烟草NbbZIP28突变体的创建及其对病毒侵染胁迫的响应

doi:10.3864/j.issn.0578-1752.2018.14.006

Abstract

Abstract: 【Objective】 bZIP 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 qRT-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 PlantCARE. 【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 (BiP, 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 S1P and S2P as AbZIP28. Western blot detection found that TMV or CMV infection significantly promoted the full length of NbbZIP28-GFP to be cleaved at sites of S1P and S2P 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.

Key words: endoplasmic reticulum stress (ERs), unfolded protein response (UPR), NbbZIP28, virus infection, Nicotiana benthamiana

HE QingYun, LIU XiaoWei, JIAO YuBing, YU FangFei, SHEN LiLi, YANG JinGuang, WANG FengLong . Targeted Mutagenesis of NbbZIP28 and Its Stress Response to Virus Infection in Nicotiana benthamiana[J].Scientia Agricultura Sinica, 2018, 51(14): 2689-2699.

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References

[1] 张计育, 渠慎春, 郭忠仁, 杜小丽, 都贝贝, 章镇. 植物bZIP转录因子的生物学功能. 西北植物学报, 2011, 31(5): 1066-1075.

ZHANG J Y, QU S C, GUO Z R, DU X L, DU B B, ZHANG Z. Biology function of bZIP transcription factors in plants. Acta Botanica Boreali-Occidentalia Sinica, 2011, 31(5): 1066-1075. (in Chinese)

[2] LIU J X, SRIVASTAVA R, CHE P, HOWELL S H. An endoplasmic reticulum stress response in Arabidopsis is mediated by proteolytic processing and nuclear relocation of a membrane-associated transcription factor, bZIP28. The Plant Cell, 2007, 19(12): 4111-4119.

[3] LIU J X, HOWELL S H. bZIP28 and NF-Y transcription factors are activated by ER stress and assemble into a transcriptional complex to regulate stress response genes in Arabidopsis. The Plant Cell, 2010, 22(3): 782-796.

[4] IWATA Y, KOIZUMI N. An Arabidopsis transcription factor, AtbZIP60, regulates the endoplasmic reticulum stress response in a manner unique to plants. Proceedings of the National Academy of Sciences of the United States of America, 2005, 102(14): 5280-5285.

[5] LU D, CHRISTOPHER D A. Endoplasmic reticulum stress activates the expression of a sub-group of protein disulfide isomerase genes and AtbZIP60 modulates the response in Arabidopsis thaliana. Molecular Genetics and Genomics, 2008, 280: 199-210.

[6] 孙玲. 拟南芥内质网胁迫应答中转录因子NAC103的功能分析[D]. 上海: 复旦大学, 2013.

SUN L. Functional characterization of a transcription factor NAC103 in the ER stress response in Arabidopsis [D]. Shanghai: Fudan University, 2013. (in Chinese)

[7] SRIVASTAVA R, CHEN Y, DENG Y, BRANDIZZI F, HOWELL S H. Elements proximal to and within the transmembrane domain mediate the organelle-to-organelle movement of bZIP28 under ER stress conditions. The Plant Journal, 2012, 70: 1033-1042.

[8] SRIVASTAVA R, DENG Y, HOWELL S H. Stress sensing in plants by an ER stress sensor/transducer, bZIP28. Frontiers in Plant Science, 2014, 5: Article 59.

[9] SUN L, ZHANG S S, LU S J, LIU J X. Site-1 protease cleavage site is important for the ER stress-induced activation of membrane- associated transcription factor bZIP28 in Arabidopsis. Science China Life Sciences, 2015, 58(3): 270-275.

[10] 杨月. 圆斑病菌胁迫下玉米ZmbZIP 28的响应及其抗逆分析[D]. 大庆: 黑龙江八一农垦大学, 2016.

YANG Y. Responses of ZmbZIP 28 under Bipolaris carbohum and the analysis of stress resistance[D]. Daqing: Heilongjiang Bayi Agricultural University, 2016. (in Chinese)

[11] TATEDA C, OZAKI R, ONODERA Y, TAKAHASHI Y, YAMAGUCHI K, BERBERICH T, KOIZUMI N, KUSANO T. NtbZIP60, an endoplasmic reticulum-localized transcription factor, plays a role in the defense response against bacterial pathogens in Nicotiana tabacum. Journal of Plant Research, 2008, 121: 603-611.

[12] ZhANG L, CHEN H, BRANDIZZI F, VERCHOT J, WANG A. The UPR branch IRE1-bZIP60 in plants plays an essential role in viral infection and is complementary to the only UPR pathway in yeast. PLoS Genetics, 2015, 11(4): e1005164.

[13] 沈川. 小麦内质网应激相关bZIP基因的鉴定与功能分析[D]. 杨凌: 西北农林科技大学, 2016.

SHEN C. Identification and functional analysis of bzip transcription factor genes related to UPR in wheat[D]. Yangling: Northwest A & F University, 2016. (in Chinese)

[14] YANG Z T, WANG M J, SUN L, BI D L, SUN L, SONG Z T, ZHANG S S, ZHOU S F, LIU J X. The membrane-associated transcription factor NAC089 controls ER-stress-induced programmed cell death in plants. PLoS Genetics, 2014, 10(3): e1004243.

[15] ZHANG L R, WANG A M. Virus-induced ER stress and the unfolded protein response. Frontiers in Plant Science, 2012, 3: Article 293.

[16] JHENG J R, HO J Y, HORNG J T. ER stress, autophagy, and RNA viruses. Frontiers in Microbiology, 2014, 5: Article 388.

[17] YE C, DICKMAN M B, WHITHAM S A, PAYTON M, VERCHOT J. The unfolded protein response is triggered by a plant viral movement protein. Plant Physiology, 2011, 156(2): 741-755.

[18] SUN Z T, YANG D, XIE L, SUN L Y, ZHANG S L, ZHU Q S, LI J M, WANG X, CHEN J P. Rice black-streaked dwarf virus P10 induces membranous structures at the ER and elicits the unfolded protein response in Nicotiana benthamiana. Virology, 2013, 447(1/2): 131-139.

[19] LU Y, YIN M, WANG X, CHEN B, YANG X, PENG J, ZHENG H, ZHAO J, LIN L, YU C, MACFARLANE S, HE J, LIU Y, CHEN J, DAI L, YAN F. The unfolded protein response and programmed cell death are induced by expression of Garlic virus X p11 in Nicotiana benthamiana. Journal of General Virology, 2016, 97: 1462-1468.

[20] LI F F, SUN H J, JIAO Y B, WANG F L, YANG J G, SHEN L L. Viral infection-induced endoplasmic reticulum stress and a membrane-associated transcription factor NbNAC089 are involved in resistance to virus in Nicotiana benthamiana. Plant Pathology, 2018, 67: 233-243.

[21] SHEN L L, LI F F, DONG W F, LIU W, QIAN Y M, YANG J G, WANG F L, WU Y H. Nicotiana benthamiana NbbZIP28, a possible regulator of unfolded protein response, plays a negative role in viral infection. European Journal of Plant Pathology, 2017, 149(4): 831-843.

[22] CONG L, RAN F A, COX D, LIN S, BARRETTO R, HABIB N, HSU P D, WU X, JIANG W, MARRAFFINI L A, ZHANG F. Multiplex genome engineering using CRISPR/Cas systems. Science, 2013, 339(6121): 819-823.

[23] GAO J P, WANG G H, MA S Y, XIE X D, WU X W, ZHANG X T, WU Y Q, ZHAO P, XIA Q Y. CRISPR/Cas9-mediated targeted mutagenesis in Nicotiana tabacum. Plant Molecular Biology, 2015, 87(1/2): 99-110.

[24] 聂梦云, 高军平, 罗培, 陈晓乐, 易小惠, 王根洪, 夏庆友. 基于CRISPR/Cas9技术的烟草NtDXR基因敲除及功能分析. 烟草科技, 2016, 49(6): 1-7.

NIE M Y, GAO J P, LUO P, CHEN X L, YI X H, WANG G H, XIA Q Y. CRISPR/Cas9-mediated targeted mutagenesis and function analysis of DXR in Nicotiana tabacum. Tobacco Science & Technology, 2016, 49(6): 1-7. (in Chinese)

[25] 郑晓瑜, 郭晋艳, 张毅, 李秋莉. 植物非生物胁迫诱导启动子顺式作用元件的研究方法. 植物生理学报, 2011, 47(2): 129-135.

ZHENG X Y, GUO J Y, ZHANG Y, LI Q L. Research methods of cis-acting elements in plant abiotic stress inducible promoters. Plant Physiology Journal, 2011, 47(2): 129-135. (in Chinese)

[26] 李田. 绒毛白蜡表达谱分析及MYB基因的克隆和功能研究[D]. 济南: 山东师范大学, 2014.

Li T. Expression profile analysis, cloning and functional research of MYB genes from Fraxinus velutina Torr[D]. Ji’nan: Shandong Normal University, 2014. (in Chinese)

[27] Liu J X, SRIVASTAVA R, CHE P, HOWELL S H. Salt stress responses in Arabidopsis utilize a signal transduction pathway related to endoplasmic reticulum stress signaling. The Plant Journal, 2007, 51(5): 897-909.

[28] 曹红利, 岳川, 王新超, 杨亚军. bZIP转录因子与植物抗逆性研究进展. 南方农业学报, 2012, 43(8): 1094-1100.

CAO H L, YUE C, WANG X C, YANG Y J. Advance in bZIP transcription factors related with plant stress resistance. Journal of Southern Agriculture, 2012, 43(8): 1094-1100. (in Chinese)

[29] 王楠, 向凤宁, 李朔. 植物膜结合转录因子与胁迫响应. 生命科学, 2016, 28(7): 799-806.

WANG N, XIANG F N, LI S. Advance in plant membrane-bound transcription factors and plant stress response. Chinese Bulletin of Life Sciences, 2016, 28(7): 799-806. (in Chinese)

[30] 刘宝玲, 张莉, 孙岩, 薛金爱, 高昌勇, 苑丽霞, 王计平, 贾小云, 李润植. 谷子bZIP转录因子的全基因组鉴定及其在干旱和盐胁迫下的表达分析. 植物学报, 2016, 51(4): 473-487.

LIU B L, ZHANG L, SUN Y, XUE J A, GAO C Y, YUAN L X, WANG J P, JIA X Y, LI R Z. Genome-wide characterization of bZIP transcription factors in foxtail millet and their expression profiles in response to drought and salt stresses. Chinese Bulletin of Botany, 2016, 51(4): 473-487. (in Chinese)

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