Identification, Characterization, and vsiRNAs of Cymbidium Mosaic/Odontoglossum Ringspot Viruses Co-infection in Phalaenopsis equestris
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摘要:
目的 探明蝴蝶兰和病毒（建兰花叶病毒、齿兰环斑病毒）之间的相互作用，进而为制定蝴蝶兰病毒性病害的有效防控措施提供理论基础。 方法 首先通过RT-PCR特异扩增建兰花叶病毒（cymbidium mosaic virus, CymMV）和齿兰环斑病毒（odontoglossum ringspot virus, ORSV）外壳蛋白基因片段，通过电镜负染和切片技术明确CymMV和 ORSV在蝴蝶兰细胞中的存在；然后通过小RNA深度测序技术鉴定分析病毒来源的小干扰RNA（vsiRNAs）的丰度、长度、碱基偏好性和正负义来源等特征。 结果 RT-PCR能够特异地扩增到外壳蛋白基因片段，电镜负染和超薄切片结果中都能够观察到长约300 nm棒状的CymMV病毒粒子和长约500 nm线性的ORSV病毒粒子的存在；小RNA深度测序分别获得7 563 892和6 133 689个读数的CymMV和 ORSV来源的vsiRNAs，vsiRNAs在丰度、长度、碱基偏好性和正负义来源等方面具有一定的普遍性和特异性等特征。 结论 CymMV和ORSV两种病毒在蝴蝶兰植株中的存在复合侵染；CymMV和ORSV viRNAs的丰度、长度、悬挂、碱基偏好性、正负义链分布、Hotspot和Coldspot等特征具有普遍性和特异性，病毒复合侵染鉴定及其病毒来源vsiRNAs特征的分析加深了病毒与蝴蝶兰之间互作的理解，对于开发蝴蝶兰病毒性病害的防治措施具有重要的理论意义。 Abstract:Objective Interactions between Phalaenopsis equestris and cymbidium mosaic virus (CymMV) and odontoglossum ringspot virus (ORSV) were studied to aid the effort in developing effective preventive and control means against the diseases caused by the pathogens. Method The coat protein (CP) genes of CymMV and ORSV were amplified using RT-PCR. Under an electron microscope, viral morphology and size of CymMV and ORSV particles in P. equestris cells were examined. Abundance, length, base preference, and origin of virus-derived vsiRNAs were analyzed applying the small RNA deep sequencing technology. Result The amplifications of CP genes of CymMV and ORSV were specifically obtain by RT-PCR. The electron microscopy revealed the lengths of the rod-like CymMV to be approximate 300nm, while the linear ORSV, 500nm. The small RNA deep sequencing yielded 7,563,892 CymMV-derived and 6,133,689 ORSV-derived vsiRNAs exhibiting the universality and specificity in abundance, length, base preference, and sense strand distribution. Conclusion Co-infections of CymMV and ORSV on P. equestris were clearly demonstrated in this study. The vsiRNAs of CymMV and ORSV displayed characteristic patterns in abundance, length, base preferences, and sense strand distribution, which provided insights into the pathogen-plant interactions between the viruses and P. equestris as well as the information for the disease prevention and control. -
Key words:
- Phalaenopsis equestris /
- virus /
- vsiRNAs /
- interaction
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图 1 CymMV和ORSV在蝴蝶兰叶片的共侵染的鉴定
A：病毒侵染的蝴蝶兰样品；B：CymMV和ORSV外壳蛋白基因的RT-PCR扩增产物的琼脂糖凝胶电泳， M为DNA marker MD102; 1和2分别为 CymMV和ORSV外壳蛋白基因产物； C：CymMV和ORSV病毒电镜观察的负染图；D：CymMV和ORSV病毒电镜观察的超薄切片图。
Figure 1. Identification of CymMV/ORSV co-infections in P. equestris
A: P. equestris plant infected by viruses; B: agarose gel electrophoresis of RT-PCR products of CP genes of CymMV and ORSV; M: DNA marker MD102; 1 and 2: RT-PCR products of CP genes of CymMV and ORSV, respectively; C: electron microscopic negative staining images of CymMV and ORSV; D: electron microscopic images on thin sections of CymMV and ORSV.
图 2 蝴蝶兰细胞中CymMV和ORSV来源的vsiRNAs特征
A：健康蝴蝶兰和病毒侵染蝴蝶兰中siRNAs长度分布；B： 病毒侵染蝴蝶兰中vsiRNAs长度分布； C： 21 nt vsiRNAs 5'或3'端的碱基overhang；D： Total vsiRNAs和21 nt vsiRNAs 5'端第一个碱基偏好性。
Figure 2. Characterization of CymMV- and ORSV-derived vsiRNAs in P. equestris
A: Size distribution of total small RNAs in virus-infected (blue line) and viruses-free (red line) P. equestris plants; B: size distribution of vsiRNAs matching CymMV and ORSV genomes in virus-infected P. equestris plants; C: reads of 21-nt vsiRNAs with 1-21 nt distance between 5′ ends from CymMV (red line) and ORSV (black line) genome in P. equestris plants; D: relative frequency of 5′-terminal nucleotide of vsiRNAs from CymMV and ORSV genome in P. equestris plants.
图 3 CymMV和ORSV vsiRNAs的生成前体分析
A：CymMV和ORSV vsiRNAs的正负义链分布；B：vsiRNAs在CymMV基因组上高低频切割位点；C：vsiRNAs在ORSV基因组上高低频切割位点； D：CymMV正链基因组的二级结构预测；E：ORSV正链基因组的二级结构预测。方框I为图3B和图3D中高频切割位点和基因组茎环结构的对应关系。方框II为图3C和图3E中高频切割位点和基因组茎环结构的对应关系。
Figure 3. Biogenesis precursors of CymMV and ORSV vsiRNAs
A: Polarity distribution of vsiRNAs matching CymMV and ORSV genomes from co-infected P. equestris plants; B and C: vsiRNAs hotspots and cold spots distribution along CymMV and ORSV genomes, respectively; D and E: secondary structures of CymMV and ORSV RNAs predicted with RNAfold server.
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[1] 庄西卿. 中国国兰产业化发展的问题与对策 [J]. 福建热作科技, 2004, 29(1):23−25. doi: 10.3969/j.issn.1006-2327.2004.01.013ZHUANG X Q. The development for the production of Chinese orchids (Cymbidium) in large quantity [J]. Fujian Science & Technology of Tropical Crops, 2004, 29(1): 23−25. (in Chinese) doi: 10.3969/j.issn.1006-2327.2004.01.013 [2] KADO C I , ENSEN D D. Cymbidium mosaic virus in Phalaenopsis [J]. Phytopathology, 1964, 54: 4-947. [3] INOUYE N. Host range and properties of a strain of Odontoglossum ringspot virus in Japan [J]. Nogaku kenkyu, 1983, 60(2): 53−67. [4] ZETTLER F W. Viruses of orchids and their control [J]. Plant Disease, 1990, 74(9): 621−626. doi: 10.1094/PD-74-0621 [5] BAKER C A, DAVISON D, JONES L. Impatiens necrotic spot virus and Tomato spotted wilt virus Diagnosed in Phalaenopsis Orchids from Two Florida Nurseries [J]. Plant Disease, 2007, 91(11): 1515. [6] ZHENG Y X, CHEN C C, YANG C J, et al. Identification and characterization of a tospovirus causing chlorotic ringspots on Phalaenopsis orchids [J]. European Journal of Plant Pathology, 2008, 120(2): 199−209. doi: 10.1007/s10658-007-9208-7 [7] ZHANG Q, DING Y M, LI M. First Report of Impatiens necrotic spot virus Infecting Phalaenopsis and Dendrobium Orchids in Yunnan Province, China [J]. Plant Disease, 2010, 94(7): 915. [8] LESEMANN D E. Long, filamentous virus-like particles associated with vein necrosis of Dendrobium phalaenopsis [J]. Journal of Phytopathology, 1977, 89(4): 330−339. doi: 10.1111/j.1439-0434.1977.tb02873.x [9] ZHENG Y X, CHEN C C, CHEN Y K, et al. Identification and characterization of a potyvirus causing chlorotic spots on Phalaenopsis orchids [J]. European Journal of Plant Pathology, 2008, 121(1): 87−95. doi: 10.1007/s10658-008-9281-6 [10] ZHENG Y X, CHEN C C, JAN F J. First Report of Carnation mottle virus in Phalaenopsis Orchids [J]. Plant Disease, 2011, 95(3): 354. [11] LESEMANN D, BEGTRUP J. Elektronenmikroskopischer Nachweis eines bazilliformen Virus in Phalaenopsis [J]. Journal of Phytopathology, 1971, 71(3): 257−269. doi: 10.1111/j.1439-0434.1971.tb03162.x [12] 施农农, 徐莺, 王慧中, 等. 复合感染建兰花叶病毒和齿兰环斑病毒的兰花超微结构观察及病原物快速鉴定 [J]. 分子细胞生物学报, 2007, 40(2):153−163.SHI N N, XU Y, WANG H Z, et al. Molecular identification of Cymbidium mosaic potexvirus and Odontoglossum ringspot tobamovirus complex infected Phalaenopsis and its pathological ultrastructural alteration [J]. Journal of Molecular Cell Biology, 2007, 40(2): 153−163. (in Chinese) [13] 张建军, 谢为龙. 兰花病毒病研究进展 [J]. 植物检疫, 1999, 13(2):47−49.ZHANG J J, XIE W L. Research progress of orchid virus disease [J]. Plant Quarantine, 1999, 13(2): 47−49. (in Chinese) [14] 刘黎卿, 林志楷, 郭莺. 蝴蝶兰病毒病研究进展及防治对策综述 [J]. 安徽农学通报, 2010, 16(24):21−23,126. doi: 10.3969/j.issn.1007-7731.2010.24.012LIU L Q, LIN Z K, GUO Y. Progress on molecule biology of Phalaenopsis virus and the corresponding prevention measures [J]. Anhui Agricultural Science Bulletin, 2010, 16(24): 21−23,126. (in Chinese) doi: 10.3969/j.issn.1007-7731.2010.24.012 [15] DING S W. RNA-based antiviral immunity [J]. Nature Reviews Immunology, 2010, 10(9): 632−644. doi: 10.1038/nri2824 [16] BLEVINS T, RAJESWARAN R, SHIVAPRASAD P V, et al. Four plant Dicers mediate viral small RNA biogenesis and DNA virus induced silencing [J]. Nucleic Acids Research, 2006, 34(21): 6233−6246. doi: 10.1093/nar/gkl886 [17] GUO Z X, LI Y, DING S W. Small RNA-based antimicrobial immunity [J]. Nature Reviews Immunology, 2019, 19(1): 31−44. doi: 10.1038/s41577-018-0071-x [18] LAN H H, LU L M. Characterization of Hibiscus Latent Fort Pierce Virus-Derived siRNAs in Infected Hibiscus rosa-Sinensis in China [J]. The Plant Pathology Journal, 2020, 36(6): 618−627. doi: 10.5423/PPJ.OA.09.2020.0169 [19] PAI H, JEAN W H, LEE Y S, et al. Genome-wide analysis of small RNAs from Odontoglossum ringspot virus and Cymbidium mosaic virus synergistically infecting Phalaenopsis [J]. Molecular Plant Pathology, 2020, 21(2): 188−205. doi: 10.1111/mpp.12888 [20] LIU C, CHEN Z, HU Y, et al. Complemented palindromic small RNAs first discovered from SARS coronavirus [J]. Genes, 2018, 9(9): 442. doi: 10.3390/genes9090442 [21] NIU X R, SUN Y, CHEN Z, et al. Using small RNA-seq data to detect siRNA duplexes induced by plant viruses [J]. Genes, 2017, 8(6): 163. doi: 10.3390/genes8060163 [22] BAULCOMBE D. RNA silencing in plants [J]. Nature, 2006, 431(7006): 356−363. [23] MI S J, CAI T, HU Y G, et al. Sorting of small RNAs into Arabidopsis argonaute complexes is directed by the 5' terminal nucleotide [J]. Cell, 2008, 133(1): 116−127. doi: 10.1016/j.cell.2008.02.034 [24] DONAIRE L, BARAJAS D, MARTÍNEZ-GARCÍA B, et al. Structural and genetic requirements for the biogenesis of tobacco rattle virus-derived small interfering RNAs [J]. Journal of Virology, 2008, 82(11): 5167−5177. doi: 10.1128/JVI.00272-08 [25] XU D L, ZHOU G H. Characteristics of siRNAs derived from Southern rice black-streaked dwarf virus in infected rice and their potential role in host gene regulation [J]. Virology Journal, 2017, 14(1): 27. doi: 10.1186/s12985-017-0699-3 [26] HO T, WANG H, PALLETT D, et al. Evidence for targeting common siRNA hotspots and GC preference by plant Dicer-like proteins [J]. FEBS Letters, 2007, 581(17): 3267−3272. doi: 10.1016/j.febslet.2007.06.022 [27] LI Y Q, DENG C L, SHANG Q X, et al. Characterization of siRNAs derived from cucumber green mottle mosaic virus in infected cucumber plants [J]. Archives of Virology, 2016, 161(2): 455−458. doi: 10.1007/s00705-015-2687-5 [28] XIA Z H, PENG J, LI Y Q, et al. Characterization of small interfering RNAs derived from Sugarcane mosaic virus in infected maize plants by deep sequencing [J]. PLoS One, 2014, 9(5): e97013. doi: 10.1371/journal.pone.0097013 [29] YANG J, ZHENG S L, ZHANG H M, et al. Analysis of small RNAs derived from Chinese wheat mosaic virus [J]. Archives of Virology, 2014, 159(11): 3077−3082. doi: 10.1007/s00705-014-2155-7 [30] FLYNT A, LIU N, MARTIN R, et al. Dicing of viral replication intermediates during silencing of latent Drosophila viruses [J]. Proceedings of the National Academy of Sciences of the United States of America, 2009, 106(13): 5270−5275. [31] MOLNÁR A, CSORBA T, LAKATOS L, et al. Plant virus-derived small interfering RNAs originate predominantly from highly structured single-stranded viral RNAs [J]. Journal of Virology, 2005, 79(12): 7812−7818. doi: 10.1128/JVI.79.12.7812-7818.2005 -
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