Rapid Construction of Infectious Clone of Pepper mottle virus Hangzhou Isolate Using an Escherichia coli-Agrobacterium tumefaciens-Saccharomyces cerevisiae Shuttle Vector
ZHAO Wei1,2, ZHANG Can2, FU Kang2, MA Ning2, GE Jia-Cheng3, SUN Kai2,*, YU Xiao-Ping2,*
1 College of Metrology Measurement and Instrument, China Jiliang University, Hangzhou 310018, China; 2 Key Laboratory State Administration for Market Regulation (Microbiological Metrology, Measurement & Bio-product Quality Security), College of Life Sciences, China Jiliang University, Hangzhou 310018, China; 3 Hailir Pesticides and Chemicals Group Co., Ltd., Qingdao 266109, China
Abstract：Pepper mottle virus (PepMoV) is a member of the genus Potyvirus, capable of infecting important Solanaceous crops such as pepper (Capsicum annuum) and tomato (Solanum lycopersicum). In this study, PepMoV was successfully detected in pepper samples from Hangzhou, Zhejiang province, using small RNA sequencing technology. The complete genome, 9 640 nucleotides in length, was amplified using RT-PCR. To overcome difficulties in constructing infectious clones of PepMoV, such as large fragment assembly challenges and lethal genes for Escherichia coli, yeast (Saccharomyces cerevisiae) homologous recombination technology was employed, which enabled the assembly 5 DNA fragments into the full-length infectious clone of PepMoV in a single step in yeast. Importantly, the yeast plasmid can be directly transformed into (Agrobacterium tumefaciens) for infiltration, thereby avoiding the E. coli cloning step and its associated toxicity issues. Results indicated that, compared with traditional enzyme ligation methods, this construction strategy reduced the required time from several months to just one week. The obtained infectious clone of PepMoV-HZ effectively infected Nicotiana benthamiana, exhibiting clear symptoms. The successful construction of the PepMoV infectious clone not only provides a positive sample control for the rapid detection method of the virus but also offers crucial experimental tools for in-depth studies of the virus's gene functions and its interaction mechanisms with the host.
[1] 冯兰香, 张宝玺, 谢丙炎, 等. 2005. 3种辣椒新病毒的发生与血清学鉴定[J]. 中国蔬菜, (12): 33-34. (Feng L X, Zhang B X, Xie B Y, et al., 2015. Occurrence and serological identification of three new pepper viruses[J]. Chinese Vegetables, (12): 33-34.) [2] 高苇, 王勇, 张春祥, 等. 2016. 天津地区辣椒病毒病调查及毒源种类初步鉴定[J]. 山东农业科学, 48(03): 91-94. (Gao W, Wang Y, Zhang C X, et al.2016. Investigation of pepper virus disease in Tianjin and preliminary identification of virus source species[J]. Shandong Agricultural Sciences, 48(03): 91-94.) [3] 郭思瑶, 童艳, 黄娅, 等. 2015. 重庆辣椒病毒病病原初步鉴定和分析[J]. 园艺学报, 42(02): 263-270. (Guo S Y, Tong Y, Huang Yet al., 2015. Preliminary identification and analysis of the pathogen of pepper virus disease in Chongqing[J]. Horticultural Journal, 42(02): 263-270. [4] 刘勇, 李凡, 李月月, 等. 2019. 侵染我国主要蔬菜作物的病毒种类、分布与发生趋势[J]. 中国农业科学, 52(02): 239-261. (Liu Y, Li F, Li Y Y, et al.2019. Types, distribution and occurrence trends of viruses infecting major vegetable crops in China[J]. Chinese Agricultural Sciences, 52(02): 239-261.) [5] 张艳超, 侯明生, 蔡丽. 2017. 湖北省蔬菜病毒病主要毒原种类检测. 华中农业大学学报, 36(6): 31-38. (Zhang Y C, Hou M S, Cai L.2017. Detection of main virus species of vegetable virus diseases in Hubei province[J]. Journal of Huazhong Agricultural University, 36(6): 31-38.) [6] Ali M C, Omar A S, Natsuaki T.2011. An infectious full-length cDNA clone of Potato virus Y (NTN-NW), a recently reported strain of PVY that causes potato tuber necrotic ringspot disease[J]. Archives of Virology, 156(11): 2039-2043. [7] Blawid R, Nagata T.2015. Construction of an infectious clone of a plant RNA virus in a binary vector using one-step Gibson Assembly[J]. Journal of Virological Methods, 222: 11-15. [8] Bordat A, Houvenaghel M C, German-Retana S.2015. Gibson assembly: an easy way to clone potyviral full-length infectious cDNA clones expressing an ectopic VPg[J]. Virology Journal, 12(1): 89. [9] Cui T, Bin Y, Yan Jet al.2018. Development of infectious cDNA clones of citrus Yellow vein clearing virus using a novel and rapid strategy[J]. Phytopathology, 108(10): 1212-1218. [10] Gao R, Tian Y P, Wang Jet al.2012. Construction of an infectious cDNA clone and gene expression vector of Tobacco vein banding mosaic virus (genus Potyvirus)[J]. Virus Research, 169(1): 276-281. [11] Gibson D G, Glass J I, Lartigue Cet al.2010. Creation of a bacterial cell controlled by a chemically synthesized genome[J]. Science, 329(5987): 52-56. [12] Johansen I E.1996. Intron insertion facilitates amplification of cloned virus cDNA in Escherichia coli while biological activity is reestablished after transcription in vivo[J]. Proceedings of the National Academy of Sciences of the USA, 93(22): 12400-12405. [13] Kim Y J, Jonson M G, Choi H Set al.2009. Molecular characterization of Korean Pepper mottle virus isolates and its relationship to symptom variations[J]. Virus Research, 144(1-2): 83-88. [14] Kumar S, Stecher G, Tamura K.2016. MEGA7: Molecular evolutionary genetics analysis version 7. 0 for bigger datasets[J]. Molecular Biology and Evolution, 2016(7): 1870. [15] Larionov V, Kouprina N, Graves Jet al.1996. Specific cloning of human DNA as yeast artificial chromosomes by transformation-associated recombination[J]. Proceedings of the National Academy of Sciences of the USA, 93(1): 491-496. [16] López-Moya JJ, García JA.2000. Construction of a stable and highly infectious intron-containing cDNA clone of Plum pox potyvirus and its use to infect plants by particle bombardment[J]. Virus Research, 68(2): 99-107. [17] Melzer M J, Sugano J S, Cabanas Det al.2012. First report of Pepper mottle virus infecting tomato in Hawaii[J]. Plant Disease, 96(6): 917-917. [18] Mizutani K.2015. High-throughput plasmid construction using homologous recombination in yeast: Its mechanisms and application to protein production for X-ray crystallography[J]. Bioscience, Biotechnology, and Biochemistry, 79(1): 1-10. [19] Moury B, Morel C, Johansen Eet al.2004. Mutations in Potato virus Y genome-linked protein determine virulence toward recessive resistances in Capsicum annuum and Lycopersicon hirsutum.[J]. Molecular Plant-Microbe Interactions, 17(3): 322. [20] Natalay K, Vladimir L.2016. Transformation-associated recombination (TAR) cloning for genomics studies and synthetic biology[J]. Chromosoma, 125(4): 1-12. [21] Oldenburg K R, Vo K T, Susan Met al.1997. Recombination-mediated PCR-directed plasmid construction in vivo in yeast[J]. Nucleic Acids Research, 25(2): 451-452. [22] Singh M V, Weil P A.2002. A method for plasmid purification directly from yeast[J]. Analytical Biochemistry, 307(1): 13-17. [23] Sun K, Zhao D, Liu Yet al.2017. Rapid construction of complex plant RNA virus infectious cDNA clones for agroinfection using a yeast-E. coli-Agrobacterium shuttle vector[J]. Viruses, 9(11): 332. [24] Thao T, Labroussaa F, Ebert Net al.2020. Rapid reconstruction of SARS-CoV-2 using a synthetic genomics platform[J]. Nature, 582: 561-565. [25] Tuo D, Fu L, Shen Wet al.2017. Generation of stable infectious clones of plant viruses by using Rhizobium radiobacter for both cloning and inoculation[J]. Virology, 510: 99-103. [26] Yang S, Revers F, Souche Set al.1998. Construction of full-length cDNA clones of Lettuce mosaic virus (LMV) and the effects of intron-insertion on their viability in Escherichia coli and on their infectivity to plants[J]. Archives of Virology, 143(12): 2443.
