OsERF7 Negatively Regulates Resistance to Sheath Blight Disease by Inhibiting Phytoalexin Biosynthesis

doi:10.1016/j.rsci.2024.12.014

Abstract

Abstract:

Ethylene response factors (ERFs) are plant transcription agents that play a pivotal role in disease resistance through the ethylene signaling pathway. However, whether and how ERFs regulate resistance to sheath blight (ShB), caused by Rhizoctonia solani in rice, remains largely unknown. Here, we demonstrated that OsERF7 negatively regulates rice resistance to ShB by inhibiting phytoalexin biosynthesis. Overexpression of OsERF7 (OsERF7OE) significantly decreased ShB resistance, whereas knockout of OsERF7 (oserf7) enhanced it. Mechanistically, antioxidant enzyme activities are significantly reduced in OsERF7OE plants, but increased in oserf7 plants. Furthermore, transcriptome analysis revealed that oserf7 plants exhibited significant upregulation of pathogenesis-related (PR) and phytoalexin biosynthesis genes upon R. solani infection. Consistently, transcript levels of phytoalexin biosynthesis genes, including OsKSL7, OsKSL8, OsKOL5, and OsCPS4, were significantly elevated in oserf7 plants, but reduced in OsERF7OE plants in response to R. solani infection. Electrophoretic mobility shift assays and dual-luciferase (LUC) reporter assays further confirmed that OsERF7 directly binds to the promoters of OsKSL8, OsKOL5, and OsCPS4, thereby repressing their expression. In summary, our study revealed that OsERF7 negatively regulated rice resistance to ShB primarily by inhibiting phytoalexin biosynthesis.

Key words: rice, sheath blight, OsERF7, phytoalexin

Xie Yuhao, Xie Wenya, Zhao Jianhua, Xue Xiang, Cao Wenlei, Shi Xiaopin, Wang Zhou, Wang Yiwen, Wang Guangda, Feng Zhiming, Hu Keming, Chen Xijun, Chen Zongxiang, Zuo Shimin. OsERF7 Negatively Regulates Resistance to Sheath Blight Disease by Inhibiting Phytoalexin Biosynthesis[J]. Rice Science, 2025, 32(3): 367-379.

Figures/Tables 6

Fig. 1. Tissue-specificity and inducible expression of OsERF7. A, Gene expression atlas of OsERF7 in two representative indica varieties (Minghui 63, MH63, and Zhenshan 97, ZS97), retrieved from the CREP database (http://crep.ncpgr.cn/crep-cgi/home.pl). Expression values are all log2-transformed using the original signal values from the CREP database. Sheaths 1 and 2 indicate the sheath tissues collected from secondary-branch primordium differentiation at development stages and 4-5 cm young panicle development stage, respectively; Stems 1 and 2 indicate 5 d before the heading stage and at the heading stage, respectively; Panicles 1-5 represent panicles at the secondary-branch primordium differentiation stage, pistil and stamen primordium differentiation stage, pollen-mother cell formation stage, 4-5 cm young panicle, and the heading stage, respectively.B and C, RNA expression levels of OsERF7 in different tissues (B) at the maturity stage and in leaf sheath at different development stages (C) of Xudao 3. The OsActin gene was used as the internal control.D and E, RNA expression levels of OsERF7 in Xudao 3 in response to Rhizoctonia solani inoculation at the booting stage (D) and treatment with ethylene (ET) or aminooxyacetic acid (AOA) at the seedling stage (E). The OsActin gene was used as the internal control.In B to E, data are Mean ± SD (n = 3). Different lowercase letters above bars indicate statistically significant differences determined by one-way analysis followed by Tukey’s multiple test (P < 0.05). ** indicates significant differences at P < 0.01 determined by one-tailed Student’s t-test.

Fig. 2. Nuclear localization and transcriptional repression activity of OsERF7. A, Subcellular localization of OsERF7 in rice protoplasts. The NLS-RFP protein was used as a nuclear marker to localize the OsERF7-GFP fusion protein. GFP, Green fluorescent protein; RFP, Red fluorescent protein. Scale bars, 10 µm. B, Yeast one-hybrid assay for OsERF7. OsERF65, a transcriptional activator, was used as a positive control. BD, DNA-binding domain; AD, Activation domain; SD, Synthetic defined medium.C, Repression effects of OsERF7 on VP16-mediated transcriptional activation of 4× UAS-LUC in rice protoplasts. UAS, Upstream activating sequence; LUC, Firefly luciferase; REN, Renilla luciferase; 35S, CaMV35S promoter; GAL4, Yeast transcriptional activator. VP16 was used as a transcriptional activator. Data are Mean ± SD (n = 3). ** indicates significant differences at P < 0.01 determined by one-tailed Student’s t-test.

Fig. 3. OsERF7 negatively regulates rice resistance to Rhizoctonia solani. A and C, Disease levels of wild type (WT), OsERF7 overexpression lines (OsERF7OE-1 and OsERF7OE-2), and knockout (oserf7-1 and oserf7-2) mutants caused by R. solani in whole-plant (A) and detached leaf sheath (C). Pictures were taken and data were collected at 12 d post-inoculation (dpi) in A and 5 dpi in C. Scale bars, 10 cm in A and 5 cm in C, respectively. The arrowhead indicates the upper and lower boundaries of lesions.B and D, Quantitative lesion lengths of WT, OsERF7OE (OsERF7OE-1 and OsERF7OE-2), and oserf7 (oserf7-1 and oserf7-2) plants in whole plants (B) and detached leaf sheaths (D) after R. solani inoculation. Data are Mean ± SD (n = 12 in B; n = 15 in D). E, Lactophenol cotton blue staining for hyphae of R. solani in leaf sheaths of WT, OsERF7OE (OsERF7OE-1 and OsERF7OE-2), and oserf7 (oserf7-1 and oserf7-2) plants at 48 h post-inoculation. Scale bars, 300 µm. F, Relative abundance of R. solani biomass in infected leaf sheaths of WT, OsERF7OE (OsERF7OE-1 and OsERF7OE-2), and oserf7 (oserf7-1 and oserf7-2) plants at 48 h post inoculation, measured by qRT-PCR using specific primers for fungal 18S rRNA gene. The OsActin gene was used as the internal control. Data are Mean ± SD (n = 3). In B, D, and F, ** indicates significant differences at P < 0.01 determined by one-tailed Student’s t-test.

Fig. 4. Disease lesion length and activity of oxidoreductase enzymes after inoculation with Rhizoctonia solani. A and B, Disease lesions on detached leaf sheaths (A) and flag leaves (B) of wild type (WT), OsERF7 overexpression lines (OsERF7OE-1 and OsERF7OE-2), and knockout (oserf7-1 and oserf7-2) mutants at 72 h post-inoculation (hpi) with R. solani. C and D, Detached leaf sheaths (C) and flag leaves (D) from A and B were stained with 3,3′-diaminobenzidine to visualize H2O2 accumulation at the inoculation site. E-J, H2O2 content (E), peroxidase (POD) activity (F), glutathione-S-transferase (GST) activity (G), superoxide dismutase (SOD) activity (H), ascorbate peroxidase (APX) (I), and catalase (CAT) activity (J) in leaves of WT, OsERF7OE, and oserf7 lines at 72 hpi. Data are Mean ± SD (n = 3). * and ** indicate significant differences at P < 0.05 and P < 0.01 determined by one-tailed Student’s t-test.

Fig. 5. Transcriptome analysis for changes in oserf7 plants in response to Rhizoctonia solani. A, Volcano plot for differentially expressed genes (DEGs) in oserf7 mutant relative to wild type (WT) plants at 12 h post-inoculation (hpi). Genes that are significantly upregulated and downregulated are depicted in red and green, respectively [Cut-off values: Padj < 0.05 and |log2(Fold change)| > 1]. B, Heatmap for the expression levels of DEGs involved in plant-pathogen interaction, diterpenoid biosynthesis pathway, or characterized as transcription factors in oserf7 and WT plants at 12 hpi with R. solani.C, Dual-luciferase reporter assay showed that OsERF7 suppresses the expression of three phytoalexin biosynthesis genes expression in rice protoplast. Data are Mean ± SD (n = 3). ** indicates significant differences at P < 0.01 determined by one-tailed Student’s t-test. REN, Renilla luciferase; LUC, Firefly luciferase; 35S, CaMV35S promoter; TF, Trigger factor; POsKSL8, OsKSL8 promoter; POsKOL5, OsKOL5 promoter; POsCPS4, OsCPS4 promoter. D, Electrophoretic mobility shift assay identified the binding ability of OsERF7 to the GCC-box on the promoters of OsKSL8, OsKOL5, and OsCPS4 genes, respectively. The probes are derived from a 30-bp sequence of their promoters that contains the GCC-box element, and the mutated probe has the GCC sequence mutated to GTT.

Fig. 6. Agronomic traits of wild type (WT), OsERF7 overexpression lines (OsERF7OE-1 and OsERF7OE-2), and knock out (oserf7-1 and oserf7-2) mutants. A and B, Whole plant morphology (A) and panicle morphology (B). Scale bar, 20 cm in A and 5 cm in B. C-K, Statistical analysis of plant height (C), tiller number (D), panicle length (E), flag leaf length (F), flag leaf width (G), primary branch number (H), secondary branch number (I), 1000-grain weight (J), and yield per plant (K) in WT, OsERF7OE (OsERF7OE-1 and OsERF7OE-2), and oserf7 (oserf7-1 and oserf7-2) plants. Data are Mean ± SD (n = 10). ** indicates significant difference at P < 0.01 determined by one-tailed Student’s t-test. ns, Not significant.

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