Characterization of Plant Factors Interacting with Cucumber Mosaic Virus Replication Protein : Characterization of Plant Factors Interacting with Cucumber Mosaic Virus Replication Protein
- 발행기관 고려대학교
- 발행년도 2006
- 학위수여년월 2006. 2
- 학위명 박사
- 학과 대학원 생명공학과 분자생물학전공
- 식별자(기타) DL:000017000121
- 서지제어번호 000045282739
초록/요약
Cucumber mosaic virus (CMV) is a member of the genus Cucumovirus in the family Bromoviridae. Unlike most plant viruses, CMV has a very broad host range and the replication is membrane associated. The CMV-encoded 1a protein has been implicated to play a role in replication of the viral genome along with 2a and one or more host factors. To identify the host cell factors interacting with CMV 1a, I used the yeast two-hybrid system using tobacco cDNA library. Initially fourteen candidate genes were isolated. Among the candidate genes, three; Nicotiana tabacum thaumatin-like protein 1 (NtTLP1), Tcoi1 (Tobacco CMV 1a interacting protein 1) and Tcoi2 (Tobacco CMV 1a interacting protein 2) were selected for further study. NtTLP1 is homologous to the plant TLP gene encoding a member of the PR-5 group of PR proteins. The recombinant GST-NtTLP1 protein, which was expressed and purified in E. coli, bound tightly to CMV 1a in vitro. In planta interaction was also verified via co-immunoprecipitation. Additionally, NtTLP1 specifically interacted with the CMV movement-related proteins, movement protein (MP) and coat protein (CP), in yeast. Real-time quantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis showed that the expression of NtTLP1 increased as the result of CMV inoculation. I have identified Tcoi1 encodes for a putative methyltransferase that interacts with the CMV 1a protein. Consistent with this observation, bacterially expressed and purified recombinant GST-Tcoi1 protein bound tightly to CMV 1a in vitro and GST-Tcoi1 directly methylated the two separated domain of CMV 1a; methyltransferase (MT) domain and helicase (Hel) domain. RT-PCR analysis showed that the expression of Tcoi1 was increased by CMV inoculation. The Tcoi1 antisense transgenic plants showed reduced accumulation of CMV RNA 3. These results suggest that Tcoi1 controls CMV propagation through an interaction with the CMV 1a protein. One of the cDNA clones encoded a protein homologus to the Arabidopsis putative protein kinase and was designated Tcoi2. Tcoi2 specifically interacted with MT domain of CMV 1a protein in yeast cell. In vitro analyses using recombinant proteins showed that Tcoi2 also specifically interacted with CMV 1a MT domain. Tcoi2 did not have autophosphorylation activity but phosphorylated CMV 1a MT domain. Analysis of the subcellular localization of the Tcoi2 fused to GFP demonstrated that it is targeted to the endoplasmic reticulum (ER). These results suggest Tcoi2 as a host factor that is capable of interacting and phosphorylating MT domain of CMV 1a protein. CMV replication complex is associated with cellular membranes. However, it is unknown whether any host factors participate in this process. In this study, five groups of Arabidopsis tonoplast intrinsic protein (AtTIP) genes were isolated and their interaction with CMV proteins was tested. TIP1 and TIP2 proteins interacted more strongly with CMV 1a protein in the yeast two-hybrid system than proteins from the other three subgroups. The interaction of CMV 1a with TIP1 and TIP2 proteins was confirmed in the Sos-recruitment system and co-immunoprecipitation assays, whereas interactions with the other three subgroups were not observed in these two assays. CMV 1a co-localized with TIP1 and TIP2 in transfected Arabidopsis protoplasts. In addition, the accumulation of CMV CP was slightly delayed in tip mutant plants and the viral RNA accumulation was decreased than in wild type Col-0. These results suggest that the two TIP groups could affect the formation of replication complex as in the tonoplast by interacting with CMV 1a.
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Table of contents
List of Tables
List of Figures
List of Abbreviations
Abstract
Chapter Ι. In vitro and in planta interaction evidence between Nicotiana tabacum thaumatin-like protein 1 (TLP1) and Cucumber mosaic virus proteins
1. Abstract
2. Introduction
3. Results
4. Discussion
5. Materials and methods
6. References
Chapter Ⅱ. Identification of novel methyltransferases of Nicotiana tabacum that interact with Cucumber mosaic virus 1a protein
1. Abstract
2. Introduction.
3. Results.
4. Discussion
5. Materials and methods
6. References
Chapter Ⅲ. Novel protein kinase interacts with the Cucumber mosaic virus 1a methyltransferase domain
1. Abstract
2. Introduction.
3. Results
4. Discussion
5. Materials and methods
6. References
Chapter Ⅳ. Arabidopsis tonoplast protein TIP1 and TIP2 interact with the Cucumber Mosaic Virus 1a replication protein
1. Abstract
2. Introduction
3. Results
4. Discussion.
5. Materials and methods.
6. References.
Chapter V. Isolation of plant factors participate in Cucumber mosaic virus replication using reverse genetics
1. Abstract
2. Introduction
3. Results and Discussion
4. References.
APPENDIX
Abstract in Korean
Acknowledgement
List of Tables
Table 1-1. Interaction of NtTLP1 and CMV 1a in yeast cells.
Table 3-1. Interaction of Tcoi2 and CMV 1a in yeast cells.
List of Figures
Figure 1-1. In vitro interaction test of recombinant NtTLP1 protein and the CMV 1a protein.
Figure 1-2. In planta interaction test of NtTLP1::GFP and CM
1a::2xHA.
Figure 1-3. Subcellular localization of NtTLP1::GFP in onion cells and Arabidopsis.
Figure 1-4. Interactions of TLPs and CMV viral proteins in yeast cells.
Figure 1-5. Real-time quantitative RT-PCR analysis of three different kinds of TLP genes upon CMV-Kor inoculation.
Figure 1-6. Genomic DNA gel blot analysis.
Figure 2-1. Interactions of Tcoi1 and CMV viral proteins in yeast cells.
Figure 2-2. Nucleotide sequence and protein sequence comparison of Tcoi1.
Figure 2-3. Southern blot ananlysis and comparrision of Tcoi1 expression in different organs.
Figure 2-4. RT-PCR analysis of Tcoi1 genes upon CMV-Kor inoculation.
Figure 2-5. Identification the region of Tcoi1 that is necessary for the interaction with CMV 1a protein and in vitro interaction analysis.
Figure 2-6. Methylation of the CMV 1a MT domain and CMV 1a Hel domain by purified Tcoi1.
Figure 2-7. Subcellular localization of Tcoi1::GFP in Arabidopsis protoplasts.
Figure 2-8. Expression of Tcoi1 gene in transgenic tobacco plants and the accumulations of the CMV RNA 3 in wild type control and transgenic plants.
Figure 3-1. Nucleotide and predicted amino acid sequences of Tcoi2 (GenBank accession number DQ202472).
Figure 3-2. Comparison of the predicted amino acid sequence of Tcoi2 with a putative kinase of Arabidopsis thaliana (GenBank accession number AAM91792) and PERK1 of Oryza sativa (GenBank accession number XP468388).
Figure 3-3. Genomic DNA gel blot analysis.
Figure 3-4. In vitro interaction test of recombinant Tcoi2 protein and the CMV 1a MT domain-containing protein.
Figure 3-5. Assay of CMV 1a MT protein phosphorylation by Tcoi2.
Figure 3-6. Subcellular localization of Tcoi2::GFP in Arabidopsis.
Figure 4-1. Interactions of Arabidopsis TIPs and CMV 1a using yeast two-hybrid assay.
Figure 4-2. Interaction analysis between TIPs and CMV 1a using SRS.
Figure 4-3. In vitro interaction test of TIPs and CMV 1a protein.
Figure 4-4. Expression patterns of TIP1 and 2 group genes upon CM
inoculation.
Figure 4-5. Subcellular localization of TIP1;2::GFP and TIP 2;1::GFP in onion cells.
Figure 4-6. Subcellular co-localization of TIPs and CMV 1a in Arabidopsis protoplasts.
Figure 4-7. Comparison of CMV-Kor coat protein accumulation in Col-0 and tip mutant plants upon CMV inoculation.
Figure 4-8. Time course of the accumulation of CMV-Kor related RNAs in wild type Col-0 and tip protoplasts.
Figure 5-1. Screen for loss-of susceptl mutants of Arabps
Figure 5-2. Accumulation of CMV coat protein in Col-0 and selected mutant lines and the results of Southern blotting.
Figure 5-3. Comparison of the CMV infectivity on Col-0 and rep mutant and the position of T-DNA insertion.
Figure 5-4. Comparison of the CMV coat protein accumulation in Col-0 and rbp mutant.
Figure 5-5. The phenotype of AGL6 activation lines and comparison of the CMV coat protein accumulation in Col-0 and AGL6 activation lines.
