| Summary: | 博士 === 國立中興大學 === 植物病理學系所 === 97 === Production of oriental melon (Cucumis melo L.) worldwide is often limited by infection by two potyviruses, the watermelon infecting Zucchini yellow mosaic virus (ZYMV) and Papaya ringspot virus W type (PRSV W) and. In order to engineer melon lines resistant to these potyviruses, a construct containing the coat protein (CP) sequences of these viruses was generated and used to transform an elite cultivar of oriental melon (Silver light) mediated by Agrobacterium using an improved cotyledon-cutting method. Altogether, our results indicated that RNA-mediated post-transcriptional gene silencing (PTGS) was the underlying mechanism of virus resistance of the transgenic melon lines. In order to further understand the relationship between plant defense and virus counteraction, we investigated the role of gene silencing suppressor, potyviral HC-Pro, which affects small RNA pathways involving in pathogenicity and symptom development. This dissertation is divided into five (including appendix) chapters as described below.
Chapter 1, “Literature review” describes references relevant to this study.
Chapter 2, 3 and 4 comprise the main text of this thesis, describing the engineering of transgenic melon lines resistant to ZYMV and PRSV W (Chapter 2 and 3) to solve the potyvirus problem in the field. We used binary vector harboring single (ZYMV) or double (ZYMV and PRSV W) CP constructs to transform an elite cultivar of oriental melon (Silver light) mediated by Agrobacterium using an improved cotyledon-cutting method. Removal of 1 mm portion from the proximal end of cotyledon greatly increased the frequency of transgenic regenerants by significantly decreasing the incidence of false positive and aberrant transformants. Southern hybridization analysis of transgenic lines revealed random insertion of the transgene in host genome, with insert numbers differing among transformants. Northern hybridization analysis evidenced an inverse correlation of the levels of accumulation of transgene transcript to the degrees of virus resistance, indicating post-transcriptional gene silencing (PTGS)-mediated transgenic resistance.
Chapter 4 describes “Discriminating mutations of HC-Pro of Zucchini yellow mosaic virus with differential effects on small RNA pathways involving viral pathogenicity and symptom development”. It is well known that the potyviral HC-Pro is a gene silencing suppressor. We sought to obtain molecular evidence on the roles of the three highly conserved amino acids, R180I (mutation A), F205L (B), and E396N (C) of HC-Pro in microRNA (miRNA) and small interfering RNA (siRNA) pathways related to viral pathogenicity and symptom development using transgenic Arabidopsis plants system. We demonstrated that amino acid residues 180, 205 and 396 of HC-Pro are critical in suppression of miRNA, trans-acting siRNA (ta-siRNA) and virus induced gene silencing (VIGS) pathways, but not sense-post transcriptional gene silencing (s-PTGS). Because the R180I/ E396N HC-Pro mutant does not interfere with miRNA and tasiRNA pathways the ZGAC mutant elicits only attenuated symptoms. Furthermore, the recovery seen on ZGAC-infected plants likely results from the weak VIGS suppression by the double AC mutations of HC-Pro. The findings of this study are useful to protect high levels of transgene expression and to genereate an attenuated potyvirus for control of virus by cross protection..
Chapter 5 (Appendix) entitled “Molecular evolution of a viral non-coding sequence under the selective pressure of amiRNA-mediated silencing” was carried out at Prof. Nam-Hai Chua’s laboratory, Rockefeller University, during my Ph.D program (2006-2008). The published paper is included as an appendix, because all the experiments of this project were carried out at Prof. Chua’s laboratory and this study was not directly connected to thesis. The main objective of this chaper was directed to determine, through artificial mutagnesis, the criticality of single nucleotide positions in the amiRNA-targeted sequence of 21 nucleotides, which were constructed in a potyvirual vector. Furthermore, the evolution of the virus through deletion and substitution in amiRNA-targrted sequence to escape amiRNA recognition was also investigated.
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