Dark green islands and gene silencing

Abstract

Dark green islands (DGIs) form within the chlorotic leaves of plants systemically infected with a mosaic virus. These DGIs are regions of leaf tissue that are dark green in colour, free of virus and resistant to superinfection by the initiating virus. Cytological studies have shown that DGIs resemble healthy tissue and contain none of the developmental and chloroplastic abnormalities that are typical of virus-infected tissue. Evidence presented in this thesis supports the hypothesis that the recently discovered phenomenon of post-transcriptional gene silencing (PTGS) is active in the formation and maintenance of DGIs. In plants transgenic for the tamarillo mosaic virus (TaMV) coat protein (CP), the TaMV CP RNA was degraded in DGIs induced by infection with TaMV but not in the surrounding virus-infected tissue. Additionally, co-infection of non-transgenic plants with TaMV and white clover mosaic potexvirus (WCIMV) containing the coding region of the TaMV CP (WTamCP) produced DGIs from which both viruses were excluded. Conversely, WCIMV that did not include the TaMV CP sequence was easily detected within TaMV-induced DGIs. A theory for the formation of DGIs by PTGS is proposed. The theory explains their random and relatively infrequent occurrence by linking their initiation to the breakdown of the nuclear membrane during mitosis and accounts for their limited size by the dilution of a silencing signal. Plants transgenic for the TaMV CP express the transgene RNA at levels easily detected by Northern analysis, so it was initially supposed that no PTGS against the TaMV CP was occurring in these plants. However, long-term infections of WTamCP in these plants produced a surprising number of WTamCP derivatives where portions of the TaMV CP sequence had been excised. This contrasted with a single major derivative present in wild-type plants infected with WTamCP. Analysis of the WTamCP derivatives provided evidence to support their generation via a series of similarity-assisted RNA recombination events. Nucleotide frequencies were analysed and several significant trends were detected. A model for the increase in recombination events in the transgenic line is proposed, based upon low levels of small RNAs complementary to the TaMV CP sequence increasing the frequency of template-switching by the viral RNA polymerase.