Identification and Functional Characterization of TPL/TPR Genes in Rice Disease Resistance

doi:10.1016/j.rsci.2025.12.002

Abstract

Abstract:

TOPLESS/TOPLESS-RELATED (TPL/TPR) proteins are transcriptional corepressors that play pivotal roles in plant development, hormone signaling, and stress responses. Although TPL/TPR proteins have been identified in various organisms, their functions in rice disease resistance remain largely unexplored. Here, we conducted a comprehensive analysis of the three rice TPL/TPR proteins, designated OsTPR1, OsTPR2, and OsTPR3, examining their evolutionary relationships, expression patterns, and subcellular localization, and assessing their roles in disease resistance. Phylogenetic analysis revealed that the three OsTPRs belonged to distinct evolutionary clades. Expression analysis demonstrated tissue-specific patterns and responsiveness to jasmonate (JA), with all three genes being induced upon infection with Xanthomonas oryzae pv. oryzae (Xoo). Consistent with their roles as transcriptional corepressors, all three OsTPRs localized to the nucleus. Disease resistance assays showed that, after inoculation with Xoo, lesion lengths on ostpr2 and ostpr3 mutants were significantly shorter than those on wild-type plants. Protein interaction assays demonstrated that OsTPR2 interacted with JA ZIM-domain protein (OsJAZ12), whose expression is also induced by Xoo. Furthermore, haplotype analysis of OsTPRs revealed natural variation, leading to the identification of superior allelic variants that confer improved resistance to bacterial blight without a yield penalty. Collectively, our findings provide a systematic characterization of TPL/TPR proteins in rice, highlight their potential roles in resistance to bacterial leaf blight, and identify valuable allelic resources for molecular breeding aimed at improving both disease resistance and yield.

Key words: TOPLESS and TOPLESS-RELATED (TPL/TPR) protein, transcriptional corepressor, rice, disease resistance, breeding

Zhan Chengfang, Lu Xueli, Chen Yingtong, Li Shunyuan, Zhang Xiaoyan, Chen Siqi, Xie Huan, Jin Lei, Ding Lin, Ge Yi, Yang Ting, Dai Liping, Cao Junfeng, Wang Mengcen, Tang Zhengbin, Zeng Dali. Identification and Functional Characterization of TPL/TPR Genes in Rice Disease Resistance[J]. Rice Science, 2026, 33(2): 232-244.

Figures/Tables 6

Fig. 1. Expression pattern analysis of the OsTPR genes. A, Expression analysis of OsTPR genes (OsTPR1, OsTPR2, and OsTPR3) in different tissues under developmental stages based on RNA-seq data from the RiceXPro database (https://ricexpro.dna.affrc.go.jp/). The heatmap displays low and high expression in white and green, respectively. DAF, Days after flowering. B-D, Tissue-specific expression profiles of OsTPR1 (B), OsTPR2 (C), and OsTPR3 (D) in root, stem, internode, flag leaf, sheath, panicles at different developmental stages (1-2 cm, 2-5 cm, 5-10 cm, and > 10 cm), and grain-filling panicle. OsUBQ was used as an internal control. Data are mean ± SD (n = 4). Different lowercase letters above bars indicate signiﬁcant differences at P < 0.05 by one-way analysis of variance with Tukey’s honestly significant difference test.

Fig. 2. Pathogen-induced expression of OsTPR genes and subcellular localization of OsTPR proteins. A-C, Relative expression levels of OsTPR1 (A), OsTPR2 (B), and OsTPR3 (C) in Nipponbare leaves after inoculation with Xanthomonas oryzae pv. oryzae (Xoo). OsUBQ was used as an internal control. Data are mean ± SD (n = 4). Different lowercase letters above bars indicate signiﬁcant differences at P < 0.05 by one-way analysis of variance with Tukey’s honestly significant difference test. D-E, Subcellular localization of OsTPR1, OsTPR2, and OsTPR3 in rice protoplasts at 12 h post-inoculation (D) and Nicotiana benthamiana leaf cells at 2 d post-agroinfiltration (E). GFP alone served as a control. GFP, Green fluorescent protein. NLS, Nuclear localization signal. Scale bars in D and E, are 10 and 20 μm, respectively.

Fig. 3. Functional characterization of OsTPR2 and OsTPR3 in bacterial blight resistance. A and B, CRISPR/Cas9 editing of OsTPR2 (A) and OsTPR3 (B). Blue and green boxes represent coding regions, and red letters or red dotted line indicate successfully edited sites. C and D, Bacterial blight symptoms (C) and lesion lengths (D) in WT and ostpr2 and ostpr3 knockout lines (ostpr2-1, ostpr2-2, ostpr3-1, and ostpr3-2) at 14 d post-inoculation (dpi) with Xanthomonas oryzae pv. oryzae (Xoo) inoculation. Scale bar, 5 cm. E, Expression levels of jasmonate (JA)-responsive genes (JAZs, COI1a, COI1b, and MYC2) and JA-biosynthesis genes (AOS2, LOX1, and JMT1) in 10-day-old seedlings of WT and ostpr2 and ostpr3 knockout lines (ostpr2-1 and ostpr3-1). OsUBQ was used as an internal control. WT, Wild type. In D and E, data are mean ± SD (n > 10). Asterisks above bars represent significant differences compared with WT (* and **, P < 0.05 and P < 0.01 by Student’s t-test).

Fig. 4. OsTPR2 interacts with OsJAZ12. A, Yeast two-hybrid (Y2H) assay demonstrating the interaction between OsTPR2 and OsJAZ12. Cotransformation with pGADT7-T and pGBKT7-p53 served as the positive control, whereas cotransformation with pGADT7-T and pGBKT7-Lam was used as the negative control. B, Bimolecular fluorescence complementation (BiFC) assay confirming interaction between OsTPR2 and OsJAZ12 in Nicotiana benthamiana leaf cells at 48 h post-inoculation. Confocal microscopy was used to visualize fluorescence signals. Scale bars, 20 μm. C, Predicted structural interaction between OsTPR2 and OsJAZ12 based on AlphaFold3 modeling. D, Relative transcript levels of OsJAZ12 in Nipponbare leaves after inoculation with Xanthomonas oryzae pv. oryzae (Xoo). OsUBQ was used as an internal control. Data are mean ± SD (n = 4). Different lowercase letters above bars indicate signiﬁcant differences at P < 0.05 by one-way analysis of variance with Tukey’s honestly significant difference test.

Fig. 5. Haplotype variations and phenotypic association of OsTPR2. A, Schematic representation of four major haplotypes in the coding sequence region of OsTPR2. Blue boxes indicate coding sequences. SNP2 is frameshift variant with ‘C’ representing the reference allele; SNP3 is frameshift variant with ‘A’ representing the reference allele. B, Subpopulation distribution frequencies of the four OsTPR2 haplotypes (OsTPR2H1-H4) across different rice subgroups in the 3K Rice Genome SNP dataset. C, Box plots comparing bacterial blight lesion lengths among OsTPR2 haplotypes after inoculation with four Xanthomonas oryzae pv. oryzae (Xoo) strains (Z173, GD1358, V, and PXO339). D, Box plots comparing yield-related traits including filled-grain number per panicle, panicle number per plant, 1000-grain weight, and grain yield among the four OsTPR2 haplotypes under normal field conditions. In C and D, values above boxes represent the number of accessions per haplotype. In C, values in red represent the mean lesion length. In D, values below boxes represent the mean value of each yield-related trait. The red dashed box highlights superior haplotype. Different lowercase letters above bars indicate signiﬁcant differences at P < 0.05 by one-way analysis of variance with Tukey’s honestly significant difference test.

Fig. 6. Haplotype variation and phenotypic association of OsTPR3. A, Schematic representation of four major haplotypes in the coding sequence region of OsTPR3. Green boxes indicate coding sequences. SNP3, Splice region variant, while ‘A’ represents the reference allele. B, Subpopulation distribution frequencies of the four OsTPR3 haplotypes (OsTPR3H1-H4) across different rice subgroups in the 3K RG SNP dataset. C, Box plots comparing bacterial blight lesion lengths among OsTPR3 haplotypes after inoculation with four Xanthomonas oryzae pv. oryzae (Xoo) strains (Z173, GD1358, V, and PXO339). D, Box plots comparing yield-related traits including filled-grain number per panicle, panicle number per plant, 1000-grain weight, and grain yield among the four OsTPR3 haplotypes under normal field conditions. In C and D, values above boxes represent the number of accessions per haplotype. In C, values in red represent the mean lesion length. In D, values below boxes represent the mean value of each yield-related trait. The red dashed box highlights superior haplotype. Different lowercase letters above bars indicate signiﬁcant differences at P < 0.05 by one-way analysis of variance with Tukey’s honestly significant difference test.

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