研發柑桔鱗砧類病毒整合性診斷偵測技術與探討類病毒與寄主間交互作用

Development of an integrative diagnostic protocol for Citrus exocortis viroid (CEVd) and study of the viroid-host interactions

林之昫(Chih-Hsu Lin)

指導教授 ： 沈湯龍

共同指導教授 ： 林詩舜(Shih-Shun Lin)

國立臺灣大學/生物資源暨農學院/植物病理與微生物學研究所/碩士(2010年)

https://doi.org/10.6342/NTU.2010.10036

摘要

無資料

關鍵字

柑橘鱗砧類病毒（CEVd） ； 類病毒衍生的小片段RNA ； 蕃茄 ； bio-index ； dot blot ； RT-PCR ； real-time RT-PCR ； 病程相關基因 (pathogenesis-related genes) ； 水楊酸 ； 乙烯 ； 茉莉酸

並列摘要

Citrus exocortis viroid (CEVd) is the casual agent of citrus exocortis disease, which has great impact on citrus crops. Easily spread by mechanical means, control of viroid diseases largely depends on the quarantine inspection and field eradication. Furthermore, without encoding any protein or helper virus, viroids can replicate, traffic systemically and cause disease symptoms. Recent studies reported that viroid-derived small RNAs (vd-sRNAs), about 18-24 nt, were detected in viroid-infected plants and might be related to the cause of symptom through RNA silencing pathway. In order to achieve control of CEVd, we developed an integrative diagnostic protocol comprising bio-index, dot-blot hybridization, one-step RT-PCR, and real-time RT-PCR techniques for Citrus exocortis viroid (CEVd) detection. After infection by in vitro transcripts of CEVd dimer, tomato cultivar (cv.) Rutgers showed epinasity, vein necrosis and stunting, which were used to set up bio-index for CEVd, but cv. Double-Fortune showed much milder symptoms and lower bio- index scores compared to Rutgers. Similarly, CEVd isolated from citrus in field could cause typical viroid symptoms on cv. Rutgers. Through in vitro transcript of CEVd monomer mixed with healthy tomato RNA, sensitivity limitations of dot-blot hybridization, one-step RT-PCR, and real-time RT- PCR were found at which were 109, 108, 107 copy number of CEVd, respectively. Also, by these methods, CEVd signals could be detected from total RNA extracted from suspected CEVd-infected citrus in field. These methods provide consistent, and sensitive results for future quarantine and prevention applications of this disease. On the other hand, for studying viroid-host interaction, we found that distinguishable symptoms of tomato plants might not be related to the levels of CEVd genomic RNA and CEVd-sRNAs. However, expression of pathogenesis-related genes (PR) 1a, PR- 1b, PR-2b and an ethylene-synthesizing enzyme, 1-aminocyclopropane-1-carboxylic acid oxidase (ACO1), were found up-regulated after CEVd infection; whereas, LoxD, involved in jasmonic acid (JA) synthesis pathway, did not change; however, proteinase inhibitor II (PI-II), a JA-responsive gene, was down-regulated. The results implied that distinguishable viroid symptom might be resulted from other factors, and that salicylic acid (SA) and ethylene pathway might be involved in defense responses to CEVd, but JA might be suppressed. This study developed diagnosis and detection methods of CEVd and further helped understanding the molecular mechanism of viroid- host interactions.

並列關鍵字

Citrus Exocortis Viroid (CEVd) ； viroid-derived small RNAs (vd-sRNAs) ； tomato ； bio-index ； dot blot ； RT-PCR ； real-time RT-PCR ； pathogenesis-related genes (PR genes) ； salicylic acid (SA) ； ethylene ； jasmonic acid (JA)

參考文獻

Bernad, L., and Duran-Vila, N. (2006). A novel RT-PCR approach for detection and characterization of citrus viroids. Mol Cell Probes 20, 105-113.

Bleecker, A.B., and Kende, H. (2000). Ethylene: a gaseous signal molecule in plants. Annu Rev Cell Dev Biol 16, 1-18.

Branch, A.D., and Robertson, H.D. (1984). A replication cycle for viroids and other small infectious RNA's. Science 223, 450-455.

Breijo, F., Garro, R., and Conejero, V. (1990). C7(P32) and C6(P34) PR proteins induced in tomato leaves by Citrus exocortis viroid infection are chitinase. Physiol Mol Plant P 36, 249-260.

Brodersen, P., and Voinnet, O. (2006). The diversity of RNA silencing pathways in plants. Trends in Genetics 22, 268-280.