Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (p. 212-238). Also available in print.

Thesis (Ph. D.)--University of Hong Kong, 2010. / Includes bibliographical references (p. 212-238). Also available online.

Modern agriculture is dependent on both global supply chains and crop monocultures. These features aid the evolution and spread of novel plant pathogens. Limited genetic diversity in commercial crop lines can result in widespread susceptibility to emerging pathogens. Pathogen resistance may be developed through conventional breeding approaches, or a number of transgenic strategies. This thesis focuses on the characterisation of an emerging maize disease, Maize lethal necrosis (MLN), and engineering resistant maize lines using an artificial microRNA (amiRNA) approach. MLN is a synergistic viral disease caused by the interaction of Maize chlorotic mottle virus (MCMV) with any maize-infecting member of the potyviridae. I used next-generation RNA sequencing to characterise the MLN outbreak in East Africa, discovering that local and Chinese strains of the potyvirus Sugarcane mosaic virus (SCMV) typically coinfect with MCMV. A first global MCMV phylogeny was constructed using these samples combined with new Sanger sequencing of samples in Ecuador and Hawaii. The phylogeny supported previous hypotheses of a link between the Chinese and African outbreaks, and suggested a novel link between the Hawaiian and Ecuadorian outbreaks. The SCMV sequences generated demonstrated strong evidence of extensive recombination, in line with previous reports on SCMV and potyviruses. These data also produced first reports of a number of RNA viruses in East Africa, and five novel viral-like sequences, with their presence confirmed by RT-PCR. RNA silencing is an important component of the plant immune response to viral infection. amiRNAs can be used to generate specific and effective viral resistance through Watson- Crick base pairing between the amiRNA and the (RNA) viral genome. Previous amiRNA approaches have targeted invariable genomic regions using consensus sequences. However, the high mutation rate of RNA viruses means single cells contain a variety of mutant genomes, collectively called a quasispecies. To deter the evolution of resistance breaking I devised a novel strategy to include intra-sample variation from NGS data in amiRNA design, and constructs, each containing five of these amiRNAs, were transformed into tropical maize lines. RNA silencing may be hampered by the expression of viral suppressors of silencing (VSRs). Local VSR assays demonstrated that there are no local VSRs in the MCMV genome, while systemic VSR assays showed a possible systemic VSR role for the unique P32 protein, and an interesting link between photoperiod and systemic silencing more generally.

Human cytomegalovirus (HCMV) is a widespread human herpesvirus pathogen and prototypical member of the β-herpesvirus subfamily. Like all herpesviruses, the virus establishes a lifelong latent infection following host exposure, which has the potential to reactivate periodically and contribute to recurrent disease processes. In individuals with weak or compromised immune systems, such reactivation can lead to profound pathology. Understanding how latent infections are maintained is important for uncovering how HCMV causes disease. The study of viral genes that are expressed during latent infection grants insight into how latency is regulated and how it could be therapeutically targeted. To that end, this project has sought to evaluate the functional significance of one such viral gene termed LUNA in the context of latency. In models of experimental latent infection based on primary myeloid cells, levels of viral gene transcription were found to be significantly reduced following infection with LUNA deletion mutant viruses, consistent with corresponding observable changes in post-translational histone modifications over the viral promoters of latency-associated genes. Additionally, using luciferase reporter systems, latency-associated viral gene promoters became activated in response to the expression of wild-type LUNA. Together, these findings argue for a role of LUNA in regulating viral gene expression during latent HCMV infection. One possible mechanism by which LUNA may fulfil its role is by targeting cellular ND10 structures, known intrinsic inhibitors of herpesvirus gene expression, for disruption. In support of this, latently infected cells were found to be devoid of ND10, a phenotype that was recapitulated by the direct expression of wild-type LUNA. Furthermore, mutation studies confirmed the identification of a novel deSUMOylase activity encoded by LUNA that was responsible for mediating ND10 disruption. Use of a catalytically inactive LUNA mutant in transcriptional analyses of latent infection also generated similar results as with the LUNA deletion viruses. Overall, these data support the hypothesis that LUNA serves as an important regulator of viral gene expression during latency, which is likely linked to its ability to target ND10 structures for disruption, thus raising the possibility that inhibition of deSUMOylation may serve as a novel therapeutic strategy to target latent HCMV infection.