CBNAC expression was revealed by comparing the transcript abundance in SA-treated leaves of wild-type and cbnac1 plants (Supplementary Figure S1B). Attempts to identify additional independent cbnac mutants were unsuccessful. Thus, a 35S:CBNAC con- struct was used to transform the cbnac1 mutant, and transgenic lines (cbnac1/CBNAC) exhibiting CBNAC ex- pression levels similar to wild-type plants were chosen for complementation analysis (Supplementary Figure S2A). Transgenic lines constitutively overexpressing CBNAC Figure 1. CBNAC expression is induced by pathogen- and SA. (A) Induction of CBNAC gene expression by PstDC3000. Leaves of 4-week-old Arabidopsis plants (Col-0) were inﬁltrated with a bacterial suspension (OD600 = 0.001 in 10 mM MgCl2). Inﬁtrated leaves were har- vested at the indicated times after inoculation. The gel blot analysis of total RNA that was performed with a 32P-labeled CBNAC probe is shown. Ethidium bromide-stained rRNA is shown as loading control. (B) Induction of CBNAC gene expression by SA. Leaves of 4-week-old Arabidopsis plants (Col-0) were treated with 1 mM SA. Leaf collection, RNA isolation and RNA gel blot analysis was performed as in (A). Nucleic Acids Research, 2012, Vol. 40, No. 18 9185 (35S:Flag-CBNAC) were generated in the wild-type back- ground. Western blot analysis using anti-FLAG antibody revealed several transgenic plants contained elevated levels of CBNAC protein regardless of SA treatment (Supplementary Figure S1C). There were no obvious differences in growth or devel- opment characteristics of the wild-type, cbnac1 or 35S:Flag-CBNAC plants. The effect of CBNAC expres- sion level on susceptibility to the virulent bacterial pathogen PstDC3000 was then examined. Three days after inoculation, bacterial growth in inﬁltrated leaves was slightly lower in the cbnac1 mutant and slightly higher in 35S:Flag-CBNAC overexpression lines compared with the wild type (Figure 2A). There was no signiﬁcant difference in bacterial growth in leaves of the wild-type plant and a cbnac1/CBNAC complementation line (Supplementary Figure S2B). As loss of CBNAC function is associated with resistance and overexpression of CBNAC is associated with susceptibility in un-induced plants, CBNAC is a negative regulator of basal defense against the bacterial pathogen PstDC3000. Induction of PR1 is a marker of SA-mediated defense signaling that leads to Arabidopsis resistance to bacterial pathogens (2). PR1 transcript accumulation was not observed in leaves of wild-type, cbnac1, cbnac1/CBNAC or 35S:Flag-CBNAC plants before inoculation with PstDC3000 (Figure 2B and Supplementary Figure S2C). At 12 h post-inoculation when the expression of CBNAC was expected to peak in wild-type plants (Figure 1A), ac- cumulation of PR1 transcript was signiﬁcantly suppressed in 35S:Flag-CBNAC plants, induced in the cbnac1 mutant and unchanged in the cbnac1/CBNAC complementation line in comparison with wild type (Figure 2B and Supplementary Figure S2C). Thus, the level of CBNAC expression correlates inversely with resistance to pathogen infection and PR1 expression, suggesting that PR1 may be a direct target of CBNAC. Figure 2. CBNAC negatively regulates resistance to PstDC3000 and PR1 expression. Leaves of wild type (WT), 35S:Flag-CBNAC and cbnac1 plants were inoculated with a bacterial suspension (OD600 = 0.001 in 10 mM MgCl2). (A) Growth of PstDC3000 in inoculated leaves at 0 and 3 dpi. Mean bacterial densities ± SE were calculated from six to eight replicate plants are shown. 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. (B) qRT-PCR analysis of PR1 expression in inoculated leaves. Values were normalized using the expression level of Tubulin 2 and expressed relative to the expression level in WT at 12 hpi, which is arbitrarily set at 100. Mean relative expression values ± SE 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).