A NAC transcription factor and SNI1 cooperatively suppress basal pathogen resistance in Arabidopsis thaliana - PubMed (original) (raw)

. 2012 Oct;40(18):9182-92.

doi: 10.1093/nar/gks683. Epub 2012 Jul 22.

Hyeong Cheol Park, Kyung Eun Kim, Mi Soon Jung, Hay Ju Han, Sun Ho Kim, Young Sang Kwon, Sunghwa Bahk, Jonguk An, Dong Won Bae, Dae-Jin Yun, Sang-Soo Kwak, Woo Sik Chung

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A NAC transcription factor and SNI1 cooperatively suppress basal pathogen resistance in Arabidopsis thaliana

Ho Soo Kim et al. Nucleic Acids Res. 2012 Oct.

Abstract

Transcriptional repression of pathogen defense-related genes is essential for plant growth and development. Several proteins are known to be involved in the transcriptional regulation of plant defense responses. However, mechanisms by which expression of defense-related genes are regulated by repressor proteins are poorly characterized. Here, we describe the in planta function of CBNAC, a calmodulin-regulated NAC transcriptional repressor in Arabidopsis. A T-DNA insertional mutant (cbnac1) displayed enhanced resistance to a virulent strain of the bacterial pathogen Pseudomonas syringae DC3000 (PstDC3000), whereas resistance was reduced in transgenic CBNAC overexpression lines. The observed changes in disease resistance were correlated with alterations in pathogenesis-related protein 1 (PR1) gene expression. CBNAC bound directly to the PR1 promoter. SNI1 (suppressor of nonexpressor of PR genes1, inducible 1) was identified as a CBNAC-binding protein. Basal resistance to PstDC3000 and derepression of PR1 expression was greater in the cbnac1 sni1 double mutant than in either cbnac1 or sni1 mutants. SNI1 enhanced binding of CBNAC to its cognate PR1 promoter element. CBNAC and SNI1 are hypothesized to work as repressor proteins in the cooperative suppression of plant basal defense.

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Figures

Figure 1.

Figure 1.

CBNAC expression is induced by pathogen- and SA. (A) Induction of CBNAC gene expression by _Pst_DC3000. 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 harvested 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).

Figure 2.

Figure 2.

CBNAC negatively regulates resistance to _Pst_DC3000 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 _Pst_DC3000 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).

Figure 3.

Figure 3.

CBNAC interacts with the E0-1-1 element of the PR1 promoter. (A) Nucleotide sequence of the native and mutated (M1–M4) E0-1-1 elements used in EMSA. (B) Analysis of binding specificity. EMSA was performed using 32P-labeled native E0-1-1 as probe as above except that GST-CBNAC protein was preincubated with 50- (lane 4), 100- (lane 5) or 200- (lane 6) fold molar excess of cold native E0-1-1 (competitor) before addition of probe. (C) EMSA of CBNAC binding. 32P-labeled native (lanes 1–3) and mutated (lanes 4–7) E0-1-1 probes were incubated with equal amounts of _E. coli_-expressed GST-CBNAC (lanes 3 to 7) or GST alone (lanes 1 and 2) before electrophoresis.

Figure 4.

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. β-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.

Figure 5.

Figure 5.

Altered responses of the cbnac1 sni1 double mutant to _Pst_DC3000. (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).

Figure 6.

Figure 6.

SNI1 enhances binding of CBNAC to the E0-1-1 element. (A) Nucleotide sequence of the PR1 promoter indicating E0-1-1 element. The numbers indicate the position of the element relative to the PR1 translation start site. (B) EMSA analysis of the effect of SNI1. EMSA was performed using 32P-labeled E0-1-1 element (lanes 1 to 5), without (lane 1) or with the addition GST (lane 2; negative control), CBNAC (lanes 4 and 5) and SNI1 (lane 5). Equal amounts of CBNAC were used in the two lanes.

Figure 7.

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 translocation of a large amount of active NPR1 to the nucleus and its interaction with TGA transcription factors. The SNI1/CBNAC protein complex can be removed by NPR1, CaM or other unknown mechanisms.

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