OsOPR10 Positively Regulates Rice Blast and Bacterial Blight Resistance

doi:10.16819/j.1001-7216.2024.231215

Abstract

Abstract:

【Objective】 Jasmonic acid (JA) plays crucial roles in plant growth and development, and responses to both biotic and abiotic stresses. OPR (12-oxo-phytodienoic acid reductase) serves as a pivotal enzyme in the JA biosynthetic pathway. In this study, transgenic rice plants overexpressing OsOPR10 were generated to assess their resistance to rice blast and bacterial blight. The research delved into the molecular mechanisms through which OsOPR10 regulates the defense response to these diseases.【Method】 Methodologically, the study involved the construction of OsOPR10 CRISPR/Cas9 knockout and overexpression vectors. These vectors were then utilized for Agrobacterium-mediated genetic transformation to obtain OsOPR10 knockout (OsOPR10-KO) and overexpressed (OsOPR10-OE) transgenic rice plants, using Nipponbare (NPB) as the wild-type parent. The transgenic plants underwent various assays to evaluate disease resistance, reactive oxygen species (ROS) burst, and the expression of genes related to the salicylic acid (SA) and jasmonic acid (JA) pathways. Additionally, the subcellular localization of OsOPR10 was examined using a laser confocal microscopy, and the interacting protein of OsOPR10 was identified through yeast two-hybrid screening and luciferase complementation experiments.【Result】 The results of the study indicated the successful generation of homozygous plants with OsOPR10 knockout and overexpression. Plants overexpressing OsOPR10 exhibited enhanced resistance to rice blast and bacterial blight. Upon induction with chitin and bacterial flagellin (flg22), ROS accumulation in OsOPR10-OE plants was notably higher than that in the wild type. Furthermore, qRT-PCR analysis revealed up-regulation of JA pathway genes (OsAOS2, OsAOC) and SA pathway genes (OsPR1a, OsPAL1) in OsOPR10-OE plants compared to NPB at 12 hours post-inoculation with Magnaporthe oryzae. Subcellular localization studies demonstrated that the OsOPR10 protein was localized in chloroplasts. The interaction protein OsCYP28 of OsOPR10 was identified through yeast two-hybrid assays and luciferase complementation experiments 【Conclusion】 In conclusion, OsOPR10 plays a significant role in responding to infections by Magnaporthe oryzae and Xanthomonas oryzae pv. oryzea, as well as to the application of exogenous methyl jasmonate (MeJA) and SA. OsOPR10 is involved in the pathogen molecular pattern-triggered immune pathway and positively regulates rice resistance to rice blast and bacterial blight through the JA and SA pathways. Additionally, OsOPR10 protein localizes in chloroplasts and interacts with the OsCYP28 protein.

Key words: rice, jasmonic acid, OsOPR10, CRISPR/Cas9, overexpression, rice blast, bacterial blight

摘要：

【目的】 茉莉酸(Jasmonic acid, JA)在植物生长发育和应对（非）生物胁迫中发挥重要功能，12-氧-植物二烯酸还原酶（12-oxo-phytodienoic acid reductase, OPR）是JA生物合成途径中的关键酶。创制OsOPR10转基因水稻，进行稻瘟病和白叶枯病抗性分析,揭示OsOPR10调控水稻抗病的分子机制，有望为水稻抗病育种提供种质资源。【方法】 构建OsOPR10的CRISPR/Cas9敲除载体和过表达载体，并分别利用农杆菌介导转化野生型水稻日本晴（Nipponbare, NPB），获得OsOPR10敲除和过表达转基因水稻。对转基因后代进行抗病性、活性氧（ROS）爆发、水杨酸（salicylic acid, SA）与茉莉酸（JA）途径基因表达模式分析。利用激光共聚焦显微镜观察OsOPR10的亚细胞定位，酵母双杂交实验和荧光素酶互补实验获取OsOPR10的互作蛋白。【结果】 获得OsOPR10纯合的敲除和过表达植株。接菌试验结果表明，OsOPR10过表达植株对稻瘟病和白叶枯病抗性更强。在几丁质（chitin）和细菌鞭毛蛋白(flg22)诱导后，OsOPR10过表达植株的ROS积累量明显高于野生型。qRT-PCR结果显示，在接种稻瘟菌12 h后，OsOPR10过表达植株中JA通路基因(OsAOS2、OsAOC)和SA通路基因(OsPR1a、OsPAL1)较NPB表达上调。亚细胞定位结果显示，OsOPR10蛋白定位于叶绿体。通过酵母双杂交试验获取OsOPR10的互作蛋白OsCYP28。【结论】 OsOPR10受稻瘟病菌和白叶枯菌以及外源茉莉酸甲酯与SA诱导。OsOPR10参与了病原菌分子模式触发的免疫途径（PTI），并且正调控水稻对稻瘟病和白叶枯病的抗性。此外，OsOPR10可能通过JA和SA途径介导水稻抗病反应。OsOPR10蛋白定位于叶绿体，与OsCYP28蛋白存在互作。

关键词: 水稻, 茉莉酸, OsOPR10, CRISPR/Cas9, 过表达, 稻瘟病, 白叶枯病

XU Danjie, LIN Qiaoxia, LI Zhengkang, ZHUANG Xiaoqian, LING Yu, LAI Meiling, CHEN Xiaoting, LU Guodong. OsOPR10 Positively Regulates Rice Blast and Bacterial Blight Resistance[J]. Chinese Journal OF Rice Science, 2024, 38(4): 364-374.

许丹洁, 林巧霞, 李正康, 庄小倩, 凌宇, 赖美玲, 陈晓婷, 鲁国东. OsOPR10正调控水稻对稻瘟病和白叶枯病的抗性[J]. 中国水稻科学, 2024, 38(4): 364-374.

Figures/Tables 12

References 37

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引物名称 Primer name	序列（5’→3’） Primer sequence（5’→3’）	用途 Purpose
OsOPR10-F	ATGAAGTCAAGTCCAAATCAGCTG	CDS扩增
OsOPR10-R	ACGAGTTGCTAGTTCTGAATTCTGAT	CDS amplification
OsOPR10-OE-F	CTTCTGCAGCCCGGGGGATCCATGAAGTCAAGTCCAAATCAGCTG	过表达载体构建
OsOPR10-OE-R	CGATCGGGGAAATTCGGATCCTCAAAGATCTTCTTCAGAAATCAACTTTTGTTCACGAGTTGCTAGTTCTGAATTCTGAT	Construction of overexpression vector
UbiP-seq	TTTTAGCCCTGCCTTCATACGC	过表达载体测序
NosR-seq	AGACCGGCAACAGGATTCAATC	Overexpression vector sequencing
OsOPR10-gRT1	CGGCCGCCTGCTTCCGTTCGTTTTAGAGCTAGAAAT	CRISPR/Cas9第一个靶点扩增
OsOPR10-OsU3T1	GAACGGAAGCAGGCGGCCGTGCCACGGATCATCTGC	Amplification of the first CRISPR/Cas9 target
OsOPR10-gRT2	AGATCGGCGCGCACCGCCTGTTTTAGAGCTAGAAAT	CRISPR/Cas9第二个靶点扩增
OsOPR10-U6aT2	AGGCGGTGCGCGCCGATCTCGGCAGCCAAGCCAGCA	Amplification of the second CRISPR/Cas9 target
OsOPR10-T1-F	AGAGAACCGGTGCCGCTTC	CRISPR/Cas9第一个靶点验证
OsOPR10-T1-R	GGTCGGAGTCGTGGCAGTC	First CRISPR/Cas9 target validation
Actin-QF	GAGTATGATGAGTCGGGTCCAG	Actin 定量 PCR
Actin-QR	ACACCAACAATCCCAAACAGAG	qRT-PCR of Actin
OsUG-F	TTCTGGTCCTTCCACTTTCAG	泛素融合蛋白定量PCR
OsUG-R	ACGATTGATTTAACCAGTCCATGA	qRT-PCR of ubiquitin fusion protein OsUG
MoPot2-F	ACGACCCGTCTTTACTTATTTGG	MoPot2 定量 PCR
MoPot2-R	AAGTAGCGTTGGTTTTGTTGGAT	qRT-PCR of MoPot2
OsOPR10-BD-F	TCAGAGGAGGACCTGCATATGATGAAGTCAAGTCCAAATCAGCTG	OsOPR10-BD载体构建
OsOPR10-BD-R	TCGACGGATCCCCGGGAATTCTCAACGAGTTGCTAGTTCTGAATTC	Construction of OsOPR10-BD vector
OsCYP28-AD-F	GTACCAGATTACGCTCATATGATGGTGTTGCCTTCATCAAA	OsCYP28-AD载体构建
OsCYP28-AD-R	ATGCCCACCCGGGTGGAATTCTTATGGCAGACTTGGAGGGG	Construction of OsCYP28-AD vector
OsOPR10-pGDG-F	gcttcgaattctgcagtcgacATGAAGTCAAGTCCAAATCAGCTG	OsOPR10-pGDG载体构建
OsOPR10-pGDG-R	ttatctagatccggtggatccTCAACGAGTTGCTAGTTCTGAATTC	Construction of OsOPR10-pGDG vector
OsOPR10-PHF223-F	CGGGATCGATGAAGTCAAGTCCAAATCAG	OsOPR10-PHF223载体构建
OsOPR10-PHF223-R	GGGGTACCACGAGTTGCTAGTTCTGAATT	Construction of OsOPR10-PHF223 vector
OsPR1a-QF	CGTCTTCATCACCTGCAACTACTC	OsPR1a 定量 PCR
OsPR1a-QR	CATGCATAAACACGTAGCATAGCA	qRT-PCR of OsPR1a
OsAOS2-QF	CAATACGTGTACTGGTCGAATGG	OsAOS2 定量 PCR
OsAOS2-QR	AAGGTGTCGTACCGGAGGAA	qRT-PCR of OsAOS2
OsPAL1-QF	AGGAGCTCGGCTGCGTATT	OsPAL1 定量 PCR
OsPAL1-QR	ATGCCGAGGAACACCTTGTT	qRT-PCR of OsPAL1
OsAOC-QF	CCAAGGTGCAGGAGATGTT	OsAOC 定量 PCR
OsAOC-QR	TACAGCTTGTTGGTGAAGGG	qRT-PCR of OsAOC
OsPBZ1-QF	CCCTGCCGAATACGCCTAA	OsPBZ1 定量 PCR
OsPBZ1-QR	CTCAAACGCCACGAGAATTTG	qRT-PCR of OsPBZ1

引物名称 Primer name	序列（5’→3’） Primer sequence（5’→3’）	用途 Purpose
OsOPR10-F	ATGAAGTCAAGTCCAAATCAGCTG	CDS扩增
OsOPR10-R	ACGAGTTGCTAGTTCTGAATTCTGAT	CDS amplification
OsOPR10-OE-F	CTTCTGCAGCCCGGGGGATCCATGAAGTCAAGTCCAAATCAGCTG	过表达载体构建
OsOPR10-OE-R	CGATCGGGGAAATTCGGATCCTCAAAGATCTTCTTCAGAAATCAACTTTTGTTCACGAGTTGCTAGTTCTGAATTCTGAT	Construction of overexpression vector
UbiP-seq	TTTTAGCCCTGCCTTCATACGC	过表达载体测序
NosR-seq	AGACCGGCAACAGGATTCAATC	Overexpression vector sequencing
OsOPR10-gRT1	CGGCCGCCTGCTTCCGTTCGTTTTAGAGCTAGAAAT	CRISPR/Cas9第一个靶点扩增
OsOPR10-OsU3T1	GAACGGAAGCAGGCGGCCGTGCCACGGATCATCTGC	Amplification of the first CRISPR/Cas9 target
OsOPR10-gRT2	AGATCGGCGCGCACCGCCTGTTTTAGAGCTAGAAAT	CRISPR/Cas9第二个靶点扩增
OsOPR10-U6aT2	AGGCGGTGCGCGCCGATCTCGGCAGCCAAGCCAGCA	Amplification of the second CRISPR/Cas9 target
OsOPR10-T1-F	AGAGAACCGGTGCCGCTTC	CRISPR/Cas9第一个靶点验证
OsOPR10-T1-R	GGTCGGAGTCGTGGCAGTC	First CRISPR/Cas9 target validation
Actin-QF	GAGTATGATGAGTCGGGTCCAG	Actin 定量 PCR
Actin-QR	ACACCAACAATCCCAAACAGAG	qRT-PCR of Actin
OsUG-F	TTCTGGTCCTTCCACTTTCAG	泛素融合蛋白定量PCR
OsUG-R	ACGATTGATTTAACCAGTCCATGA	qRT-PCR of ubiquitin fusion protein OsUG
MoPot2-F	ACGACCCGTCTTTACTTATTTGG	MoPot2 定量 PCR
MoPot2-R	AAGTAGCGTTGGTTTTGTTGGAT	qRT-PCR of MoPot2
OsOPR10-BD-F	TCAGAGGAGGACCTGCATATGATGAAGTCAAGTCCAAATCAGCTG	OsOPR10-BD载体构建
OsOPR10-BD-R	TCGACGGATCCCCGGGAATTCTCAACGAGTTGCTAGTTCTGAATTC	Construction of OsOPR10-BD vector
OsCYP28-AD-F	GTACCAGATTACGCTCATATGATGGTGTTGCCTTCATCAAA	OsCYP28-AD载体构建
OsCYP28-AD-R	ATGCCCACCCGGGTGGAATTCTTATGGCAGACTTGGAGGGG	Construction of OsCYP28-AD vector
OsOPR10-pGDG-F	gcttcgaattctgcagtcgacATGAAGTCAAGTCCAAATCAGCTG	OsOPR10-pGDG载体构建
OsOPR10-pGDG-R	ttatctagatccggtggatccTCAACGAGTTGCTAGTTCTGAATTC	Construction of OsOPR10-pGDG vector
OsOPR10-PHF223-F	CGGGATCGATGAAGTCAAGTCCAAATCAG	OsOPR10-PHF223载体构建
OsOPR10-PHF223-R	GGGGTACCACGAGTTGCTAGTTCTGAATT	Construction of OsOPR10-PHF223 vector
OsPR1a-QF	CGTCTTCATCACCTGCAACTACTC	OsPR1a 定量 PCR
OsPR1a-QR	CATGCATAAACACGTAGCATAGCA	qRT-PCR of OsPR1a
OsAOS2-QF	CAATACGTGTACTGGTCGAATGG	OsAOS2 定量 PCR
OsAOS2-QR	AAGGTGTCGTACCGGAGGAA	qRT-PCR of OsAOS2
OsPAL1-QF	AGGAGCTCGGCTGCGTATT	OsPAL1 定量 PCR
OsPAL1-QR	ATGCCGAGGAACACCTTGTT	qRT-PCR of OsPAL1
OsAOC-QF	CCAAGGTGCAGGAGATGTT	OsAOC 定量 PCR
OsAOC-QR	TACAGCTTGTTGGTGAAGGG	qRT-PCR of OsAOC
OsPBZ1-QF	CCCTGCCGAATACGCCTAA	OsPBZ1 定量 PCR
OsPBZ1-QR	CTCAAACGCCACGAGAATTTG	qRT-PCR of OsPBZ1