BackgroundThe 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.ResultsWe 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.ConclusionsThese 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.

SUMMARYRice (Oryza sativa) produces diterpenoid phytoalexins (DPs), momilactones and phytocassanes as major phytoalexins. Accumulation of DPs is induced in rice by blast fungus infection, copper chloride or UV light. Here, we describe a rice transcription factor named diterpenoid phytoalexin factor (DPF), which is a basic helix-loop-helix (bHLH) transcription factor. The gene encoding DPF is expressed mainly in roots and panicles, and is inducible in leaves by blast infection, copper chloride or UV. Expression of all DP biosynthetic genes and accumulation of momilactones and phytocassanes were remarkably increased and decreased in DPF over-expressing and DPF knockdown rice, respectively. These results clearly demonstrated that DPF positively regulates DP accumulation via transcriptional regulation of DP biosynthetic genes, and plays a central role in the biosynthesis of DPs in rice. Furthermore, DPF activated the promoters of COPALYL DIPHO-SPHATE SYNTHASE2 (CPS2) and CYTOCHROME P450 MONOOXYGENASE 99A2 (CYP99A2), whose products are implicated in the biosynthesis of phytocassanes and momilactones, respectively. Mutations in the Nboxes in the CPS2 upstream region, to which several animal bHLH transcription factors bind, decreased CPS2 transcription, indicating that DPF positively regulates CPS2 transcription through the N-boxes. In addition, DPF partly regulates CYP99A2 through the N-box. This study demonstrates that DPF acts as a master transcription factor in DP biosynthesis.

Plant activators such as benzothiadiazole (BTH) protect plants against diseases by priming the salicylic acid (SA) signaling pathway. In rice, the transcription factor WRKY45 plays a central role in this process. To investigate the mechanism involved in defense-priming by BTH and the role of WRKY45 in this process, we analyzed the transcripts of biosynthetic genes for diterpenoid phytoalexins (DPs) during the rice–Magnaporthe oryzae interaction. The DP biosynthetic genes were barely upregulated in BTH-treated rice plants, but were induced rapidly after M. oryzae infection in a WRKY45-dependent manner. These results indicate that the DP biosynthetic genes were primed by BTH through WRKY45. Rapid induction of the DP biosynthetic genes was also observed after M. oryzae infection to WRKY45-overexpressing (WRKY45-ox) plants. The changes in gene transcription resulted in accumulation of DPs in WRKY45-ox and BTH-pretreated rice after M. oryzae infection. Previously, we reported that cytokinins (CKs), especially isopentenyladenines, accumulated in M. oryzae-infected rice. Here, we show that DP biosynthetic genes are regulated by the SA/CK synergism in a WRKY45-dependent manner. Together, we propose that CK plays a role in mediating the signal of M. oryzae infection to trigger the induction of DP biosynthetic genes in BTH-primed plants.Electronic supplementary materialThe online version of this article (doi:10.1007/s11103-014-0221-x) contains supplementary material, which is available to authorized users.

WRKY62 is a transcriptional repressor regulated downstream of WRKY45, a central transcription factor of the salicylic acid signaling pathway in rice. Previously, WRKY62 was reported to regulate defense negatively. However, our expressional analysis using WRKY62-knockdown rice indicated that WRKY62 positively regulates defense genes, including diterpenoid phytoalexin biosynthetic genes and their transcriptional regulator DPF. Blast and leaf blight resistance tests also showed that WRKY62 is a positive defense regulator. Yeast two-hybrid, co-immunoprecipitation and gel-shift assays showed that WRKY45 and WRKY62 can form a heterodimer, as well as homodimers, that bind to W-boxes in the DPF promoter. In transient assays in rice sheaths, the simultaneous introduction of WRKY45 and WRKY62 as effectors resulted in a strong activation of the DPF promoter:hrLUC reporter gene, whereas the activity declined with excessive WRKY62. Thus, the WRKY45-WRKY62 heterodimer acts as a strong activator, while the WRKY62 homodimer acts as a repressor. While benzothiadiazole induced equivalent numbers of WRKY45 and WRKY62 transcripts, consistent with heterodimer formation and DPF activation, submergence and nitrogen replacement induced only WRKY62 transcripts, consistent with WRKY62 homodimer formation and DPF repression. Moreover, WRKY62 positively regulated hypoxia genes, implying a role forWRKY62 in the modulation of the 'trade-off' between defense and hypoxia responses.

A class-C floral homeotic gene of Petunia, pMADS3, is specifically expressed in the stamen and carpels of developing flowers. We had previously reported the ect-pMADS3 phenomenon in which introduction of a part of the pMADS3 genomic sequence, including intron 2, induces ectopic expression of endogenous pMADS3. Unlike transcriptional or posttranscriptional gene silencing triggered by the introduction of homologous sequences, this observation is unique in that the gene expression is up-regulated. In this study, we demonstrated that the ect-pMADS3 phenomenon is due to transcriptional activation based on RNA-directed DNA methylation (RdDM) occurring in a particular CG in a putative cis-element in pMADS3 intron 2. The CG methylation was maintained over generations, along with pMADS3 ectopic expression, even in the absence of RNA triggers. These results demonstrate a previously undescribed transcriptional regulatory mechanism that could lead to the generation of a transcriptionally active epiallele, thereby contributing to plant evolution. Our results also reveal a putative negative cis-element for organ-specific transcriptional regulation of class-C floral homeotic genes, which could be difficult to identify by other approaches. DNA methylation and histone modifications have been implicated in many biological processes including transcriptional gene silencing (TGS), genome imprinting, and paramutation in plants and animals. In many instances, RNA molecules play a crucial role as a trigger to induce a series of reactions leading to the modulation of gene expression mediated by DNA methylation and/or histone modifications. Small doublestranded RNAs (dsRNAs) of 21-24 bp trigger RNA-directed DNA methylation (RdDM) of homologous DNA sequences, leading to TGS in plants (1, 2). Induction of TGS by small RNAs has also been reported in human cells (3,4). Involvement of dsRNAs due to RNA-dependent RNA polymerase (RdRP) and possibly small RNAs has been shown in paramutation of the b1 locus in maize (5, 6). These epigenetic regulations reported so far have been the down-regulation of transcription. Recently, however, 2 research groups have reported that the addition of small dsRNAs homologous to transcriptional regulatory sequences of certain endogenous genes up-regulates the transcription of target genes in human cultured cells (7,8). This finding is striking because it has shown the existence of a previously undescribed type of small dsRNA-mediated regulation, a regulation opposite to that of TGS. However, only limited numbers of observations have been reported and their underlying mechanisms are still unclear.We had previously reported that the introduction of a part of the genomic sequence of pMADS3, a class-C floral homeotic gene, induces ectopic up-regulation of endogenous pMADS3 in the flowering plant Petunia hybrida (9). This gene is a petunia ortholog of AGAMOUS (AG), an Arabidopsis class-C floral homeotic gene involved in the specification of stamens and carpels (10). AG contains a long second intron (intron 2), which ...