S-Space College of Agriculture and Life Sciences (농업생명과학대학) Dept. of Plant Science (식물생산과학부) Theses (Master's Degree_식물생산과학부)
Isolation and Characterization of Pepper Genes Interacting with CMV-P1 Helicase Domain : CMV-P1 Helicase domain과 상호작용하는 고추 유전자 동정 및 기능 분석
- 농업생명과학대학 원예학과
- Issue Date
- 서울대학교 대학원
- 학위논문 (석사)-- 서울대학교 대학원 : 식물생산과학부 원예학 전공, 2013. 2. 강병철.
- Cucumber mosaic virus (CMV), which has the broadest host range among plant viruses, is a very destructive pathogen in pepper production. Capsicum annuum Bukang contains a single dominant resistance gene, Cmr1 (Cucumber mosaic resistance 1) to CMV. C. annuum Bukang is resistant to most of CMV strains, but susceptible to CMV-P1. CMV-P1 is a new strain overcoming Cmr1recently identified in South Korea. Previous study reported that CMV-P1 RNA1 helicase domain is responsible for overcoming Cmr1. To identify plant host factors involved in CMV-P1 replication and movement, a yeast two-hybrid system derived from C. annuum Bukang cDNA library was used. A total of 156 potential clones interacting with the CMV-P1 RNA1 helicase domain were isolated from about 100,000 clones in the first screening. Beta-galactosidase filter lift assay, PCR screening, and sequencing analysis narrowed the candidates to 10 host genes. These genes are known to be involved in virus infection, replication, or virus movement. To elucidate functions of these genes, the host genes were silenced in Nicotiana benthamiana, which were then inoculated with CMV-P1 expressing the green fluorescent protein (GFP). Gene silencing was confirmed by semi-quantitative RT-PCR. Plants silenced for 7 genes showed normal development comparable to N. benthamiana wild type, whereas plants silenced for the other three genes showed developmental defects including stunting and severe distortion. Virus accumulation in silenced plants was assessed by monitoring GFP fluorescence and enzyme-linked immunosorbent assay (ELISA). Among the 7 candidates, silencing the cysteine synthase gene showed rapid and high accumulation of CMV while silencing two host genes, formate dehydrogenase and calreticulin-3 precursor, showed reduced virus accumulation. In the case of the cysteine synthase-silenced plants, infection foci were observed in both inoculated and upper leaves, and GFP signals were detected earlier than TRV::00. Formate dehydrogenase-silenced plants showed local infection in inoculated leaves, and there were no GFP signal in upper leaves. In the case of calreticulin-3 precursor, no GFP signals were observed in both the inoculated and upper leaves. ELISA results confirmed the confocal observation results. These results demonstrate that formate dehydrogenase and calreticulin-3 precursor are required for CMV-P1 infection