Introduction

In response to attacks by a wide variety of microbial pathogens and insect herbivores, plants activate a complex series of defense responses. These induced responses are expressed not only locally but also in plant parts distant from the site of infection, thereby protecting plants from secondary infection. Systemic acquired resistance (SAR) is one of the best-studied induced defense responses in plants; it is long-lasting and effective against a broad spectrum of microbial pathogens (Ryals et al. 1996). In dicots such as Arabidopsis and tobacco, the onset of SAR is accompanied by an increase in endogenous levels of salicylic acid (SA) and concomitant upregulation of a large number of genes, including pathogenesis-related (PR) genes.

NPR1 is a central positive regulator of SAR in Arabidopsis, and transduces the SA signal to downstream PR gene activation (Cao et al. 1994; Delaney et al. 1995; Shah et al. 1997). Nuclear localization of NPR1 is required for the upregulation of PR genes (Kinkema et al. 2000); its nucleocytoplasmic localization is regulated by changes in cellular redox potentials (Mou et al. 2003). Under uninduced conditions, NPR1 forms an oligomer and is localized in the cytoplasm. After SA accumulation triggered by pathogen infection, cellular redox potential changes, resulting in the translocation of NPR1 to the nucleus as an active monomeric form and the interaction with TGA family members of basic domain/Leu zipper (bZIP) transcription factors (TFs) (Mou et al. 2003; Zhang et al. 1999). These interactions stimulate the binding of TGA factors to SA-responsive elements in the promoter of PR genes and regulate the expression of defense-related genes (Despres et al. 2003; Fan and Dong 2002; Johnson et al. 2003). Recently, proteasome-mediated turnover of NPR1 was found to play an important role in modulating transcription of its target genes (Spoel et al. 2009). Role of NPR1 in antagonistic interaction of SA signaling on abscisic acid signaling has also been reported (Yasuda et al. 2008).

Rice (Oryza sativa) is one of the most important food crops and is a model in monocots for investigating the molecular mechanisms behind defense responses. Unlike dicots, in which basal levels of SA are low and the levels increase substantially after a pathogen attack, rice plants accumulate high levels of SA under uninfected conditions, and those levels are apparently insensitive to pathogen infection (Silverman et al. 1995); this implies that the rice defense mechanism is different from those in dicots. However, several studies have demonstrated that rice also has an SA-mediated defense-signaling pathway. SA-deficient transgenic plants overexpressing the nahG gene, encoding a salicylate hydroxylase, exhibit a reduced resistance to blast disease, accompanied by an increased susceptibility to oxidative damages incurred by biotic and abiotic stresses (Yang et al. 2004). Overexpression of Arabidopsis NPR1 in rice confers an enhanced resistance to bacterial blight disease and fungal pathogens (Chern et al. 2001; Fitzgerald et al. 2004; Quilis et al. 2008). Moreover, the overexpression of OsNPR1/NH1, a rice ortholog of NPR1, confers an enhanced resistance to bacterial blight disease (Chern et al. 2005; Yuan et al. 2007), while OsNPR1 knockdown renders rice plants more susceptible to this disease (Yuan et al. 2007). Yuan et al. (2007) suggested that the intracellular localization of OsNPR1 is regulated by changes in cellular redox potential as Arabidopsis NPR1. These observations suggest the presence of a defense-signaling pathway in rice similar to the SA/NPR1-mediated pathway in Arabidopsis, and that NPR1 function is at least in part conserved between dicots and monocots.

Benzothiadiazole (BTH) is a so-called plant activator that protects plants from infectious diseases. BTH is a functional analog of SA, and when applied exogenously, it induces PR gene expression and disease resistance in plants (Gorlach et al. 1996; Lawton et al. 1996). BTH induces plant resistance by priming the SA-signaling pathway mediated by NPR1 in Arabidopsis (Conrath et al. 2002; Lawton et al. 1996). According to Wang et al. (2006), the expression of 99% of BTH-responsive genes depends on NPR1. Several WRKY TFs act as both positive and negative regulators of SAR, downstream of NPR1 (Wang et al. 2006). BTH enhances resistance to various diseases in rice, including blast and leaf-blight diseases. We recently reported that a BTH-upregulated WRKY TF in rice, WRKY45, plays an essential role in BTH-induced blast resistance (Shimono et al. 2007). Interestingly, rice WRKY45 acts in the SA-signaling pathway, independent of OsNPR1, rather than acting downstream of OsNPR1; this suggests that the SA-signaling pathways of rice and Arabidopsis are evolutionally divergent (Shimono et al. 2007).

In this study, we characterized the role of OsNPR1 in the regulation of BTH-induced defense responses in rice. We showed that OsNPR1 is essential for BTH-inducible blast resistance and that an overexpression of OsNPR1 cDNA confers blast resistance to rice. A genome-wide analysis of BTH-responsive genes in rice and of their OsNPR1-dependence revealed that OsNPR1 plays a role in the coordination of BTH-induced downregulation of genes, such as those involved in photosynthesis, chloroplastic transcription and translation, and abscisic acid (ABA)-responsive genes. Taken together with the results of the determination of photosynthetic activities, we discuss the role of OsNPR1 in BTH-induced disease resistance.

Results

OsNPR1 is essential for BTH-inducible blast resistance

To investigate the role of OsNPR1 in BTH-inducible disease resistance, we generated OsNPR1-kd rice plants by expressing an inverted-repeat sequence of the 3′ region of OsNPR1 cDNA. As shown in Fig. 1a, the basal expression of OsNPR1 was reduced in the transformants, in comparison to wild-type (WT) plants, whereas two homologs of OsNPR1 (OsNPR2 and OsNPR3) remained unaffected, demonstrating the specificity of gene-silencing in those OsNPR1-kd lines. In addition, expression of OsNPR1 after BTH treatment was also reduced in transformant lines (Fig. 1b). These transformant lines were then tested for resistance to a compatible race of blast fungus (Magnaporthe grisea; race 007) after BTH application, to examine the role of OsNPR1 in BTH-inducible blast resistance. As shown in Fig. 1c, d, BTH application drastically reduced the number of susceptible-type lesions in WT plants. In contrast, BTH application did not cause a major reduction in the number of blast lesions in OsNPR1-kd lines (Fig. 1d). These results demonstrate that OsNPR1 is essential for BTH-inducible blast resistance.

Fig. 1
figure 1

OsNPR1 is essential for rice blast resistance. a Expression of OsNPR1 and 2 homologous genes in OsNPR1-kd transgenic rice plants. OsNPR1 expression levels in wild-type (WT) rice and 3 T3 OsNPR1-kd transgenic lines (lines #1, #7, and #14) were analyzed by semiquantitative RT–PCR. Transcript levels of OsNPR2 and OsNPR3 were also examined. The expression of Rubq1 was used as an internal control. b qRT-PCR analysis of OsNPR1 expression after mock (M) or BTH (B) treatment in wild-type (WT) rice and T3 OsNPR1-kd transgenic lines (lines #7 and #14). Rubq1 was used as an internal control. Mean ± standard error of mean (SEM) values from 3 independent experiments are shown. c Disease symptoms on the fourth leaves of mock-treated WT, BTH-treated WT, and BTH-treated OsNPR1-kd plants. d Numbers of susceptible-type lesions in the central 10-cm region of the fourth leaves of T3 OsNPR1-kd and WT rice plants. Mean and SEM were calculated from 3 independent experiments. e Blast resistance of OsNPR1-ox rice plants. Average blast lesion numbers of 20–25 plants counted as in d are shown with SD. f In a separate blast-infection experiment, ratios of M. grisea 28S rDNA versus rice Ubi-1 DNA were determined by qRT-PCR. DNA was extracted from each of 20 pooled plants. Averages of triplicate determinations are shown with SD

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We also examined whether OsNPR1 is involved in basal resistance to blast fungus in the absence of BTH treatment. However, we did not observe any significant difference in disease symptoms between WT and OsNPR1-kd rice plants (data not shown) after inoculation with three races of blast fungus, race 007, Ken54-04, and 5203-R-21. We also generated transformant rice plants overexpressing OsNPR1 (OsNPR1-ox) and tested them for blast resistance. The results showed that the 2 lines of OsNPR1-ox plants showed strong resistance against a compatible blast fungus (race 007, Fig. 1e, f), further supporting the positive role of OsNPR1 in blast resistance. Yuan et al. (2007) reported the absence of blast resistance in OsNPR1-ox plants. Differences in experimental conditions—such as expression levels of transgene, growth conditions, plant ages, and the blast fungal races used—may have influenced the blast-resistance phenotype.

Identification of OsNPR1-dependent, BTH-responsive genes using a rice whole-genome oligo microarray

The fact that OsNPR1 is essential for BTH-induced disease resistance prompted us to investigate the details of the defense reactions that OsNPR1 mediates in BTH-treated rice plants. To this end, we analyzed global gene-expression profiles in response to BTH in WT and OsNPR1-kd rice plants, using an Agilent rice oligo microarray comprising 29,923 rice genes. A statistical analysis of the results of 4 biological replicate experiments revealed that a total of 8,567 genes were differentially expressed between mock- and BTH-treated WT rice plants with a q-value of <0.05, after applying a false-discovery rate (FDR) multiple-testing correction, according to the method of Benjamini and Hochberg (1995). We selected 2,297 genes that showed >2-fold change and defined them as being BTH-responsive, of which 1,228 and 1,069 genes were upregulated and downregulated, respectively. Thus, the numbers of BTH-upregulated and BTH-downregulated genes identified herein were roughly comparable to those previously reported for Arabidopsis: Of the approximate 24,000, 1,147 and 1,133 genes were upregulated and downregulated, respectively, after BTH treatments (Wang et al. 2006). We performed two-way analysis of variance (ANOVA; WT vs. OsNPR1-kd, mock vs. BTH) for the microarray results with WT and 2 lines (#7 and #14) of OsNPR1-kd plants after mock and BTH treatments, to identify OsNPR1-dependently BTH-responsive genes (Nettleton 2006; Wang et al. 2006). We then selected the genes that either showed a genotype-treatment interaction or were affected by genotype and treatment in both OsNPR1-kd lines after applying an FDR multiple testing correction with a criteria of q < 0.1 (Benjamini and Hochberg 1995). The genes that passed these filters represented those whose BTH-responsiveness was at least partially negated by OsNPR1-knockdown, and therefore defined as OsNPR1-dependent BTH-responsive genes. Consequently, the BTH-responsiveness of 358 genes out of the 1,228 BTH-upregulated genes was found to be OsNPR1-dependent (29%); meanwhile, that of 724 genes out of the 1,069 BTH-downregulated genes was OsNPR1-dependent (68%).

Detailed lists of BTH-responsive and OsNPR1-dependent genes are available in the supplemental data (Tables S1 and S2). Quantitative real-time polymerase chain reaction (qRT–PCR) analysis for several arbitrarily chosen representative genes completely reproduced the expression patterns in the microarray data with respect to both BTH-responsiveness and OsNPR1-dependence, thus validating the reliability of the microarray data (Fig. 2).

Fig. 2
figure 2

Validation of microarray data by qRT-PCR. Expression data of arbitrarily chosen representative genes from the microarray (left) and qRT-PCR (right) experiments are shown in a pairwise manner. T3 homozygous OsNPR1-kd (lines #7-1 and #14-1) and wild-type rice plants were treated with either DMSO (mock, M) or 10 μM BTH (B) in DMSO. The third and fourth leaves were harvested 24 h after BTH treatment. RNAs extracted from these samples, which were also analyzed by microarray, were analyzed by qRT-PCR for some arbitrary chosen representative genes to validate the BTH-responsiveness and OsNPR1-dependence of expression in microarray analysis. Expression of endogenous OsNPR1 was also examined. Mean ± SEM values from 3 independent experiments are shown. Rubq1 expression was used as an internal control. Putative functions of analyzed genes are as follows: Os07g0677100, peroxidase; Os02g0232900, aquaporin NIP1-1 (OsNIP1;1); Os03g0364400, protein kinase domain containing protein; Os10g0502000, thylakoid lumenal 17.4 kDa protein, chloroplast precursor (P17.4); Os07g0577600, Lhca2 protein; Os02g0814400, cytochrome c domain containing protein; Os02g0754300, 50S ribosomal protein L29, chloroplast precursor. Experiments were repeated three times with similar results

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Functional categorization of BTH-responsive and OsNPR1-dependent genes

To deduce the effects of BTH application on various biological processes from the (predicted) functions of BTH-regulated genes, we analyzed the BTH-responsive genes using Gene Ontology terms (Gene-Ontology-Consortium 2006; Harris et al. 2004). This analysis showed that BTH regulated a broad range of genes with diverse functions classified into several categories (Fig. 3a). Among the BTH-responsive genes, upregulated genes outnumbered the downregulated genes, in nine out of 13 categories; downregulated genes dominated only two categories, i.e., “photosynthesis” and “transcription.” The genes in each category were further analyzed with respect to the OsNPR1-dependence of their BTH-responsiveness (Fig. 3b, c). In contrast to all the BTH-responsive genes, OsNPR1-dependent genes were dominated by downregulated genes in many categories: Upregulated and downregulated genes dominated three and eight categories, respectively (Fig. 3, 3c).

Fig. 3
figure 3

Functional classification of OsNPR1-dependent and BTH-responsive genes. a Total number of BTH-upregulated and BTH-downregulated rice genes in each functional category. The Y-axis shows the number of genes that exhibited >2-fold or <0.5-fold differential expression, after BTH treatments. Genes in different functional categories according to the Gene Ontology database were analyzed. b Total number of OsNPR1-dependent rice genes among BTH-responsive genes. The Y-axis shows the number of genes whose expression is OsNPR1-dependent. c Percentage of OsNPR1-dependent rice genes among the BTH-upregulated and BTH-downregulated genes in each functional category. d Total number of BTH-upregulated and -downregulated Arabidopsis genes in each functional category. Y-axis shows the number of genes that exhibit >2- or <0.5-fold differential expression, after BTH treatments. Functional categories are based on the Gene Ontology database in the Arabidopsis Information Resource (http://www.arabidopsis.org/)

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NPR1-dependent genes in Arabidopsis (Wang et al. 2006) were analyzed in a similar manner for comparison (Fig. 3d). Results showed that the genes categorized as photosynthesis and translation-related genes were dominated by downregulated genes, as is the case with OsNPR1-dependent genes in rice; thus, this is a common feature of (Os)NPR1-dependent genes in rice and Arabidopsis. Among Arabidopsis NPR1-dependent genes in many categories, the proportions of upregulated genes were greater than or similar to those of downregulated genes, unlike the rice OsNPR1-dependent genes. Another evident difference was noted in the genes categorized under “defense response.” While all of the BTH-upregulated genes categorized under “defense response” were NPR1-dependent in Arabidopsis, only 5.9% of those were OsNPR1-dependent in rice.

We further categorized (Os)NPR1-dependently BTH-responsive genes both in rice and Arabidopsis using BioMaps (Hayano-Kanashiro et al. 2009). As shown in Tables S5 and S6, genes involved in various photosynthetic processes were significantly enriched in OsNPR1-dependently BTH-downregulated genes in rice. On the other hand, genes including those involved in translation, amino acid and polysaccharide metabolic processes, and those encoding proteins with chitinase activity and glutathione transferase activity were enriched in OsNPR1-independently BTH-upregulated genes (Table S7). In Arabidopsis, genes involved in defense response and protein phosphorylation were significantly enriched in NPR1-upregulated genes (Table S8), whereas genes involved in response to abiotic stimulus, lipid metabolic process, carbohydrate metabolic process and photosynthesis were enriched in NPR1-downregulated genes (Table S9). These results reveal some differences in the categories of genes that are regulated by (Os)NPR1 in rice and Arabidopsis.

OsNPR1 mediates BTH-induced downregulation of photosynthesis and chloroplast-related genes

We further inspected the functions of BTH-responsive and OsNPR1-dependent genes in reference to known metabolic pathways by using KEGG pathway maps (Kanehisa et al. 2008; Kanehisa et al. 2004). This analysis revealed that the genes involved in various photosynthesis processes were coordinately downregulated by BTH in an OsNPR1-dependent manner (Table 1). These included several genes that encoded components of photosystems I and II, as well as the light-harvesting chlorophyll complex (Table 1, Figs. S2 and S3). These genes also included those involved in the Calvin cycle and in the biosynthesis of porphyrin and chlorophyll (Table 1; Fig. S1).

Table 1 List of OsNPR1-dependently BTH-downregulated genes involved in photosynthesis
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A number of genes involved in chloroplastic translation and transcription were found to be downregulated by BTH in an OsNPR1-dependent manner. These included the genes for the components of 50S and 30S subunits of chloroplastic ribosome; their BTH responses were exclusively OsNPR1-dependent (Table 2). The genes for chloroplastic ribosomes were significantly enriched in OsNPR1-dependently BTH-downregulated genes (P-value: 9.33E-32); chloroplastic ribosomal genes were also enriched in NPR1-dependently BTH-downregulated genes in Arabidopsis (P-value: 6.95E-12). Nuclear-encoded sigma factors are known to regulate organelle transcription (Ishizaki et al. 2005; Kanamaru et al. 2001); the genes for 3 rice sigma factors (Kasai et al. 2004) were downregulated by BTH in an OsNPR1-dependent manner (Table 2). Thus, OsNPR1 mediates the downregulation of the genes for chloroplastic translation and transcription. Of note, several genes for the 60S and 40S subunits of cytoplasmic ribosome were upregulated by BTH (Table 2)—some of them (26%) OsNPR1-dependently. Collectively, OsNPR1 plays a role in the suppression of chloroplastic translation and transcription while activating cytoplasmic translation, thereby coordinating overall cellular activity upon SA-pathway-mediated defense responses.

Table 2 List of BTH-responsive and OsNPR1-dependent genes involved in translation and transcription
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To examine whether the changes in gene expression of photosynthetic genes and those involved in chloroplastic translation and transcription were reflected in photosynthetic activities, we determined the changes in leaf maximum PSII quantum yield (Fv/Fm), which is the maximum quantum yield of photosystem II, after BTH treatment in WT and OsNPR1 knockdown rice plants (Fig. 4). Before the treatments, there was no significant difference in Fv/Fm values between WT and the 2 OsNPR1-kd lines (0.80–0.82). Some decreases in Fv/Fm were observed even in control mock-treated WT plants after control treatment, presumably reflecting suboptimal growth conditions. Notably, the decreases in Fv/Fm values were markedly faster in BTH-treated than in the control plants, indicating that the BTH treatment reduced photosynthetic activity. In addition, in OsNPR1-knockdown lines, the BTH-induced decreases in Fv/Fm values were negated; the values were even higher than those in the control plants. These results are essentially consistent with the downregulation of photosynthetic (and chloroplastic translational and transcriptional) genes by BTH and its negation by OsNPR1 knockdown.

Fig. 4
figure 4

Effects of BTH and OsNPR1 knockdown on leaf maximum PSII quantum yield (Fv/Fm). The Fv/Fm values after BTH (closed circles) and mock (open circles) treatments were expressed as relative to those measured before the treatments. Fv/Fm values on day 0 (i.e., before treatments) were 0.810 ± 0.004 (mean ± S.E., n = 4) and 0.810 ± 0.005 for mock and BTH-treated wild type, respectively. The values were 0.812 ± 0.007 (control) and 0.818 ± 0.003 (BTH) in OsNPR1-kd #7, and 0.810 ± 0.004 (mock) and 0.799 ± 0.006 (BTH) in OsNPR1-kd #14

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The reason for the higher Fv/Fm in OsNPR1-kd lines than in WT is unclear. It could be an indirect effect of the BTH-induced activation of the WRKY45-dependent pathway, which is basically independent of OsNPR1 (Shimono et al. 2007).

OsNPR1 regulates BTH-downregulation of ABA-responsive genes

We also noted that OsNPR1-dependently BTH-downregulated genes include those bearing annotations suggestive of ABA-responsiveness (Table S2). Of these, Os01g0348900 (SalT) and Os01g0705200 (OsWSI18) have been reported to be indeed ABA-responsive (Shobbar et al. 2008). Os06g0324400 encodes late embryogenesis-abundant (LEA) protein. We then examined the ABA responses of these genes in leaf-disc assays by using qRT–PCR, demonstrating that all these genes were indeed ABA-inducible (Fig. 5). Moreover, ABA-induced upregulation of these genes was negated by the simultaneous addition of SA (Fig. 5). However, the suppression of their ABA-induced upregulation by SA was not affected in OsNPR1-kd plants. Collectively, these results suggest that SA-induced downregulation of these ABA-responsive genes from their basal expression level is OsNPR1-dependent, but antagonistic effect of SA on their ABA-induced upregulation is through OsNPR1-independent mechanism.

Fig. 5
figure 5

ABA-responsive expression of OsNPR1-regulated genes, and their suppression by SA. Expression of genes in leaf discs from wild type (white bar), OsNPR1-kd lines #7 (black bar), and #14 (gray bar) at 4 h after treatments with SA, ABA, SA, and ABA or control H2O. The concentrations of applied SA and ABA solutions were 1 mM and 50 μM, respectively. Responses of WRKY45 were examined as control of treatments. Expression levels in qRT-PCR relative to those of Rubq1 as an internal control are shown. Averages of 3 determinations are shown, along with SD. The primer pairs used are listed in Table S4

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BTH-responsiveness and OsNPR1-dependence of TF genes

The BTH-responsive genes included the genes for a number of TFs, some of which were OsNPR1-dependent. Particular families of TFs are often associated with specific biological processes; we inspected the BTH-responsiveness and OsNPR1-dependence of the TF genes in different classes with this view (Table 3).

Table 3 List of BTH-responsive and OsNPR1-dependent genes for transcription factors
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A total of 8 AUX/IAA-type TFs were downregulated by BTH—5 of them in an OsNPR1-dependent manner (Table 3). Two ARF genes were also downregulated by BTH in an OsNPR1-dependent manner. Both AUX/IAAs and ARFs are associated with auxin-related regulation. The downregulation of several AUX/IAA and ARF genes has also been reported in Arabidopsis; it was presumably indirectly NPR1-dependent (Wang et al. 2007). Therefore, (Os)NPR1 suppresses the auxin-related regulatory pathway in response to BTH in rice and Arabidopsis, in a similar manner.

BTH-responsiveness and OsNPR1-dependence of other genes

We also inspected the BTH-responsiveness and OsNPR1-dependence of the genes involved in some metabolic pathways. The genes involved in the TCA cycle, and the shikimate, aromatic-acid biosynthetic, and phenylpropanoid pathways were upregulated by BTH. The upregulation of these genes should lead to the biosynthesis of phenylpropanoid compounds, such as lignins and phytoalexins, which may play a role in plant defense. Interestingly, unlike in Arabidopsis, only a few of them were OsNPR1-dependent.

Discussion

In previous studies, it has been reported that rice shares a defense-signaling pathway similar to the NPR1-mediated pathway in dicots, and that components of this pathway largely share functions common to both rice and dicots (Chern et al. 2005; Yuan et al. 2007). In addition to the role of OsNPR1 in bacterial leaf-blight resistance (Chern et al. 2005; Yuan et al. 2007), in this study, we demonstrated that OsNPR1 plays an essential role in BTH-induced resistance against fungal blast disease by showing that BTH-inducible blast resistance was compromised in OsNPR1-kd rice plants and that an overexpression of OsNPR1 conferred blast resistance to rice (Fig. 1). These results indicate that the role of OsNPR1 in the (chemical-induced) resistance to biotrophic and hemibiotrophic pathogens is consistent with rice sharing a SA-signaling pathway similar to those observed in dicots. We showed in our previous study that the SA-signaling pathway of rice branches into 2 subpathways downstream of SA—that is, the OsNPR1-dependent and WRKY45-dependent ones (Shimono et al. 2007). The WRKY45-dependent pathway is also essential for BTH-induced resistance to blast disease (Shimono et al. 2007). The question then arises as to how OsNPR1 and WRKY45 share their functions in the SA pathway in rice. In Arabidopsis, nearly all the BTH-responsive genes are NPR1-dependent (Wang et al. 2006), although larger proportion of genes is NPR-independent in early response to SA (Blanco et al. 2009). In contrast, we showed in this study that only a subset of BTH-responsive genes were OsNPR1-dependent in rice. The genes upregulated by BTH were less OsNPR1-dependent than those downregulated by BTH (Fig. 3c). These results imply that the role of OsNPR1 in the SA-signaling pathway of rice is rather limited as compared to that of NPR1 in Arabidopsis, and thus reflects the branched SA pathway in rice (Shimono et al. 2007). We cannot completely rule out the possibility that the partialness of the OsNPR1-dependence is (partly) due to incomplete suppression of OsNPR1 and/or the possibility that our analysis of the microarray data is somehow biased toward BTH-downregulated genes due to statistical factors (Fig. 2). Nevertheless, the extensively compromised blast resistance phenotype in OsNPR1-knockdown rice suggests that OsNPR1 function is substantially impaired in these plants and hence the validity of the microarray results seems to be supported by this observation (Fig. 1c, d).

Many studies have shown that biotrophic pathogens, such as rusts, mildews, and powdery mildews, cause a reduction in the rate of photosynthesis and a loss of chlorophylls from infected leaves as a whole (Schloles 1992). It has also been recognized that reductions in photosynthesis transcripts/proteins are common in defense responses (Somssich and Hahlbrock 1998). In rice, too, a coordinated repression of transcripts for chloroplast-related genes, including photosynthetic genes, has been observed after infection with the rice dwarf virus (Shimizu et al. 2007). The biological significance of the suppression of photosynthetic activities vis-à-vis pathogen infection has been discussed in relation to the reallocation of resources from plant growth to defense reactions as part of defense responses (Lerdau 1992). This is relevant to fitness cost that defense reactions impose on plant growth and development. Production of defensive proteins during SAR requires large amounts of resources and consequently reduces plant fitness, in particular under nutrition-limited conditions (Heidel et al. 2004; Heidel and Dong 2006). Contribution of NPR1 in reducing the fitness cost of SAR has been reported in Arabidopsis (Heidel et al. 2004; Heidel and Dong 2006; van Hulten et al. 2006). We found in this study that photosynthesis-related genes, as well as chloroplastic ribosomal genes, were transcriptionally repressed OsNPR1-dependently when the SA-signaling pathway was activated (Tables 1, 2), which led to a reduction of photosynthetic activities (Fig. 4). Arabidopsis NPR1 was also found to have similar functions (Fig. 3d). Because photosynthetic proteins account for high proportion of cellular proteins in plants, this regulation presumably saves substantial amounts of amino acids to be used for production of defensive proteins. Thus, the allocation of resources from household activities to defensive activities during SAR seems to be one of common functions of (Os)NPR1 in rice and Arabidopsis.

In Arabidopsis, the NPR1-mediated suppression of ABA-responsive gene expression has been reported (Yasuda et al. 2008). Expression of some ABA-responsive genes is suppressed by BTH/SA also in rice (Jiang et al. 2010). However, our data shows that this regulation is independent of OsNPR1, unlike in Arabidopsis. Thus, the SA pathway acts antagonistically upon the ABA-signaling pathway in the two plant species in common, but the antagonistic interactions in them differ in their (Os)NPR1-dependence. We previously proposed that the SA pathway in rice is branched into WRKY45-dependent and OsNPR1-dependent pathways downstream of SA (Shimono et al. 2007), and ABA has negative impacts on both pathways (Jiang et al. 2010). The branched nature of the SA pathway may account for the difference in (Os)NPR1 dependency in the SA-ABA interaction between rice and in Arabidopsis.

In summary, our results demonstrate that OsNPR1 plays a crucial role in SA-mediated defense signaling, which is also effective against blast disease. Our data shed new light on the role of OsNPR1 including its suppression of chloroplast activities and photosynthesis. The information should help advance an understanding of the SA/BTH-signaling pathway in rice, a major staple food worldwide.

Materials and methods

Plant materials, chemicals, and pathogen treatments

All experiments were carried out with rice (Oryza sativa) cv. Nipponbare. Plants were grown in a growth chamber as described previously (Shimono et al. 2007). For BTH treatments, rice seedlings at the four-leaf stage were excised about 5 mm above the soil surface and held for 24 h in a solution containing 10 μM BTH in 0.01% dimethyl sulfoxide (DMSO) or the solvent only (mock treatment). For M. grisea inoculations, spores were suspended in 0.02% Tween 20 at a density of 105/mL and sprayed onto rice plants as described previously (Shimono et al. 2007). Disease symptoms were evaluated by lesion numbers and fungal DNA contents. Fungal DNA contents were determined by RT–PCR-based quantification of M. grisea 28S rDNA in rice leaves (Qi and Yang 2002). Fungal growth was evaluated by the ratio of M. grisea 28S rDNA versus rice Ubi-1 DNA. Mann–Whitney U test was used to determine if the effects of the OsNPR1 mutants on disease symptoms were statistically significant.

Plasmid construction and plant transformation

To construct a plasmid for OsNPR1 RNAi, part of the OsNPR1 cDNA was amplified by PCR, using the primers NL0RiF2 (5′-CGAGGACAAGGAGGAGAATG-3′) and NL0RiR2 (5′-ACGAGCAAACCACTGGAAAT-3′); it was then inserted into the pANDA vector, as described previously (Miki et al. 2005; Miki and Shimamoto 2004; Shimono et al. 2007). Rice was transformed by an Agrobacterium tumefaciens (strain EHA105)-mediated procedure, as described previously (Toki et al. 2006).

Microarray analysis

Total RNA was isolated from the fourth leaves of BTH- or mock-treated rice plants and labeled with Cy3, as described previously (Shimono et al. 2007). Cy3-labeled probes (1 mg each) were hybridized to an Agilent Rice Oligo Microarray (44 K, custom-made; Agilent Technologies). Four biological replicate sample sets from BTH- and mock-treated leaves were analyzed. All microarray experiments, including data analysis, were carried out as described previously (Shimono et al. 2007; Wang et al. 2006). Briefly, BTH-responsive genes in WT were identified on the basis of statistical significance with an ANOVA q-value <0.05, after applying an FDR multiple-testing correction (Benjamini and Hochberg 1995) and fold change (>2). These genes were filtered through a two-way ANOVA, with consideration for both genotype (WT vs. OsNPR1-kd) and treatment (mock vs. BTH) effects. We then selected the genes that either showed a genotype-treatment interaction or were affected by genotype and treatment in both OsNPR1-kd lines #7 and #14 with q < 0.1 (Benjamini and Hochberg 1995). For the functional assignment of BTH-responsive genes, Gene Ontology terms (http://www.geneontology.org/) were used. We obtained Gene Ontology and annotations of rice (Oryza sativa) cv. Nipponbare and Arabidopsis thaliana genomes from RAP-DB (http://rapdb.dna.affrc.go.jp/index.html) and TAIR (http://www.arabidopsis.org/), respectively. For the analysis of microarray data in conjunction with KEGG pathway maps (http://www.genome.jp/kegg/pathway.html), the KeggArray tool (0.5.3 beta; http://www.genome.jp/kegg/kegg4.html) was used. For functional classification of differentially expressed transcripts, the microarray data was analyzed using BioMaps at the Virtual-Plant site (http://virtualplant.bio.nyu.edu). Statistic significance of differential expression of the genes for functional category of ribosome was examined by a Chi-squared test based on the Gene Ontology terms obtained from the Virtual-Plant site.

RT-PCR analysis

RT-PCR and qRT-PCR was performed as described previously (Shimono et al. 2007). Rice ubiquitin 1 (Rubq1; AK121590) was used as an internal standard. PCR primers used in this study are listed in Table S4; the primer sets were tested by dissociation curve analysis and verified for the absence of nonspecific amplification.

Determination of chlorophyll fluorescence

Rice plants at the seventh-leaf emergence stage were transferred from a greenhouse to a growth chamber and grown during the taking of chlorophyll fluorescence measurements. The condition in the growth chamber were 16/8 h of day/night cycle, day/night temperatures of 28/23°C and a light intensity at plant height of 300 μmol m−2 s−1. WT and OsNPR1-kd rice plants were supplied with 50 μM BTH in 0.05% (v/v) DMSO or solvent only (mock) from the roots by bathing the roots with sufficient solution. Maximum PSII quantum yield (Fv/Fm) was measured on the fifth leaves, after 30 min-2 h dark adaptation (PAM-2100; Walz, Germany). Four plants were used for each treatment.

ABA and SA treatments of leaf discs

Stock solutions of ABA (10 mM, in ethanol) and SA (1 M, aqueous) were diluted with distilled water containing 0.01% (v/v) Silwet L-77 (NIPPON Genetics, Tokyo, Japan) to prepare the test solutions. Rice leaves were cut to 5-mm lengths and soaked in test solutions containing 0.5% (v/v) ethanol in a 12-well microtiter plate. The samples were incubated for 4 h at room temperature, weighed, and stored at −80°C.