WRKY45-dependent priming of diterpenoid phytoalexin biosynthesis in rice and the role of cytokinin in triggering the reaction

Abstract

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.

Fig. 1

Biosynthetic pathway of diterpenoid phytoalexins in rice

Fig. 2

Transcript levels of DP biosynthetic genes in WRKY45-ox and NPR1-ox rice lines. Transcript levels of biosynthetic genes for momilactones (CPS4, KSL4, CYP99A2, CYP99A3, and MAS), phytocassanes (CPS2, KSL7, KOL4, and CYP71Z7), and oryzalexins (KSL10) in WRKY45-ox (#15 and #21) and OsNPR1 (#14 and #17) lines were determined by qRT-PCR. RNAs were extracted from fourth leaves of three rice seedlings per line at the four-leaf stage. Means of three technical repeats are shown with standard deviations (SD). We obtained similar results in another independent experiment

Fig. 3

Upregulation of DPs biosynthetic genes by DEX-induced WRKY45. A and B Induction of WRKY45. GVG-WRKY45-myc transformants at the three-leaf stage were treated with DEX to induce WRKY45-myc. Whole seedlings were harvested at 2, 5, 10, and 24 h after treatments. Transcript levels of WRKY45 transgene and WRKY45 endogenous gene (A) were determined by qRT-PCR using specific primers for rbcs-3A terminator (T3A) and WRKY45 3′-UTR sequences, respectively. Protein levels of WRKY45-myc (B) were determined by western blotting using anti-myc antibody. C Transcript levels of DP biosynthetic genes. Transcript levels of genes involved in the biosynthesis of momilactones (CPS4, KSL4, CYP99A2, CYP99A3, and MAS), phytocassanes (CPS2, KSL7, KOL4, and CYP71Z7), and oryzalexins (KSL10) were determined by qRT-PCR. Means of three determinations are shown with SD

Fig. 4

Priming of momilactone and phytocassane biosynthetic genes by BTH via WRKY45. A Experimental scheme. Nipponbare (NB) and WRKY45-kd lines were treated with BTH and/or spray-inoculated with M. oryzae (1.0 × 10⁵ conidia/ml) at four-leaf stage. Transcript levels of DP biosynthetic genes were determined using fourth leaves harvested and pooled from three seedlings in each treatment/line. Transcript levels of WRKY45 (B) and DP biosynthetic genes (C) at 1 dpi were determined by qRT-PCR after treatments. Means of three determinations are shown with standard deviations (SD). We obtained similar results in another independent experiment

Fig. 5

Upregulation of momilactone biosynthetic genes in WRKY45-ox rice after M. oryzae infection. A Nipponbare (NB) and WRKY45-ox (#15 and #21) plants were inoculated with M. oryzae conidia (10⁵/ml) and the transcript levels of CPS4 in fifth leaves from three seedlings each were determined by qRT-PCR. B Nipponbare (gray lines) and WRKY45-ox (#21, black lines) plants were inoculated with M. oryzae conidia (solid lines, 10⁵/ml) or mock-treated by spraying solvent only (dashed lines) at four-leaf stage. Transcript levels of momilactone biosynthetic genes (CPS4, KSL4, CYP99A2, and MAS) in fifth leaves from three seedlings in each treatment/line were determined by qRT-PCR.Means of three determinations are shown with SD. All the experiments were performed twice independently and obtained similar results

Fig. 6

Accumulation of momilactones and phytocassanes in response to M. oryzae infection in WRKY45-ox rice plants.Plants at four-leaf-stage were spray-inoculated with conidia of M. oryzae (1.5 × 10⁵/ml). Fourth leaves from the seedlings in each treatment were harvested into six pools of three plants each (0.05 g) at each time point. DPs were determined for the replicate samples and means are shown with standard errors (SE). *p < 0.05 in student’s t test

Fig. 7

Synergism between SA and CK in inducing DP biosynthetic genes. A Induction of DP biosynthetic genes in rice by co-treatments of CK and SA. Roots of Nipponbare rice plants were supplied with kinetin or IP (50 µM) and/or BTH (90 µM) for 12 or 24 h. Transcript levels of DP biosynthetic genes were determined by qRT-PCR. B WRKY45 dependence of induction of DP biosynthetic genes by CK/SA synergism. Nipponbare (NB) and WRKY45-kd rice plants were treated with an aqueous solution containing 50 µM IP and/or 90 µM BTH from roots for 8 h. Then, transcript levels of DP biosynthetic genes were determined by qRT-PCR. C Induction of DP biosynthetic genes in WRKY45-ox rice triggered by CK treatment. Nipponbare (NB) and WRKY45-ox rice lines were treated with 50 µM kinetin (K) or IP for 12 h. Transcript levels of DP biosynthetic genes were determined by qRT-PCR. Means of three determinations are shown with SD. We obtained similar results in another independent experiment

Fig. 8

Proposed model for WRKY45-dependent regulation of DP biosynthesis. BTH acts on the rice SA signaling pathway, which is split into WRKY45- and OsNPR1 subpathways. BTH primes the expression of DP biosynthetic genes via WRKY45. M. oryzae infection triggers early accumulation of CKs, which in turn act synergistically with the WRKY45-mediated SA signaling pathway to activate expression of DP biosynthetic genes, leading to accumulation of DPs. MKK4-dependent pathways are also shown