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OsWRKY28, a PAMP-responsive transrepressor, negatively regulates innate immune responses in rice against rice blast fungus

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Abstract

WRKY transcription factors form a large family of plant-specific transcription factors and participate in plant defense responses either as positive or negative regulators. In this study, we comprehensively analyzed the role of one of the group IIa WRKY transcription factors in rice, OsWRKY28, in the regulation of basal defense responses to a compatible race of the rice blast fungus Magnaporthe oryzae, strain Ina86-137. The expression analyses of the group IIa WRKY transcription factors in rice revealed that OsWRKY28, together with OsWRKY71, exhibit an early-induced expression prior to the late-induced expressions of OsWRKY62 and OsWRKY76. The GFP–OsWRKY28 fusion protein localized mainly in the nuclei of onion epidermal cells, and the maltose-binding protein–fused OsWRKY28 recombinant protein specifically bound to W-box elements. A transient reporter gene assay clearly showed that OsWRKY28 functions as a transcriptional repressor. Overexpression of OsWRKY28 in rice plants resulted in enhanced susceptibility to Ina86-137. Finally, transcriptome analysis revealed that the induction of several defense-related genes in the wild type after Ina86-137 infection was counteracted in OsWRKY28-overexpressing rice plants. These results strongly suggest that OsWRKY28 is a negative regulator of basal defense responses against Ina86-137 and acts as a modulator to maintain the responses at an appropriate level by attenuating the activation of defense-related gene expression levels.

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Acknowledgments

We thank Dr. M. Takagi and Dr. M. Shikata for the 35S:GAL4 (430T1.2) plasmids, Dr. H. Takatsuji for the pUCAP-Ubi-NT and pZH1 plasmids, Dr. K. Shimamoto for the p2KG plasmid, and Dr. T. Nakagawa for the pGWB2 and pGWB6 plasmids. We also thank Dr. Y. Nagamura and Ms. R. Motoyama of the Rice Genome Resource Center for technical support with the microarray analysis. This work was supported by the Program for Promotion of Basic and Applied Researches for Innovations in Bio-oriented Industry.

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Author notes

  1. Tetsuya Chujo

    Present address: Institute of Molecular BioSciences, Massey University, Private Bag 11 222, Palmerston North, 4442, New Zealand

  2. Takafumi Shimizu

    Present address: Botanical Gardens, Graduate School of Science, Osaka City University, 2000 Kisaichi, Katano, Osaka, 576-0004, Japan

Authors and Affiliations

  1. Biotechnology Research Center, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo, 113-8657, Japan

    Tetsuya Chujo, Koji Miyamoto, Takeo Shimogawa, Takafumi Shimizu, Hideaki Nojiri, Hisakazu Yamane & Kazunori Okada

  2. Division of Plant Sciences, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki, 305-8602, Japan

    Yuko Otake, Naoki Yokotani, Yoko Nishizawa & Eiichi Minami

  3. Department of Life Sciences, Faculty of Agriculture, Meiji University, 1-1-1 Higashi-mita, Tama-ku, Kawasaki, Kanagawa, 214-8571, Japan

    Naoto Shibuya

  4. Department of Biosciences, Teikyo University, 1-1 Toyosatodai, Utsunomiya, Tochigi, 320-8551, Japan

    Hisakazu Yamane

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  1. Tetsuya Chujo
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Correspondence to Kazunori Okada.

Additional information

Tetsuya Chujo, Koji Miyamoto, Takeo Shimogawa and Takafumi Shimizu contributed equally to this work.

Electronic supplementary material

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11103_2013_32_MOESM1_ESM.eps

Supplementary Fig. S1. Alignment of the deduced amino acid sequences of group IIa WRKYs in rice and their homologs from monocots and dicots. The deduced amino acid sequences of OsWRKY28/62/71/76, barley HvWRKY1/2, and AtWRKY18/40/60 were aligned using the CLUSTAL W program. Highly conserved residues are shaded in black, and similar residues are shaded in gray. LxLxL(I), putative repressor motif; LZ, leucine zipper motif; NLS, putative nuclear localization signal; WRKY, WRKY DNA-binding motif. (EPS 2346 kb)

11103_2013_32_MOESM2_ESM.eps

Supplementary Fig. S2. Expression profiles of group IIa WRKY genes in rice leaves infected with the rice blast fungus. Total RNA was isolated from rice leaves 48 h after infection of M. oryzae strain Ina86-137 (137) or mock treatment. mRNA levels were analyzed by SYBR green-based real-time PCR. Values indicate relative mRNA levels normalized to the expression of the UBQ gene. Expression levels are shown relative to the mock treated rice leaves. The results are the average of at least 3 independent experiments; bars indicate the standard deviation of the mean. Mock, mock treatment; 137, M. oryzae Ina86-137 infection. (EPS 939 kb)

11103_2013_32_MOESM3_ESM.eps

Supplementary Fig. S3. Transrepression activity of OsWRKY28.The scheme represents effector constructs fused to the GAL4 DNA-binding domain (GAL4DB) and reporter construct. A non-fused construct was used as a negative control, and the GAL4DB-SRDX construct was used as a positive control. LUC activity was normalized against that of Renilla LUC. The bars represent relative LUC activity after incubation of the rice cells. The values are mean ± SE of 3 replicates. Significantly lower LUC activity affected by either GAL4DB-OsWRKY28 or GAL4DB-SRDX compared to GAL4DB control are denoted by asterisks (P < 0.001 by t test). (EPS 420 kb)

11103_2013_32_MOESM4_ESM.eps

Supplementary Fig. S4. Summary of the genes regulated by OsWRKY28 in rice plants. Effect of OsWRKY28 on both steady-state gene expression and the rice blast fungus–induced gene expression changes were analyzed using the Agilent rice 44 k oligoarray with the one-color method. Venn diagrams show the number of overlapping and unique genes. Genes that were downregulated or upregulated more than twofold by mock treatment or rice blast fungus infection on OsWRKY28-overexpressing rice plants compared to WT rice plants are shown. (EPS 414 kb)

11103_2013_32_MOESM5_ESM.eps

Supplementary Fig. S5. Leaf lesion lengths of OsWRKY28-overexpressing rice plants challenged with the blight bacteria X. oryzae pv. oryzae. The youngest fully expanded sixth leaves were inoculated, and lesion lengths were measured 10 d afterwards. Each bar represents the average and standard error of at least six leaves. (EPS 824 kb)

11103_2013_32_MOESM6_ESM.eps

Supplementary Fig. S6. Chloroplast-related genes were downregulated in OsWRKY28-overexpressing rice plants. Bar graphs represent gene expression levels derived from the microarray data set; bars indicate the standard deviation of the mean. WT, wild-type rice plants; W28OX, OsWRKY28-overexpressing rice plants; Mock, mock treatment; 137, M. oryzae Ina86-137 infection. (EPS 472 kb)

11103_2013_32_MOESM7_ESM.eps

Supplementary Fig. S7. Upregulated genes in OsWRKY28-overexpressing rice plants infected with the rice blast fungus organized by molecular function GO categories. Organization of the 282 upregulated genes in OsWRKY28-overexpressing rice plants infected with the rice blast fungus compared to WT rice plants by molecular function GO categories. Categories are as follows: binding (Binding; GO:0005488), catalytic activity (Catalytic; GO:0003824), chloroplast-related genes (Chloroplast; non-GO categories), defense-related genes (Defense; non-GO categories), nucleic acid binding transcription factor activity (Transcription; GO:0001071), transporter activity (Transporter; GO:0005215), electron carrier activity (e-carrier; GO:0009055), and molecular function unknown (Unknown; non-GO category). (EPS 3339 kb)

11103_2013_32_MOESM8_ESM.xlsx

Supplementary Table S1. List of genes whose expression is significantly downregulated in OsWRKY28-overexpressing rice plants infected with the compatible rice blast fungus strain (corresponding to Fig. 4) (XLSX 62 kb)

11103_2013_32_MOESM9_ESM.xlsx

Supplementary Table S2. List of genes whose expression is significantly upregulated in OsWRKY28-overexpressing rice plants infected with the compatible rice blast fungus strain. (XLSX 49 kb)

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Chujo, T., Miyamoto, K., Shimogawa, T. et al. OsWRKY28, a PAMP-responsive transrepressor, negatively regulates innate immune responses in rice against rice blast fungus. Plant Mol Biol 82, 23–37 (2013). https://doi.org/10.1007/s11103-013-0032-5

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