Expression and silencing of cowpea mosaic virus transgenes

Plant viruses are interesting pathogens because they can not exist without their hosts and exploit the plant machinery for their multiplication. Fundamental knowledge on viral processes is of great importance to understand, prevent and control virus infections which can cause drastic losses in crops. In this thesis, cowpea mosaic virus (CPMV) was studied. This virus consists of two, icosahedral particles that each carry a distinct single stranded RNA molecule of positive polarity. Several years of research have revealed much information on the genomic organisation, the strategy of gene expression and the multiplication processes of CPMV, which are described in Chapter 1, but also many aspects remain to be elucidated.To study individual viral processes, like replication, encapsidation or cell to cell movement, transgenic plants can be generated that express individual viral genes like the replicase, coat protein or movement protein gene. A prerequisite in this approach is the presence of an efficient and reliable plant regeneration and transformation system. (CPMV) 5 natural host is the tropical grain legume cowpea, Vigna unguiculata, a plant species that is recalcitrant at regeneration. Although in experiments described in Chapter 2 fertile plants could be regenerated from nodal thin cell layer segments, the explants were not competent for Agrobacterium-mediated transformation. Possibly in further studies, these nodal explants could prove suited for another transformation method.Therefore, tobacco, which is also a host for CPMV and highly competent for regeneration and transformation, was preferred as the species to generate transgenic plants carrying CPMV specific genes. Especially the CPMV movement proteins (MP) genes appealed to us for overexpression studies. CPMV cell to cell movement is enabled by the CPMV MPs that act to modify plasmodesmata. They are assumed to channel plasmodesmata with MP-containing tubular structures and through or with these tubules virus particles are transported to adjacent cells. To obtain more information on the plasmodesmatal modifications brought about by the MPs, transgenic tobacco plants were generated that carried the MP gene under the control of either a constitutive or an inducible 35S promoter. However, in none of these plants the MPs were expressed to detectable levels (Chapter 3). Using the potato virus X (PVX)-based expression vector, accumulation of CPMV MPs was observed in the form of tubular structures extending from the surface of infected protoplasts into the medium. These PVX-derivatives look promising for providing effective tools in future studies on the effects of the CPMV MPs in plants.Studies on MP functioning could involve complementation experiments with a CPMV mutant that is defective in cell to cell movement. In experiments described in Chapter 4 is was analysed by a molecular approach whether the CPMV mutant N123, that was first described in 1976, could be used to this effect. As the basis of the N123 specific phenotype was found not only to rest in the movement protein gene but also in one of the two coat protein genes, this mutant seemed not very suitable for complementation studies. Presumably a recently developed CPMV mutant in which the MP gene has been replaced by the fluorescent marker protein GFP (green fluorescent protein), will be a more appropriate tool.Transgenic Nicotiana benthamiana plants that were expressing either the CPMV MP or the replicase gene under the control of a constitutive promoter, were found to exhibit a resistant phenotype when inoculated with CPMV (Chapter 5). Protoplast studies revealed that the resistance occurred as full immunity and was maintained in the cell. Resistance was specific to viruses highly homologous to CPMV, and in addition it was found to be specifically directed against the replication of the CPMV segment of which the transgene was derived (Chapter 5). Pathogen derived resistance can be mediated either by the protein encoded by the transgene or by the transcribed mRNA. Protein -mediated resistance generally offers moderate protection against a broad range of viruses, while RNA-mediated resistance results in immunity at the cellular level. Resistance obtained in transgenic plants transformed with defective genes confirmed that an RNA-based mechanism was underlying the highly specific transgenic resistance against CPMV (Chapter 6).Specifically in the resistant lines, the transgene mRNA steady state levels were low compared to the relative transgene nuclear transcription rates (Chapter 6). This indicated that resistance occurs from a specific, cytoplasmic RNA turnover mechanism. This process can be regarded as a post- transcriptional gene-silencing process, that is primarily induced on the transgene mRNAs but to which also incoming, homologous CPMV genomes fall victim. In addition, heterologous RNA molecules, like PVX genomes, that contain the sequences corresponding to the transgene, are eliminated (Chapter 6). By inserting sequences homologous to only parts of the transgene in the genome of PVX and studying the fate of these recombinant genomes, it was shown that the degradation process is primarily targeted to a defined region of the transgene mRNA, the 3' region. Further analyses revealed that degradation can occur at various sites within this 3' region and that not a specific sequence or structure is of predominant importance. We observed that small inserts, like of only 60 nucleotides, can tag recombinant PVX molecules for the elimination process, albeit with reduced efficiency, which suggested that the RNA turnover process carries quantitative features.On the intruiging question why post-transcriptional gene-silencing is induced in only some of the transgenic lines, we revealed (Chapter 6) that the organisation of integrated transgene sequences has an important role. Transformation with a transgene containing a directly repeated MP gene, increased the frequency at which resistant lines arise to 60%, compared to 20% of resistant lines that occur upon transformation with a transgene with a single MP gene. Thus, the resistance process seems influenced by qualitative features of the integrated transgenes. Also, it was observed that resistance concurred with extensive methylation at the transcribed transgene sequences (Chapter 6), which could indicate an essential role of methylation at transcribed sequences in obtaining RNA-mediated pathogen derived resistance.From these observations and from data described in literature, a model for RNA-mediated virus resistance was made and presented in Chapter 6. In Chapter 7, the post-transcriptional gene-silencing phenomenon is discussed in more details and in addition an approach is presented by which the process could be exploited to efficiently engineer virus resistance or study plant gene expression

英語母語話者と日本人学習者の英語動詞運用

博士（学術）神戸大

FDSTools: A software package for analysis of massively parallel sequencing data with the ability to recognise and correct STR stutter and other PCR or sequencing noise

Molecular Technology and Informatics for Personalised Medicine and Healt

Modeling recursive RNA interference.

An important application of the RNA interference (RNAi) pathway is its use as a small RNA-based regulatory system commonly exploited to suppress expression of target genes to test their function in vivo. In several published experiments, RNAi has been used to inactivate components of the RNAi pathway itself, a procedure termed recursive RNAi in this report. The theoretical basis of recursive RNAi is unclear since the procedure could potentially be self-defeating, and in practice the effectiveness of recursive RNAi in published experiments is highly variable. A mathematical model for recursive RNAi was developed and used to investigate the range of conditions under which the procedure should be effective. The model predicts that the effectiveness of recursive RNAi is strongly dependent on the efficacy of RNAi at knocking down target gene expression. This efficacy is known to vary highly between different cell types, and comparison of the model predictions to published experimental data suggests that variation in RNAi efficacy may be the main cause of discrepancies between published recursive RNAi experiments in different organisms. The model suggests potential ways to optimize the effectiveness of recursive RNAi both for screening of RNAi components as well as for improved temporal control of gene expression in switch off-switch on experiments

An RIG-I-Like RNA Helicase Mediates Antiviral RNAi Downstream of Viral siRNA Biogenesis in Caenorhabditis elegans

Dicer ribonucleases of plants and invertebrate animals including Caenorhabditis elegans recognize and process a viral RNA trigger into virus-derived small interfering RNAs (siRNAs) to guide specific viral immunity by Argonaute-dependent RNA interference (RNAi). C. elegans also encodes three Dicer-related helicase (drh) genes closely related to the RIG-I-like RNA helicase receptors which initiate broad-spectrum innate immunity against RNA viruses in mammals. Here we developed a transgenic C. elegans strain that expressed intense green fluorescence from a chromosomally integrated flock house virus replicon only after knockdown or knockout of a gene required for antiviral RNAi. Use of the reporter nematode strain in a feeding RNAi screen identified drh-1 as an essential component of the antiviral RNAi pathway. However, RNAi induced by either exogenous dsRNA or the viral replicon was enhanced in drh-2 mutant nematodes, whereas exogenous RNAi was essentially unaltered in drh-1 mutant nematodes, indicating that exogenous and antiviral RNAi pathways are genetically distinct. Genetic epistatic analysis shows that drh-1 acts downstream of virus sensing and viral siRNA biogenesis to mediate specific antiviral RNAi. Notably, we found that two members of the substantially expanded subfamily of Argonautes specific to C. elegans control parallel antiviral RNAi pathways. These findings demonstrate both conserved and unique strategies of C. elegans in antiviral defense

The ERI-6/7 Helicase Acts at the First Stage of an siRNA Amplification Pathway That Targets Recent Gene Duplications

Endogenous small interfering RNAs (siRNAs) are a class of naturally occuring regulatory RNAs found in fungi, plants, and animals. Some endogenous siRNAs are required to silence transposons or function in chromosome segregation; however, the specific roles of most endogenous siRNAs are unclear. The helicase gene eri-6/7 was identified in the nematode Caenorhabditis elegans by the enhanced response to exogenous double-stranded RNAs (dsRNAs) of the null mutant. eri-6/7 encodes a helicase homologous to small RNA factors Armitage in Drosophila, SDE3 in Arabidopsis, and Mov10 in humans. Here we show that eri-6/7 mutations cause the loss of 26-nucleotide (nt) endogenous siRNAs derived from genes and pseudogenes in oocytes and embryos, as well as deficiencies in somatic 22-nucleotide secondary siRNAs corresponding to the same loci. About 80 genes are eri-6/7 targets that generate the embryonic endogenous siRNAs that silence the corresponding mRNAs. These 80 genes share extensive nucleotide sequence homology and are poorly conserved, suggesting a role for these endogenous siRNAs in silencing of and thereby directing the fate of recently acquired, duplicated genes. Unlike most endogenous siRNAs in C. elegans, eri-6/7–dependent siRNAs require Dicer. We identify that the eri-6/7–dependent siRNAs have a passenger strand that is ∼19 nt and is inset by ∼3–4 nts from both ends of the 26 nt guide siRNA, suggesting non-canonical Dicer processing. Mutations in the Argonaute ERGO-1, which associates with eri-6/7–dependent 26 nt siRNAs, cause passenger strand stabilization, indicating that ERGO-1 is required to separate the siRNA duplex, presumably through endonucleolytic cleavage of the passenger strand. Thus, like several other siRNA–associated Argonautes with a conserved RNaseH motif, ERGO-1 appears to be required for siRNA maturation

特集 生活習慣病

Molecular Technology and Informatics for Personalised Medicine and Healt

The DNA/RNA-Dependent RNA Polymerase QDE-1 Generates Aberrant RNA and dsRNA for RNAi in a Process Requiring Replication Protein A and a DNA Helicase

The Neurospora RNA-dependent RNA polymerase QDE-1 is an RNA polymerase that can use both RNA and DNA as templates, suggesting a new mechanism for small RNA production

PIWI Associated siRNAs and piRNAs Specifically Require the Caenorhabditis elegans HEN1 Ortholog henn-1

Small RNAs—including piRNAs, miRNAs, and endogenous siRNAs—bind Argonaute proteins to form RNA silencing complexes that target coding genes, transposons, and aberrant RNAs. To assess the requirements for endogenous siRNA formation and activity in Caenorhabditis elegans, we developed a GFP-based sensor for the endogenous siRNA 22G siR-1, one of a set of abundant siRNAs processed from a precursor RNA mapping to the X chromosome, the X-cluster. Silencing of the sensor is also dependent on the partially complementary, unlinked 26G siR-O7 siRNA. We show that 26G siR-O7 acts in trans to initiate 22G siRNA formation from the X-cluster. The presence of several mispairs between 26G siR-O7 and the X-cluster mRNA, as well as mutagenesis of the siRNA sensor, indicates that siRNA target recognition is permissive to a degree of mispairing. From a candidate reverse genetic screen, we identified several factors required for 22G siR-1 activity, including the chromatin factors mes-4 and gfl-1, the Argonaute ergo-1, and the 3′ methyltransferase henn-1. Quantitative RT–PCR of small RNAs in a henn-1 mutant and deep sequencing of methylated small RNAs indicate that siRNAs and piRNAs that associate with PIWI clade Argonautes are methylated by HENN-1, while siRNAs and miRNAs that associate with non-PIWI clade Argonautes are not. Thus, PIWI-class Argonaute proteins are specifically adapted to associate with methylated small RNAs in C. elegans