OsTGAP1, a bZIP transcription factor, coordinately regulates the inductive production of diterpenoid phytoalexins in rice

Abstract

Production of major diterpenoid phytoalexins, momilactones and phytocassanes, is induced in rice upon recognition of pathogenic invasion as plant defense-related compounds. We recently showed that biosynthetic genes for momilactones are clustered on rice chromosome 4 and co-expressed after elicitation, mimicking pathogen attack. Because genes for most metabolic pathways in plants are not organized in gene clusters, examination of the mechanism(s) regulating the expression of such clustered genes is needed. Here, we report a chitin oligosaccharide elicitor-inducible basic leucine zipper transcription factor, OsTGAP1, which is essential for momilactone biosynthesis and regulates the expression of the five genes in the cluster. The knock-out mutant for OsTGAP1 had almost no expression of the five clustered genes (OsCPS4, OsKSL4, CYP99A2, CYP99A3, and OsMAS) or production of momilactones upon elicitor treatment. Inductive expression of OsKSL7 for phytocassane biosynthesis was also largely affected in the ostgap1 mutant, although phytocassane accumulation still occurred. Conversely, OsTGAP1-overexpressing lines exhibited enhanced expression of the clustered genes and hyperaccumulation of momilactones in response to the elicitor. Furthermore, enhanced expression of OsKSL7 and hyperaccumulation of phytocassanes was also observed. We also found that OsTGAP1 overexpression can influence transcriptional up-regulation of OsDXS3 in the methylerythritol phosphate pathway, eventually leading to inductive production of diterpenoid phytoalexins. These results indicate that OsTGAP1 functions as a key regulator of the coordinated transcription of genes involved in inductive diterpenoid phytoalexin production in rice and mainly exerts an essential role on expression of the clustered genes for momilactone biosynthesis.

FIGURE 1.

A, proposed biosynthetic pathway for the diterpenoid phytoalexins in rice. The enzymes involved in momilactone and phytocassane biosyntheses are indicated. The dashed arrows show steps required for multiple reactions. B, map of the momilactone biosynthetic gene cluster on rice chromosome 4. The black boxes indicate the five momilactone biosynthetic genes.

FIGURE 2.

A, deletion analysis of the OsKSL4 promoter between −1928 and −414 bp. The promoter fragment located −1928 bp upstream of the transcription start site (TSS) of OsKSL4 and the 5′-untranslated region (UTR; 59 bp) were successively deleted from −1928 to −414, and the resulting fragments were fused to the firefly LUC gene. A putative TATA box is located at −39 to −31 bp. B, nucleotide sequence of the region from −1224 to −991 upstream of the transcription start site of OsKSL4 and known cis-acting elements existing in this region. C, mutation analysis of candidate cis-acting elements in the promoter region from −1224 to −991. Positions of mutated TGAC sequences, in which TGAC was altered to CCTA, are indicated by X. LUC activity was normalized against that of Renilla LUC. The black and open bars indicate the relative LUC activities of the deletion derivatives after 15 h of incubation of the rice cells with or without the elicitor, respectively. The data are means ± S.D. of three replicates. *, significant differences (p < 0.01) between the relative LUC activities in the elicited and non-elicited cells.

FIGURE 3.

A, heat map of elicitor-inducible bZIP transcription factor genes isolated from microarray data after elicitor treatment. The results of two independent experiments are shown. The ratio of the level of gene expression at various time points in untreated cells compared with elicitor-treated cells was used to compile the heat map, which represents increases (red) and decreases (green) in expression relative to the untreated levels. The color bar represents the range of ratios. Three TGA-type transcription factor genes are indicated. B, transcription profiling of AK073715, AK102690, and OsKSL4 after elicitation, using qRT-PCR. Values indicate relative mRNA levels of AK073715 (red diamond), AK102690 (blue circles), and OsKSL4 (black box), normalized to the expression level of the UBQ gene. The maximal value in each experiment using different primers was arbitrarily set at 1.0. The results are the average of at least three independent experiments; bars indicate the means ± S.D. C, nuclear localization of the green fluorescent protein (GFP)-fused AK073715 or AK102690 protein in onion epidermal cells. Top, localization of the green fluorescent protein control. Middle, localization of AK073715 tagged with green fluorescent protein at its N terminus. Bottom, localization of AK102690 tagged with green fluorescent protein at its N terminus. The pictures represent green fluorescence (left) or visible light (right). Bars, 100 μm.

FIGURE 4.

Physiological function of AK073715 in momilactone biosynthesis. A, accumulation of momilactones in wild-type and H0155 mutant cells after elicitor treatment. B, accumulation of phytocassanes in wild-type and H0155 mutant cells after elicitor treatment. Phytoalexin levels in the culture medium collected 0, 48, and 72 h after elicitor treatment were determined by LC-MS/MS. The results are the average of at least three independent experiments. Bars, mean ± S.D. C, expression analysis of OsKSL4 in elicitor-treated cells, by qRT-PCR. Total RNA was prepared 0, 6, 12, and 24 h after elicitor treatment. The results are the average of at least three independent experiments, and values for OsKSL4 mRNA expression were normalized to the expression of the UBQ gene. Bars, means ± S.D. D, expression analysis of OsKSL7 in elicitor-treated cells after qRT-PCR as in C. E, expression analysis of OsDXS3 in elicitor-treated cells after qRT-PCR as in C. F, GC-MS analysis of accumulated diterpene hydrocarbons in the mutant and wild-type cells after elicitation. Selected ion chromatograms at a mass/charge ratio of 272 showing 9βH-pimara-7,15-diene and ent-cassa-12,15-diene are indicated.

FIGURE 5.

A, sequence-specific DNA-binding activity of OsTGAP1. Electrophoretic mobility shift assays show the binding of recombinant OsTGAP1 protein to probes (P) containing the TGACG motif (TGACGT), but not to the mutated probe (Pm, with the TGAC sequence mutated to CCTA). For the assay, 240 ng of GST-OsTGAP1 were loaded with the probes. Nonlabeled probe was added in a 250-fold molar excess. B, transactivation activity of OsTGAP1. The scheme represents effector constructs fused to the GAL4 DNA-binding domain (GAL4-DBD) and reporter construct. A non-fused construct was used as a negative control. LUC activity was normalized against that of Renilla LUC. The bar represents relative LUC activity after 15 h of incubation of the rice cells with or without elicitor. The data are means ± S.D. of three replicates.

FIGURE 6.

Phytoalexin accumulation in T7-OsTGAP1-overexpressing lines. A, immunoblot analysis of T7-OsTGAP1-overexpressing lines using an anti-T7 antibody. Two overexpressing lines with different expression levels are indicated. Shown is LC-MS/MS analysis of momilactone (B) and phytocassane (C) production in T7-OsTGAP1-overexpressing lines after elicitation. Phytoalexin accumulation before elicitation is indicated in the inset. qRT-PCR analyses indicate constitutive expression of the OsKSL4 gene (D) and OsKSL7 (E) in the overexpressing lines without elicitation (inset). Hyperinduction of OsKSL4 and OsKSL7 expression is observed in the overexpressing lines after elicitation. F, expression of OsDXS3 involved in production of the geranylgeranyl diphosphate substrate through the MEP pathway, which is located upstream of the momilactone biosynthetic pathway in plastids. The expression profiles at the indicated time points after elicitation in wild-type and T7-OsTGAP1-overexpressing lines were determined by qRT-PCR. Expression levels are shown relative to the levels of the UBQ gene (n > 3).

FIGURE 7.

OsTGAP1 regulates expression of the momilactone biosynthetic gene cluster. Expression of the five momilactone biosynthetic genes in the cluster and two neighboring genes located outside of the cluster that is illustrated in Fig. 1B are shown. The expression profiles at the indicated time points after elicitation in the ostgap1 mutant, wild type, and T7-OsTGAP1-overexpressing line (#5) were determined by qRT-PCR. Values for each mRNA expression were normalized to expression of the UBQ gene. Expression levels are shown relative to the levels 6 h after elicitation in wild-type cells (n > 3).