RNA silencing is a form of post-transcriptional gene regulation, in which a small RNA guides a member of the Argonaute protein family in an effector complex to repress target gene expression. piRNAs, found in germ cells, are the most recently discovered major subset of small RNAs. A key known function of piRNA is to repress the transposable elements in the germline and maintain the germline genome integrity. The defining features of the piRNAs are 1) they are ubiquitously methylated at the 3’-end of the 2’-OH group by methyltransferase Hen1; 2) they associate exclusively with the Piwi subfamily Argonaute proteins. Much is not understood about the biogenesis and the regulation of the piRNA pathway. One of the fundamental questions is how the 3’-end of the piRNA is generated and recognised specifically by Piwi but not by Ago subfamily Argonaute proteins. In this thesis, the high resolution crystal structure of the Aubergine PAZ domain, a domain from a Piwi subfamily Argonaute, bound to a 7 mer single-stranded methylated piRNA ‘mimic’, reveals the mode of recognition for the 3’-end of piRNAs by Piwi subfamily Argonautes. The structure provides the molecular basis for why Piwi but not Ago PAZ domains preferentially bind to RNAs with 2’-O-methylation at the 3’-end, thus conferring substrate specificity. The structural results are confirmed by biochemical studies. Biochemical and biophysical studies on the methyltransferase Hen1 have provided insights into substrate specificity for piRNA 3’-end methylation and revealed a potential regulatory role for the C-terminal region of the protein. Extensive biochemical analysis defined a minimal active Hen1/short RNA complex, though crystallisation screening yielded no crystals for structure determination. Overall, this study provides insights into the generation and molecular recognition of the piRNA in animals.

Plant viruses can cause many plant diseases, which result in substantial damage to crop production. To overcome viral infections, plants evolved RNA silencing which can recognise viral RNAs during their replications and slice them into small RNA (sRNA) using antiviral nucleases called DICER or Dicer-like (DCL). The resulting virus-derived small interfering RNA (vsiRNA, 21-24 nucleotides) then guides effector nucleases, namely ARGONAUTE (AGO), to cleave viral RNAs in the cytoplasm in a nucleotide-specific manner. However, the activity of vsiRNA is not restricted to the control of viral RNA accumulation. Virus-derived sRNAs can regulate host gene expression if host mRNAs share sequence complementarity with vsiRNAs. Interestingly, vsiRNAs are also able to target and methylate homologous DNA sequences in the nucleus indicating that vsiRNAs have potential to regulate endogenous genes at transcriptional level by modifying the epigenetic status of gene promoter sequences. This mechanism is referred to as transcriptional gene silencing (TGS). Thus, RNA silencing opens up new strategies to stably and heritably alter gene expression in plants. However, the mechanisms and efficacy of plant virus-induced TGS are largely unknown. The aim of my PhD was to investigate the molecular and environmental factors that are involved in virus-induced epigenetic modifications in the infected plants and in their progeny. First, I examined the required sequence complementary between sRNAs and their nuclear target sequence. I demonstrated for the first time that nuclear-imported vsiRNAs can induce RNA-directed DNA methylation (RdDM) and subsequently heritable virus-induced transcriptional gene silencing (ViTGS) even when they do not share 100% nucleotide sequence complementarity with the target DNA. This finding reveals a more dynamic interaction between viral RNAs and the host epigenome than previously thought. Secondly, I explored how environmental stimuli such as light and temperature can affect the efficacy of ViTGS. I found that ViTGS is greatly inhibited at high temperature. Using RNA-seq, I established that inefficient ViTGS at high temperature is due to the limited production of secondary sRNAs that may limit the initiation, amplification and spreading of virus-induced DNA methylation to neighbouring cells and down generations. Lastly, I studied the link between the viral suppressors of RNA silencing (VSRs): viral proteins that can interfere with plant RNA silencing and ViTGS. I established that VSRs of certain viruses can impair TGS in infected tissues, suggesting that viruses may alter the epigenome and consequently plant gene expression in the infected plants and their progeny. Collectively, my work reveals how viruses can re-program the epigenome of infected plants, and deepens our knowledge of how we can harness pathogens to modify the epigenome for plant breeding.

The basidiomycete cereal pathogens Ustilago hordei and U. maydis are closely related and possess genomes with a high degree of homology and synteny. I report on the disparity of the RNAi phenomenon between U. hordei and U. maydis. Using an RNAi expression vector I targeted both a GUS transgene and an endogenous mating-type gene and confirmed the presence of double-stranded (ds)RNA in transgenic cells of both species. However, down-regulation of the GUS gene and production of siRNAs were seen only in U. hordei. The biological effect was a reduction in GUS protein and activity, and reduced mating only in U. hordei. In support of this experimental evidence, homologs to Dicer and Argonaute were found in the U. hordei genome but not in the published U. maydis genome. Interestingly, preliminary U. hordei sequences reveal conservation and synteny in U. maydis in the regions spanning these loci, with the only noticeable difference being the lack of Dicer and Argonaute genes in U. maydis. U. maydis also appears to differ from U. hordei with respect to genes presumed to be involved in transcriptional gene silencing and also has far fewer transposons in its genome. Efforts to clone the avirulent gene UhAvr1 led to a locus containing a large number of small proteins predicted to be secreted. This locus appears to be heterochromatic and is orthologous to the largest cluster of secreted proteins in U. maydis. Other laboratories have reported that deletion of this cluster in U. maydis results in a dramatic reduction in virulence. Genetic evidence for an avirulence gene at this locus in U. hordei suggests that the locus may also be important for U. hordei. Differences between these two smut fungi at this locus and at others identified in this study point to key differences in gene regulation and genome evolution.

The basidiomycete cereal pathogens Ustilago hordei and U. maydis are closely related and possess genomes with a high degree of homology and synteny. I report on the disparity of the RNAi phenomenon between U. hordei and U. maydis. Using an RNAi expression vector I targeted both a GUS transgene and an endogenous mating-type gene and confirmed the presence of double-stranded (ds)RNA in transgenic cells of both species. However, down-regulation of the GUS gene and production of siRNAs were seen only in U. hordei. The biological effect was a reduction in GUS protein and activity, and reduced mating only in U. hordei. In support of this experimental evidence, homologs to Dicer and Argonaute were found in the U. hordei genome but not in the published U. maydis genome. Interestingly, preliminary U. hordei sequences reveal conservation and synteny in U. maydis in the regions spanning these loci, with the only noticeable difference being the lack of Dicer and Argonaute genes in U. maydis. U. maydis also appears to differ from U. hordei with respect to genes presumed to be involved in transcriptional gene silencing and also has far fewer transposons in its genome. Efforts to clone the avirulent gene UhAvr1 led to a locus containing a large number of small proteins predicted to be secreted. This locus appears to be heterochromatic and is orthologous to the largest cluster of secreted proteins in U. maydis. Other laboratories have reported that deletion of this cluster in U. maydis results in a dramatic reduction in virulence. Genetic evidence for an avirulence gene at this locus in U. hordei suggests that the locus may also be important for U. hordei. Differences between these two smut fungi at this locus and at others identified in this study point to key differences in gene regulation and genome evolution.

Viruses are ubiquitous in natural growth environments and cause severe losses to crop yields, globally. Approximately 30% of plant viruses described to date are grouped within the family Potyviridae, making it one of the largest plant virus families. Furthermore, certain potyvirus species can cause devastating diseases in several agriculturally and economically important crops. Hence, gaining insight into potyvirus resistance and recovery mechanisms in plants is an important research focus. This thesis firstly explores how environmental cues can modulate the activity of a central form of viral defence, namely RNA silencing. Specifically, high temperatures and low light intensities were found to increase the efficacy of viral RNA silencing in Arabidopsis, resulting in recovery from infection by Turnip Mosaic Virus. The biological context and potential for agricultural exploitation of these phenomena are discussed. Secondly, this thesis explores the ability to engineer resistance alleles using the latest genome editing techniques. Specifically, resistance to Turnip Mosaic Virus was successfully engineered in Arabidopsis by CRISPR/Cas9-induced deletion of a known susceptibility factor eIF(iso)4E. Biotechnological methods to implement this proof of concept research in crop species were also investigated.

The basidiomycete cereal pathogens Ustilago hordei and U. maydis are closely related and possess genomes with a high degree of homology and synteny. I report on the disparity of the RNAi phenomenon between U. hordei and U. maydis. Using an RNAi expression vector I targeted both a GUS transgene and an endogenous mating-type gene and confirmed the presence of double-stranded (ds)RNA in transgenic cells of both species. However, down-regulation of the GUS gene and production of siRNAs were seen only in U. hordei. The biological effect was a reduction in GUS protein and activity, and reduced mating only in U. hordei. In support of this experimental evidence, homologs to Dicer and Argonaute were found in the U. hordei genome but not in the published U. maydis genome. Interestingly, preliminary U. hordei sequences reveal conservation and synteny in U. maydis in the regions spanning these loci, with the only noticeable difference being the lack of Dicer and Argonaute genes in U. maydis. U. maydis also appears to differ from U. hordei with respect to genes presumed to be involved in transcriptional gene silencing and also has far fewer transposons in its genome. Efforts to clone the avirulent gene UhAvr1 led to a locus containing a large number of small proteins predicted to be secreted. This locus appears to be heterochromatic and is orthologous to the largest cluster of secreted proteins in U. maydis. Other laboratories have reported that deletion of this cluster in U. maydis results in a dramatic reduction in virulence. Genetic evidence for an avirulence gene at this locus in U. hordei suggests that the locus may also be important for U. hordei. Differences between these two smut fungi at this locus and at others identified in this study point to key differences in gene regulation and genome evolution. / Science, Faculty of / Botany, Department of / Graduate

No description available.

RNA silencing is a cellular mechanism of post-transcriptional gene regulation which is highly conserved among the plant and animal kingdoms of life, and plays a critical part of developmental biology, maintenance of homeostasis, and host-pathogen interactions. The pathway is engaged by small double-stranded (ds)RNA molecules (small RNAs), which effect sequence specific gene silencing by targeting complementary RNA sequences. There are several classes of small RNAs which engage the pathway. MicroRNAs (miRNAs) are expressed in the genome as endogenous regulators of gene expression. Short-interfering RNAs (siRNAs) are usually from exogenous sources such as viral-derived short-interfering RNAs, or synthetic siRNAs which are applied to cells or organisms to inhibit expression of specific genes. The p19 protein is a viral suppressor of RNA silencing (VSRS) endogenous to tombusviruses, which binds small RNA duplexes of any sequence with extremely high affinity. Because of its unique binding properties, recombinant p19 proteins are an excellent platform for tool development surrounding the RNA silencing pathway and are used extensively in novel applications for modulating the activity of small RNAs in living systems and for detecting small RNAs in biological samples. Herein we present work that has increased the breadth of p19’s utility as a biotechnology tool in three distinct realms. First, we present a chemical biology approach which combines p19 and small molecules for potent inhibition of the RNA silencing pathway in human cells. Secondly, we present the development of a novel fusion protein between p19 and a cell penetrating peptide (CPP), which functions as an siRNA delivery agent to allow gene knockdown in human cells. Thirdly, we have improved the utility of p19 for detecting and sequestering human miRNAs through rationally designing the binding surface; we describe mutations which dramatically enhance p19's affinity for human miRNA-122. The work presented here adds to the growing repertoire of engineered RNA binding proteins (RBPs) as tools for studying small RNA molecules and modulating their activity for applications in human therapeutics.

Chez les plantes, le RNA silencing (RNAi) est le principal mécanisme de défense antivirale. Il est opéré par de petites molécules d’ARN (siRNA), de 21-22nt de long, générées à partir de l’ARN viral par DCL4 et DCL2, respectivement. Ces siRNA confèrent la séquence-spécificité des réactions de défense intracellulaire et peuvent se déplacer à longue distance pour immuniser les cellules saines. En conséquence, les virus ont développé des protéines (VSRs) capables de supprimer ces deux aspects du RNAi. Au cours de cette thèse, j’ai pu démontrer in vivo que la protéine P15 du Peanut clump virus (PCV) est capable de séquestrer les siRNA de 21 et 22nt et qu’elle bloque le mouvement de ces derniers plus efficacement que ceux de 21nt. Pour compenser cette faiblesse, au cours de l’infection par le PCV, P15 est transportée à l’intérieur des peroxisomes en association avec les siRNA qu’elle séquestre. Le confinement des siRNA mobiles de 21nt à l’intérieur de ces organelles conduit à une inhibition du RNAi systémique et stimule fortement la propagation du PCV à travers la plante. Ces travaux définissent une nouvelle stratégie de pathogénèse virale au cours de laquelle une organelle est utilisé pour neutraliser des molécules de défense produites par l’hôte. / In plants, RNA interference (RNAi) is the main antiviral defense mechanism. It is initiated through the processing of viral RNA into 21-22nt long siRNA by DCL4 and DCL2, respectively. These siRNA can mediate sequence-specific local defense reactions (cell-autonomous RNAi) or move to distant tissues to prime defenses in naive cells (systemic RNAi). Consequently, viruses have evolved proteins (VSRs) to suppress both aspects of RNAi. In this in vivo study, I show that P15, the VSR of Peanut clump virus (PCV), binds and sequesters both 21nt and 22nt siRNA. Importantly, it stops the movement of 22nt siRNA more efficiently than 21nt siRNA. During infection, P15 is shuttled into peroxisomes, and is able to « piggyback » siRNA into these organelles. By confining mobile DCL4-dependent antiviral 21nt siRNA within peroxisomes, P15 is able to shut down systemic RNAi and strongly promote PCV movement. This work describes a novel pathogenic strategy in which an organelle is used to neutralize host defensive molecules.

Meiosis, the core engine of sexual reproduction, is a complex process that results in the production of recombinant haploid genomes. In the meioses of Neurospora, worms and mice, gene expression from DNA that lacks a pairing partner is silenced. We posit that this is a two-step process. First, a process called meiotic trans-sensing compares the chromosomes from each parent and identifies significant differences as unpaired DNA. Second, if unpaired DNA is identified, a process called meiotic silencing inhibits expression of genes within the unpaired region and regions sharing sequence identity. Meiotic silencing is mechanistically most likely related to RNAi in other eukaryotes. We used a combination of forward and reverse genetic strategies aimed at understanding the mechanisms of meiotic trans-sensing and meiotic silencing. Here, we present genetic evidence that arguably differentiates the meiotic transsensing step from meiotic silencing, by demonstrating that DNA methylation affects sensing of specific allele-types without interfering with silencing in general. We also determined that DNA sequence is an important parameter scrutinized during meiotic trans-sensing. This, and other observations, led us to hypothesize meiotic recombination as the mechanism for meiotic trans-sensing. However, we find that mutants of key genes required for recombination and chromosome pairing are not required for locus-specific meiotic silencing. We conclude that two interesting possibilities remain: meiotic trans-sensing occurs through a previously uncharacterized recombination pathway or chromosomal regions are carefully compared in the absence of recombination. Finally, forward genetics revealed a novel component of meiotic silencing, Sms-4, encoding the Neurospora ortholog of mammalian mRNP component ELG protein. Unlike previous loss-of-function mutants that abate meiotic silencing by unpaired DNA, Sms-4 is not required for successful meiosis, showing that meiosis and meiotic silencing are distinct, yet overlapping, phenomena. Intriguingly, SMS-4 is the first component to be localized with bulk chromatin in the nucleus, presumably the site of trans-sensing. Finally, we carried out a critical examination of the current evidence in the field and present alternative models for meiotic trans-sensing and meiotic silencing in Neurospora.