Interaction of NPR1 with basic leucine zipper protein transcription factors that bind sequences required for salicylic acid induction of the PR-1 gene

Abstract

The Arabidopsis thaliana NPR1 has been shown to be a key regulator of gene expression during the onset of a plant disease-resistance response known as systemic acquired resistance. The npr1 mutant plants fail to respond to systemic acquired resistance-inducing signals such as salicylic acid (SA), or express SA-induced pathogenesis-related (PR) genes. Using NPR1 as bait in a yeast two-hybrid screen, we identified a subclass of transcription factors in the basic leucine zipper protein family (AHBP-1b and TGA6) and showed that they interact specifically in yeast and in vitro with NPR1. Point mutations that abolish the NPR1 function in A. thaliana also impair the interactions between NPR1 and the transcription factors in the yeast two-hybrid assay. Furthermore, a gel mobility shift assay showed that the purified transcription factor protein, AHBP-1b, binds specifically to an SA-responsive promoter element of the A. thaliana PR-1 gene. These data suggest that NPR1 may regulate PR-1 gene expression by interacting with a subclass of basic leucine zipper protein transcription factors.

Figure 1

Yeast two-hybrid assay of interactions between NPR1 and bZIP transcription factors. (A) Yeast cells (EGY48) containing TomNPR1 and NIF1, NPR1 and AHBP-1b, NPR1 and TGA6, or NPR1 and OBF5 were grown for 2 days at 30°C on galactose medium lacking leucine for detection of the LEU2 reporter expression. (B) β-Galactosidase activity was calculated as described previously (33). Three independent colonies were taken for each combination, and four measurements were performed on a culture grown from each colony. The mean ± SD is presented.

Figure 2

Sequence alignment of tomato NIF1 and A. thaliana AHBP-1b, TGA6, and OBF5. The tomato NIF1 sequence was aligned with AHBP-1b (residues 96–330) (34), TGA6 (residues 90–325) (35), and OBF5 (residues 90–324) (36) by using multalin (37). Red letters represent the residues of high consensus, blue letters indicate the amino acids with low consensus, and black letters highlight the residues with no consensus.

Figure 3

In vitro analysis of interactions between NPR1 and AHBP-1b or OBF5. Immunoblot analysis was performed on protein preparations purified by using Ni-NTA resin to detect copurification of NPR1 with His-tagged AHBP-1b or OBF5. The blot was probed with an antiserum generated against the carboxyl-terminal 16 aa of NPR1. Lanes: 1, crude extract (0.2 μl) of Sf9 insect cells expressing NPR1 by baculovirus; 2, NPR1 copurified with AHBP-1b; 3, NPR1 copurified with OBF5; 4, NPR1 copurified with AHBP-1b from a separate purification; 5, NPR1 purified with Ni-NTA resin alone.

Figure 4

Interactions between mutant npr1 proteins and the bZIP transcription factors. (A) Truncated versions of the NPR1 protein were tested for their interactions with AHBP-1b by measuring the β-galactosidase activity resulting from the interactions in yeast. Three independent colonies were taken for each combination, and four measurements were performed on a culture grown from each colony. The mean ± SD is presented. (B) Yeast two-hybrid assay performed by using npr1–1 and npr1–2 point mutants as bait. Yeast cells carrying mutant npr1 and either AHBP-1b or TGA6 were grown for 2 days at 30°C on galactose medium lacking leucine for detection of the LEU2 reporter expression (Left). The same yeast cells also were grown for 2 days at 30°C in medium containing 5-bromo-4-chloro-3-indolyl β-d-galactoside for detection of the β-galactosidase reporter expression (Right). (C) Immunoblot analysis of the expression of npr1 mutant proteins in yeast. Ten micrograms of total yeast protein was loaded in each lane, and immunoblot analysis was carried out by using the NPR1 antiserum.

Figure 5

AHBP-1b interacts specifically with the A. thaliana PR-1 promoter sequence containing an as-1-like element. All binding reactions contain 4 × 10⁴ cpm ³²P-labeled probe, incubated with 1 μg of either a control protein preparation (lane 1) or AHBP-1b (lanes 2–10), in the absence (lanes 1 and 2) or presence of specific (lanes 3–6) or nonspecific (lanes 7–10) competitor probes. In lanes 3–6, 1×, 10×, 40×, and 100× molar excess of the unlabeled as-1-like probe were added, respectively. In lanes 7–10, 1×, 10×, 40×, and 100× molar excess of the unlabeled probe containing point mutations in the as-1-like element were added, respectively. S, specific band shifting; ∗, nonspecific banding; FP, free probe.