转水稻条纹病毒NS3本氏烟的抗病性

doi:10.3864/j.issn.0578-1752.2019.10.005

摘要/Abstract

摘要：

【目的】NS3编码的蛋白是水稻条纹病毒（Rice stripe virus,RSV）RNA沉默抑制子。笔者前期研究发现,转NS3本氏烟（Nicotiana benthamiana）对RSV有一定的抗性。为深入揭示NS3在寄主体内的作用机制,本文将进一步探究转NS3本氏烟对其他烟草病害的抗性。【方法】以野生型本氏烟和转NS3本氏烟为试验材料,通过农杆菌浸润法接种马铃薯X病毒（Potato virus X,PVX）侵染性克隆,观察病毒症状,抽提系统发病叶片总RNA,并利用RT-qPCR方法检测病毒的相对积累量;采用灌根接种法分别接种烟草青枯病菌（Ralstonia solanacearum）、烟草黑胫病菌（Phytophthora parasitica var. nicotianae）,观察植株表型并统计发病率和病情指数。【结果】PVX侵染本氏烟后,植株叶片主要表现为花叶、褪绿等症状,NS3表达植株与野生型植株具有相同的PVX症状表现。在接种PVX 10 d后,RT-qPCR检测发现与野生型植株病毒积累量相比,NS3表达抑制了PVX在本氏烟体内的积累水平,两个转NS3株系（NS3-6、NS3-9）中PVX的相对积累量分别为野生型植株的68.17%和81.01%。青枯病菌在野生型植株和转基因植株上均引起萎蔫、猝倒等典型的病害症状,且两者的症状无明显差异。在青枯病菌侵染早期,NS3表达在一定程度上利于病菌的侵染,表现出对青枯病较为敏感;而在接种14 d,野生型植株的发病率为100.00%,病情指数为78.67,而NS3-6、NS3-9株系的发病率分别为95.00％、98.33％,病情指数分别为70.00、73.00;14 d之后发病率和病情指数逐渐升高,而NS3表达株系的病情指数均低于野生型植株。黑胫病菌在野生型植株和转基因植株上均为在茎基部出现黑褐色坏死斑等典型病害症状,且NS3表达也不影响本氏烟的症状表现。在黑胫病菌的侵染初期,NS3-6株系的发病率高于野生型植株;但在整个黑胫病发生过程中,转NS3株系的病情指数均低于野生型植株,在接种12 d,野生型本氏烟的发病率为96.67％,病情指数为77.50,而NS3-6、NS3-9株系的发病率分别为90.00％、86.67％,病情指数分别为64.17、62.08。【结论】通过病原侵染过程观察表明NS3表达在一定程度上影响了烟草病害在本氏烟上的侵染和严重程度,且对不同病原菌的影响不同。

关键词: 水稻条纹病毒, NS3, 转基因本氏烟, 抗病性

Abstract:

【Objective】The protein encoded by NS3 is a suppressor of RNA silencing of Rice stripe virus (RSV). In previous study, it was found that NS3 expression enhanced the resistance of Nicotiana benthamiana to RSV using transgenic technology. In order to reveal the detailed function of NS3 in host plants, the resistance of NS3-transgenic N. benthamiana to other tobacco diseases will be analyzed in this study.【Method】Wild-type and NS3-transgenic N. benthamiana were used to study the infection of Potato virus X (PVX), Ralstonia solanacearum, and Phytophthora parasitica var. nicotianae. PVX infectious clone was inoculated by agro-infiltration, viral symptoms were observed. The total RNA of infected leaves was extracted, and viral relative accumulation was detected by RT-qPCR. The method of filling root was used to inoculate R. solanacearum and P. parasitica var. nicotianae, respectively, disease symptoms were observed, and the incidence and disease index were analyzed.【Result】The main symptoms of PVX in N. benthamiana leaves were mosaic and chlorosis, and the NS3-transgenic N. benthamiana plants had the same PVX symptoms as those of wild-type plants. At 10 days past inoculation (dpi), the results of RT-qPCR showed that NS3 expression inhibited the accumulation of PVX in N. benthamiana, the relative accumulation of PVX in NS3-6 line and NS3-9 line was 68.17% and 81.01% of that of wild-type N. benthamiana, respectively. After the infection of R. solanacearum, the symptoms both in wild-type and NS3-transgenic N. benthamiana plants were wilting and damping-off. In the early stage of disease, the expression of NS3 was beneficial to the infection of R. solanacearum to a certain extent, which showed that it was more sensitivity to bacterial wilt. At 14 dpi, the incidence and disease index of bacterial wilt of wild-type N. benthamiana were 100.00% and 78.67, while the incidence of NS3-6, NS3-9 line was 95.00%, 98.33%, and the disease index was 70.00, 73.00, respectively. After 14 dpi, the incidence and disease index increased gradually, while the disease index of NS3-transgenic lines was lower than that of wild-type plants. The black-brown necrotic patches at the base of stem were observed both in wild-type and transgenic plants after P. parasitica var. nicotianae infection, and the expression of NS3 did not affect the symptoms of N. benthamiana. At the early stage of P. parasitica var. nicotianae infection, the incidence of black shank disease in NS3-6 was higher than that of wild-type N. benthamiana, however, during the whole process of black shank disease, the disease index of NS3-transgenic lines was lower than that of wild-type N. benthamiana. At 12 dpi, the incidence and disease index of black shank disease in wild-type N. benthamiana were 96.67% and 77.50, while the incidence of NS3-6, NS3-9 line was 90.00%, 86.67%, and the disease index was 64.17, 62.08, respectively.【Conclusion】The observation of pathogen infection process showed that the expression of NS3 affected the infection and severity of tobacco diseases on N. benthamiana to a certain extent, and the effects on different pathogens were also different.

Key words: Rice stripe virus (RSV), NS3, transgenic Nicotiana benthamiana, disease resistance

吴根土, 陈广香, 张珈源, 胡桥, 马明鸽, 窦彦霞, 李明骏, 青玲. 转水稻条纹病毒NS3本氏烟的抗病性[J]. 中国农业科学, 2019, 52(10): 1710-1720.

WU GenTu, CHEN GuangXiang, ZHANG JiaYuan, HU Qiao, MA MingGe, DOU YanXia, LI MingJun, QING Ling. Disease Resistance of Rice stripe virus NS3-Transgenic Nicotiana benthamiana[J]. Scientia Agricultura Sinica, 2019, 52(10): 1710-1720.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2019.10.005

https://www.chinaagrisci.com/CN/Y2019/V52/I10/1710

图/表 9

图1

图2

图3

图4

图5

图6

图7

图8

图9

参考文献 38

[1]	COGONI C, MACINO G . Post-transcriptional gene silencing across kingdoms. Current Opinion in Genetics and Development, 2000,10(6):638-643. doi: 10.1016/S0959-437X(00)00134-9
[2]	AZEVEDO J, GARCIA D, PONTIER D, OHNESORGE S, YU A, GARCIA S, BRAUN L, BERGDOLL M, HAKIMI M A, LAGRANGE T, VOINNET O . Argonaute quenching and global changes in Dicer homeostasis caused by a pathogen-encoded GW repeat protein. Genes and Development, 2010,24(9):904-915. doi: 10.1101/gad.1908710
[3]	CHEN J, LI W X, XIE D, PENG J R, DING S W . Viral virulence protein suppresses RNA silencing-mediated defense but upregulates the role of microRNA in host gene expression. The Plant Cell, 2004,16(5):1302-1313. doi: 10.1105/tpc.018986
[4]	CHIU M H, CHEN I H, BAULCOMBE D C, TSAI C H . The silencing suppressor P25 of Potato virus X interacts with Argonaute1 and mediates its degradation through the proteasome pathway. Molecular Plant Pathology, 2010,11(5):641-649.
[5]	MALLORY A C, ELY L, SMITH T H, MARATHE R, ANANDALAKSHMI R, FAGARD M, VAUCHERET H, PRUSS G, BOWMAN L, VANCE V B . HC-Pro suppression of transgene silencing eliminates the small RNAs but not transgene methylation or the mobile signal. The Plant Cell, 2001,13(3):571-583. doi: 10.1105/tpc.13.3.571
[6]	DUNOYER P, LECELLIER C H, PARIZOTTO E A, HIMBER C, VOINNET O . Probing the microRNA and small interfering RNA pathways with virus-encoded suppressors of RNA silencing. The Plant Cell, 2004,16(5):1235-1250. doi: 10.1105/tpc.020719
[7]	JAY F, WANG Y, YU A, TACONNAT L, PELLETIER S, COLOT V, RENOU J P, VOINNET O . Misregulation of AUXIN RESPONSE FACTOR 8 underlies the developmental abnormalities caused by three distinct viral silencing suppressors in Arabidopsis. PLoS Pathogens, 2011,7(5):e1002035.
[8]	SHEN W J, RUAN X L, LI X S, ZHAO Q, LI H P . RNA silencing suppressor Pns11 of Rice gall dwarf virus induces virus-like symptoms in transgenic rice. Archives of Virology, 2012,157(8):1531-1539.
[9]	WU G T, WANG J Y, YANG Y, DONG B, WANG Y L, SUN G C, YAN C Q, YAN F, CHEN J P . Transgenic rice expressing Rice stripe virus NS3 protein, a suppressor of RNA silencing, shows resistance to rice blast disease. Virus Genes, 2014,48(3):566-569.
[10]	WU G T, ZHENG G X, HU Q, MA M G, LI M J, SUN X C, YAN F, QING L . NS3 protein from Rice stripe virus affects the expression of endogenous genes in Nicotiana benthamiana. Virology Journal, 2018,15:105.
[11]	YAN F, ZHANG H M, ADAMS M J, YANG J, PENG J J, ANTONIW J F, ZHOU Y J, CHEN J P . Characterization of siRNAs derived from Rice stripe virus in infected rice plants by deep sequencing. Archive of Virology, 2010,155(6):935-940.
[12]	ZHU Y, HAYAKAWA T, TORIYAMA S . Complete nucleotide sequence of RNA 4 of Rice stripe virus isolate T, and comparison with another isolate and with Maize stripe virus. The Journal of General Virology, 1992,73(5):1309-1312.
[13]	WU G T, LU Y W, ZHENG H Y, LIN L, YAN F, CHEN J P . Transcription of ORFs on RNA2 and RNA4 of Rice stripe virus terminate at an AUCCGGAU sequence that is conserved in the genus Tenuivirus. Virus Research, 2013,175(1):71-77.
[14]	XIONG R Y, WU J X, ZHOU Y J, ZHOU X P . Characterization and subcellular localization of an RNA silencing suppressor encoded by Rice stripe tenuivirus. Virology, 2009,387(1):29-40.
[15]	XU Y, WU J X, FU S, LI C Y, ZHU Z R, ZHOU X P . Rice stripe tenuivirus nonstructural protein 3 hijacks the 26S proteasome of the small brown planthopper via direct interaction with regulatory particle non-ATPase subunit 3. Journal of Virology, 2015,89(8):4296-4310. doi: 10.1128/JVI.03055-14
[16]	ZHENG L J, ZHANG C, SHI C N, WANG Y, ZHOU T, SUN F, WANG H, ZHAO S S, QIN Q Q, QIAO R, DING Z M, WEI C H, XIE L H, WU J G, LI Y . Rice stripe virus NS3 protein regulates primary miRNA processing through association with the miRNA biogenesis factor OsDRB1 and facilitates virus infection in rice. PLoS Pathogens, 2017,13(10):e1006662.
[17]	姜良良 . 水稻条纹病毒p3蛋白与寄主核定位蛋白NbPip3的互作研究[D]. 南京: 南京农业大学, 2017.
	JIANG L L . Studies on interaction between RSV p3 protein and a host nucleus-localized protein Nbpip3[D]. Nanjing: Nanjing Agricultural University, 2017. (in Chinese)
[18]	DASGUPTA I, MALATHI V G, MUKHERJEE S K . Genetic engineering for virus resistance. Current Science, 2003,84(3):341-354.
[19]	RUBINO L, CAPRIOTTI G, LUPPO R, RUSSO M . Resistance to cymbidium ringspot tombusvirus infection in transgenic Nicotiana benthamiana plants exprssing the virus coat protein gene. Plant Molecular Biology, 1993,21(4):665-672.
[20]	余晓红, 朱玉贤, 应华澄, 陈章良 . 烟草花叶病毒运动蛋白基因的cDNA克隆、序列测定及植物转化. 生物工程学报, 1996,12(2):211-214.
	YU X H, ZHU Y X, YING H C, CHEN Z L . Molecular cloning, nucleotide sequencing and plant transformation of a cDNA coding for the Tobacco mosaic virus movement protein. Chinese Journal of Biotechnology, 1996,12(2):211-214. (in Chinese)
[21]	PRUSS G J, LAWRENCE C B, BASS T, LI Q Q, BOWMAN L H, VANCE V . The potyviral suppressor of RNA silencing confers enhanced resistance to multiple pathogens. Virology, 2004,320(1):107-120. doi: 10.1016/j.virol.2003.11.027
[22]	郑桂贤 . RSV四川分离物p3基因原核表达及其影响本氏烟内源基因表达研究[D]. 重庆: 西南大学, 2018.
	ZHENG G X . The study about prokaryotic expression of p3 from Sichuan RSV isolate and p3 affects on endogenous gene expression of Nicotiana benthamiana[D]. Chongqing: Southwest University, 2018. (in Chinese)
[23]	王羽, 肖崇刚 . 番茄青枯病病菌无致病力菌株的分离和控病研究. 西南农业大学学报(自然科学版), 2004,26(4):426-428.
	WANG Y, XIAO C G . Isolation of avirulent strains of bacterial wilt of tomato (Ralstonia solanacearum) and its application in biocontrol of the disease. Journal of Southwest Agricultural University (Natural Science), 2004,26(4):426-428. (in Chinese)
[24]	方中达 . 植病研究方法. 3版. 北京: 中国农业出版社, 2007.
	FANG Z D. Plant Disease Research Methods. 3rd ed. Beijing: China Agriculture Press, 2007. (in Chinese)
[25]	王海波 . 两株烟草黑胫病和青枯病拮抗菌的诱变选育[D]. 重庆: 西南大学, 2009.
	WANG H B . Study on mutation breeding of two antagonistic bacterium strains against Phytophthora parasitica and Ralstonia solanacearum[D]. Chongqing: Southwest University, 2009. (in Chinese)
[26]	马国胜, 高智谋 . 烟草黑胫病菌培养性状的研究. 中国农业科学, 2007,40(3):512-517.
	MA G S, GAO Z M . Cultural characteristics of Phytophthora nicotianae var. nicotianae pathogen of tobacco blank shank. Scientia Agricultura Sinica, 2007,40(3):512-517. (in Chinese)
[27]	王岩 . 烟草黑胫病菌、青枯病菌拮抗放线菌的筛选及控病研究[D]. 重庆: 西南大学, 2009.
	WANG Y . Screening and biocontrol effect of antagonistic actinomycetes against Phytophthora parasitica and Ralstonia solannacearum[D]. Chongqing: Southwest University, 2009. (in Chinese)
[28]	KASSCHAU K D, XIE Z, ALLEN E, LLAVE C, CHAPMAN E J, KRIZAN K A, CARRINGTON J C . P1/HC-Pro, a viral suppressor of RNA silencing, interferes with Arabidopsis development and miRNA function. Developmental Cell, 2003,4(2):205-217.
[29]	TAKAGI K, NISHIZAWA K, HIROSE A, KURAUCHI T, SENDA M, MASUTA C, ISHIMOTO M . Seed coat pigmentation in transgenic soybean expressing the silencing suppressor 2b gene of Cucumber mosaic virus. Plant Cell Reports, 2013,32(12):1903-1912.
[30]	刘玉乐, 王振东, 朱锋, 康良仪, 田波 . 表达黄瓜花叶病毒卫星RNA的转基因烟草耐烟草花叶病毒. 微生物学报, 1994,34(5):403-405.
	LIU Y L, WANG Z D, ZHU F, KANG L Y, TIAN B . Tolerance of the transgenic tobacco expressing satellite RNA of Cucumber mosaic virus to Tobacco mosaic virus. Acta Microbiologica Sinica, 1994,34(5):403-405. (in Chinese)
[31]	刘晓玲, 宋云枝, 刘红梅, 温孚江, 朱常香, 白庆荣 . 马铃薯X病毒25 kD运动蛋白基因和外壳蛋白基因介导的抗病性研究. 作物学报, 2005,31(7):827-832.
	LIU X L, SONG Y Z, LIU H M, WEN F J, ZHU C X, BAI Q R . Virus resistance mediated by the cDNAs encoding for the movement and coat proteins of Potato Virus X. Acta Agronomica Sinica, 2005,31(7):827-832. (in Chinese)
[32]	郭兴启, 韩宏岩, 张杰道, 王洪刚 . 转不可翻译PVY ^N CP基因烟草的抗病性分析 . 实验生物学报, 2003,36(3):176-184.
	GUO X Q, HAN H Y, ZHANG J D, WANG H G . The viral resistance of transgenic tobacco plants containing untranslatable PVY ^N coat protein gene . Acta Biologiae Experimentalis Sinica, 2003,36(3):176-184. (in Chinese)
[33]	ZHANG G, CHEN M, LI L, XU Z, CHEN X, GUO J, MA Y . Overexpression of the soybean GmERF3 gene, an AP2/ERF type transcription factor for increased tolerances to salt, drought, and diseases in transgenic tobacco. Journal of Experimental Botany, 2009,60(13):3781-3796. doi: 10.1093/jxb/erp214
[34]	ZHANG G, CHEN M, CHEN X, XU Z, LI L, GUO J, MA Y . Isolation and characterization of a novel EAR-motif-containing gene GmERF4 from soybean(Glycine max L.). Molecular Biology Reports, 2010,37(2):809-818.
[35]	JAYNES J M, NAGPALA P, DESTEFANO-BELTRAN L, HUANG J H, KIM J, DENNY T, CETINER S . Expression of a Cecropin B lytic peptide analog in transgenic tobacco confers enhanced resistance to bacterial wilt caused by Pseudomonas solanacearum. Plant Science, 1993,89(1):43-53.
[36]	徐莺, 白永延, 卫志明, 许智宏 . Shiva A基因在转基因烟草中的表达及其对烟草青枯病菌(Pseudomonas solanacearum pv. tabaci)的抗性. 实验生物学报, 1999,32(1):73-76.
	XU Y, BAI Y Y, WEI Z M, XU Z H . Enhanced resistance of transgenic tobacco expressing Shiva A gene against bacterial wilt disease (Pseudomonas solanacearum pv. tabaci). Acta Biologiae Experimentalis Sinica, 1999,32(1):73-76. (in Chinese)
[37]	HASSAN M, SINDEN S L, KOBAYASHI R S, NORDEEN R O, OWENS L D . Transformation of potato (Solanum tuberosum) with a gene for an anti-bacterial protein, ceropin. Acta Horticulture, 1993,336:127-132.
[38]	MUKHTAR M S, DESLANDES L, AURIAC M C, MARCO Y, SOMSSICH I E . The Arabidopsis transcription factor WRKY27 influences wilt disease symptom development caused by Ralstonia solanacearum. The Plant Journal, 2008,56(6):935-947.