Osa-miR166i-3 Positively Regulates Resistance to Sheath Blight Through Mediating the Accumulation of Reactive Oxygen Species

doi:10.16819/j.1001-7216.2025.240107

Abstract

Abstract:

【Objective】Sheath blight is a major disease in rice, leading to significant yield and quality losses. Osa-miR166i-3p responds to Rhizoctonia solani infection in both the susceptible japonica rice variety Xudao 3 and the resistant indica-japonica hybrid variety YSBR1. This study aimed to clarify the role and molecular mechanism of Osa-miR166i-3p in rice resistance to sheath blight and to explore the potential pathways involving downstream genes. 【Method】Vectors containing Osa-miR166i-3p were constructed, and transgenic plants were generated in the susceptible variety Xudao 3 using Agrobacterium-mediated transformation. The authenticity of the transgenic plants was confirmed by sequencing and detecting the expression level of Osa-miR166i-3p. The plants were inoculated with R. solani in a greenhouse, and lesion lengths were measured. Additionally, the main agronomic traits of the transgenic and control plants grown under field conditions were investigated. Leaf sheath tissues collected at 0 h, 8 h, and 16 h post-inoculation were used for RNA-seq analysis to study the biological function of Osa-miR166i-3p. 【Results】Compared with Xudao 3, the expression level of Osa-miR166i-3p was significantly reduced in knockout plants and significantly increased in overexpressed plants. After inoculation with R. solani, the lesion length of Osa-miR166i-3p knockout plants increased, indicating enhanced susceptibility, while the lesion length of overexpression plants decreased, indicating enhanced resistance. Statistical analysis of agronomic traits, including plant height, panicle length, number of branches per panicle, and 1000-grain weight, showed no significant differences between transgenic and control plants, suggesting that overexpression or knockout of Osa-miR166i-3p did not affect rice agronomic traits. Enrichment analysis revealed that multiple peroxidase genes were induced in Osa-miR166i-3p overexpression plants 8 hours after inoculation with R. solani. 【Conclusion】This study demonstrates that Osa-miR166i-3p positively regulates rice resistance to sheath blight by modulating the expression of class III peroxidase-related genes and influencing reactive oxygen species (ROS) accumulation. These findings provide new insights for improving rice disease resistance.

Key words: rice, sheath blight, Osa-miR166i-3p, biological function, RNA-seq

摘要：

【目的】纹枯病是水稻上的重要病害，严重影响稻米品质和产量。在感病的粳型常规水稻品种徐稻3号和高抗籼粳交后代群体YSBR1中，Osa-miR166i-3p响应立枯丝核菌（Rhizoctonia solani）的侵染。明确Osa-miR166i-3p在水稻抗纹枯病过程中发挥的作用及其分子机制，同时探究下游基因可能涉及的通路具有重要意义。【方法】通过构建Osa-miR166i-3p的敲除和过表达载体，使用农杆菌转化法创制徐稻3号背景下的转基因植株，通过测序及检测Osa-miR166i-3p表达水平验证转基因植株真实性。在温室环境对筛选后的植株进行纹枯病菌接种，统计病斑长度，同时对大田环境下正常生长的转基因植株及对照进行主要农艺性状考察。选取立枯丝核菌接种后0 h、8 h、16 h的水稻叶鞘组织构建文库进行RNA-seq分析，对Osa-miR166i-3p的生物学功能进行研究。【结果】与徐稻3号相比，在敲除植株中Osa-miR166i-3p表达水平明显降低，在过表达植株中则明显升高。接种立枯丝核菌后，Osa-miR166i-3p敲除植株病斑长度增加，对纹枯病的抗性下降；过表达植株病斑长度减小，对纹枯病的抗性增强。大田中转基因植株及对照的株高、穗长、每穗枝梗数、千粒重的统计结果没有显著差异，表明过表达和敲除Osa-miR166i-3p不影响水稻的农艺性状。富集分析结果显示，在接种立枯丝核菌8 h后的Osa-miR166i-3p过表达植株中，多个过氧化物酶基因被诱导表达。【结论】综上所述，Osa-miR166i-3p主要通过调节植物第三类过氧化物酶相关基因的表达，影响水稻中活性氧的积累，正调控水稻纹枯病抗性，可为提高水稻抗病性提供新思路。

关键词: 水稻, 纹枯病, Osa-miR166i-3p, 生物学功能, RNA-seq

FENG Tao, ZHANG Zhaoyang, HUANG Xinni, WANG Yue, ZHONG Xuzhi, FENG Zhiming, LIU Xin, ZUO Shimin, OUYANG Shouqiang. Osa-miR166i-3 Positively Regulates Resistance to Sheath Blight Through Mediating the Accumulation of Reactive Oxygen Species[J]. Chinese Journal OF Rice Science, 2025, 39(2): 187-196.

冯涛, 张朝阳, 黄新妮, 王月, 钟旭志, 冯志明, 刘欣, 左示敏, 欧阳寿强. Osa-miR166i-3p介导活性氧积累途径正调控水稻纹枯病抗性[J]. 中国水稻科学, 2025, 39(2): 187-196.

Figures/Tables 7

Table 1. Primers used in this study.

引物 Primer	序列 Sequence（5’-3’）
OsmiR166KO F	GCCGATTGAGAGAGATAGGTGTT	构建Osa-miR166i-3p敲除载体
OsmiR166KO R	AAACAACACCTATCTCTCTCAAT	Constructing a knockout vector for Osa-miR166i-3p
OsmiR166OE F	GATATCCGTTGTGGTCGTAACCTTCT GCACTAGGTACC	构建Osa-miR166i-3p过表达载体 Constructing an overexpression vector for Osa-miR166i-3p
OsmiR166OE R	CTCGAAGTAAGGAATGAGCCGCTCG ACCTGCAGGTACC
OsmiR166 Fq	CGTTAGCTTTGCCTTTTGTT	转基因及对照植株中Osa-miR166i-3p表达
OsmiR166 Rq	GGGCTGGTTTCACTTTCATA	Expression of Osa-miR166i-3p in transgenic and control plants
OsmiR166 RT	GTCGTATCCAGTGCAGGGTCCGAGG TATTCGCACTGGATACGACGAGGAA	水稻基因组DNA反转录为Osa-miR166i-3p Reverse transcription of rice genomic DNA into Osa-miR166i-3p
OsmiR166-F	GCGGCGGTCGGATCAGGCTTCA
Universal primer	GTGCAGGGTCCGAGGT
Os18S rRNA F	CTACGTCCCTGCCCTTTGTACA	差异表达基因水平检测的内参基因
Os18S rRNA R	ACACTTCACCGGACCATTCAA	Internal reference genes for detection of differentially expressed gene levels
Os06g35520 Fq	GATTGCTTCGTCAATGGATG	转录组测序中差异表达基因表达水平检测 Detection of differentially expressed gene levels in transcriptome sequencing
Os06g35520 Rq	GCCTCCACCTGCGTCTTGAT
Os01g73200 Fq	ACTTCCACGACTGCTTCGTC
Os01g73200 Rq	GGAGCAGGACACGACGGTGT
Os07g47990 Fq	AGTCCTGTTGCTCTTGTGTC
Os07g47990 Rq	AAGCAGTCATGGAAGTGAAG
Os12g02080 Fq	AGCTTCTACTCGTACTCGTG
Os12g02080 Rq	GATGGCGTCGATCACCTCAA

Fig. 1. Expression level of Osa-miR166i-3p was suppressed by R. solani infection A, Symptoms of Xudao 3 and YSBR1 infected by R. solani. B, Lesion length of Xudao 3 and YSBR1 at different days after R. solani infection. C, Northern Blot of Osa-miR166i-3p. The sheath tissues of Xudao 3 and YSBR1 infected by R. solani at 5, 10, and 20 hours were designated as XT5, XT10, XT20 and YT5, YT10, YT20, respectively. The sheath tissues of Xudao 3 and YSBR1 treated with water at 5, 10, and 20 hours were designated as XC5, XC10, XC20 and YC5, YC10, YC20, respectively.

Fig. 2. Generation and expression analysis of Osa-miR166i-3p transgenic lines A, Sequencing results of Osa-miR166i-3p knockout lines. Blue box represents predicted protospacer adjacent motifs (PAM), and red boxes represent mature miRNA sequence. B, Relative expression levels of Osa-miR166i-3p in overexpression(OE_1-OE_4) and knockout lines(KO_1-KO_4). Error bars represent SD, n=3. Different lowercase letters indicate significant difference (P<0.05).

Fig. 3. Overexpression of Osa-miR166i-3p enhances sheath blight resistance of Xudao 3 A, Lesion of Osa-miR166i-3p lines as compared to Xudao 3 at 5, 10, 15 day after infection by R. solani. Bars=10 cm. B and C, Lesion length of Osa-miR166i-3p_OE(B) and Osa-miR166i-3p_KO(C) as compared to Xudao 3 at 5, 10,15 days after infection by R. solani. Error bars represent SD, n=20, Different lowercase letters indicate significant difference (P<0.05).

Fig. 4. Comparison of main agronomic traits of Osa-miR166i-3p lines and Xudao 3 A-E, Plant height(A), panicle length(B), No. of primary branches per panicle(C), panicle number(D) and 1000-grain weight(E) of Osa-miR166i-3p_OE, Osa-miR166i-3p_KO and Xudao 3. Error bars represent SD, n=20; Common lowercase letters indicate no significant difference (P≥0.05). F and G, Photograph of panicle length (F) and No. of primary branches per panicle (G) of Osa-miR166i-3p_OE, Osa-miR166i-3p_KO and Xudao 3. H, Photograph of plant height of Osa-miR166i-3p_OE and Xudao 3. I, Photograph of plant height of Osa-miR166i-3p_KO and Xudao 3.

Fig. 5. Transcriptome analysis of Osa-miR166i-3p_OE lines for differentially expressed genes KEGG enrichment analysis of differentially expressed genes in the Osa-miR166i-3p overexpression lines infected by R. solani at 8 h and 16 h.

Fig. 6. Expression levels of multiple peroxidase related genes induced by R. solani in the Osa-miR166i-3p _OE lines A, Number of differentially expressed genes in Osa-miR166i-3p_OE lines infected by R. solani at 0 h, 8 h, and 16 h. B, Relative expression level of Os07g47990, Os01g73200, Os06g35520 and Os12g02080 in Osa-miR166i-3p_OE lines infected by R. solani at 0 h, 8 h, and 16 h.

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