transcription factors such as WRKY7, WRKY11 and WRKY17 (18–21). SNI1 was identiﬁed in a screen for suppressors of npr1 (18). SNI1 encodes a protein with structural similarity to Armadillo-repeat proteins that are involved in scaffolding or protein–protein interactions. The mechanism by which NPR1 and SNI1 interact to control PR1 expression is not clear. SA-inducible PR gene expression and resistance are restored in the npr1 sni1 double mutant suggesting that there is an NPR1-independent pathway of SA activation of PR1 transcription and that NPR1 blocks SNI1 activity. The deoxyribonucleic acid (DNA) recombination protein RAD15 seems to be involved in the regulation of PR1 expression by the NPR1-independent pathway (22). Both SNI1 and RAD51D were found to play roles in PR gene transcription and DNA recombination (22). Histone modiﬁcations are involved in SNI1-mediated re- pression (23). Calcium signaling is another component of the defense response. Calcium signals are transduced in many ways including the binding of calcium to calmodulins (CaMs) or CaM-like proteins (24). The Ca2+/CaM complex modulates immune responses by repressing or activating transcription. Transcription of genes involved in SA bio- synthesis is modulated by Ca2+/CaM (25). TGA and WRKY transcription factors are involved in controlling PR1 expression, and some members of these families of transcription factors are known to bind Ca2+/CaM. Details of CaM regulation of defense gene expression are not well-understood. We show here a novel connection between SNI1 and Ca2+/CaM control of PR1 expression. We demonstrate that a previously identiﬁed CaM-binding NAC transcrip- tion repressor designated CBNAC (26) binds to cis- elements on the PR1 promoter that contain a GCTT core sequence and also interacts physically with SNI1. Genetic analyses showed that CBNAC functions as a negative regulator of pathogen-induced PR1 expression and basal resistance to a virulent strain of P. syringae. CBNAC and SNI1 were found to function synergistically as negative regulators of both PR1 expression and disease resistance. MATERIALS AND METHODS Plant and bacterial materials All Arabidopsis plants used in this study were of the Columbia (Col-0) ecotype. The virulent bacterial patho- gen, P. syringae pv. tomato (Pst) DC3000 was used for disease response tests. Escherichia coli BL21 (DE3) pLysS was used to express and produce recombinant GST-CBNAC protein. Arabidopsis transformation was performed as described previously (27). Generation of transgenic plants To generate transgenic plants, CBNAC complementary DNA (cDNA) with or without the FLAG tag was placed under the control of the CaMV 35S promoter. These constructs were cloned into pCAMBIA 1300 and transformed into Agrobacterium tumefaciens GV3101. Nucleic Acids Research, 2012, Vol. 40, No. 18 9183 Arabidopsis wild-type plants were transformed with the 35S:Flag-CBNAC construct according to a published protocol (27), and T3 progeny lines overexpressing CBNAC were selected for experiments. The 35S:CBNAC construct was used to transform cbnac1 plants and T3 progeny lines (cbnac1/CBNAC) expressing approximately the same level of CBNAC as wild-type plants in 1 mM SA-treated leaves were selected for experiments. MS medium containing 40 mg/ml hygromycin was used for se- lection of transformants. Plant growth conditions Arabidopsis thaliana plants were grown in growth chambers at 22 C and under 120 mEm 2 s 1 light intensity and 16-h-light/8-h-dark photoperiod. Isolation of the cbnac1 and cbnac1 sni1 mutant lines The cbnac1 (Salk_065051) T-DNA insertion mutant was identiﬁed from the Salk Arabidopsis T-DNA population (28). The T-DNA insertion was conﬁrmed by polymerase chain reaction (PCR) using a T-DNA-speciﬁc primer (T-DNA) and a CBNAC-speciﬁc primer (CBNAC-S). A homozygous cbnac1 line was identiﬁed by PCR using a pair of primers corresponding to T-DNA ﬂanking se- quences (F1 and F2). The sni1 mutant was provided by Dr Xinnian Dong. The cbnac1 sni1 double mutant was obtained by crossing cbnac1 and sni1, selﬁng the F1 progeny and screening of the F2 population. The F2 population was screened for an absence of both genes by gene-speciﬁc PCR using the following primers: SNI1- speciﬁc primers (SNI1-S1 and SNI1-S2) and CBNAC- speciﬁc primers (F1 and F2). The primers used for PCR are listed in Supplementary Table S1. Pseudomonas infection Pseudomonas infection was carried out as described previ- ously (29). Pseudomonas syringae DC3000 (PstDC3000) carrying empty vector (pVSP61) was grown at 28 C on King’s agar plates supplemented with 50 mg/ml rifampicin and 50 mg/ml kanamycin. In brief, bacteria were sus- pended in 10 mM MgCl2, adjusted to optical density (OD)600 = 0.001 and pressure inﬁltrated into leaves using a needleless syringe. Leaf discs from four independ- ent plants were combined, ground in 10 mM MgCl2, serial-diluted 1:10 and plated onto King’s B medium con- taining the appropriate antibiotics. Plates were incubated at 28 C for 2 or 4 d, after which the colonies were counted. Quantitative PCR and ribonucleic acid gel blot analysis Total ribonucleic acid (RNA) was isolated using the guanidinium thiocyanate-phenol-chloroform extraction method with subsequent ultracentrifugation (29). Arabidopsis RNA was extracted using LiCl method, and cDNA was synthesized using the SuperScriptTM II RNase-Reverse Transcriptase (Invitrogen). Quantitative PCR (qPCR) was performed using the SsoFast EvaGreen Supermix (Bio-Rad) in a CFX96TM Real- Time PCR System (Bio-Rad). The primers used for qPCR are listed in Supplementary Table S2. Expression