Distinguished Research Fellow

林納生 LIN, Na-Sheng Distinguished Research Fellow Ph.D., Department of Plant Pathology, University of Nebraska -Lincoln, USA (1984) TEL： +886-2-2787-1128 (Office) +886-2-2787-1022 (Lab) E-mail： nslin@sinica.edu.tw Research Interests Publications Lab Members   MOLECULAR BASIS OF VIRUS, SATELLITE RNA AND HOST PLANT INTERACTIONS Satellite RNAs are subviral RNAs that have little sequence homology with their helper viruses, but depend on the viral genomic RNAs for replication, encapsidation and movement. We have used a satellite RNA associated with bamboo mosaic virus (BaMV) as a model (1) to develop the satellite RNA-based plant expression vector, (2) to study the satBaMV sequence and satBaMV-encoded products involved in the satellite RNA replication and movement, and (3) to analyze the interactions between the virus, subviral RNAs and host plant that lead to virus resistance. GENERAL OUTLINE BaMV, a member of the potexvirus group, consists of single-stranded and positive-sense RNA genome. Satellite RNAs of BaMV (satBaMV) is the only one found to be associated with the potexvirus group. It is a linear molecule with 836 nucleotides in length and encodes a protein of 20 kDa (P20). The open reading frame (ORF) of P20 is not essential for satBaMV replication, however, the ORF of P20 is present in all isolates of satBaMV that we have sequenced. The P20 was only transiently expressed at the early stage of infection and was mainly located in the cytoplasm and nuclei of infected cells by immunoelectron microscopy. The ORF of P20 can be replaced with the reporter gene chloramphenicol transferase. Thus, satBaMV has potentially become the satellite-based plant expression vector. With the wide collection of satBaMV isolates domestically and abroad, we found that two isolates of satBaMV exhibit extreme differences in phenotypes. They differ greatly in their abilities to reduce the helper virus accumulation, to attenuate the virus-induced symptoms, and to move systemically in the infected host plants. Thus, these two satBaMVs provide excellent materials for studying the molecular basis of the mRNA type satellite RNA-mediated interaction with virus and host plants. PROGRESS AND ACCOMPLISHMENTS 1. Mapping the subgenomic RNA (sgRNA) core promoter sequences by the use of satBaMV vector: Insertion of subgenomic promoter-like sequences (SGP) of BaMV capsid protein (CP) gene into the satBaMV vector gave rise to the synthesis of sgRNA of satBaMV in protoplasts of Nicotiana benthamiana and leaves of Chenopodium quinoa when co- inoculated with BaMV-V genomic RNA. By deletion analysis, we have concluded that the subgenomic promoter-like sequences of CP gene contains one core promoter sequence and two upstream and one downstream enhancers. 2. Characterization of satBaMV-encoded P20 protein: Experiments of gel retardation, UV crosslinking, and Northwestern hybridization demonstrated that the P20 is a RNA-binding protein, preferentially binding to the 5" and 3" untranslated regions (UTRs) of satBaMV. The N-terminal arginine motif of P20 is the RNA-binding domain. Furthermore, we have shown that P20 protein assists the systemic movement of satBaMV. Mutants of satBaMV which blocked the P20 gene expression did not significantly affect the accumulation level of satBaMV in the inoculated leaves, but dramatically reduced the efficiency of systemical movement in the co-inoculated Nicotiana benthamiana, indicating the important role(s) of P20 for the long-distance movement of satBaMV. 3. Identification of determinants of BSL6 satBaMV responsible for its phenotypes: By the construction of chimeric satBaMVs between BSF4 and BSL6 satBaMVs, we have mapped that the determinants are located in the 5" UTR of satBaMV. Insertion of 5" UTR of BSL6 satBaMV into the BaMV genome confers the resistance to BaMV infection in C. quinoa plants. The molecular interaction among 5" UTR of satBaMV, BaMV and host plants are under investigation.   Research Interests Publications Lab Members   All publication list Selected publication list Lin, N. S., Lee, Y. S., Lin, B. Y., Lee, C. W., and Hsu, Y. H. 1996. The open reading frame of bamboo mosaic virus satellite RNA is not essential for its replication and can be replicated with a bacterial gene. Proc. Natl. Acad. Sci. USA 93: 3138-3142. Liu, J. S., Hsu, Y. H., Huang, T. Y., and Lin, N. S. 1997. Molecular evolution and phylogeny of satellite RNA associated with bamboo mosaic potexvirus. J. Mol. Evol. 44: 207-213. Lee, Y. S., Lin, B. Y., Hsu, Y. H., Chang, B. Y., and Lin, N. S. 1998. Subgenomic RNAs of bamboo mosaic potexvirus-V isolate are packaged into virions. J. Gen. Virol. 79: 1825-1832. Hsu, Y. H., Lee, Y. S., Liu, J. S., and Lin, N. S. 1998. Differential interactions of bamboo mosaic potexvirus satellite RNAs, helper virus and host plants. Mol. Plant-Microbe Interact. 11: 1207-1213. Tsai, M. S., Hsu, Y. H., and Lin, N. S. 1999. Characterization of bamboo mosaic potexvirus satellite RNA-encoded protein: a cooperative, sequencespecific RNA binding protein. J. Virol. 73: 3032-3039. Hsu, Y. H, Annamalai, P., Lin, C. S., Chen, Y. Y., Chang, W. C., and Lin, N. S. 2000. A sensitive method for detecting bamboo mosaic virus (BaMV) and establishment of BaMV-free meristem tip cultures. Plant Pathology 49: 101-107. Lee, Y. S., Hsu, Y. H., and Lin, N. S. 2000. Generation of subgenomic RNA directed by a satellite RNA associated with bamboo mosaic potexvirus: analyses of potexvirus subgenomic RNA promoter. J. Virol. 74: 10341-10348. Annamalai, P., Hsu, Y. H., Tsai, C. H., and Lin, N. S. 2003. Structural and mutational analyses of cis-acting sequences in the 5"-untranslated region of satellite RNA of bamboo mosaic potexvirus. Virology 311: 229-239. Yeh, W. B., Hsu, Y. H., Chen, H. C., and Lin, N. S. 2004. Conserved secondary structure in the hypervariable region at the 5" end of Bamboo mosaic virus satellite RNA is functionally interchangeable. Virology 330: 105-115. Chang, C., Chen, Y. C., Hsu, Y. H., Wu, J. T., Hu, C. C., Chang, W. C., and Lin, N. S. 2005. Transgenic resistance to Cymbidium Mosaic Virus in Dendrobium expressing the viral capsid protein gene. Transgenic Res. 14: 41-46. Vijaya Palani, P., Kasiviswanathan, V., Chen, J. C., Chen, W., Hsu, Y. H., and Lin, N. S. 2006. The arginine-rich motif of Bamboo mosaic virus satellite RNA-encoded P20 mediates self-interaction, intracellular targeting, and cell-to-cell movement. Mol. Plant-Microbe Interact. 19:758-67. Lin, M. K., Hu, C. C., Lin, N. S., Chang, B. Y., and Hsu, Y. H. 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. 87:1357-67. Lin, M. K, Hu, C. C., Lin, N. S., Chang, B. Y, and Hsu, Y. H. 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. 87:1357-67. Hsu, Y. H., Chen, H. C., Cheng, J., Annamalai, P., Lin, B. Y., Wu, C. T., Yeh, W. B., Lin N. S. 2006. Crucial role of the 5" conserved structure of bamboo mosaic virus satellite RNA in downregulation of helper viral RNA replication. J. Virol. 80:2566-74. Vijaya palani, P., and Lin, N. S. 2007. Northern analysis of viral plus- and minus-strand RNAs. Current Protocols in Microbiology. Chapter 16E.3.1-16E.3.32. John Wilely & Sons. Inc., USA. C. D. Yang, J. T. Liao, C. Y. Lai, M. H. Jong, C. M. Liang, Y. L. Lin, Lin, N. S. *, Y. H. Hsu* and S. M. Liang*. 2007. Induction of protective immunity in swine by recombinant bamboo mosaic virus expressing foot-and-mouth disease virus epitopes. BMC Biotechnol. 7: 62. Chen, H. C., Hsu, Y. H. and Lin, N. S.*. 2007. Downregulation of Bamboo mosaic virus replication requires the 5’ apical hairpin stem loop structure and sequence of satellite RNA. VIROLOGY. 365: 271-84. Palani, P. V., Chiu, M., Chen, W., Wang, C.C., Lin, C.C., Hsu, C.C., Cheng, C.C., Chen, C. M., Hsu, Y. H., Lin, N. S.*. 2009. Subcellular localization and expression of Bamboo mosaic virus satellite RNA- encoded protein. JOURNAL OF GENERAL VIROLOGY. 90: 507-518. Huang, Y.W., Hu, C.C., Lin, C.A., Liu, Y.P., Tsai, C.H., Lin, N. S.*, Hsu, Y.H. 2009. Structural and functional analyses of the 3 ' untranslated region of Bamboo mosaic virus satellite RNA. VIROLOGY. 386(1): 139-153. Wu, F.H., Kan, D.P., Lee, S.B., Daniell, H., Lee, Y.W., Lin, C.C., Lin, N. S., Lin, C.S. 2009. Complete nucleotide sequence of Dendrocalamus latiflorus and Bambusa oldhamii chloroplast genomes. Tree Physiology. 29(6): 847-856. Wang, W.C., Lin, F.M., Chang, W.C., Lin, K.Y., Huang, H.D., Lin, N. S. 2009. miRExpress: Analyzing high-throughput sequencing data for profiling microRNA expression. BMC Bioinformatics. 10. Tseng, Y.H., Hsu, H.T., Chou, Y.L., Hu, C.C., Lin, N. S., Hsu, Y.H., Chang, B.Y. 2009. The Two Conserved Cysteine Residues of the Triple Gene Block Protein 2 Are Critical for Both Cell-to-Cell and Systemic Movement of Bamboo mosaic virus. Molecular Plant-Microbe Interactions. 22(11): 1379-1388. Hu, C. C., Hsu, Y. H., Lin, N. S. 2010. Satellite RNAs and Satellite Viruses of Plants. Viruses ( in press).   Research Interests Publications Lab Members   Postdoctoral Research Associates Hsin-Chuan Chen, Hsin-Ting Yang, Ping Lan, Cheng-Chung Liu, Lih-Ren Kong, Ying-Wen Huang Research Assistant Shu-Hsuan Hu, Chun-Yen Lin, Ting-Yu Chen, Sih-Min Yen, Chi-An Wang Assistant Alice Liao Graduate Students Ming-Ru Liou, Kuan-Yu Lin, Mazen ALAZEM, C.C.-Chen   ‧CHEN, Chung-Mong ‧CHEN, Long-Fang ‧CHENG, Wan-Hsing ‧CHU, Hsiu-An ‧CHUNG, Mei-Chu ‧DAI, Hwa ‧FENG, Teng-Yung ‧FU, Hong-yong ‧HSIEH, Ming-Hsiun ‧HSING, Yue-Ie ‧HUANG, Li-Chun ‧JAUH, Guang-Yuh ‧JU, Yu-Ming ‧LAI, Erh-Min ‧LIN, Na-Sheng ‧LO, Wan-Sheng ‧Schmidt, Wolfgang ‧TSOU, Chih-Hua ‧TU, Jenn ‧TU, Shih-Long ‧Verslues, Paul E. ‧WANG, Chao-Wen ‧WANG, Long-Chi ‧WU, Shu-Hsing ‧YU, Tien-Shin