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GhROP6通过调控茉莉酸合成与木质素代谢参与棉花抗黄萎病反应
周雪慧, 高二林, 王钰静, 李焱龙, 袁道军, 朱龙付
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GhROP6通过调控茉莉酸合成与木质素代谢参与棉花抗黄萎病反应
GhROP6 involved in cotton resistance to Verticillium wilt through regulating jasmonic acid synthesis and lignin metabolism
【目的】 克隆抗病相关基因GhROP6,解析其作用机制,为开展棉花抗病分子育种提供理论基础。【方法】 利用生物信息学方法系统分析了陆地棉Rho鸟苷三磷酸酶基因（Rho-related guanosine triphosphatase from plants, ROP）家族成员在染色体上的分布及组织表达模式。克隆了GhROP6 （Gh_A01G1392.1）基因,通过实时荧光定量聚合酶链式反应、病毒诱导的基因沉默（virus-induced gene silencing, VIGS）技术、拟南芥遗传转化技术及代谢物测定等对该基因进行功能分析。【结果】 在陆地棉中鉴定到28个ROP,其编码的多肽均含有ROP结构,包括4个GTP/GDP结合域、与下游靶蛋白结合的效应结构域和C末端的可变区域。染色体定位分析发现陆地棉ROP家族中24个基因对称分布在A亚基因组和D亚基因组中,另有3个基因分布在D亚基因组。实时荧光定量聚合酶链式反应分析发现,GhROP6在棉花不同器官表达量不同,在花瓣、柱头、开花后10 d的纤维中表达量较高,并受茉莉酸甲酯诱导上调表达。抑制GhROP6表达会降低茉莉酸生物合成相关基因GhLOX1、GhOPR3-1、GhOPR3-3、GhAOC1、GhAOS和茉莉酸信号通路相关基因GhMYC2的表达水平,削弱木质素合成相关基因GhCCR-1、GhF5H-1、GhCCoAOMT-2和GhCCoAOMT-3的表达,从而降低棉花对黄萎病的抗性;超表达组成型激活的GhROP6能增加转基因拟南芥茉莉酸-异亮氨酸含量和木质素含量,增强其对黄萎病菌抗性。【结论】 GhROP6可能通过茉莉酸合成和信号通路以及木质素合成代谢参与棉花抗黄萎病反应。
[Objective] This study aims to characterize the GhROP6 and study its roles of resistance to Verticillium wiltin upland cotton (Gossypium hirsutum L.). [Method] The bioinformatics analysis was used to identify Rho-related guanosine triphosphatase from plants (ROP) genes in upland cotton. The chromosome distributions, expression pattern analysis of GhROP genes were investigated. The function of GhROP6 gene was studied by quantitative real-time polymerase chain reaction (qRT-PCR), virus-induced gene silencing (VIGS), plant genetic transformation and metabolism analysis. [Result] Totally, 28 ROP genes were identified in upland cotton. And the corresponding amino acid sequence contained the ROP protein specific structures, including four GTP/GDP binding domains, effector domain binding to downstream target proteins and variable C-terminal regions. Chromosomal mapping analysis showed that 24 ROP genes were symmetrically distributed in subgenome A and subgenome D, and 3 genes specifically distributed in subgenome D. qRT-PCR analysis showed that the transcript levels of GhROP6 varied in different organs, and showed higher expression level in petals, stigma, fiber of 10 days post anthesis. Meanwhile, the transcript level of GhROP6 was upregulated in cotton by methyl jasmonate (MeJA). Knock-down of GhROP6 through VIGS weakened the cotton resistance to Verticillium wilt, and reduced the expression of GhLOX1, GhOPR3-1, GhOPR3-3, GhAOC1, GhAOS involved in jasmonic acid (JA) synthesis, and the expression of GhMYC2 involved in JA signaling pathway, and the expression of GhCCR-1, GhF5H-1, GhCCoAOMT-2, and GhCCoAOMT-3 genes involved in lignin synthesis. However, constitutively activated GhROP6 in Arabidopsis enhanced the plants resistantce to V. dahliae. Further analysis showed that the contents of JA-isoleucine and lignin in transgenic Arabidopsis were higher than those of wild type. [Conclusion] GhROP6 may involve in the resistance of cotton to Verticillium wilt through JA synthesis and signaling pathway and lignin synthesis.
棉花 / 黄萎病 / GhROP6 / 抗病性 / 茉莉酸 / 木质素 / 信号通路 {{custom_keyword}} /
cotton / Verticillium wilt / GhROP6 / disease resistance / jasmonic acid / lignin / signaling pathway {{custom_keyword}} /
图1 棉花ROP基因的系统进化分析及转录水平分析A：28个陆地棉ROP蛋白和11个拟南芥ROP蛋白的系统进化分析;B：ROP基因在陆地棉不同器官中的表达热图,其中DPA表示开花后时间（d）。Fig. 1 Phylogenetic analysis and transcript level profiles of ROP genes in upland cotton A: Phylogenetic analysis of 28 ROP proteins in upland cotton and 11 ROP proteins in A. thaliana; B: Heatmap of ROP genes transcript level profiles in different organs in upland cotton, DPA: day post anthesis. |
图2 GhROP6在不同器官和不同激素处理下的表达模式分析A：GhROP6在棉花不同器官中的表达分析;B：SA和MeJA处理后GhROP6的表达分析。*和**分别表示激素处理与清水处理（mock）相比在0.05和0.01水平上差异显著。Fig. 2 Expression profile analysis of GhROP6 in cotton organs and different phytohormone treatments A: Expression analysis of GhROP6 in different organs; B: Expression analysis of GhROP6 after SA and MeJA treatments. * and **: Significant difference compared with water treatmen (mock) at the 0.05 and 0.01 probability level, respectively. |
图4 抑制GhROP6表达削弱棉花对黄萎病菌的抗性A：农杆菌侵染后14 d,注射TRV:CLA1植株出现叶片白化表型;B：RT-PCR分析棉花根中GhROP6的表达水平。 C：接种V991后12 d的TRV:00和TRV:GhROP6幼苗表型,以水处理为对照。D：接种V991后15 d统计的病情指数, **表示在0.01水平上差异显著。E：接种V991后15 d,TRV:00和TRV:GhROP6植株茎秆纵切面。F：真菌恢复培养试验。Fig. 4 Silencing of GhROP6 impairs cotton resistance to V. dahlia A: The plants showed leaf albinism phenotype inoculated with TRV:CLA at 14 days post inoculation; B: The expression level of GhROP6 in cotton roots by RT-PCR; C: The phenotypes of TRV:00 and TRV:GhROP6 seedlings 12 days after inoculation with V991, with water as mock; D: The disease index determined at 15 d after inoculated with V991, **: Significant diffe-rence at the 0.01 probability level; E: Longitudinal cross-section of darkened vascular tissues dissected at 15 d after inoculation; F: The fungal recovery assay. |
图5 JA信号通路相关基因的表达量和JA-Ile含量A：JA合成相关基因的表达分析;B：GhMYC2的表达分析;C：TRV:00和TRV:GhROP6植株接种黄萎病菌后JA-Ile含量。Mock为水处理。*和**分别表示与对照植株相比在0.05和0.01水平上差异显著。Fig. 5 The expression level of JA-related genes and JA-Ile content A: Expression analysis of genes involved in JA synthesis; B: Expression analysis of GhMYC2; C: The JA-Ile content in TRV:00 and TRV:GhROP6 plants inoculated with V. dahliae. Mock：water treatment. * and **: Significant difference compared with TRV:00 at the 0.05 and 0.01 probability level, respectively. |
图6 木质素含量及木质素合成相关基因表达量A：TRV:00和TRV:GhROP6植株根中木质素含量;B：TRV:00和TRV:GhROP6植株茎秆中木质素的组织化学分析,图中标尺为200 μm;C：木质素合成相关基因的表达分析。Mock为水处理,TRV:00为对照,TRV:GhROP6为沉默GhROP6植株。**表示同一处理下,TRV:GhROP6植株与对照植株相比在0.01水平上差异显著。Fig. 6 Lignin content and the expression level of genes-involved in lignin biosynthesis A: Lignin content in the roots of TRV:00 and TRV:GhROP6 plants; B: Histochemical analysis of lignin in the stems of TRV:00 and TRV:GhROP6 plants, scale bar: 200 μm; C: Expression analysis of genes involved in lignin synthesis. Mock：water treatment. **: Significant difference compared with TRV:00 in the same treatment at the 0.01 probability level. |
图7 超表达GhROP6提高转基因拟南芥的抗病性A：转基因拟南芥接种黄萎病菌后18 d的表型;B：接种黄萎病菌后的病情指数;C：接种黄萎病菌后JA-Ile含量; D：JA信号通路相关基因的表达水平分析;E：接种黄萎病菌后木质素含量;F：木质素合成相关基因的表达分析。*和**分别表示与野生型相比在0.05和0.01水平上差异显著。Fig. 7 Overexpression of GhROP6 enhances A. thaliana resistance to V. dahlia A: The phenotype of transgenic lines 18 days after inoculated with V. dahliae; B: Disease index after inoculation with V. dahliae; C: JA-Ile contents measured after inoculation with V. dahliae; D: The expression levels of JA signaling pathway genes; E: Ligin contents measured after inoculation with V. dahliae; F: Expression analysis of genes involved in lignin synthesis.* and **: Significant difference compared with wild type (WT) at the 0.05 and 0.01 probability level, respectively. |
附表1 本研究所用的引物序列Table S1 List of primers used in this study |
| 引物名称Primer name | 序列Sequence | 用途Purpose |
|---|---|---|
| qGhROP6-F | GCTCATCTCCTACACCAGCAATAC | 检测相应基因的表达水平 detect gene expression by qRT-PCR |
| qGhROP6-R | CAGCAGTATCCCACAATCCAAG | |
| GhLOX1-F | TAGAGAGGACATTTTGCCCTGG | |
| GhLOX1-R | GGTCAAGGTCGTCCAGAGATTTTA | |
| GhAOS1-F | CGGATTAGAGCCTCAGTGTCGG | |
| GhAOS1-R | ATCTTGAGAAATGAAAGGACCAGG | |
| GhAOS2-F | TGCCACCTGGTCCTTTCATTTC | |
| GhAOS2-R | GCGTGTTTGGGCTCGGAAGGGTCG | |
| GhAOC1-F | CAACCCCTTCACTACCACTGCC | |
| GhAOC1-R | AGGGCTGCTTCTGTCTCTCTCG | |
| GhOPR3-1-F | ATGCTGTTCATGCCAAAGGAGG | |
| GhOPR3-1-R | TTTCTGATGTTTCCAGGGGTCG | |
| GhMYC2-F | GCTCCGCCACTACCGTGCTC | |
| GhMYC2-R | CTCGAAGCACTTTTTTACGGTGTTC | |
| GhCCR-1-F | ATTGTTATGGGAAGGCAGTGGC | |
| GhCCR-1-R | ACGAGAAGGTGTGCTAATGCG | |
| GhF5H-1-F | TTGGAGGCAGATGCGAAAGATT | |
| GhF5H-1-R | TCCTCTTGCCCGTGTTTGTTAC | |
| GhCCoAOMT-2-F | TACGACAACACGCTGTGGAATGGG | |
| GhCCoAOMT-2-R | CATCGCCGACAGGAAACATACAGA | |
| GhCCoAOMT-3-F | GAGACCAGTGTGTATCCGAGGG | |
| GhCCoAOMT-3-R | CAAGGGCAGTGGCTAAGAGAGA | |
| GhUB7-F | GAAGGCATTCCACCTGACCAAC | |
| GhUB7-R | CTTGACCTTCTTCTTCTTGTGCTTG | |
| AtPAL1-F | CTTGTCAGGAGCAACACCATCA | |
| AtPAL1-R | AACGAGCAAGGCATATTTGAAGAG | |
| AtPAL2-F | CTACCCCATCAACCTGAACCCA | |
| AtPAL2-R | TATGGTCCAGAAGCGGATGTGT | |
| AtC4H-F | GCTTAGCAACAATGGTGGAATG | |
| AtC4H-R | CATCCTTGGTATTACTTTGGGTCG | |
| AtF5H-F | TCCTCTTGCCCGTGTTTGTTAC | |
| AtF5H-R | TTGGAGGCAGATGCGAAAGATT | |
| AtAOS1-F | TTTTATCGCCGAGAATCCAC | |
| AtAOS1-R | CCTCCGCTAATCGGTTATGA | |
| AtAOC1-F | AACTGAGCGTGTACGAAATCAAT | |
| AtAOC1-R | CAAACATACTGCATTCACAAGGA | |
| AtOPR3-F | CGGCGTTGGCAGAGTATTAT | |
| AtOPR3-R | GCGAGCTTTGAGCCATTAAC | |
| AtMYC2-F | ACGACTGAAACAACTCCGACG | |
| AtMYC2-R | AACCGTCGTATGATTTCTCCG | |
| RT-GhROP6-F | TCCTACACCAGCAATACTTTC | RT-PCR检测GhROP6表达水平 Detect the expression of GhROP6 by RT-PCR |
| RT-GhROP6-R | GGAGGAATTAAGAAAGCTGAT | |
| VIGS-GhROP6-F | CAGTGCCCATTACCACAGCC | 构建GhROP6沉默载体Construction of GhROP6 silencing vector |
| VIGS-GhROP6-R | AGGAAAGTGTGAGAACACAAAGGG | |
| GhROP6-F | ATGAGTGCATCAAGGTTCATCA | 扩增 GhROP6 基因 Amplification of full-length GhROP6 gene |
| GhROP6-R | TCACAATATCGAGCAGGCCTT | |
| CA1-GhROP6-F | ACAATAATTGAAGCAAGAG | 构建组成型激活的GhROP6载体 Construction of constitutively active GhROP6 vector |
| CA1-GhROP6-R | GTGACGTGTGCCGTCGGCAAGA | |
| CA2-GhROP6-F | TCACTGTTGGTGACGGTGCA | |
| CA2-GhROP6-R | ATTCCGGAGTTGAGTCATACTT | |
| DN1-GhROP6-F | ATTACTTTCACTTTTGCAGCAT | 构建组成型失活的GhROP6载体Construction of dominant negative GhROP6 vector |
| DN1-GhROP6-R | CGGCAAGAGAATGCATGCT | |
| DN2-GhROP6-F | AAGGAGTGCATGCTCATCTC | |
| DN2-GhROP6-R | ACCCTGGAGCAGTGCCCATT |
附表2 陆地棉中ROP基因基本信息Table S2 The information of ROP genes in upland cotton (Gossypium hirsutum) |
| 基因ID Gene ID | 长度 length/bp | 染色体位置 Chromosome localization | 基因ID Gene ID | 长度 length/bp | 染色体位置 Chromosome localization |
|---|---|---|---|---|---|
| Gh_A01G1392.1 | 597 | A01 | Gh_D03G0072 | 591 | D03 |
| Gh_A02G0857 | 588 | A02 | Gh_D05G0243 | 603 | D05 |
| Gh_A05G0179 | 630 | A05 | Gh_D05G1765 | 588 | D05 |
| Gh_A05G1588 | 588 | A05 | Gh_D05G2437 | 591 | D05 |
| Gh_A06G0026 | 591 | A06 | Gh_D06G0618 | 714 | D06 |
| Gh_A06G0551 | 630 | A06 | Gh_D06G1409 | 645 | D06 |
| Gh_A06G2039 | 594 | A06 | Gh_D06G2288 | 591 | D06 |
| Gh_A08G0520 | 639 | A08 | Gh_D08G0612 | 630 | D08 |
| Gh_A08G1258 | 627 | A08 | Gh_D08G1547 | 627 | D08 |
| Gh_A11G1595 | 594 | A11 | Gh_D10G0271 | 603 | D10 |
| Gh_A12G0343 | 597 | A12 | Gh_D11G1753 | 564 | D11 |
| Gh_A12G2499 | 636 | A12 | Gh_D12G0319 | 597 | D12 |
| Gh_D01G1636 | 552 | D01 | Gh_D12G2627 | 666 | D12 |
| Gh_D02G0984 | 588 | D02 | Gh_Sca006742G01 | 591 |
附图1 AtROP6和GhROP6氨基酸序列比对Fig. S1 Sequence alignment analysis between AtROP6 and GhROP6 |
附图4 GhROP6与CA、DN突变体氨基酸序列比对分析Fig. S4 Sequence alignment analysis between GhROP6、CA and DN mutant |
附图5 不同转基因拟南芥株系中GhROP6基因表达分析A:超表达CA-GhROP6的拟南芥与野生型拟南芥中GhROP6基因的表达水平, B:超表达DN-GhROP6的拟南芥与野生型拟南芥中GhROP6基因的表达水平; C:转基因拟南芥的PCR检测结果。Fig. S5 Expression of GhROP6 in different transgenic Arabidopsis lines A: Expression of GhROP6 in CA-GhROP6 transgenic and wild type (WT) Arabidopsis; B: Expression of GhROP6 in DN-GhROP6 transgenic and WT Arabidopsis; C: Identification of GhROP6 by PCR in transgenic Arabidopsis. |
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Cell death plays important roles in the development and defense of plants as in other multicellular organisms. Rapid production of reactive oxygen species often is associated with plant defense against pathogens, but their molecular mechanisms are not known. We introduced the constitutively active and the dominant negative forms of the small GTP-binding protein OsRac1, a rice homolog of human Rac, into the wild type and a lesion mimic mutant of rice and analyzed H(2)O(2) production and cell death in transformed cell cultures and plants. The results indicate that Rac is a regulator of reactive oxygen species production as well as cell death in rice.
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Long noncoding RNAs (lncRNAs) have several known functions in plant development, but their possible roles in responding to plant disease remain largely unresolved. In this study, we described a comprehensive disease-responding lncRNA profiles in defence against a cotton fungal disease Verticillium dahliae. We further revealed the conserved and specific characters of disease-responding process between two cotton species. Conservatively for two cotton species, we found the expression dominance of induced lncRNAs in the Dt subgenome, indicating a biased induction pattern in the co-existing subgenomes of allotetraploid cotton. Comparative analysis of lncRNA expression and their proposed functions in resistant Gossypium barbadense cv. '7124' versus susceptible Gossypium hirsutum cv. 'YZ1' revealed their distinct disease response mechanisms. Species-specific (LS) lncRNAs containing more SNPs displayed a fiercer inducing level postinfection than the species-conserved (core) lncRNAs. Gene Ontology enrichment of LS lncRNAs and core lncRNAs indicates distinct roles in the process of biotic stimulus. Further functional analysis showed that two core lncRNAs, GhlncNAT-ANX2- and GhlncNAT-RLP7-silenced seedlings, displayed an enhanced resistance towards V. dahliae and Botrytis cinerea, possibly associated with the increased expression of LOX1 and LOX2. This study represents the first characterization of lncRNAs involved in resistance to fungal disease and provides new clues to elucidate cotton disease response mechanism.© 2017 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.
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Verticillium dahliae is a phytopathogenic fungal pathogen that causes vascular wilt diseases responsible for considerable decreases in cotton yields. The lignification of cell wall appositions is a conserved basal defence mechanism in the plant innate immune response. However, the function of laccase in defence-induced lignification has not been described. Screening of an SSH library of a resistant cotton cultivar, Jimian20, inoculated with V. dahliae revealed a laccase gene that was strongly induced by the pathogen. This gene was phylogenetically related to AtLAC15 and contained domains conserved by laccases; therefore, we named it GhLAC15. Quantitative reverse transcription-polymerase chain reaction indicated that GhLAC15 maintained higher expression levels in tolerant than in susceptible cultivars. Overexpression of GhLAC15 enhanced cell wall lignification, resulting in increased total lignin, G monolignol and G/S ratio, which significantly improved the Verticillium wilt resistance of transgenic Arabidopsis. In addition, the levels of arabinose and xylose were higher in transgenic plants than in wild-type plants, which resulted in transgenic Arabidopsis plants being less easily hydrolysed. Furthermore, suppression of the transcriptional level of GhLAC15 resulted in an increase in susceptibility in cotton. The content of monolignol and the G/S ratio were lower in silenced cotton plants, which led to resistant cotton cv. Jimian20 becoming susceptible. These results demonstrate that GhLAC15 enhances Verticillium wilt resistance via an increase in defence-induced lignification and arabinose and xylose accumulation in the cell wall of Gossypium hirsutum. This study broadens our knowledge of defence-induced lignification and cell wall modifications as defence mechanisms against V. dahliae.© 2018 The Authors. Molecular Plant Pathology published by BSPP and John Wiley & Sons Ltd.
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OsRac1, one of the Rac/Rop family of small GTPases, plays important roles in defense responses, including a role in the production of reactive oxygen species mediated by NADPH oxidase. We have identified an effector of OsRac1, namely rice (Oryza sativa) cinnamoyl-CoA reductase 1 (OsCCR1), an enzyme involved in lignin biosynthesis. Lignin, which is polymerized through peroxidase activity by using H(2)O(2) in the cell wall, is an important factor in plant defense responses, because it presents an undegradable mechanical barrier to most pathogens. Expression of OsCCR1 was induced by a sphingolipid elicitor, suggesting that OsCCR1 participates in defense signaling. In in vitro interaction and two-hybrid experiments, OsRac1 was shown to bind OsCCR1 in a GTP-dependent manner. Moreover, the interaction of OsCCR1 with OsRac1 led to the enzymatic activation of OsCCR1 in vitro. Transgenic cell cultures expressing the constitutively active OsRac1 accumulated lignin through enhanced CCR activity and increased reactive oxygen species production. Thus, it is likely that OsRac1 controls lignin synthesis through regulation of both NADPH oxidase and OsCCR1 activities during defense responses in rice.
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PDF(15032 KB)
图1 棉花ROP基因的系统进化分析及转录水平分析图2 GhROP6在不同器官和不同激素处理下的表达模式分析图3 GhROP6及其突变型的亚细胞定位图4 抑制GhROP6表达削弱棉花对黄萎病菌的抗性图5 JA信号通路相关基因的表达量和JA-Ile含量图6 木质素含量及木质素合成相关基因表达量图7 超表达GhROP6提高转基因拟南芥的抗病性
附表1 本研究所用的引物序列附表2 陆地棉中ROP基因基本信息附图1 AtROP6和GhROP6氨基酸序列比对附图2 陆地棉ROP蛋白序列比对附图3 GhROP基因的染色体分布附图4 GhROP6与CA、DN突变体氨基酸序列比对分析附图5 不同转基因拟南芥株系中GhROP6基因表达分析/
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