Scientia Agricultura Sinica ›› 2021, Vol. 54 ›› Issue (19): 4110-4120.doi: 10.3864/j.issn.0578-1752.2021.19.007

• PLANT PROTECTION • Previous Articles     Next Articles

Cloning of Nicotiana benthamiana NAC062 and Its Inhibitory Effect on Potato Virus Y Infection

QU XiaoLing(),JIAO YuBing,LUO JianDa,SONG LiYun,LI Ying,SHEN LilLi(),YANG JinGuang(),WANG FengLong   

  1. Tobacco Research Institute, Chinese Academy of Agricultural Sciences, Qingdao 266101, Shandong
  • Received:2021-02-22 Accepted:2021-04-14 Online:2021-10-01 Published:2021-10-12
  • Contact: LilLi SHEN,JinGuang YANG E-mail:812718023@qq.com;shenlili@caas.cn;yangjinguang@caas.cn

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Abstract:

【Objective】 Potato Y virus (PVY) is one of the most important viruses that endanger the tobacco production in China. NAC transcription factors are closely related to plant disease resistance and stress resistance. The objective of this study is to clone NbNAC062, analyze its bioinformatics and research its role in the process of PVY infection, and to provide a target for the development of tobacco antiviral agents. 【Method】 Nicotiana benthamiana was used as the material to clone NbNAC062, and MEGA, UniProt, SMART, TMHMM Server 2.0, Sol Genomics Network, PlantCARE and other technologies were used for bioinformatics analysis. Laser confocal microscope and quantitative real-time PCR (qRT-PCR) were used to clarify the localization of NbNAC062 protein and the change of NbNAC062 mRNA expression before and after PVY infection. Based on virus-induced gene silencing (VIGS) technology and over-expression technology, the pTRV::NbNAC062 silencing vector and the pEarleyGate100::RFP::NbNAC062 over-expression vector were constructed. qRT-PCR and Western blot were used to detect the changes of PVY accumulation and the expression of unfolded protein response (UPR) related gene BiP after silencing and over-expression in N. benthamiana.【Result】NbNAC062 encodes 646 amino acids, the N-terminal 28-179 aa is the NAC domain, 129-185 aa is the DNA binding region, and the C-terminal 621-643 aa is a hydrophobic transmembrane structure. Phylogenetic tree and protein sequence analysis show that N. benthamiana NbNAC062 is closely related to N. attenuata NaNAC062. The NbNAC062 promoter contains a variety of cis-acting elements related to abscisic acid, methyl jasmonate, salicylic acid and stress response. PVY infection activates NbNAC062 to transfer from cell membrane to nucleus and induces NbNAC062 up-regulation of expression. For 5 and 7 days after PVY infection, the NbNAC062 mRNA level in the treatment group was 2.52 and 1.95 times of that of the control group, respectively. For 3 days after PVY infection, the BiP mRNA expression was 2.39 times of that of the control group, and for 7 days after PVY infection, the expression of BiP was significantly lower than that of the control group, which was down-regulated by 56.77%. NbNAC062 was silenced and PVY was inoculated, compared with the control group, the expression of PVY CP mRNA was up-regulated in the silence group at 3, 5, and 7 days after inoculation, which was 2.12, 2.41, and 1.38 times of that of the control group, respectively. However, the expression of BiP mRNA was down-regulated by 28.19%, 58.11%, and 10.77%, respectively. The PVY CP protein content of the silence group was also significantly higher than that of the control group at 5 and 7 days after vaccination. NbNAC062 was over-expressed and PVY was inoculated, compared with the control group, the expression of PVY CP mRNA in the over-expression group at 24, 48, 72 hours after inoculation was down-regulated by 22.60%, 34.51%, and 36.21%, respectively, and BiP mRNA was up-regulated at 48 and 72 hours after inoculation, which was 1.56 and 1.35 times of that of the control group, respectively. The content of PVY CP in the over-expression group was also lower than that of the control group.【Conclusion】NbNAC062 belongs to the NAC class of membrane-bound transcription factors, which can be activated by PVY infection and transferred to the nucleus. It may regulate the expression of the UPR-related gene BiP to promote cell survival and inhibit early PVY infection.

Key words: NbNAC062, potato virus Y (PVY), gene silencing, transient over-expression

Table 1

Primers used in this study"

引物Primer 序列Sequence
NbNAC062 F ATGATGGCAGTACTTCCTGG
NbNAC062 R TACTCGCACTCTAAAGTATTCCC
TRV-NbNAC F TAAGGTTACCGAATTCTTGGATGGATCACACCCTGGC
TRV-NbNAC R AGACGCGTGAGCTCGGTACCTTCTGTATCATCAGCAATACAGC
Fu-NbNAC F CTTTAGATCTTCTAGAATGATGGCAGTACTTCCTGG
Fu-NbNAC R AGGAGGCCATGAATTCTACTCGCACTCTAAAGTATTCCC
PVY-CP-F GATGAATGGGCTTATGGTTTGGTG
PVY-CP-R GATTTGCCTAAGGGTTGGTTTCG
Actin-F CAAGGAAATCACCGCTTTGG
Actin-R AAGGGATGCGAGGATGGA
qPCR-NbNAC062 F TGGACAAGAATTGGCATCGC
qPCR-NbNAC062 R AACACCTCGGGCTCAAAGAAG
qPCR-BiP F GCCACAGAAGAAGCTACCAAGTTG
qPCR-BiP R GGTCCTCTCTGGGTTAACAGCG

Fig. 1

Phylogenetic analyses of NbNAC062"

Fig. 2

NbNAC062 protein sequence and domain analysis Comparative analysis of NbNAC062 protein amino acid sequence. Dark blue indicates that the amino acids are identical, pink indicates that there is only one amino acid difference, light blue indicates that there are two amino acids differences. The red, yellow, and green arrow lines represent the NAC domain, the DNA binding region, and the TMD transmembrane domain in turn; Protein domain analysis of NbNAC062"

Fig. 3

Changes of NbNAC062 and BiP expression after PVY infecting N. benthamiana * indicates that difference is significant at the 0.05 level, P<0.05. ** indicates that difference is significant at the 0.01 level, P<0.01. The same as below"

Fig. 4

Subcellular localization of NbNAC062 protein before and after PVY infection"

Fig. 5

Phenotype analysis and silencing efficiency of NbNAC062 silencing"

Fig. 6

Changes in PVY and BiP accumulation after silence of NbNAC062"

Fig. 7

Changes in PVY and BiP accumulation after overexpression of NbNAC062"

[1] HU X, KARASEV A V, BROWN C J, LORENZEN J H. Sequence characteristics of potato virus Y recombinants. Journal of General Virology, 2009, 90(12):3033-3041.
doi: 10.1099/vir.0.014142-0
[2] 朱贤朝, 王彦亭, 王智发. 中国烟草病害. 北京: 中国农业出版社, 2001: 210.
ZHU X C, WANG Y T, WANG Z F. Tobacco diseases of China. Beijing: China Agriculture Press, 2001: 210. (in Chinese)
[3] 王凤龙, 周义和, 任广伟. 中国烟草病害图鉴. 北京: 中国农业出版社, 2019: 6-7.
WANG F L, ZHOU Y H, REN G W. Illustrated Book of Tobacco Diseases in China. Beijing: China Agriculture Press, 2019: 6-7. (in Chinese)
[4] CHEN S, LI F, LIU D, JIANG C, CUI L, SHEN L, LIU G, YANG A. Dynamic expression analysis of early response genes induced by potato virus Y in PVY-resistant Nicotiana tabacum. Plant Cell Reports, 2017, 36(2):297-311.
doi: 10.1007/s00299-016-2080-1
[5] 姜瀚林, 田延平, 郭兆奎, 刘永中, 万秀清, 刘文涛, 李现道, 李向东, 张永春, 孟凡武. 抗马铃薯Y病毒(PVY)和烟草花叶病毒(TMV)单联弱毒疫苗的研制及防效测定. 中国烟草学报, 2020, 26(2):65-70.
JIANG H L, TIAN Y P, GUO Z K, LIU Y Z, WAN X Q, LIU W T, LI X D, LI X D, ZHANG Y C, MENG F W. Preparation and control effect determination of mild vaccines against potato virus Y and tobacco mosaic virus. Acta Tabacaria Sinica, 2020, 26(2):65-70. (in Chinese)
[6] 万秀清, 乔婵, 赵淑娟, 李若, 李丽杰, 郭振楠. 黑龙江烟区烟草马铃薯Y病毒株系的分子鉴定. 烟草科技, 2015, 48(10):13-18, 25.
WAN X Q, QIAO C, ZHAO S J, LI R, LI L J, GUO Z N. Molecular identification of tobacco potato virus Y strains in heilongjiang tobacco planting areas. Tobacco Science and Technology, 2015, 48(10):13-18, 25. (in Chinese)
[7] 陈德鑫, 王凤龙, 李多川, 钱玉梅, 申莉莉. 山东烟草病毒病发生特点及防治对策. 中国烟草科学, 2007, 28(1):25-28.
CHEN D X, WANG F L, LI D C, QIAN Y M, SHEN L L. Epidemic characteristics of tobacco virus disease and control measures in Shandong Province. Chinese Tobacco Science, 2007, 28(1):25-28. (in Chinese)
[8] 吉璐. 南荻抗逆相关NAC转录因子的克隆及功能鉴定[D]. 长沙: 湖南农业大学, 2013.
JI L. Cloning and function identification of stress resistance-related NAC transcription factors from Miscanthus lutarioriparius (Poaceae)[D]. Changsha: Hunan Agricultural University, 2013. (in Chinese)
[9] 付长春. NAC类转录因子参与调控番木瓜果实后熟过程中类胡萝卜素代谢的机制研究[D]. 广州: 华南农业大学, 2017.
FU C C. Mechanism analysis of NAC transcription factors in regulation of carotenoid biosynthesis during papaya fruit ripening[D]. Guangzhou: South China Agricultural University, 2017. (in Chinese)
[10] PENG H, CHENG H Y, CHEN C, YU X W, YANG J N, GAO W R, SHI Q H, ZHANG H, LI J G, MA H. A NAC transcription factor gene of chickpea (Cicer arietinum), CarNAC3, is involved in drought stress response and various developmental processes. Journal of Plant Physiology, 2009, 166(17):1934-1945.
doi: 10.1016/j.jplph.2009.05.013
[11] ZHONG R, LEE C, YE Z H. Global analysis of direct targets of secondary wall NAC master switches in Arabidopsis. Molecular Plant, 2010, 3(6):1087-1103.
doi: 10.1093/mp/ssq062
[12] ODA-YAMAMIZO C, MITSUDA N, SAKAMOTO S, OGAWA D, OHME-TAKAGI M, OHMIYA A. The NAC transcription factor ANAC046 is a positive regulator of chlorophyll degradation and senescence in Arabidopsis leaves. Scientific Reports, 2016, 6:23609.
doi: 10.1038/srep23609
[13] MENG C, YANG D Y, MA X C, ZHAO W Y, LIANG X Q, MA N N, MENG Q W. Suppression of tomato SlNAC1 transcription factor delays fruit ripening. Journal of Plant Physiology, 2016, 193:88-96.
doi: 10.1016/j.jplph.2016.01.014
[14] DELESSERT C, KAZAN K, WILSON I W, VAN DER STRAETEN D, MANNERS J, DENNIS E S, DOLFERUS R. The transcription factor ATAF2 represses the expression of pathogenesis-related genes in Arabidopsis. The Plant Journal, 2005, 43(5):745-757.
doi: 10.1111/tpj.2005.43.issue-5
[15] JENSEN M K, LINDEMOSE S, DE MASI F, REIMER J J, NIELSEN M, PERERA V, WORKMAN C T, TURCK F, GRANT M R, MUNDY J, PETERSEN M, SKRIVER K. ATAF1 transcription factor directly regulates abscisic acid biosynthetic gene NCED3 in Arabidopsis thaliana. FEBS Open Bio, 2013, 3:321-327.
doi: 10.1016/j.fob.2013.07.006
[16] YOSHII M, YAMAZAKI M, RAKWAL R, KISHI-KABOSHI M, MIYAO A, HIROCHIKA H. The NAC transcription factor RIM1 of rice is a new regulator of jasmonate signaling. The Plant Journal, 2010, 61(5):804-815.
doi: 10.1111/tpj.2010.61.issue-5
[17] MARCINIAK S J, RON D. Endoplasmic reticulum stress signaling in disease. Physiological Reviews, 2006, 86(4):1133-1149.
doi: 10.1152/physrev.00015.2006
[18] 杨正婷, 刘建祥. 植物内质网胁迫应答研究进展. 生物技术通报, 2016, 32(10):84-96.
YANG Z T, LIU J X. Endoplasmic reticulum stress response in plants. Biotechnology Bulletin, 2016, 32(10):84-96. (in Chinese)
[19] KIM M J, PARK M J, SEO P J, SONG J S, KIM H J, PARK C M. Controlled nuclear import of the transcription factor NTL6 reveals a cytoplasmic role of SnRK2.8 in the drought-stress response. The Biochemical Journal, 2012, 448(3):353-363.
doi: 10.1042/BJ20120244
[20] SEO P J, KIM M J, SONG J S, KIM Y S, KIM H J, PARK C M. Proteolytic processing of an Arabidopsis membrane-bound NAC transcription factor is triggered by cold-induced changes in membrane fluidity. The Biochemical Journal, 2010, 427(3):359-367.
doi: 10.1042/BJ20091762
[21] SEO P J, KIM M J, PARK J Y, KIM S Y, JEON J, LEE Y H, KIM J, PARK C M. Cold activation of a plasma membrane-tethered NAC transcription factor induces a pathogen resistance response in Arabidopsis. The Plant Journal, 2010, 61(4):661-671.
doi: 10.1111/tpj.2010.61.issue-4
[22] BEAUCHEMIN C, BOUGIE V, LALIBERTÉ J F. Simultaneous production of two foreign proteins from a potyvirus-based vector. Virus Research, 2005, 112(1/2):1-8.
doi: 10.1016/j.virusres.2005.03.001
[23] 龚明月, 段啸天, 余婷婷, 王杰, 申莉莉, 李莹, 刘明宏, 李永亮, 吕洪坤, 章松柏, 杨金广. 烟草Hsc70-2的克隆及对马铃薯Y病毒侵染烟草的促进作用. 中国农业科学, 2020, 53(4):771-781.
GONG M Y, DUAN X T, YU T T, WANG J, SHEN L L, LI Y, LIU M H, LI Y L, LÜ H K, ZHANG S B, YANG J G. Cloning of Hsc70-2 and its promoting effect on potato virus Y infection in Nicotiana benthamiana. Scientia Agricultura Sinica, 2020, 53(4):771-781. (in Chinese)
[24] SUN H J, SHEN L L, QIN Y X, LIU X W, HAO K Q, LI Y, WANG J, YANG J G, WANG F L. CLC-Nt1 affects potato virus Y infection via regulation of endoplasmic reticulum luminal Ph. New Phytologist, 2018, 220(2):539-552.
doi: 10.1111/nph.15310
[25] KUMAR S, STECHER G, TAMURA K. MEGA7: Molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution, 2016, 33(7):1870-1874.
doi: 10.1093/molbev/msw054
[26] 罗静初. UniProt蛋白质数据库简介. 生物信息学, 2019, 17(3):131-144.
LUO J C. A brief introduction to UniProt. Chinese Journal of Bioinformatics, 2019, 17(3):131-144. (in Chinese)
[27] LETUNIC I, KHEDKAR S, BORK P. SMART: Recent updates, new developments and status in 2020. Nucleic Acids Research, 2020, 49(D1):D458-D460.
doi: 10.1093/nar/gkaa937
[28] LETUNIC I, BORK P. 20 years of the SMART protein domain annotation resource. Nucleic Acids Research, 2018, 46(D1):D493-D496.
doi: 10.1093/nar/gkx922
[29] SCHULTZ J, MILPETZ F, BORK P, PONTING C P. SMART, a simple modular architecture research tool: Identification of signaling domains. Proceedings of the National Academy of Sciences of the United States of America, 1998, 95(11):5857-5864.
[30] EL-RAMI F E, SIKORA A E. Bioinformatics workflow for gonococcal proteomics//Methods in Molecular Biology. Springer Science+ Business Media, 2019, 1997:185-205.
[31] 陈倩, 谢旗. 内质网胁迫在植物中的研究进展. 生物技术通报, 2018, 34(1):15-25.
CHEN Q, XIE Q. The research progress of the endoplasmic reticulum (ER) stress response in plant. Biotechnology Bulletin, 2018, 34(1):15-25. (in Chinese)
[32] VERCHOT J. Wrapping membranes around plant virus infection. Current Opinion in Virology, 2011, 1(5):388-395.
doi: 10.1016/j.coviro.2011.09.009
[33] LALIBERTÉ J F, SANFAÇON H. Cellular remodeling during plant virus infection. Annual Review of Phytopathology, 2010, 48:69-91.
doi: 10.1146/annurev-phyto-073009-114239
[34] SUN Z T, YANG D, XIE L, SUN L Y, ZHANG S L, ZHU Q S, LI J M, WANG X, CHEN J. 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.
doi: 10.1016/j.virol.2013.09.001
[35] WEI T, HUANG T S, MCNEIL J, LALIBERTE J F, HONG J, NELSON R S, WANG A. Sequential recruitment of the endoplasmic reticulum and chloroplasts for plant potyvirus replication. Journal of Virology, 2010, 84(2):799-809.
doi: 10.1128/JVI.01824-09
[36] 李方方. TMV、CMV诱导烟草内质网应激及调控因子NbNAC089的功能分析[D]. 北京: 中国农业科学院, 2017.
LI F F. TMV/CMV induces tobacco endoplasmic reticulum stress and functional characterization of regulator NbNAC089[D]. Beijing: Chinese Academy of Agricultural Sciences, 2017. (in Chinese)
[37] YANG Z T, LU S J, WANG M J, BI D L, SUN L, ZHOU S F, SONG Z T, LIU J X. A plasma membrane-tethered transcription factor, NAC062/ANAC062/NTL6, mediates the unfolded protein response in Arabidopsis. The Plant Journal, 2014, 79(6):1033-1043.
doi: 10.1111/tpj.12604
[38] 彭曙光. 我国烟草病毒病的发生及综合防治研究进展. 江西农业学报, 2011, 23(1):115-117.
PENG S G. Research advance in occurrence and integrated control of tobacco virus diseases in China. Acta Agriculturae Jiangxi, 2011, 23(1):115-117. (in Chinese)
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