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.
| Main Author: | |
|---|---|
| Other Authors: | |
| Format: | Doctoral thesis biblioteca |
| Language: | English |
| Published: |
Landbouwuniversiteit Wageningen
|
| Subjects: | cowpea mosaic virus, cowpeas, gene expression, genetic engineering, pleiotropy, recombinant dna, vigna, genetische modificatie, genexpressie, koebonenmozaïekvirus, pleiotropie, vignabonen, |
| Online Access: | https://research.wur.nl/en/publications/expression-and-silencing-of-cowpea-mosaic-virus-transgenes |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|