转水稻条纹病毒NS3本氏烟的抗病性
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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。
【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.
【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.
引文
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[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 GWrepeat protein.Genes and Development,2010,24(9):904-915.
[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.
[4]CHIU M H,CHEN I H,BAULCOMBE D C,TSAI C H.The silencing suppressor P25 of Potato virus X interacts with Argonaute1and 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.
[6]DUNOYER P,LECELLIER C H,PARIZOTTO E A,HIMBER C,VOINNET O.Probing the microRNA and small interfering RNApathways with virus-encoded suppressors of RNA silencing.The Plant Cell,2004,16(5):1235-1250.
[7]JAY F,WANG Y,YU A,TACONNAT L,PELLETIER S,COLOT V,RENOU J P,VOINNET O.Misregulation of AUXIN RESPONSEFACTOR 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,ANTONIWJ 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.
[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 miRNAbiogenesis factor OsDRB1 and facilitates virus infection in rice.PLoSPathogens,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.
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