臺灣博碩士論文加值系統

竹嵌紋病毒(Bamboo mosaic virus, BaMV)，分類上屬於Alphaflexiviridae科中的Potexvirus屬。是一個單一核酸的正股RNA病毒，全長約有6400個核苷酸。本研究利用本氏菸草(Nicotiana benthamiana)做為病毒寄主，探討植物Argonaute基因與竹嵌紋病毒之間的關係。真核生物具有RNAi(RNA interference)機制對抗入侵的病原體包括病毒，RNAi意指小片段核酸可以影響細胞的基因表現，最典型的方式為加速mRNA的分解或抑制其轉譯，因此植物在病毒入侵細胞後便利用RNAi系統抑制及消除病毒。通常寄主利用RNAi的系統來對抗病毒基因體的機制發生在RNA病毒複製的過程產生了雙股RNA，導致ribonuclease III-like protein (植物體或稱 Dicer-like (DCL))將其切割成21~26核苷酸長的小片段RNA，稱作siRNA(small interfering RNA)，這些siRNA再與Argonaute (AGO)及一系列相關蛋白形成RNA-induced silencing complexes (RISCs)去專一性的切割病毒的核酸基因體。在阿拉伯芥(Arabidopsis thaliana)中，已有10個AGO基因被報導，分別為Ago1~Ago10，而在本氏菸草中，目前序列已發表有AGO1, AGO2, AGO4, AGO5等。本研究將利用已知的資訊，設計引子來合成不同的AGO基因片段，並利用病毒引發之基因靜默技術(virus-induced gene silencing, VIGS)，使其基因表現量下降，然後觀察這些基因的表現量下降，是否會對竹嵌紋病毒的感染造成影響。實驗結果發現，當AGO4的表現量下降後，竹嵌紋病毒在接種葉及系統葉上的外鞘蛋白的累積皆無明顯的影響，推測AGO4可能沒有參與到植物對抗竹嵌紋病毒的機制中。而在AGO1表現量下降後，竹嵌紋病毒在接種葉及接種原生質體上的外鞘蛋白累積有增加，AGO2表現量下降後則對病毒累積量沒有明顯影響。另外我們也發現在AGO1表現量下降後，AGO2的表現量有上升，因此未來的研究方向將針對AGO1以及AGO2在抵抗竹嵌紋病毒的方式上以及AGO1及AGO2之間的關係進行更進一步的研究。

Bamboo mosaic virus (BaMV) is a single-stranded positive-sense RNA virus belonging to the genus Potexvirus of the family Alphaflexiviridae. The genome of BaMV is about 6.4 kb with a 5?cap and a 3?poly (A) tail. In this study we use Nicotiana benthamiana as a model plant to inspect the relationship between Argonautes (AGOs) and BaMV. RNA interference (RNAi) in Eukaryotes is known to play an antiviral defense role especially the RNA viruses. During the replication of viral RNAs, the double-stranded (ds) RNA accumulates and triggers the host silencing process. DICER-LIKE (DCL) proteins cleave the viral dsRNA into short interfering RNAs (siRNAs) of 21~24 nucleotides in length. These siRNAs are loaded into AGOs to form the RNA induced silencing complex (RISC). The RISC contains the specific siRNAs and targets to the viral RNAs. There are 10 AGOs identified in Arabidopsis thaliana. By contrast, only four AGOs (AGO1, AGO2, AGO4, and AGO5) were sequenced in N. benthamiana. In this thesis, I used Tobacco rattle virus (TRV)-based gene silencing technique to knock down the expression of different AGOs in N. benthamiana to reveal the relationship between AGOs and BaMV. The results indicated that reducing the expression of AGO4 did not have any effect on the accumulation of BaMV. The results also imply that AGO4 may not play a key role against BaMV in N. benthamiana. By contrast, the reduction of the expression of AGO1 could result in an increase of the accumulation of BaMV in plants and protoplasts. Furthermore, but the accumulation of BaMV has no significant change in AGO2-silencing plants. Moreover, the expression of AGO2 is increased in AGO1-knockdown plants. The results suggest that AGO2 might be controlled by AGO1 as that revealed in Arabidopsis. The relationship between AGO1 and AGO2 in N. benthamiana needs to be further explored.

中文摘要 i
Abstract ii
Introduction 1
Bamboo mosaic virus 1
RNA interference 2
Argonautes 3
Aim 4
Materials and Methods 5
Argonautes knockdown and virus infection. 5
Viral particle purification 6
Viral RNA extraction 6
Protoplast isolation and viral RNA inoculation 7
Western blotting assay 8
Total RNA extraction 8
Real-time RT-PCR 9
Results 11
AGO4 of N. benthamiana do not play any role in BaMV accumulation 11
AGO1 of N. benthamiana has a defense role on BaMV infection 11
AGO2 of N. benthamiana has no significant role in regulating the infection of BaMV 12
The expression of AGO2 in N. benthamiana is regulated by AGO1 13
Discussion 14
Figures 16
References 26

Aliyari, R., Ding, S.W., 2009. RNA-based viral immunity initiated by the Dicer family of host immune receptors. Immunol Rev 227, 176-188.

Andika, I.B., Maruyama, K., Sun, L., Kondo, H., Tamada, T., Suzuki, N., 2015a. Different Dicer-like protein components required for intracellular and systemic antiviral silencing in Arabidopsis thaliana. Plant Signal Behav 10, e1039214.

Andika, I.B., Maruyama, K., Sun, L., Kondo, H., Tamada, T., Suzuki, N., 2015b. Differential contributions of plant Dicer-like proteins to antiviral defences against potato virus X in leaves and roots. Plant J 81, 781-793.

Baumberger, N., Tsai, C.H., Lie, M., Havecker, E., Baulcombe, D.C., 2007. The Polerovirus silencing suppressor P0 targets ARGONAUTE proteins for degradation. Curr Biol 17, 1609-1614.

Bayne, E.H., Rakitina, D.V., Morozov, S.Y., Baulcombe, D.C., 2005. Cell-to-cell movement of potato potexvirus X is dependent on suppression of RNA silencing. Plant J 44, 471-482.

Bhattacharjee, S., Zamora, A., Azhar, M.T., Sacco, M.A., Lambert, L.H., Moffett, P., 2009. Virus resistance induced by NB-LRR proteins involves Argonaute4-dependent translational control. Plant J 58, 940-951.

Bologna, N.G., Voinnet, O., 2014. The diversity, biogenesis, and activities of endogenous silencing small RNAs in Arabidopsis. Annu Rev Plant Biol 65, 473-503.

Burgyan, J., 2008. Role of silencing suppressor proteins. Methods Mol Biol 451, 69-79.
Chan, S.W., Henderson, I.R., Jacobsen, S.E., 2005. Gardening the genome: DNA methylation in Arabidopsis thaliana. Nat Rev Genet 6, 351-360.

Chan, S.W., Zilberman, D., Xie, Z., Johansen, L.K., Carrington, J.C., Jacobsen, S.E., 2004. RNA silencing genes control de novo DNA methylation. Science 303, 1336.

Chang, B.Y., Lin, N.S., Liou, D.Y., Chen, J.P., Liou, G.G., Hsu, Y.H., 1997. Subcellular localization of the 28 kDa protein of the triple-gene-block of bamboo mosaic potexvirus. J Gen Virol 78 ( Pt 5), 1175-1179.

Chen, I.H., Chiu, M.H., Cheng, S.F., Hsu, Y.H., Tsai, C.H., 2013. The glutathione transferase of Nicotiana benthamiana NbGSTU4 plays a role in regulating the early replication of Bamboo mosaic virus. New Phytol 199, 749-757.

Cheng, S.F., Huang, Y.P., Wu, Z.R., Hu, C.C., Hsu, Y.H., Tsai, C.H., 2010. Identification of differentially expressed genes induced by Bamboo mosaic virus infection in Nicotiana benthamiana by cDNA-amplified fragment length polymorphism. Bmc Plant Biol 10.

Cheng, S.F., Tsai, M.S., Huang, C.L., Huang, Y.P., Chen, I.H., Lin, N.S., Hsu, Y.H., Tsai, C.H., Cheng, C.P., 2013. Ser/Thr Kinase-Like Protein of Nicotiana benthamiana Is Involved in the Cell-to-Cell Movement of Bamboo mosaic virus. Plos One 8.

Chiu, M.H., Chen, I.H., Baulcombe, D.C., Tsai, C.H., 2010. The silencing suppressor P25 of Potato virus X interacts with Argonaute1 and mediates its degradation through the proteasome pathway. Mol Plant Pathol 11, 641-649.

Chomczynski, P., Sacchi, N., 1987. Single-Step Method of Rna Isolation by Acid Guanidinium Thiocyanate Phenol Chloroform Extraction. Anal Biochem 162, 156-159.

Csorba, T., Bovi, A., Dalmay, T., Burgyan, J., 2007. The p122 subunit of Tobacco Mosaic Virus replicase is a potent silencing suppressor and compromises both small interfering RNA- and microRNA-mediated pathways. J Virol 81, 11768-11780.

Ding, S.W., Voinnet, O., 2007. Antiviral immunity directed by small RNAs. Cell 130, 413-426.

Harvey, J.J., Lewsey, M.G., Patel, K., Westwood, J., Heimstadt, S., Carr, J.P., Baulcombe, D.C., 2011. An antiviral defense role of AGO2 in plants. PLoS One 6, e14639.

Havelda, Z., Varallyay, E., Valoczi, A., Burgyan, J., 2008. Plant virus infection-induced persistent host gene downregulation in systemically infected leaves. Plant J 55, 278-288.

Huang, Y.P., Chen, J.S., Hsu, Y.H., Tsai, C.H., 2013. A putative Rab-GTPase activation protein from Nicotiana benthamiana is important for Bamboo mosaic virus intercellular movement. Virology 447, 292-299.

Huang, Y.W., Hu, C.C., Liou, M.R., Chang, B.Y., Tsai, C.H., Meng, M., Lin, N.S., Hsu, Y.H., 2012. Hsp90 interacts specifically with viral RNA and differentially regulates replication initiation of Bamboo mosaic virus and associated satellite RNA. PLoS Pathog 8, e1002726.

Hung, C.J., Huang, Y.W., Liou, M.R., Lee, Y.C., Lin, N.S., Meng, M., Tsai, C.H., Hu, C.C., Hsu, Y.H., 2014. Phosphorylation of coat protein by protein kinase CK2 regulates cell-to-cell movement of Bamboo mosaic virus through modulating RNA binding. Mol Plant Microbe Interact 27, 1211-1225.

Hutvagner, G., Simard, M.J., 2008. Argonaute proteins: key players in RNA silencing. Nat Rev Mol Cell Biol 9, 22-32.

Jaubert, M., Bhattacharjee, S., Mello, A.F., Perry, K.L., Moffett, P., 2011. ARGONAUTE2 mediates RNA-silencing antiviral defenses against Potato virus X in Arabidopsis. Plant Physiol 156, 1556-1564.

Lee, C.C., Ho, Y.N., Hu, R.H., Yen, Y.T., Wang, Z.C., Lee, Y.C., Hsu, Y.H., Meng, M.H., 2011. The Interaction between Bamboo Mosaic Virus Replication Protein and Coat Protein Is Critical for Virus Movement in Plant Hosts. Journal of Virology 85, 12022-12031.

Li, Y.I., Cheng, Y.M., Huang, Y.L., Tsai, C.H., Hsu, Y.H., Meng, M., 1998. Identification and characterization of the Escherichia coli-expressed RNA-dependent RNA polymerase of bamboo mosaic virus. J Virol 72, 10093-10099.

Li, Y.I., Shih, T.W., Hsu, Y.H., Han, Y.T., Huang, Y.L., Meng, M., 2001. The helicase-like domain of plant potexvirus replicase participates in formation of RNA 5'' cap structure by exhibiting RNA 5''-triphosphatase activity. J Virol 75, 12114-12120.

Lin, N.S., Chen, C.C., 1991. Association of Bamboo Mosaic-Virus (Bomv) and Bomv-Specific Electron-Dense Crystalline Bodies with Chloroplasts. Phytopathology 81, 1551-1555.

Lin, N.S., Lin, B.Y., Lo, N.W., Hu, C.C., Chow, T.Y., Hsu, Y.H., 1994. Nucleotide sequence of the genomic RNA of bamboo mosaic potexvirus. J Gen Virol 75 ( Pt 9), 2513-2518.

Lin, N.S., Lin, F.Z., Huang, T.Y., Hsu, Y.H., 1992. Genome Properties of Bamboo Mosaic-Virus. Phytopathology 82, 731-734.

Liu, Y., Schiff, M., Dinesh-Kumar, S.P., 2002. Virus-induced gene silencing in tomato. Plant J 31, 777-786.

Martinez de Alba, A.E., Elvira-Matelot, E., Vaucheret, H., 2013. Gene silencing in plants: a diversity of pathways. Biochim Biophys Acta 1829, 1300-1308.

Matranga, C., Tomari, Y., Shin, C., Bartel, D.P., Zamore, P.D., 2005. Passenger-strand cleavage facilitates assembly of siRNA into Ago2-containing RNAi enzyme complexes. Cell 123, 607-620.

Mlotshwa, S., Pruss, G.J., Vance, V., 2008. Small RNAs in viral infection and host defense. Trends Plant Sci 13, 375-382.

Molnar, A., Csorba, T., Lakatos, L., Varallyay, E., Lacomme, C., Burgyan, J., 2005. Plant virus-derived small interfering RNAs originate predominantly from highly structured single-stranded viral RNAs. J Virol 79, 7812-7818.

Morel, J.B., Godon, C., Mourrain, P., Beclin, C., Boutet, S., Feuerbach, F., Proux, F., Vaucheret, H., 2002. Fertile hypomorphic ARGONAUTE (ago1) mutants impaired in post-transcriptional gene silencing and virus resistance. Plant Cell 14, 629-639.

Odokonyero, D., Mendoza, M.R., Alvarado, V.Y., Zhang, J., Wang, X., Scholthof, H.B., 2015. Transgenic down-regulation of ARGONAUTE2 expression in Nicotiana benthamiana interferes with several layers of antiviral defenses. Virology 486, 209-218.

Rajamaki, M.L., Streng, J., Valkonen, J.P., 2014. Silencing suppressor protein VPg of a potyvirus interacts with the plant silencing-related protein SGS3. Mol Plant Microbe Interact 27, 1199-1210.

Rivas, F.V., Tolia, N.H., Song, J.J., Aragon, J.P., Liu, J., Hannon, G.J., Joshua-Tor, L., 2005. Purified Argonaute2 and an siRNA form recombinant human RISC. Nat Struct Mol Biol 12, 340-349.

Ruiz-Ferrer, V., Voinnet, O., 2009. Roles of plant small RNAs in biotic stress responses. Annu Rev Plant Biol 60, 485-510.

Scholthof, H.B., Alvarado, V.Y., Vega-Arreguin, J.C., Ciomperlik, J., Odokonyero, D., Brosseau, C., Jaubert, M., Zamora, A., Moffett, P., 2011. Identification of an ARGONAUTE for antiviral RNA silencing in Nicotiana benthamiana. Plant Physiol 156, 1548-1555.

Senshu, H., Ozeki, J., Komatsu, K., Hashimoto, M., Hatada, K., Aoyama, M., Kagiwada, S., Yamaji, Y., Namba, S., 2009. Variability in the level of RNA silencing suppression caused by triple gene block protein 1 (TGBp1) from various potexviruses during infection. J Gen Virol 90, 1014-1024.

Silhavy, D., Molnar, A., Lucioli, A., Szittya, G., Hornyik, C., Tavazza, M., Burgyan, J., 2002. A viral protein suppresses RNA silencing and binds silencing-generated, 21- to 25-nucleotide double-stranded RNAs. EMBO J 21, 3070-3080.

Tolia, N.H., Joshua-Tor, L., 2007. Slicer and the argonautes. Nat Chem Biol 3, 36-43.

Tsai, C.H., Cheng, C.P., Peng, C.W., Lin, B.Y., Lin, N.S., Hsu, Y.H., 1999. Sufficient length of a poly(A) tail for the formation of a potential pseudoknot is required for efficient replication of bamboo mosaic potexvirus RNA. J Virol 73, 2703-2709.

Voinnet, O., Lederer, C., Baulcombe, D.C., 2000. A viral movement protein prevents spread of the gene silencing signal in Nicotiana benthamiana. Cell 103, 157-167.

Voinnet, O., Pinto, Y.M., Baulcombe, D.C., 1999. Suppression of gene silencing: a general strategy used by diverse DNA and RNA viruses of plants. Proc Natl Acad Sci U S A 96, 14147-14152.

Wang, X.B., Jovel, J., Udomporn, P., Wang, Y., Wu, Q., Li, W.X., Gasciolli, V., Vaucheret, H., Ding, S.W., 2011. The 21-nucleotide, but not 22-nucleotide, viral secondary small interfering RNAs direct potent antiviral defense by two cooperative argonautes in Arabidopsis thaliana. Plant Cell 23, 1625-1638.

Widholm, J.M., 1972. Use of Fluorescein Diacetate and Phenosafranine for Determining Viability of Cultured Plant-Cells. Stain Technol 47, 189-&.

Yang, C.C., Liu, J.S., Lin, C.P., Lin, N.S., 1997. Nucleotide sequence and phylogenetic analysis of a bamboo mosaic potexvirus isolate from common bamboo (Bambusa vulgaris McClure). Bot Bull Acad Sinica 38, 77-84.