminal fragment of ﬁreﬂy Luc in pCAMBIA NLuc vector (CLuc-CBNAC). SNI1 was fused with the N-terminal fragment of Luc in pCAMBIA NLuc vector (SNI1-NLuc). STG1a-NLuc and CLuc-RAR1 constructs described previously were used as positive interaction controls (32). The constructs were each introduced into A. tumefaciens strain GV3101. Each bacterial strain was grown overnight in LB medium at 30 C, collected by centrifugation, then washed two times with inﬁltration buffer (10 mM MgCl2, 10 mM MES and 100 mM acetosyringone) and re-suspended in the same buffer. Equal volumes of bacterial suspensions of a CLuc and an NLuc construct were mixed and co-inﬁltrated into fully expanded leaves of the 3-week-old Nicotiana benthamiana plants using a needleless syringe. After inﬁltration, plants were placed at 23 C for 48 h. PstDC3000 (OD600 = 0.001 in 10 mM MgCl2) was treated after 24 h as Agro inﬁltration. The leaves were sprayed with luciferin solution (100 mM luciferin, 0.1% Triton X-100) and kept in the dark for 4 h to quench ﬂuorescence. Luc activity (luminescence) was observed with a low-light cooled CCD imaging ap- paratus (AndoriXon; Andor). RESULTS CBNAC transcripts are induced by pathogen and SA treatment To identify the biological function of CBNAC, we investigated the gene expression of CBNAC in response to environmental stresses. The transcript level of CBNAC was examined after exposure to several biotic and abiotic stresses including bacterial pathogen, SA, jasmonic acid (JA), ABA, drought and NaCl. Interestingly, CBNAC transcript levels were increased in leaves of wild-type Arabidopsis plants after exposure to the virulent bacterial pathogen PstDC3000 or SA (Figure 1). CBNAC tran- scripts were undetectable in untreated leaves and remained undetectable over a 48-h period in leaves after inﬁltration with 10 mM MgCl2. In leaves inﬁltrated with PstDC3000, signiﬁcant accumulation of CBNAC tran- scripts was observed at 6–12 h post-inoculation, and it returned to an undetectable level by 24 h (Figure 1A). Expression of CBNAC was also induced by SA, the inducer of systemic acquired resistance (Figure 1B). In SA-treated leaves, CBNAC transcripts reached maximum levels at 6 h, persisted for 24 h and declined to basal levels 48 h after treatment. These results suggested that CBNAC may be involved in SA-mediated pathogen resistance signaling in plants. CBNAC is a negative regulator of the plant defense The following genetic resources were developed for investigation of CBNAC function in vivo. A Salk line (Salk_065051) carrying a T-DNA insertion in CBNAC (cbnac1) was identiﬁed and homozygous F2 progeny derived from this line were used for analyses. Location of the T-DNA insertion at the third exon of the CBNAC gene was conﬁrmed by PCR analyses of genomic DNA (Supplementary Figure S1A). There was no detectable ac- cumulation of CBNAC transcripts in untreated leaves of the wild-type and cbnac1 mutant plants. Complete loss of