光敏色素类互作因子OsPIL1敲除对籼稻稻瘟病抗性的影响

Effects of phytochrome-interacting factor OsPIL1 knockout on blast disease resistance in indica rice

  • 摘要: 【目的】明确水稻(Oryza sativa)光敏色素类互作因子基因(OsPIL1)敲除对籼稻月亮谷生长、产量和抗瘟性的影响,初步探索外源茉莉酸或水杨酸对OsPIL1调控水稻稻瘟病抗性的影响,并探索OsPIL1整合JA/SA调控水稻稻瘟病抗性的作用,为解析稻瘟病抗性机制提供基础数据。【方法】以水稻品种月亮谷为研究材料,利用CRISPR/Cas9技术构建获得OsPIL1敲除转基因株系,并对其开展表型(根长、胚芽鞘长度、株高、分蘖数、穗长、穗粒数、百粒重、粒长、粒宽等)分析和稻瘟病抗性鉴定(病害调查、侵染进程观察等),同时进行外源JA/SA处理后的稻瘟病抗性鉴定。【结果】CRISPR/Cas9构建获得3个纯系的OsPIL1敲除转基因水稻株系(ΔOsPIL1 ko#7、#33和#36),OsPIL1敲除转基因水稻株系在株高、分蘖数、根长、胚芽鞘长度、茎基底到新叶叶鞘连接处长度、穗长、穗粒数、穗节点到顶叶叶鞘连接处长度、每节长度、百粒重、粒长、粒宽等农艺性状低于野生型;与野生型相比,OsPIL1敲除转基因水稻株系中过氧化物酶(POD)活性变化不大,但活性氧(ROS)含量显著增加而木质素含量显著减少(P<0.05,下同),水稻叶绿素和防御相关基因表达水平发生变化。稻瘟菌侵染OsPIL1敲除转基因水稻株系稻瘟病症状加重,与稻瘟菌接种野生型相比,OsPIL1敲除转基因水稻株系病情指数显著升高、胼胝质数变化不大、死细胞数和ROS含量增加、POD活性和木质素含量减少、防御相关基因表达水平降低;稻瘟菌活体致伤接种OsPIL1敲除转基因水稻株系引起单个病斑真菌相对生长量显著增加,进一步证实OsPIL1敲除转基因水稻株系稻瘟病抗性降低;外源JA或SA可明显提高OsPIL1敲除转基因水稻株系稻瘟病抗性,但提高效果不及JA或SA对野生型水稻。【结论】OsPIL1敲除对籼稻生长、谷粒发育和抗瘟性具有负面影响,OsPIL1可能介导JA或SA通路调控籼稻的抗瘟性。

     

    Abstract: 【Objective】To clarify the effects of knockout of phytochrome-interacting factor gene(OsPIL1) in rice(Oryza sativa) on the growth, yield and disease resistance of indica rice Yuelianggu, and to preliminarily explore effects of exogenous jasmonic acid(JA) or salicylic acid(SA) on the regulation of rice blast resistance by OsPIL1, and to explore the role of OsPIL1 integrating JA/SA in regulating rice blast resistance, which could provide basic data for analyzing the mechanism of rice blast resistance. 【Method】Rice variety Yuelianggu was as material CRISPR/Cas9 technology was used to obtain OsPIL1 knockout transgenic rice line, which was analyzed for phenotypic analysis(root length, coleoptile length, plant height, tiller number, spike length, number of solid grains per spike, 100-grain weight, grain length, grain width) and assay of rice blast resistance(observation of blast symptom and infection process) in inoculated rice plants, as well as for the analysis of rice blast resistance in inoculated rice plants after exogenous JA/SA treatment. 【Result】Three pure OsPIL1 knockout transgenic rice lines (ΔOspil1 ko #7, #33 and #36) were acquired by CRISPR/Cas9. These OsPIL1 knockout transgenic rice lines exhibited lower in various agronomic traits, including plant height, tiller number, root length, coleo-ptile length, distance from the stem base to the new leaf sheath junction, panicle length, grain number per panicle, length from the panicle node to the top leaf sheath junction, internode length, 100-grain weight, grain length and grain width than those of the wild type. Compared with the wild type, the activity of peroxidase(POD) in the OsPIL1 knockout transgenic rice lines changed slightly, but the content of reactive oxygen species(ROS) increased significantly while the content of lignin decreased significantly(P<0.05, the same below), and the expression levels of chloroplasts and defense related genes in rice changed. The symptoms of rice blast in OsPIL1 knockout transgenic rice lines infected with Magnaporthe oryzae were aggravated. Compared with the wild type inoculated with Magnaporthe oryzae, the disease index of OsPIL1 knockout transgenic rice lines was significantly increased, the number of callose changed slightly, the number of dead cells and ROS content increased, the activity of POD and the content of lignin decreased, and the expression level of defense related genes decreased. Inoculation with live injury caused by Magnaporthe oryzae into OsPIL1 knockout rice lines led to a significant increase in the relative growth of fungi in individual lesions, further confirming the reduced resistance to rice blast in OsPIL1 knockout transgenic rice lines. Exogenous JA or SA could greatly enhance the resistance of OsPIL1 knockout transgenic rice lines to rice blast, but the improvement effect was inferior to that of JA or SA on the resistance to rice blast in wild type rice. 【Conclusion】OsPIL1 knockout negatively affects rice growth, grain development and rice blast resistance, and OsPIL1 regulates JA or SA pathway to improve rice blast resistance in indica rice.

     

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