烟草NbbZIP28突变体的创建及其对病毒侵染胁迫的响应

Targeted Mutagenesis of Nbb ZIP28 and Its Stress Response to Virus Infection in Nicotiana benthamiana

【目的】b ZIP类转录因子参与植物的生长发育、激素信号、抗病性及抗逆性等多种生物胁迫过程。前期研究表明黄瓜花叶病毒(Cucumber mosaic virus,CMV)或烟草花叶病毒(Tobacco mosaic virus,TMV)侵染本氏烟(Nicotiana benthamiana)上调内质网应激(endoplasmic reticulum stress,ERs)因子NbbZIP28;NbbZIP28沉默导致病毒积累量上升,本研究旨在验证NbbZIP28对病毒侵染胁迫的响应机制。【方法】通过CRISPR/Cas9基因编辑技术和烟草遗传转染创建NbbZIP28基因突变植株,以野生型植株为对照,分别浸润接种侵染性克隆TMV-GFP、摩擦接种TMV-GFP或CMV接种液,检测突变体植株对病毒侵染胁迫的敏感性变化,接种CMV后0—48 h,采用q RT-PCR检测内质网应激相关的未折叠蛋白反应(unfolded protein response,UPR)基因的表达。本氏烟接种TMV 24 h、接种CMV 48 h后,在接种叶上浸润融合蛋白NbbZIP28-GFP,瞬时表达48 h后,采用Western blot检测病毒诱导NbbZIP28蛋白的水解激活;采用在线搜索工具Plant CARE分析NbbZIP28启动子区域中参与防卫和应激反应的顺式作用元件。【结果】CRISPR/Cas9定点敲除NbbZIP28后,目的基因靶位点缺失了10个碱基,导致翻译错误、蛋白功能变化。在正常生长条件下,转基因阳性植株与野生型无显著的表型差异。植株接种TMV-GFP后4—8 d,突变体中的病毒浸润斑亮度或初侵染点数目均显著高于野生型,扩展至新叶的速度较快。接种CMV后12—48 h,突变体中UPR相关基因Bi P、PDI、CAM及NbbZIP28下游基因NF-YC2的表达量显著低于野生型;5—7 d突变体中的病毒外壳蛋白(coat protein,CP)基因表达量显著高于野生型,花叶及皱缩症状更显著。蛋白质序列比对分析显示NbbZIP28具有与拟南芥Atb ZIP28相同的S1P和S2P蛋白酶水解位点。Western blot检测发现,与接种清水对照相比,TMV或CMV侵染显著促进了全长的融合蛋白NbbZIP28-GFP在S1P和S2P位点发生裂解。NbbZIP28启动子序列中包含5个参与热应激反应的顺式作用元件(heat stress response element,HSE)、3个参与低温反应的顺式作用元件(low temperature response element,LTR)、1个参与防卫和应激反应的顺式作用元件(TC-rich repeats)。【结论】病毒侵染促进NbbZIP28的水解激活并上调相关的UPR基因;NbbZIP28敲除导致植株对病毒的敏感性上升,病毒诱导的UPR基因表达被抑制。NbbZIP28为病毒侵染胁迫下的UPR调控因子,在病毒侵染早期通过上调UPR信号和提高寄主基础防卫反应而延缓病毒的侵染和增殖。

【Objective】 b ZIP transcriptional factors are involved in plant developing, hormonal signal, disease resistance, and abiotic stress responsiveness. Previous study showed that Tobacco mosaic virus（TMV） or Cucumber mosaic virus（CMV） infection up-regulated the endoplasmic reticulum stress（ERs） gene NbbZIP28, and knock-down of NbbZIP28 increased more susceptibility to virus than wide type（WT）. The mechanism of its stress response to virus infection still needs to be verified. 【Method】NbbZIP28 mutant plants were obtained by CRISPR/Cas9 gene editing technique and tobacco genetic transfection. The susceptibility of mutations to virus infection and the ER stress related UPR genes expression in mutations were compared to that in wide type by q RT-PCR. Then the fusion protein of NbbZIP28-GFP was expressed transiently in leaves of Nicotiana benthamiana in advance of inoculation with TMV 24 h or CMV 48 h, and its hydrolyzation induced by viral infection was checked by Western blot. The Cis-acting elements involved in defense and stress response in promoter region of NbbZIP28 were analyzed by Plant CARE. 【Result】The DNA deletion of 10 bp had been found in the target sites of NbbZIP28, leading to translation error and protein function changed. Under normal conditions, mutant plants showed no different phenotype from WT. Upon inoculation TMV-GFP, mutant plants showed more virus infected spots and faster expansion than WT. Upon inoculation CMV, the UPR related genes（Bi P, PDI, CAM and NF-YC2） showed lower expression in mutants than that in WT plants at 12-48 hours post inoculation. In addition, mutant plants showed more virus accumulation than WT at 5-7 days, the symptoms of mosaic and shrinkage were more significant. Protein sequence alignment analysis showed that NbbZIP28 had the same hydrolytic site of S1 P and S2 P as Ab ZIP28. Western blot detection found that TMV or CMV infection significantly promoted the full length of NbbZIP28-GFP to be cleaved at sites of S1 P and S2 P than inoculation water. There are five cis-acting elements involved in heat stress responsiveness（HSE）, three cis-acting elements involved in low-temperature responsiveness（LTR） and one cis-acting element involved in defense and stress responsiveness（TC-rich repeats）.【Conclusion】 Virus infection activated NbbZIP28 and up-regulated its related UPR genes. NbbZIP28 knock-out increased the susceptibility to virus than WT, and virus induced UPR gene expression is suppressed in mutant plants. NbbZIP28 is the regulating factor of UPR under virus infection stress and plays a role in suppression viral infection at early stage through up-regulating UPR signal and increasing plants basic defense response.