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. 2006 Jul;18(7):1750-65.
doi: 10.1105/tpc.105.039677. Epub 2006 Jun 9.

A comprehensive structure-function analysis of Arabidopsis SNI1 defines essential regions and transcriptional repressor activity

Rebecca A Mosher  1 , Wendy E Durrant, Dong Wang, Junqi Song, Xinnian Dong
Affiliations

A comprehensive structure-function analysis of Arabidopsis SNI1 defines essential regions and transcriptional repressor activity

Rebecca A Mosher et al. Plant Cell. 2006 Jul.

Abstract

The expression of systemic acquired resistance (SAR) in plants involves the upregulation of many Pathogenesis-Related (PR) genes, which work in concert to confer resistance to a broad spectrum of pathogens. Because SAR is a costly process, SAR-associated transcription must be tightly regulated. Arabidopsis thaliana SNI1 (for Suppressor of NPR1, Inducible) is a negative regulator of SAR required to dampen the basal expression of PR genes. Whole genome transcriptional profiling showed that in the sni1 mutant, Nonexpresser of PR genes (NPR1)-dependent benzothiadiazole S-methylester-responsive genes were specifically derepressed. Interestingly, SNI1 also repressed transcription when expressed in yeast, suggesting that it functions as an active transcriptional repressor through a highly conserved mechanism. Chromatin immunoprecipitation indicated that histone modification may be involved in SNI1-mediated repression. Sequence comparison with orthologs in other plant species and a saturating NAAIRS-scanning mutagenesis of SNI1 identified regions in SNI1 that are required for its activity. The structural similarity of SNI1 to Armadillo repeat proteins implies that SNI1 may form a scaffold for interaction with proteins that modulate transcription.

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Figures

Figure 1.
Figure 1.
GFP-SNI1 Is Localized in the Nucleus. (A) P35S:GFP-SNI1 transgenic lines (GFP-SNI1 plants 1 and 2) show complementation of the sni1-1 leaf morphology. Plants were soil-grown for 4 weeks. (B) P35S:GFP-SNI1 transgenic lines show complementation of the sni1-1 mutation in PBGL2:GUS gene expression. Seedlings were grown on MS plates with (+) or without (−) INA (10 μM) for 12 d and stained for GUS activity (Bowling et al., 1994). (C) P35S:GFP-SNI1 transgenic lines show complementation of the sni1-1 mutation in root length. Root length was measured on 8-d-old plate-grown seedlings. For each genotype, 15 seedlings were measured, and se values were calculated from results of three independent experiments. (D) GFP-SNI1 is primarily localized in the nucleus. P35S:GFP-SNI1 seedlings were grown vertically on MS plates for 5 d. Roots were treated with propidium iodide to stain cell walls before microscopy. A ×60 water-immersion lens and a 488-nm laser were used with a laser-scanning confocal microscope. GFP fluorescence was false-colored green and propidium iodide was false-colored red.
Figure 2.
Figure 2.
SNI1 Represses Transcription in a Yeast Expression System. (A) The SS38 reporter gene. (B) β-Galactosidase activity. SNI1 was fused to G4DBD in pMAN to generate G4DBD-SNI1 and transformed into yeast strain 122 carrying the integrated SS38 reporter gene. β-Galactosidase expression was measured using standard techniques (Sambrook and Russell, 2001). The values are given as averages ± se of three independent assays. (C) Immunoblot with G4DBD and G4DBD-SNI1. Yeast protein extracts were run on an 8 to 18% gradient gel and probed with anti-G4DBD antibodies (Santa Cruz Biotechnology).
Figure 3.
Figure 3.
Chromatin Modifications at the PR-1 Promoter. (A) The PR-1 promoter. Probe indicates the region amplified in chromatin immunoprecipitation experiments. LS 4, 7, and 10 are the promoter regions identified as important regulators of SAR induction (Lebel et al., 1998). (B) Chromatin immunoprecipitation. Mean amount of PR-1 promoter relative to ubiquitin associated with MeH3K4 and AcH3. Error bars indicate se of four experiments, each in triplicate. Two-tailed t tests were performed on the data. The untreated wild type values are significantly lower than all other values for both modifications (* P < 0.1, ** P < 0.05, *** P < 0.01). None of the other samples are significantly different from each other.
Figure 4.
Figure 4.
Structure and Function of SNI1 Are Conserved in Different Plant Species. (A) Genomic structure of SNI1 homologues. At, Arabidopsis; Gm, soybean; Lj, lotus; St, potato; and Os, rice. The 15 exons of each locus are indicated with black boxes, introns are indicated by dashed lines, and corresponding exons in each plant species are connected by gray lines. All introns and exons are drawn to scale. (B) P35S:Gm SNI1 and P35S:St SNI1 in the sni1-1 background restore wild-type morphology and PBGL2:GUS expression. Morphology was determined on 4-week-old, soil-grown plants. PBGL2:GUS staining was conducted on seedlings grown for 12 d on MS plates with (+) or without (−) 10 μM INA.
Figure 5.
Figure 5.
Sequence Conservation among the SNI1 Homologues and Outline of At SNI1 Functional Regions Identified by NAAIRS Mutagenesis. Alignment of cloned and predicted full-length cDNAs from Arabidopsis (At), soybean (Gm), lotus (Lj), potato (St), tomato (Sl), rice (Os), and sugarcane (So). Highly conserved residues (>70% identity) are colored red, and moderately conserved residues (>70% similarity) are colored blue. The black numbered lines indicate residues in At SNI1 substituted by NAAIRS. The colored bars above the sequence alignment outline the functional regions of SNI1 defined by NAAIRS mutagenesis (see Figure 6): blue bars, regions nonessential for SNI1 activity; red bars, regions essential for SNI1 activity.
Figure 6.
Figure 6.
Functional Analysis of the NAAIRS Mutants. Complementation of sni1-1 by NAAIRS mutants as measured by root length. Approximately 15 homozygous NAAIRS mutants were grown vertically on MS plates for 8 d, and root length was measured. Colored bars represent average root length of the best complementing transgenic line for each NAAIRS mutant (number indicated on the x axis) compared with the wild type (set as 100%). The dashed line at 65% was set to separate the functional NAAIRS mutants (blue bars) from the nonfunctional NAAIRS mutants (red bars).
Figure 7.
Figure 7.
Modeling of the SNI1 Protein. At SNI1 (amino acids 14 to 402) was threaded onto the ARMs of α-importin (M. musculus; amino acids 70 to 496). Residues identified as essential via NAAIRS mutagenesis are colored red.
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