Identification of an OsPR10a promoter region responsive to salicylic acid

Abstract

Orysa sativa pathogenesis-related protein 10a (OsPR10a) was induced by pathogens, salicylic acid (SA), jasmonic acid (JA), ethephon, abscisic acid (ABA), and NaCl. We tried to analyze the OsPR10a promoter to investigate the transcriptional regulation of OsPR10a by SA. We demonstrated the inducibility of OsPR10a promoter by SA using transgenic Arabidopsis carrying OsPR10a:GFP as well as by transient expression assays in rice. To further identify the promoter region responsible for its induction by SA, four different deletions of the OsPR10a promoter were made, and their activities were measured by transient assays. The construct containing 687-bp OsPR10a promoter from its start codon exhibited a six-fold increase of induction compared to the control in response to SA. Mutation in the W-box like element 1 (WLE 1) between 687 and 637-bp from TGACA to TGAAA completely abolished induction of the OsPR10a promoter by SA, indicating that the WLE 1 between -687 and -637 of OsPR10a promoter is important in SA-mediated OsPR10a expression. We show for the first time that the W-box like element plays a role in SA mediated PR gene expression.

Fig. 1

Expression pattern of OsPR10a in rice leaves treated with Xoo and five compounds. a Three-week-old rice seedlings were infected with Xoo and were harvested at 0, 6, 12, 24, and 48 h. b, c Three-week-old rice seedlings were treated with SA, JA, ethephon, ABA, or NaCl and were harvested at 0, 6, 12, 24, and 48 h. Total RNA was isolated from each sample, and RT-PCR was performed using OsPR10a specific primer pair. Transcript levels of OsActin show that equal amounts of RNA were used in the RT-PCR samples

Fig. 2

OsPR10a promoter activity in response to SA: a schematic representation of the OsPR10a promoter in the reporter construct. b A transient assay showing the OsPR10a promoter in response to SA. OsPR10a:LUC was bombarded into rice leaves, which were then incubated in MS medium or MS medium containing 1 mM SA at 28°C for 24 h. Protein extracts were made by dissociation in passive lysis buffer as described in “Materials and methods”. Relative luciferase activity is the ratio of the value obtained with the SA-treated OsPR10a:LUC divided by the value obtained with the buffer-treated OsPR10a:LUC. Bars indicate the standard error of three replicates

Fig. 3

Fluorescence images of Arabidopsis transgenic plants carrying OsPR10a:GFP: a schematic diagram of OsPR10a:GFP::GUS fusion construct. b Induction of OsPR10a promoter by SA. OsPR10a:GFP::GUS was introduced into Arabidopsis by Agrobacterium-mediated transformation. Transgenic Arabidopsis seedlings carrying the OsPR10a:GFP::GUS was examined using fluorescence microscopy after SA treatment at 72 h. Non-transgenic Arabidopsis seedling was used as a control (left panel at mock and SA treatments). Shown are the bright-field images (upper panel Bright), the green fluorescent images using GFP filter (middle panel GFP) and the GFPA filter (bottom panel GFPA). Images are representatives from two independent experiments. The experiments were repeated at least twice

Fig. 4

Putative cis-acting elements in 1.0 kb OsPR10a promoter. The putative cis-elements are indicated in boxes and its name is given above each element. Arrows indicate the direction of the cis-element. W-box WRKY transcription factor binding site; RAV1AAT RAV transcription factor binding site; ASF1 motif bZIP factor binding site; WLE1 putative WRKY transcription factor binding site

Fig. 5

Deletion analysis of OsPR10a promoter: a schematic diagrams of serial deletion constructs of OsPR10a promoter. The numbers to the left of each construct indicate the distance from the start codon ATG. The predicted cis-elements ( W-box, RAV1AAT, and ASF1motif) are indicated by their respective abbreviations. The start codon, ATG, is written in bold. b Luciferase activity in deletion constructs of the OsPR10a promoter. Each deletion construct OsPR10a:LUC was bombarded into rice leaves, which were incubated in MS liquid medium or MS medium containing 1 mM SA at 28°C for 24 h. Protein extracts were made by dissociation in passive lysis buffer as described in “Materials and methods”. Bars indicate the standard error of three replicates. The values are the ratio of the value obtained from each deletion constructs of OsPR10a promoter treated with SA or buffer divided by the value obtained from 1.0 kb OsPR10a promoter construct treated with buffer

Fig. 6

The effect of the mutation in the WLE1 of OsPR10a promoter region III. a Sequences of the WLE1 (the −659 to −644 bp) in the OsPR10a promoter and the mWLE1 with the TGAAA instead of TGACA. The WLE1 sequence is underlined and bolded. The asterisk represents the mutated base in the WLE1. b Luciferase activity in 687:LUC and m687:LUC in rice leaves. Bar indicates the standard error of the three replicates