Please use this identifier to cite or link to this item:
標題: The study of NbLTP1 from Nicotiana benthamiana involved in the replication of Bamboo mosaic virus
菸草蛋白NbLTP1參與 竹嵌紋病毒複製機制之研究
作者: 邱齡瑩
Ling-Ying Chiu
關鍵字: 竹嵌紋病毒;差異性表現基因;脂質傳送蛋白;攜鈣素結合蛋白;bamboo mosaic virus;differentially expressed host gene;lipid transfer protein;calmodulin binding protein
引用: Bernhard WR, Thoma S, Botella J, Somerville CR (1991) Isolation of a cDNA Clone for Spinach Lipid Transfer Protein and Evidence that the Protein Is Synthesized by the Secretory Pathway. Plant Physiol 95: 164-170 Buhot N, Gomes E, Milat ML, Ponchet M, Marion D, Lequeu J, Delrot S, Coutos-Thevenot P, Blein JP (2004) Modulation of the biological activity of a tobacco LTP1 by lipid complexation. Mol Biol Cell 15: 5047-5052 Carvalho Ade O, Gomes VM (2007) Role of plant lipid transfer proteins in plant cell physiology-a concise review. Peptides 28: 1144-1153 Chen IH, Chiu MH, Cheng SF, Hsu YH, Tsai CH (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 SF, Huang YP, Wu ZR, Hu CC, Hsu YH, Tsai CH (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: 286 Cheng SF, Tsai MS, Huang CL, Huang YP, Chen IH, Lin NS, Hsu YH, Tsai CH, Cheng CP (2013) Ser/Thr kinase-like protein of Nicotiana benthamiana is involved in the cell-to-cell movement of Bamboo mosaic virus. PLoS One 8: e62907 Chomczynski P, Sacchi N (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162: 156-159 Coutos-Thevenot P, Jouenne T, Maes O, Guerbette F, Grosbois M, Le Caer JP, Boulay M, Deloire A, Kader JC, Guern J (1993) Four 9-kDa proteins excreted by somatic embryos of grapevine are isoforms of lipid-transfer proteins. Eur J Biochem 217: 885-889 Edstam MM, Viitanen L, Salminen TA, Edqvist J (2011) Evolutionary history of the non-specific lipid transfer proteins. Mol Plant 4: 947-964 Hsu YH, Chen HC, Cheng J, Annamalai P, Lin BY, Wu CT, Yeh WB, Lin NS (2006) Crucial role of the 5' conserved structure of bamboo mosaic virus satellite RNA in downregulation of helper viral RNA replication. J Virol 80: 2566-2574 Huang YL, Han YT, Chang YT, Hsu YH, Meng M (2004) Critical residues for GTP methylation and formation of the covalent m7GMP-enzyme intermediate in the capping enzyme domain of bamboo mosaic virus. J Virol 78: 1271-1280 Huang YP, Chen JS, Hsu YH, Tsai CH (2013) A putative Rab-GTPase activation protein from Nicotiana benthamiana is important for Bamboo mosaic virus intercellular movement. Virology 447: 292-299 Hyodo K, Kaido M, Okuno T (2014) Traffic jam on the cellular secretory pathway generated by a replication protein from a plant RNA virus. Plant Signal Behav 9: e28644 Jiang S, Lu Y, Li K, Lin L, Zheng H, Yan F, Chen J (2014) Heat shock protein 70 is necessary for Rice stripe virus infection in plants. Molecular Plant Pathology: n/a-n/a Jose-Estanyol M, Gomis-Ruth FX, Puigdomenech P (2004) The eight-cysteine motif, a versatile structure in plant proteins. Plant Physiol Biochem 42: 355-365 Kader JC (1996) Lipid-Transfer Proteins in Plants. Annu Rev Plant Physiol Plant Mol Biol 47: 627-654 Kiba A, Nakatsuka T, Yamamura S, Nishihara M (2012) Gentian lipid transfer protein homolog with antimicrobial properties confers resistance to Botrytis cinerea in transgenic tobacco. Plant Biotechnology 29: 95-101 Li C, Xie W, Bai W, Li Z, Zhao Y, Liu H (2008) Calmodulin binds to maize lipid transfer protein and modulates its lipids binding ability. FEBS J 275: 5298-5308 Li C, Xie W, Wang L, Zhao Y (2011) The phosphorylation of lipid transfer protein CaMBP10. Protein Pept Lett 18: 17-22 Li YI, Chen YJ, Hsu YH, Meng M (2001) Characterization of the AdoMet-dependent guanylyltransferase activity that is associated with the N terminus of bamboo mosaic virus replicase. J Virol 75: 782-788 Li YI, Cheng YM, Huang YL, Tsai CH, Hsu YH, 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 YI, Shih TW, Hsu YH, Han YT, Huang YL, 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 JW, Chiu HN, Chen IH, Chen TC, Hsu YH, Tsai CH (2005) Structural and functional analysis of the cis-acting elements required for plus-strand RNA synthesis of Bamboo mosaic virus. J Virol 79: 9046-9053 Lin KF, Liu YN, Hsu ST, Samuel D, Cheng CS, Bonvin AM, Lyu PC (2005) Characterization and structural analyses of nonspecific lipid transfer protein 1 from mung bean. Biochemistry 44: 5703-5712 Lin MK, Hu CC, Lin NS, Chang BY, Hsu YH (2006) Movement of potexviruses requires species-specific interactions among the cognate triple gene block proteins, as revealed by a trans-complementation assay based on the bamboo mosaic virus satellite RNA-mediated expression system. J Gen Virol 87: 1357-1367 Lin NS, Chen CC (1991) Association of Bamboo mosaic virus (Bomv) and Bomv-specific electron-dense crystalline bodies with chloroplasts. Phytopathology 81: 1551-1555 Lin NS, Lin BY, Lo NW, Hu CC, Chow TY, Hsu YH (1994) Nucleotide sequence of the genomic RNA of bamboo mosaic potexvirus. J Gen Virol 75: 2513-2518 Lin NS, Lin FZ, Huang TY, Hsu YH (1992) Genome properties of Bamboo mosaic virus. Phytopathology 82: 731-734 Liu Y, Schiff M, Dinesh-Kumar SP (2002) Virus-induced gene silencing in tomato. Plant J 31: 777-786 Lucas WJ (2006) Plant viral movement proteins: agents for cell-to-cell trafficking of viral genomes. Virology 344: 169-184 Molina A, Segura A, Garcia-Olmedo F (1993) Lipid transfer proteins (nsLTPs) from barley and maize leaves are potent inhibitors of bacterial and fungal plant pathogens. FEBS Lett 316: 119-122 Nishimura S, Tatano S, Gomi K, Ohtani K, Fukumoto T, Akimitsu K (2008) Chloroplast-localized nonspecific lipid transfer protein with anti-fungal activity from rough lemon. Physiological and Molecular Plant Pathology 72: 134-140 Ostergaard J, Hojrup P, Knudsen J (1995) Amino acid sequences of three acyl-binding/lipid-transfer proteins from rape seedlings. Biochim Biophys Acta 1254: 169-179 Salcedo G, Sanchez-Monge R, Barber D, Diaz-Perales A (2007) Plant non-specific lipid transfer proteins: an interface between plant defence and human allergy. Biochim Biophys Acta 1771: 781-791 Samuel D, Liu YJ, Cheng CS, Lyu PC (2002) Solution structure of plant nonspecific lipid transfer protein-2 from rice (Oryza sativa). J Biol Chem 277: 35267-35273 Shin DH, Lee JY, Hwang KY, Kim KK, Suh SW (1995) High-resolution crystal structure of the non-specific lipid-transfer protein from maize seedlings. Structure 3: 189-199 Takishima K, Watanabe S, Yamada M, Suga T, Mamiya G (1988) Amino acid sequences of two nonspecific lipid-transfer proteins from germinated castor bean. Eur J Biochem 177: 241-249 Tchang F, This P, Stiefel V, Arondel V, Morch MD, Pages M, Puigdomenech P, Grellet F, Delseny M, Bouillon P, et al. (1988) Phospholipid transfer protein: full-length cDNA and amino acid sequence in maize. Amino acid sequence homologies between plant phospholipid transfer proteins. J Biol Chem 263: 16849-16855 Thivierge K, Nicaise V, Dufresne PJ, Cotton S, Laliberte JF, Le Gall O, Fortin MG (2005) Plant virus RNAs. Coordinated recruitment of conserved host functions by (+) ssRNA viruses during early infection events. Plant Physiol 138: 1822-1827 Thoma S, Kaneko Y, Somerville C (1993) A non-specific lipid transfer protein from Arabidopsis is a cell wall protein. Plant J 3: 427-436 Tsuboi S, Osafune T, Tsugeki R, Nishimura M, Yamada M (1992) Nonspecific lipid transfer protein in castor bean cotyledon cells: subcellular localization and a possible role in lipid metabolism. J Biochem 111: 500-508 Wang C, Xie W, Chi F, Hu W, Mao G, Sun D, Li C, Sun Y (2008) BcLTP, a novel lipid transfer protein in Brassica chinensis, may secrete and combine extracellular CaM. Plant Cell Rep 27: 159-169 Wang Z, Benning C (2012) Chloroplast lipid synthesis and lipid trafficking through ER-plastid membrane contact sites. Biochem Soc Trans 40: 457-463 Wang Z, Xie W, Chi F, Li C (2005) Identification of non-specific lipid transfer protein-1 as a calmodulin-binding protein in Arabidopsis. FEBS Lett 579: 1683-1687 Whitham SA, Wang Y (2004) Roles for host factors in plant viral pathogenicity. Curr Opin Plant Biol 7: 365-371 Widholm JM (1972) The use of fluorescein diacetate and phenosafranine for determining viability of cultured plant cells. Stain Technol 47: 189-194 Yeats TH, Rose JK (2008) The biochemistry and biology of extracellular plant lipid-transfer proteins (LTPs). Protein Sci 17: 191-198
竹嵌紋病毒(Bamboo mosaic virus, BaMV)為一隸屬於Flexiviridae科Potexvirus屬的正股RNA病毒,是感染竹子的主要病毒。本研究的主要目的是找到與竹嵌紋病毒感染過程中有關的宿主蛋白質,藉此了解病毒的繁殖機制。根據前人研究之cDNA-amplified fragment length polymorphism (cDNA-AFLP)結果,我們在菸草中找到一個宿主基因ACGT12,會在感染竹嵌紋病毒後表現量降低。利用菸草脆裂病毒(Tobacco rattle virus, TRV)誘導基因靜默(virus-induced gene silencing; VIGS)以降低基因的表現,進而以西方墨點法(western blot)偵測這些基因對於BaMV在宿主內繁殖的影響,結果發現靜默宿主基因ACGT12後,病毒鞘蛋白(coat protein)的累積量下降。再以西方墨點法偵測基因靜默後的原生質體(protoplast),發現病毒的累積量也同樣下降。因此推斷宿主基因ACGT12可能與病毒的複製而非移動有關,且ACGT12的基因表現下降時,病毒的累積量也會隨之減少。為進一步了解此基因在宿主內可能的用途,利用rapid amplification of cDNA ends (RACE)技術,延長宿主基因的上下游片段,得到全長的cDNA,將其序列與Biology WorkBench資料庫之BLASTP比對後,推測此基因應為non-specific lipid transfer protein 1 (nsLTP1),因此我們將此基因命名為NbLTP1。利用融合橘螢光蛋白(Orange fluorescent protein)的表現,觀察其於細胞的位置主要分佈於胞外(extracellular matrix),然而如將分泌的signal peptide去除,則發現大部分的蛋白會與葉綠體的自體螢光重疊。此外,在大量表現NbLTP1後感染病毒,也發現病毒鞘蛋白表現量會上升。總結以上的結果,我們可以推測NbLTP1在病毒的感染週期中扮演幫助病毒核酸的複製。與其他LTP比對後也發現,NbLTP1具有可與calmodulin (CaM)結合的保守區域,此一特性也可能為影響病毒複製的原因。

Bamboo mosaic virus (BaMV) is a single-stranded positive sense RNA virus belonging to genus Potexvirus of the family Flexiviridae. The objective of this study is to understand the relationship of the host proteins involved in the replication mechanism of BaMV. The results derived from a previous study, a downregulated gene fragment ACGT12 from Nicotiana benthamiana post BaMV infection identified by cDNA-AFLP technique is further characterized. We used the Tobacco rattle virus (TRV)-based silencing system (virus-induced gene silencing; VIGS) to knock down the expression of ACGT12 and infected the BaMV on the knockdown plants. The results indicate that the accumulation levels of BaMV coat protein in the knockdown plants are lower than that in the control plants. Similar results are observed in the knockdown protoplasts that less coat protein accumulated than that in the control protoplasts. These results suggest that ACGT12 may be involved in the replication process rather than in viral movement. The full-length cDNA of ACGT12 is obtained by rapid amplification of cDNA ends (RACE) technique. The sequence of ACGT12 is blasted to the Biology WorkBench database and matches to that of non-specific lipid transfer protein 1 (nsLTP1) and designated as NbLTP1. Furthermore, the cellular localization of NbLTP1-OFP (fused with Orange fluorescence protein) is mainly associated with the extracellular matrix. However, when the signal peptide is removed, the majority of the expressed mutant protein is associated with chloroplasts. The accumulation of BaMV coat protein is enhanced when NbLTP1 is transiently expressed in plants. Overall, the results indicate that the newly identified host protein NbLTP1 is a positive regulator for the replication of BaMV RNA. In addition to the hydrophobic pocket to accommodate the lipid, NbLTP1 has a conserved calmodulin
(CaM)-binding site at the very C-terminus. Therefore, the lipid binding and the CaM-binding properties involving in the replication of BaMV is needed to be further characterized.
Rights: 同意授權瀏覽/列印電子全文服務,2014-08-31起公開。
Appears in Collections:生物科技學研究所

Files in This Item:
File Description SizeFormat Existing users please Login
nchu-103-7102041018-1.pdf1.45 MBAdobe PDFThis file is only available in the university internal network   
Show full item record

Google ScholarTM


Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.