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Identifier 000376026
Title Μελέτη του βιολογικού κύκλου του ιοειδούς Potato spindle tuber viroid σε φυτά Nicotiana benthamiana που είναι κατεσταλμένα για τις πρωτεΐνες τύπου Dicer
Alternative Title Insights in the biological cycle of potato spindle tuber viroid in dicer-like knock down
Author Νταντάμη, Ελένη Ιω.
Thesis advisor Καλαντίδης, Κρίτων
Reviewer Τσαγρή, Ευθυμία
Αβέρωφ, Μιχάλης
Δελιδάκης, Χρήστος
Κοτζαμπάσης, Κυριάκος
Βόντας, Ιωάννης
Πυρίντσος, Στέργιος
Abstract RNA silencing is a wide spead mechanism that negatively regulates gene expression. It is involved in different developmental phenomena in higher eukaryotes like cancerogenesis in mammals and defense against viruses and transposable elements in plant kingdom. By now many different RNA silencing pathways are identified. Key players of the mechanism are the small RNAs (21-24 nuceotides long), that are produced by RNAseIII Dicer-like proteins (DCLs). The role of small RNAs is governed by the DCL that generated it. Each DCL produces a distinct size of small RNA, which target homologous RNA and DNA sequences for degradation or methylation, respectively. Viroids are minimal pathogens that infect plants systemically and locally. Their gemone consists of a single stranded, naked RNA molecule that does not code for any protein. Viroids, or their double stranded RNA intermediates are substrate for DCLs. The produced small RNAs are biologically active in trans, but most probably not in cis, since viroids infectivity and titer are not reduced. The aim of this thesis is to understand i) how RNA silencing machinery influences the biological cycle of Potato tuber spindle tuber viroid (PSTVd) and ii) how PSTVd influences RNA silencing pathways. Previous studies in our laboratory identified DCL fragments from the non sequenced organisms Nicotiana benthamiana and Lycopersicon esculentum. These enzymes are both functionally and structurally conserved in eukaryotes. The isolated DCL fragments were used for the production of constucts that suppress DCLs expression in both organisms. In this work, we produced N. benthamiana plants, which are knocked down for the four plant Dicer-like proteins. These plants were used for monitoring the infectivity of PSTVd. In more detail, i) we investigated the role of each DCL in PSTVd processing, ii) we performed time course analysis of PSTVd infection in whole plant level and iii) we analyzed PSTVd replication in cellular level. Our results indicate that DCL2, DCL3 and DCL4 process PSTVd, since we could detect 22- , 24- and 21 nt small RNAs, respectively. In addition, the detection of plus and minus polarity small RNAs suggest that double stranded PSTVd molecules are substrate for the above DCLs. The role of DCL1 on PSTVd processing was elusive. Our results for PSTVd infectivity in whole plant analysis suggest an active role for DCL1 and DCL4. This was not observed in cellular level, though. We conclude that DCL1 and DCL4 are essential, at least at the first stages of viroid´s systemic spread, indicating an unexpected auxilliary role of RNA silencing on PSTVd infection. Finally, we have shown that PSTVd does not revert already established virus induced gene silencing, but it delays its establishment. Overall, we hypothesize that although PSTVd is targeted by RNA silencing it has evolved to use the latter to its benefit. auxilliary role of RNA silencing on PSTVd infection. Finally, we have shown that PSTVd does not revert already established virus induced gene silencing, but it delays its establishment. Overall, we hypothesize that although PSTVd is targeted by RNA silencing it has evolved to use the latter to its benefit.
Language Greek
Subject RNA σίγηση
Ατράκτωση του κονδύλου της πατάτας
Ιοειδές
Μικρά RNAS
Issue date 2012-11-01
Collection   Faculty/Department--Faculty of Sciences and Engineering--Department of Biology--Doctoral theses
  Type of Work--Doctoral theses
Permanent Link https://elocus.lib.uoc.gr//dlib/c/3/2/metadata-dlib-1351670790-254861-22148.tkl Bookmark and Share
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