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Open Access Research article

Genome-wide identification of WRKY45-regulated genes that mediate benzothiadiazole-induced defense responses in rice

Akira Nakayama12, Setsuko Fukushima1, Shingo Goto1, Akane Matsushita13, Masaki Shimono14, Shoji Sugano1, Chang-Jie Jiang1, Aya Akagi15, Muneo Yamazaki1, Haruhiko Inoue1 and Hiroshi Takatsuji16*

Author Affiliations

1 Disease Resistant Crops Research Unit, National Institute of Agrobiological Sciences, Ibaraki 305-8602, Japan

2 Maebashi Institute of Technology, Maebashi 371-0816, Japan

3 Present address: Du Pont Kabushiki Kaisha, 2-11-1, Nagata-cho, Chiyoda-ku, Tokyo 100-6111, Japan

4 Present address: Department of Plant Pathology, Michigan State University, East Lansing, MI 48824, USA

5 Present address: Bayer CropScience, Tokyo 100-8262, Japan

6 Disease Resistant Crops Research Unit, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305–8602, Japan

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BMC Plant Biology 2013, 13:150  doi:10.1186/1471-2229-13-150

Published: 4 October 2013

Abstract

Background

The rice transcription factor WRKY45 plays a crucial role in salicylic acid (SA)/benzothiadiazole (BTH)-induced disease resistance. Its knockdown severely reduces BTH-induced resistance to the fungal pathogen Magnaporthe oryzae and the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo). Conversely, overexpression of WRKY45 induces extremely strong resistance to both of these pathogens. To elucidate the molecular basis of WRKY45-dependent disease resistance, we analyzed WRKY45-regulated gene expression using rice transformants and a transient gene expression system.

Results

We conducted a microarray analysis using WRKY45-knockdown (WRKY45-kd) rice plants, and identified WRKY45-dependent genes among the BTH-responsive genes. The BTH-responsiveness of 260 genes was dependent on WRKY45. Among these, 220 genes (85%), many of which encoded PR proteins and proteins associated with secondary metabolism, were upregulated by BTH. Only a small portion of these genes overlapped with those regulated by OsNPR1/NH1, supporting the idea that the rice SA pathway branches into WRKY45- regulated and OsNPR1/NH1-regulated subpathways. Dexamethazone-induced expression of myc-tagged WRKY45 in rice immediately upregulated transcription of endogenous WRKY45 and genes encoding the transcription factors WRKY62, OsNAC4, and HSF1, all of which have been reported to have defense-related functions. This was followed by upregulation of defense genes encoding PR proteins and secondary metabolic enzymes. Many of these genes were also induced after M. oryzae infection. Their temporal transcription patterns were consistent with those after dexamethazone-induced WRKY45 expression. In a transient expression system consisting of particle bombardment of rice coleoptiles, WRKY45 acted as an effector to trans-activate reporter genes in which the luciferase coding sequence was fused to upstream and intragenic sequences of WRKY62 and OsNAC4. Trans-activation of transcription occurred through a W-box-containing sequence upstream of OsNAC4 and mutations in the W-boxes abolished the trans-activation.

Conclusions

These data suggest a role of WRKY45 in BTH-induced disease resistance as a master regulator of the transcriptional cascade regulating defense responses in one of two branches in the rice SA pathway.

Keywords:
WRKY; Salicylic acid; Benzothiadiazole; Magnaporthe oryzae; OsNPR1