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

Genome-Wide RNAi of C. elegans Using the Hypersensitive rrf-3 Strain Reveals Novel Gene Functions

RNA-mediated interference (RNAi) is a method to inhibit gene function by introduction of double-stranded RNA (dsRNA). Recently, an RNAi library was constructed that consists of bacterial clones expressing dsRNA, corresponding to nearly 90% of the 19,427 predicted genes of C. elegans. Feeding of this RNAi library to the standard wild-type laboratory strain Bristol N2 detected phenotypes for approximately 10% of the corresponding genes. To increase the number of genes for which a loss-of-function phenotype can be detected, we undertook a genome-wide RNAi screen using the rrf-3 mutant strain, which we found to be hypersensitive to RNAi. Feeding of the RNAi library to rrf-3 mutants resulted in additional loss-of-function phenotypes for 393 genes, increasing the number of genes with a phenotype by 23%. These additional phenotypes are distributed over different phenotypic classes. We also studied interexperimental variability in RNAi results and found persistent levels of false negatives. In addition, we used the RNAi phenotypes obtained with the genome-wide screens to systematically clone seven existing genetic mutants with visible phenotypes. The genome-wide RNAi screen using rrf-3 significantly increased the functional data on the C. elegans genome. The resulting dataset will be valuable in conjunction with other functional genomics approaches, as well as in other model organisms

RDE-2 interacts with MUT-7 to mediate RNA interference in Caenorhabditis elegans

In Caenorhabditis elegans, the activity of transposable elements is repressed in the germline. One of the mechanisms involved in this repression is RNA interference (RNAi), a process in which dsRNA targets cleavage of mRNAs in a sequence-specific manner. The first gene found to be involved in RNAi and transposon silencing in C.elegans is mut-7, a gene encoding a putative exoribonuclease. Here, we show that the MUT-7 protein resides in complexes of ∼250 kDa in the nucleus and in the cytosol. In addition, we find that upon triggering of RNAi the cytosolic MUT-7 complex increases in size. This increase is independent of the presence of target RNA, but does depend on the presence of RDE-1 and RDE-4, two proteins involved in small interfering RNA (siRNA) production. Finally, using a yeast two-hybrid screen, we identified RDE-2/MUT-8 as one of the other components of this complex. This protein is encoded by the rde-2/mut-8 locus, previously implicated in RNAi and transposon silencing. Using genetic complementation analysis, we show that the interaction between these two proteins is required for efficient RNAi in vivo. Together these data support a role for the MUT-7/RDE-2 complex downstream of siRNA formation, but upstream of siRNA mediated target RNA recognition, possibly indicating a role in the siRNA amplification step

Functional diversification of Argonautes in nematodes:an expanding universe

In the last decade, many diverse RNAi (RNA interference) pathways have been discovered that mediate gene silencing at epigenetic, transcriptional and post-transcriptional levels. The diversity of RNAi pathways is inherently linked to the evolution of Ago (Argonaute) proteins, the central protein component of RISCs (RNA-induced silencing complexes). An increasing number of diverse Agos have been identified in different species. The functions of most of these proteins are not yet known, but they are generally assumed to play roles in development, genome stability and/or protection against viruses. Recent research in the nematode Caenorhabditis elegans has expanded the breadth of RNAi functions to include transgenerational epigenetic memory and, possibly, environmental sensing. These functions are inherently linked to the production of secondary siRNAs (small interfering RNAs) that bind to members of a clade of WAGOs (worm-specific Agos). In the present article, we review briefly what is known about the evolution and function of Ago proteins in eukaryotes, including the expansion of WAGOs in nematodes. We postulate that the rapid evolution of WAGOs enables the exceptional functional plasticity of nematodes, including their capacity for parasitism

New insights into control of arbovirus replication and spread by insect RNA interference pathways

Arthropod-borne (arbo) viruses are transmitted by vectors, such as mosquitoes, to susceptible vertebrates. Recent research has shown that arbovirus replication and spread in mosquitoes is not passively tolerated but induces host responses to control these pathogens. Small RNA-mediated host responses are key players among these antiviral immune strategies. Studies into one such small RNA-mediated antiviral response, the exogenous RNA interference (RNAi) pathway, have generated a wealth of information on the functions of this mechanism and the enzymes which mediate antiviral activities. However, other small RNA-mediated host responses may also be involved in modulating antiviral activity. The aim of this review is to summarize recent research into the nature of small RNA-mediated antiviral responses in mosquitoes and to discuss future directions for this relatively new area of research

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

The HIrisPlex-S system for eye, hair and skin colour prediction from DNA: Introduction and forensic developmental validation

Forensic DNA Phenotyping (FDP), i.e. the prediction of human externally visible traits from DNA, has become a fast growing subfield within forensic genetics due to the intelligence information it can provide from DNA traces. FDP outcomes can help focus police investigations in search of unknown perpetrators, who are generally unidentifiable with standard DNA profiling. Therefore, we previously developed and forensically validated the IrisPlex DNA test system for eye colour prediction and the HIrisPlex system for combined eye and hair colour prediction from DNA traces. Here we introduce and forensically validate the HIrisPlex-S DNA test system (S for skin) for the simultaneous prediction of eye, hair, and skin colour from trace DNA. This FDP system consists of two SNaPshot-based multiplex assays targeting a total of 41 SNPs via a novel multiplex assay for 17 skin colour predictive SNPs and the previous HIrisPlex assay for 24 eye and hair colour predictive SNPs, 19 of which also contribute to skin colour prediction. The HIrisPlex-S system further comprises three statistical prediction models, the previously developed IrisPlex model for eye colour prediction based on 6 SNPs, the previous HIrisPlex model for hair colour prediction based on 22 SNPs, and the recently introduced HIrisPlex-S model for skin colour prediction based on 36 SNPs. In the forensic developmental validation testing, the novel 17-plex assay performed in full agreement with the Scientific Working Group on DNA Analysis Methods (SWGDAM) guidelines, as previously shown for the 24-plex assay. Sensitivity testing of the 17-plex assay revealed complete SNP profiles from as little as 63 pg of input DNA, equalling the previously demonstrated sensitivity threshold of the 24-plex HIrisPlex assay. Testing of simulated forensic casework samples such as blood, semen, saliva stains, of inhibited DNA samples, of low quantity touch (trace) DNA samples, and of artificially degraded DNA samples as well as concordance testing, demonstrated the robustness, efficiency, and forensic suitability of the new 17-plex assay, as previously shown for the 24-plex assay. Finally, we provide an update of the publically available HIrisPlex website https://hirisplex.erasmusmc.nl/, now allowing the estimation of individual probabilities for 3 eye, 4 hair, and 5 skin colour categories from HIrisPlex-S input genotypes. The HIrisPlex-S DNA test represents the first forensically validated tool for skin colour prediction, and reflects the first forensically validated tool for simultaneous eye, hair and skin colour prediction from DNA