9190 Nucleic Acids Research, 2012, Vol. 40, No. 18 Figure 7. Model for the regulation of PR1 by the CBNAC-SNI1 complex. In non-induced conditions (–Pathogen), because SNI1 does not contain a known DNA-binding domain, we postulate that SNI1 binds to CBNAC and is thereby recruited the E0-1-1 element of PR1 promoter. SNI1 enhances the DNA-binding activity of CBNAC and somehow this enhances repression of PR1 by SNI1. In the presence of inducer (+Pathogen), PR1 gene expression is induced by the transloca- tion of a large amount of active NPR1 to the nucleus and its inter- action with TGA transcription factors. The SNI1/CBNAC protein complex can be removed by NPR1, CaM or other unknown mechanisms. NPR1 stimulates the DNA binding of TGA factors to the LS7 element (10). The tobacco ankyrin repeat protein ANK1 inhibits the binding of the bZIP factor BZI-1 to its cognate promoter element in EMSA without altering the complex mobility (38). Similarly, SNI1 enhanced the binding of CBNAC to its cognate promoter element in EMSA without altering the complex mobility (Figure 6B). There are two possible explanations. First, SNI1 may have been released from the complex by a con- formational change resulting from CBNAC binding to DNA. Second, other interacting proteins may be required for a stable interaction between CBNAC and SNI1. Model for PR1 regulation Based on the information generated in this and previous studies, the following model is proposed (Figure 7). The PR1 promoter contains both positive TGA-binding (TGACG) and negative CBNAC-binding (E0-1-1) elements. Genetic analyses showed that CBNAC interacts synergistically with SNI1 as a transcriptional repressor of PR1 gene expression and basal resistance to PstDC3000 (Figure 5C and D). Unlike cbnac1 plants, sni1 and cbnac1 sni1 plants exhibit constitutive PR gene expression and other pleiotropic phenotypes (Figure 5A). Therefore, in absence of pathogen induction, SNI1 is proposed to function as the major negative regulator of PR gene ex- pression whose activity is governed, in part, by its inter- action with the transcriptional repressor CBNAC at the E0-1-1 element. The constitutive binding of transcrip- tional activators (such as TGA1, TGA3 and TGA6) to the positive element in a non-induced state is still a possi- bility. However, this binding would not lead to gene ex- pression in the presence of CBNAC and SNI1. On pathogen infection, transcription of PR1 is activated and repression is terminated. Transcriptional activation of PR1 by NPR1 is well characterized and is mediated by its association with the positive TGA transcription factors. However, mechanisms by which repression of PR1 expres- sion are terminated following pathogen infection are poorly understood. On the basis of the data presented herein, three possibilities can be considered. First, CBNAC and SNI1 could be degraded by a pathogen signal, but there is no strong evidence in support of this hypothesis. Second, CBNAC and SNI1 could be removed by the binding of other regulator proteins or by covalent modiﬁcations because the interaction between CBNAC and SNI1 is reduced or removed by pathogen treatment (Supplementary Figure S9). CaM could also be involved in the termination of transcriptional repression through the PR1 promoter E0-1-1 element because CaM inter- acts with CBNAC (26), and the CaM-binding domain of CBNAC is required for interaction with SNI1 (Supplementary Figure S6). This scenario is rendered likely because inﬂux of Ca2+ followed by the activation of Ca2+/CaM signaling processes represents early and es- sential events in the response to pathogen infection (39,40). Several CaM-binding proteins have already been shown to be involved in plant defense responses (41,42). Third, CBNAC and SNI1 repression is removed by NPR1. It has been proposed that the role of NPR1 in a wild-type plant is to inactivate SNI1-mediated transcrip- tional repression of PR genes (18). As such, PR gene ex- pression is restored and is inducible in the sni1 npr1-1 double mutant. However, a physical interaction between SNI1 and NPR1 has never been demonstrated so it is unclear whether NPR1 regulates the SNI1 repression of PR genes. Further molecular and genetic studies are required to precisely characterize the regulatory mechan- isms that converge on the PR1 gene, resulting in the precise control of PR1 expression in response to environ- mental stimuli. SUPPLEMENTARY DATA Supplementary Data are available at NAR Online: Supplementary Figures 1–9 and Supplementary Tables 1 and 2. ACKNOWLEDGEMENTS The authors thank Dr. Xinnian Dong for critical reading of the manuscript and providing sni1 seed, Dr. Hans J. Bohnert for helpful discussions and critique of the manu- script and the Laboratory of JianMing Zhou (National Institute of Biological Sciences, China) for providing the CAMBIA1300-NLuc/CLuc vector. They also thank the Salk Institute Genomic Analysis Laboratory for seeds and for providing T-DNA insertion ﬂanking sequence information. FUNDING World Class University Program [R32-10148] and Basic Science Research Program [2010-0010607] of the National Research Foundation (NRF) funded by the Ministry of Education, Science and Technology, and a