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| Expressing p20 hairpin RNA of Citrus tristeza virus confers Citrus aurantium with tolerance/resistance against stem pitting and seedling yellow CTV strains |
| CHENG Chun-zhen, YANG Jia-wei, YAN Hu-bin, BEI Xue-jun, ZHANG Yong-yan, LU Zhi-ming, ZHONG Guang-yan |
1、College of Horticulture and landscape architecture, Southwest University, Chongqing 400715, P.R.China
2、Institute of Fruit Tree Research, Guangdong academy of agricultural Sciences, Guangzhou 510640, P.R.China
3、Economic Crops Station, agricultural Bureau of Rongxian, Zigong 643100, P.R.China
4、Institute of Crop Science, Shanxi academy of agricultural Sciences, Taiyuan 030031, P.R.China
5、Key laboratory of South Subtropical Fruit Biology and Genetic Resource Utilization, Ministry of agriculture, Guangzhou 510640,P.R.China |
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摘要 The Citrus tristeza virus (CTV) uses 3 silencing suppressor genes, p20, p23 and p25, to resist the attacks from its Citrus hosts. Inactivating these genes is therefore obviously a potential defensive option in addition to the current control strategies including aphid management and the use of mild strain cross protection. In this study, we cloned partial DNA fragments from the three genes, and used them to construct vectors for expressing hairpin RNAs (hpRNAs). To facilitate the formation of hpRNAs, the constructs were introduced in a loop structure. Following transformation of sour orange (Citrus aurantium) with these constructs, 8 p20 hpRNA (hp20) and 1 p25 hpRNA (hp25) expressing lines were obtained. The 7 hp20 transgenic lines were further characterized. Their reactions to CTV were tested following inoculation with CT14A and/ or TR-L514, both of which are severe strains. Results showed that 3 lines (hp20-5, hp20-6 and hp20-8) were completely resistant to TR-L514 under greenhouse conditions for no detectable viral load was found in their leaves by PCR. However, they exhibited only partial suppression of TR-L514 under screen house conditions since the virus was detected in their leaves, though 2 months later compared to non-transgenic controls. Further tests showed that hp20-5 was tolerant also to CT14A under screen house conditions. The growth of hp20-5 was much better than others including the controls that were concurrently challenged with CT14A. These results showed that expressing p20 hpRNA was sufficient to confer sour orange with CTV resistance/tolerance.
Abstract The Citrus tristeza virus (CTV) uses 3 silencing suppressor genes, p20, p23 and p25, to resist the attacks from its Citrus hosts. Inactivating these genes is therefore obviously a potential defensive option in addition to the current control strategies including aphid management and the use of mild strain cross protection. In this study, we cloned partial DNA fragments from the three genes, and used them to construct vectors for expressing hairpin RNAs (hpRNAs). To facilitate the formation of hpRNAs, the constructs were introduced in a loop structure. Following transformation of sour orange (Citrus aurantium) with these constructs, 8 p20 hpRNA (hp20) and 1 p25 hpRNA (hp25) expressing lines were obtained. The 7 hp20 transgenic lines were further characterized. Their reactions to CTV were tested following inoculation with CT14A and/ or TR-L514, both of which are severe strains. Results showed that 3 lines (hp20-5, hp20-6 and hp20-8) were completely resistant to TR-L514 under greenhouse conditions for no detectable viral load was found in their leaves by PCR. However, they exhibited only partial suppression of TR-L514 under screen house conditions since the virus was detected in their leaves, though 2 months later compared to non-transgenic controls. Further tests showed that hp20-5 was tolerant also to CT14A under screen house conditions. The growth of hp20-5 was much better than others including the controls that were concurrently challenged with CT14A. These results showed that expressing p20 hpRNA was sufficient to confer sour orange with CTV resistance/tolerance.
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Received: 25 September 2014
Accepted:
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| Fund: This work was supported by the International Science & Technology Cooperation Program of China (2012DFA30610), the National Natural Science Foundation of China (30571291) and the Special Fund for Agro-Scientific Research in the Public Interest, China (201203075-07). |
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Corresponding Authors:
ZHONG Guang-yan, E-mail: gy_zhong@163.com
E-mail: gy_zhong@163.com
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| About author: CHENG Chun-zhen, E-mail: ld0532cheng@126.com; YANG Jia-wei,E-mail: yangjiawei1010@163.com; YAN Hu-bin, E-mail: maikyhb@ 126.com; * These authors contributed equally to this study. |
Cite this article:
CHENG Chun-zhen, YANG Jia-wei, YAN Hu-bin, BEI Xue-jun, ZHANG Yong-yan, LU Zhi-ming, ZHONG Guang-yan.
2015.
Expressing p20 hairpin RNA of Citrus tristeza virus confers Citrus aurantium with tolerance/resistance against stem pitting and seedling yellow CTV strains. Journal of Integrative Agriculture, 14(9): 1767-1777.
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| Abel P P, Nelson R S, De B, Hoffmann N, Rogers S G, FraleyR T, Beachy R N. 1986. Delay of disease development intransgenic plants that express the Tobacco mosaic viruscoat protein gene. Science, 232, 738-743Ananthakrishnan G, Orbovi? V, Pasquali G, ?alovi? M, GrosserJ W. 2007. Transfer of Citrus tristeza virus (CTV)-derivedresistance candidate sequences to four grapefruit cultivarsthrough agrobacterium-mediated genetic transformation. InVitro Cellular & Developmental Biology (Plant), 43, 593-601Atta S, Zhou C Y, Zhou Y, Cao M J, Wang X F. 2012. Distributionand research advances of Citrus tristeza virus. Journal ofIntegrative agriculture, 11, 346-358Bar-Joseph M, Marcus R, Lee R F. 1989. The continuouschallenge of Citrus tristeza virus control. annual Review ofPhysiology, 27, 291-316Bar-Joseph M, Mawassi M. 2013. The defective RNAs ofClosteroviridae. Frontiers in Microbiology, 4, 132.Batuman O, Mawassi M, Bar-Joseph M. 2006. Transgenesconsisting of a dsRNA of an RNAi suppressor plus the 3´UTR provide resistance to Citrus tristeza virus sequencesin Nicotiana benthamiana but not in citrus. Virus Genes,33, 319-327Campos L, Granell P, Tárraga S, López-Gresa P, ConejeroV, Bellés J M, Rodrigo I, Lisón P. 2014. Salicylic acid andgentisic acid induce RNA silencing-related genes and plant resistance to RNA pathogens. Plant Physiology andBiochemistry, 77, 35-43Carrington J C, Kasschau K D, Johansen L K. 2001. Activationand suppression of RNA silencing by plant viruses. Virology,281, 1-5Costa M, Otoni W, Moore G. 2002. An evaluation of factorsaffecting the efficiency of agrobacterium-mediatedtransformation of Citrus paradisi (Macf.) and production oftransgenic plants containing carotenoid biosynthetic genes.Plant Cell Reports, 21, 365-373Csorba T, Bovi A, Dalmay T, Burgyán J. 2007. The p122 subunitof Tobacco mosaic virus replicase is a potent silencingsuppressor and compromises both small interfering RNAandmicroRNA-mediated pathways. Journal of Virology,81, 11768-11780Csorba T, Pantaleo V, Burgyán J. 2009. RNA silencing: Anantiviral mechanism. advances in Virus Research, 75, 35-71Dawson W O, Garnsey S M, Tatineni S, Folimonova S Y, HarperS J, Gowda S. 2013. Citrus tristeza virus-host interactions.Frontiers in Microbiology, 4, 88.Day A G, Bejarano E R, Buck K W, Burrell M, Lichtenstein C P.1991. Expression of an antisense viral gene in transgenictobacco confers resistance to the DNA virus Tomato goldenmosaic virus. Proceedings of the National academy ofSciences of the United States of america, 88, 6721-6725Domínguez A, Cervera M, Pérez R M, Romero J, Fagoaga C,Cubero J, López M M, Juárez J A, Navarro L, Peña L. 2004.Characterisation of regenerants obtained under selectiveconditions after agrobacterium-mediated transformation ofcitrus explants reveals production of silenced and chimericplants at unexpected high frequencies. Molecular Breeding,14, 171-183Domínguez A, Fagoaga C, Navarro L, Moreno P, Peña L.2002a. Regeneration of transgenic citrus plants under nonselective conditions results in high-frequency recovery ofplants with silenced transgenes. Molecular Genetics andGenomics, 267, 544-556Domínguez A, Guerri J, Cambra M, Navarro L, Moreno P, PeñaL. 2000. Efficient production of transgenic citrus plantsexpressing the coat protein gene of citrus tristeza virus.Plant Cell Reporter, 19, 427-433Domínguez A, de Mendoza A H, Guerri J, Cambra M, MorenoP, Peña L. 2002b. Pathogen-derived resistance to Citrustristeza virus (CTV) in transgenic mexican lime (Citrusaurantifolia (Christ.) Swing.) plants expressing its p25 coatprotein gene. Molecular Breeding, 10, 1-10Fagoaga C, López C, de Mendoza A H, Moreno P, Navarro L,Flores R, Peña L. 2006. Post-transcriptional gene silencingof the p23 silencing suppressor of Citrus tristeza virusconfers resistance to the virus in transgenic Mexican lime.Plant Molecular Biology, 60, 153-165Fagoaga C, López C, Moreno P, Navarro L, Flores R, PeñaL. 2005. Viral-like symptoms induced by the ectopicexpression of the p23 gene of Citrus tristeza virus arecitrus specific and do not correlate with the pathogenicityof the virus strain. Molecular Plant-Microbe Interactions,18, 435-445Fagoaga C, Pensabene-Bellavia G, Moreno P, Navarro L,Flores R, Peña L. 2011. Ectopic expression of the p23silencing suppressor of Citrus tristeza virus differentiallymodifies viral accumulation and tropism in two transgenicwoody hosts. Molecular Plant Pathology, 12, 898-910Febres V J, Lee R F, Moore G A. 2008. Transgenic resistanceto Citrus tristeza virus in grapefruit. Plant Cell Reports, 27,93-104Febres V J, Niblett C L, Lee R F, Moore G A. 2003.Characterization of grapefruit plants (Citrus paradisi Macf.)transformed with Citrus tristeza closterovirus genes. PlantCell Reports, 21, 421-428Flores R, Ruiz-Ruiz S, Soler N, Sánchez-Navarro J, FagoagaC, López C, Navarro L, Moreno P, Peña L. 2013. Citrustristeza virus p23: A unique protein mediating key virus-hostinteractions. Frontiers in Microbiology, 4, 98.Ghorbel R, Domínguez A, Navarro L, Penña L. 2000. Highefficiency genetic transformation of sour orange (Citrusaurantium) and production of transgenic trees containing thecoat protein gene of Citrus tristeza virus. Tree Physiology,20, 1183-1189Ghorbel R, López C, Fagoaga C, Moreno P, Navarro L, FloresR, Peña L. 2001. Transgenic citrus plants expressing theCitrus tristeza virus p23 protein exhibit viral-like symptoms.Molecular Plant Pathology, 2, 27-36Gillet F X, Cattoni D I, Petiot-Bécard S, Delalande F, PoignaventV, Brizard J P, Bessin Y, Van Dorsselaer A, Declerck N,Sanglier-Cianférani S, Brugidou C, Vignols F. 2013. TheRYMV-encoded viral suppressor of RNA silencing P1 is azinc-binding protein with redox-dependent flexibility. Journalof Molecular Biology, 425, 2423-2435Gillings M, Broadbent P, Indsto J, Lee R. 1993. Characterisationof isolates and strains of Citrus tristeza closterovirus usingrestriction analysis of the coat protein gene amplified by thepolymerase chain reaction. Journal of Virological Methods,44, 305-317Gmitter F G, Xiao S Y, Huang S, Hu X L, Garnsey S M, DengZ. 1996. A localized linkage map of the Citrus tristeza virusresistance gene region. Theoretical and applied Genetics,92, 688-695Gowda S, Satyanarayana T, Davis C L, Navas-Castillo J,Albiach-Martí M R, Mawassi M, Valkov N, Bar-Joseph M,Moreno P, Dawson W O. 2000. The p20 gene product ofCitrus tristeza virus accumulates in the amorphous inclusionbodies. Virology, 274, 246-254Gowda S, Tatineni S, Folimonova S Y, Hilf M E, Dawson W O.2009. Accumulation of a 5´ proximal subgenomic RNA ofCitrus tristeza virus is correlated with encapsidation by theminor coat protein. Virology, 389, 122-131Groenenboom M A C, Hogeweg P. 2012. Modelling thedynamics of viral suppressors of RNA silencing. Journal ofthe Royal Society, 9, 436-447Gutiérrez E M A, Luth D, Moore G A. 1997. Factors affectingagrobacterium-mediated transformation in citrus andproduction of sour orange (Citrus aurantium L.) plantsexpressing the coat protein gene of Citrus tristeza virus.Plant Cell Reports, 16, 745-753 Helliwell C, Waterhouse P. 2003. Constructs and methodsfor high-throughput gene silencing in plants. Methods, 30,289-295Helliwell C A, Waterhouse P M. 2005. Constructs and methodsfor hairpin RNA-mediated gene silencing in plants. Methodsin Enzymology, 392, 24-35Holzberg S, Brosio P, Gross C, Pogue G P. 2002. Barley stripemosaic virus-induced gene silencing in a monocot plant.The Plant Journal, 30, 315-327Karasev A V, Boyko V P, Gowda S, Nikolaeva O V, Hilf ME, Koonin E V, Niblett C L, Cline K, Gumpf D J, Lee RF, Garnsey S M, Lewandowski D J, Dawson W O. 1995.Complete sequence of the Citrus tristeza virus RNAgenome. Virology, 208, 511-520Kasschau K D, Xie Z, Allen E, Llave C, Chapman E J, KrizanK A, Carrington J C. 2003. P1/HC-Pro, a viral suppressorof RNA silencing, interferes with arabidopsis developmentand miRNA function. Developmental Cell, 4, 205-217Lee R F, Keremane M L. 2013. Mild strain cross protection oftristeza: A review of research to protect against decline onsour orange in Florida. Frontiers in Microbiology, 4, 259.López C, Cervera M, Fagoaga C, Moreno P, Navarro L, FloresR, Peña L. 2010. Accumulation of transgene-derivedsiRNAs is not sufficient for RNAi-mediated protectionagainst Citrus tristeza virus in transgenic Mexican lime.Molecular Plant Pathology, 11, 33-41Mette M F, Matzke A J, Matzke M A. 2001. Resistance of RNAmediatedTGS to HC-Pro, a viral suppressor of PTGS,suggests alternative pathways for dsRNA processing.Current Biology, 11, 1119-1123Moreno P, Ambrós S, Albiach-Martí M R, Guerri J, Peña L. 2008.Citrus tristeza virus: A pathogen that changed the course ofthe citrus industry. Molecular Plant Pathology, 9, 251-268Muniz F R, Souza A J, Stipp L C L, Schinor E, Freitas W,Harakava R, Stach-Machado D R, Rezende J A M, MourãoFilho F A A, Mendes B M J. 2012. Genetic transformationof Citrus sinensis with Citrus tristeza virus (CTV) derivedsequences and reaction of transgenic lines to CTV infection.Biologia Plantarum, 56, 162-166Peña L, Cervera M, Juárez J, Navarro A, Pina J A, Durán-VilaN, Navarro L. 1995. Agrobacterium-mediated transformationof sweet orange and regeneration of transgenic plants. PlantCell Reports, 14, 616-619Piestun D, Batuman O, Che X, Gofman R, Filatov V, Zypman S,Gafny R, Bar Joseph M, Goren R, Goldschmidt E E. 1998.Truncated verions of the Citrus tristeza virus (CTV) replicaseand basta resistance genes incorporated in transgenicTroyer citrange. First International Citrus BiotechnologySymposium, 1998, 223-230Rai M. 2006. Refinement of the Citrus tristeza virus resistancegene (Ctv) positional map in Poncirus trifoliata andgeneration of transgenic grapefruit (Citrus paradisi) plantlines with candidate resistance genes in this region. PlantMolecular Biology, 61, 399-414Sanford J C, Johnston S A. 1985. The concept of parasitederivedresistance-deriving resistance genes from theparasite’s own genome. Journal of Theoretical Biology,113, 395-405Satyanarayana T, Gowda S, Ayllón M A, Albiach-Martí M R,Rabindran S, Dawson W O. 2002. The p23 protein of Citrustristeza virus controls asymmetrical RNA accumulation.Journal of Virology, 76, 473-483Schmittgen T D, Livak K J. 2008. Analyzing real-time PCRdata by the comparative C(T) method. Nature Protocols,3, 1101-1108Smith N A, Singh S P, Wang M B, Stoutjesdijk P A, Green A G,Waterhouse P M. 2000. Gene expression: Total silencing byintron-spliced hairpin RNAs. Nature, 407, 319-320Soler N, Plomer M, Fagoaga C, Moreno P, Navarro L, FloresR, Peña L. 2012. Transformation of Mexican lime with anintron-hairpin construct expressing untranslatable versionsof the genes coding for the three silencing suppressors ofCitrus tristeza virus confers complete resistance to the virus.Plant Biotechnology Journal, 10, 597-608Tomari Y, Zamore P D. 2005. Perspective: Machines for RNAi.Genes & Development, 19, 517-529Tzfira T, Citovsky V. 2006. agrobacterium-mediated genetictransformation of plants: Biology and biotechnology. CurrentOpinion in Biotechnology, 17, 147-154Voinnet O, Lederer C, Baulcombe D C. 2000. A viral movementprotein prevents spread of the gene silencing signal inNicotiana benthamiana. Cell, 103, 157-167Yan H B. 2008. Construction of RNAi/PTGS vectors of CTVand transformation in sour orange. MSc thesis, SouthwestUniversity, Chongqing. (in Chinese)Yang J W. 2010. Construction of RNAi vectors of Citrus tristezavirus, genetic transformation and evaluation of transgenicplant. MSc thesis, Southwest University, Chongqing. (inChinese)Ye K, Malinina L, Patel D J. 2003. Recognition of smallinterfering RNA by a viral suppressor of RNA silencing.Nature, 426, 874-878Zhang J Z, Li Z M, Liu L, Mei L, Yao J L, Hu C G. 2008.Identification of early-flower-related ESTs in an earlyfloweringmutant of trifoliate orange (Poncirus trifoliata)by suppression subtractive hybridization and macroarrayanalysis. Tree Physiology, 28, 1449-1457Zhou Y, Zhou C Y, Song Z, Liu K H, Yang F Y. 2007a.Characterization of Citrus tristeza virus isolates by indicatorsand molecular biology methods. agricultural Sciences inChina, 6, 573-579Zhou Y, Zhou C Y, Zhao X Y, Jiang Y H, Wang X F. 2007b.Influence of six citrus cultivars on the composition of citrustristeza virus isolste TR-L514. acta Phytopathologica Sinica,37, 131-137 (in Chinese)Zhou Y, Zhou C Y, Li Z A, Wang X F, Liu K H. 2008. Mildstrains cross protection against stem-pitting tristeza ofsweet orange. Scientia agricultura Sinica, 41, 4085-4091(in Chinese)Ziebell H, Carr J P. 2010. Cross-protection: a century of mystery.advances in Virus Research, 76, 211–264. |
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