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

Abstract

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.