Nucleic Acids Research, 2012, Vol. 40, No. 18 9187 Figure 4. CBNAC interacts with SNI1. (A) Yeast two-hybrid analysis. Transformants of yeast strain pJ69-4A were grown as indicated (upper left) on minimal medium with (+Ade) or without (–Ade) selection. Adenine prototrophy indicates positive interaction. b-Galactosidase activity in the colonies grown in +Ade medium was determined by ﬁlter-lift assay (LacZ). (B) LCI assay for detecting interaction in planta. Tobacco leaves were transformed by Agrobacterium inﬁltration using a needleless syringe. The indicated NLuc and CLuc construct pairs were used for transformation. Shown are luminescence images (upper panel) and quantitative luminescence measurements (lower panel) depicting luciferase activity in inoculated leaves at 48 hpi. used for each primer pair. As expected, immunopre- cipitation reactions lacking anti-FLAG antibody did not result in the recovery of chromatin fragments containing the PR1 promoter (Supplementary Figure S5C, No Ab). Only the E0 (–1209 to –970 bp) region was ampliﬁed in immunoprecipitates obtained with anti-FLAG antibody (Supplementary Figure S5C, a-Flag Ab), conﬁrming that CBNAC directly binds to a region of the PR1 promoter that contains the GCTT core sequence. CBNAC physically interacts with SNI1 Similar to CBNAC, SNI1 is a negative regulator of PR1 expression. However, unlike CBNAC, SNI1 does not have a DNA-binding domain. Therefore, the possibility of interaction between CBNAC and SNI1 was examined in a yeast two-hybrid assay. The bait construct (pAS2-1:: CBNAC) contained full-length CBNAC cDNA fused to the GAL4 DNA-binding domain. The prey construct (pGAD424::SNI1) contained full-length SNI1 cDNA fused to the GAL4 activation domain. As shown in Figure 4A, yeast cells expressing either bait or prey con- struct alone were not able to grow on selection media. Growth on selective medium and expression of the LacZ reporter gene was observed only if yeast contained both pAS2-1::CBNAC and pGAD424::SNI1, indicating speciﬁc interaction between CBNAC and SNI1. Interaction of CBNAC with SNI1 in planta was then examined using the LCI assay in N. benthamiana leaves using transient expression (32). The positive control com- bination of STG1a-NLuc and CLuc-RAR1 resulted in strong Luc activity as previously reported (32) (Figure 4B). The negative control combinations of CLuc-CBNAC/NLuc vector and SNI1-NLuc/CLuc vector did not show Luc activity (Figure 4B). Luc activity was detected with the combination of SNI1- NLuc/CLuc-CBNAC showing that CBNAC interacts with SNI1 in planta. Additional yeast two-hybrid assays were performed to deﬁne the CBNAC domain responsible for inter- action with SNI1. A schematic diagram of the CBNAC deletion constructs used for these assays is shown in Supplementary Figure S6A. The NAC domain of CBNAC was not required for interaction with SNI1 (Supplementary Figure S6B). The C-terminal region (301–512 amino acids) of CBNAC interacted with SNI1. The interaction between CBNAC and SNI1 required the CaM-binding domain as well as other sequences. CBNAC and SNI1 function synergistically as negative regulators of disease resistance A cbnac1 sni1 double mutant was generated through genetic crossing and analyzed for potential interactions between CBNAC and SNI1. Morphological phenotypes of the cbnac1 sni1 mutant resembled those of the sni1 mutant rather than the cbnac1 mutant. Both sni1 and cbnac1 sni1 plants had smaller rosettes compared with the wild type (Figure 5A). Similar to the sni1 mutant, cbnac1 sni1 plants exhibited pleiotropic phenotypes, including decreased leaf size and altered leaf texture (Figure 5A and B). In contrast, the cbnac1 plants showed no differences in growth, development or morph- ology in comparison with the wild type (Figure 5A and B). As shown in Figure 5C, the cbnac1 and sni1 mutants had similar levels of basal resistance to PstDC3000 (Figure 5C). Bacterial counts in inoculated leaves of both lines were slightly lower than in leaves of the wild type 3 days post-inoculation. Disease symptoms were also less severe in the cbnac1 and sni1 mutants than in wild-type leaves (Figure 5B). The cbnac1 sni1 double mutant was markedly more resistant to PstDC3000 than either the cbnac1 or sni1 single mutants (Figure 5C). Bacterial counts in inoculated leaves of the cbnac1 sni1 mutant were 10-fold lower than in leaves of the wild type at 3 days post- inoculation. This marked reduction in bacterial growth