9188 Nucleic Acids Research, 2012, Vol. 40, No. 18 Figure 5. Altered responses of the cbnac1 sni1 double mutant to PstDC3000. (A) Morphology of 5-week-old wild-type (WT), cbnac1, sni1 and cbnac1 sni1 plants grown on MS agar plates. (B–D) Disease resistance responses in leaves inoculated with bacterial suspension as in Figure 3. Disease symptoms in inoculated leaves at 5 dpi are depicted (B). Bacterial growth in inoculated leaves at 0 and 3 dpi are compared (C). Mean bacterial densities ± SE were calculated from six to eight replicate plants. Signiﬁcant differences as calculated by Student’s t test (P < 0.05) are indicated by unique letters. The experiment was repeated at least three times with similar results. PR1 expression was monitored in inoculated leaves by qRT-PCR (D). Values were normalized using the expression level of Tubulin 2 and expressed relative to the expression level in WT at 12hpi, which is arbitrarily set at 100. Mean relative expression values ±SE values from three independent experiments are shown. Data were analyzed by Student’s t test. Different letters indicate statistically signiﬁcant differences between genotypes (P < 0.05). in inoculated leaves of the cbnac1 sni1 mutant compared with the cbnac1 or sni1 mutants was accompanied by the substantially reduced disease symptom development (Figure 5B). To investigate the enhanced basal resistance of the double mutant, PR1 expression was analyzed following bacterial pathogen infection. The level of PR1 mRNA was comparable in uninoculated leaves of wild type and cbnac1 plants (Figure 5D). SNI1 has been reported to repress basal PR1 expression (18). Accordingly, the level of PR1 mRNA was higher in uninoculated leaves of the sni1 mutant than in wild type. At 12 h post-inoculation, PR1 transcript abundance was elevated in the cbnac1 and sni1 mutants compared with wild type, as expected if they function as transcriptional repressors. The PR1 levels in uninoculated and inoculated leaves of the cbnac1 sni1 double mutant were signiﬁcantly higher than in leaves of cbnac1 or sni1 single mutants under the same condition, suggesting a synergistic effect of the combined cbnac1 and sni1 mutations on PR1 expression (Figure 5D). Together, these results show that CBNAC and SNI1 function syner- gistically as negative regulators of basal resistance to the virulent bacterial pathogen PstDC3000 due, in part, to their overlapping activities as negative regulators of PR1 expression. SNI1 enhances the DNA-binding activity of the CBNAC Although SNI1 functions as a transcriptional repressor of PR1 expression, it lacks a DNA-binding domain (23). Therefore, the possibility that SNI1 could inﬂuence the DNA-binding activity of CBNAC toward the PR1 promoter cis-element (E0-1-1) was investigated because SNI1 interacts with CBNAC. The PR1 promoter element (E0-1-1), previously identiﬁed by EMSA, contains the preferred GCTT core binding sequence for CBNAC (Figure 6A). Incubation of SNI1 with the E0-1-1 probe did not result in a retarded band in an EMSA (Figure 6B, lane 3) indicating that SNI1 does not directly bind to this element. Addition of SNI1 to the CBNAC-binding mixture increased CBNAC binding to the E0-1-1 element without producing a supershift band, indicating that the DNA–protein complex contained CBNAC but not SNI1 (Figure 6B, lanes 5 and 4). These results suggested that SNI1 enhances the binding of CBNAC to the E0-1-1 element. DISCUSSION CBNAC acts as a negative regulator of plant defense responses Discrepancies exist in several studies that had reported on the biochemical properties of CBNAC/NTL9 (26,33). First, CBNAC/NTL9 had been reported to contain a C-terminal transmembrane domain and that CBNAC/ NTL9 was localized in the plasma membrane (33). However, the predicted C-terminal transmembrane domain of CBNAC/NTL9 was identiﬁed as a CaM- binding domain (26). Furthermore, the GFP-tagged CBNAC/NTL9 protein was considered to be dominantly localized in nuclei (26). Finally, on a different level of function, CBNAC/NTL9 was reported to be involved in regulating signaling during leaf senescence (33). The biological function of CBNAC/NTL9 was based on