|RNA silencing in the infection of DNA and negative-strand RNA viruses
Prins, M.W. ; Ribeiro, S.G. ; Hemmes, J.C. ; Bucher, E.C. ; Goldbach, R.W. - \ 2006
In: Suppression and Circumvention of Host Defence by Plant Viruses, Haikko, Finland, 1 - 5 July, 2007. - Helsinki : - p. 34 - 34.
Multiple virus resistance at a high frequency using a single transgene construct
Bucher, E.C. ; Lohuis, H. ; Poppel, P.J. van; Dimitriadou, C. ; Goldbach, R.W. ; Prins, M.W. - \ 2006
Journal of General Virology 87 (2006)12. - ISSN 0022-1317 - p. 3697 - 3701.
rna-mediated resistance - spotted wilt virus - tospovirus resistance - interfering rnas - plants - sequence - length
RNA silencing is a natural antiviral defence in plants, which can be exploited in transgenic plants for preprogramming virus recognition and ensuring enhanced resistance. By arranging viral transgenes as inverted repeats it is thus possible to obtain strong repression of incoming viruses. Due to the high sequence specificity of RNA silencing, this technology has hitherto been limited to the targeting of single viruses. Here it is shown that efficient simultaneous targeting of four different tospoviruses can be achieved by using a single small transgene based on the production of minimal sized chimaeric cassettes. Due to simultaneous RNA silencing, as demonstrated by specific siRNA accumulation, the transgenic expression of these cassettes rendered up to 82% of the transformed plant lines heritably resistant against all four viruses. Thus RNA silencing can be further improved for high frequency multiple virus resistance by combining small RNA fragments from a series of target viruses
Antiviral RNA silencing viral counter defense in plants
Bucher, E.C. - \ 2006
Wageningen University. Promotor(en): R.W. Goldbach, co-promotor(en): M.W. Prins. - Wageningen : Wageningen Universiteit - ISBN 9789085044482 - 115
transgene planten - rna - plantenvirussen - ziekteresistentie - genexpressie - antiviruseigenschappen - interferentie - rna-virussen - transgenic plants - rna - interference - plant viruses - disease resistance - gene expression - antiviral properties - rna viruses
The research described in this thesis centres around the mechanism of RNA silencing in relation to virus-host interaction, an area of increasing importance. It shows how this recently disclosed mechanism can be used to produce virus-resistant plants. Based on the activity of the RNA silencing machinery, which is an important line of defense of plants against viruses, plants can be pre-programmed to produce so-called small-interfering RNAs (siRNAs) that specifically target the genome or transcripts of incoming viruses. This principle works like an antivirus software package on a computer, in which signatures recognizing specific viruses are included. Any incoming file potential virus) is scanned for the recognition sequence much like an siRNA does on an incoming viral RNA.
In order to produce multiple virus resistance the work presented in this thesis combined the very high silencing activity of double stranded RNA (dsRNA) with a fusion strategy involving small, 150 basepairs long segments of the nucleocapsid (N) gene of four different tomato-infecting tospoviruses. The virus-derived sequences were arranged in an inverted repeat leading to the production of dsRNA. Transgenic plants stably expressing these dsRNAs were shown to process them into siRNAs thereby pre-programming the plants to fight incoming tospoviruses. Indeed, these transgenic plants showed multiple virus resistance at a high frequency. The high frequency is important for the production of transgenic plants that are difficult to transform.
As RNA silencing is an important antiviral defense mechanism of plants, viruses have to overcome this hurdle and they do this by encoding proleins capable of blocking this pathway at certain steps. At the onset of this research project a number of such ("RNA si!encing suppressor") proteins had been characterized for positive strand RNA viruses and DNA viruses. In Chapter 3 it is shown that also negative strand RNA viruses, i.e. Tomato spotted wilt virus (TSWV) and Rice hoja blanca virus (RHBV), encode suppressors of RNA silencing QSISs and NS3, respectively). Interestingly, while the silencing suppressors are encoded on analogous genomic positions, they show different silencing suppression activities.
Having identified the first negative strand RNA viral silencing suppressors the next step was to investigate whether also mammalian negative strand RNA viruses could also encode such proteins. This was a logical next step to take for two reasons. 1. TSWV is a member of the Bunyaviridae, a virus family of which most other members infect mammals, and 2. viruses of this family are propagated in their insect vectors while it has been shown that silencing suppressors can be active in insects. To test this possibility the well characterized NSl protein of the Influenza A virus was chosen to be tested, indeed, it could be demonstrated that NSl is capable of suppressing RNA silencing in plants, possibly by binding siRNAs. These findings were strengthened by the observation that NSl enhances the virulence of Potato virus X when it was incorporated into its genome. The chimeric PVXMSl virus induced more pronounced disease symptoms, the NSI still being capable of suppressing RNA silencing when expressed from that viral vector. Whether NSl also has RNA silencing suppressor activity in mammalian cell systems though, still awaits to be confirmed. An intriguing fact is that NSl is an inhibitor of the interferon pathway, a mammalian antiviral mechanism in which - again - dsRNA plays a key role. This may indicate that in mammalian cells the interferon response and RNA silencing are somehow interlinked to act in concert. It is also possible that NSl binds long dsRNA to hide it from the interferon response and that the resulting silencing suppression in plants is only a side effect caused by that activity.
A further aim of this thesis was to better understand the interactions between viral silencing suppressors and the host. The suppressors of RNA silencing used were the previously mentioned NSs, NS3, NSl and additionally HC-Pro of Cowpea aphid borne mosaic virus and 2b of Cucumber mosaic virus. For the analysis, cDNA-amplified fragment length polymorphism (AFLP) technology was used. This technique allows a very broad analysis of transcriptional changes in plant tissues upon certain treatments. This combined with the A. tumefaciens mediated transient gene expression allowed monitoring of transcription profile changes caused by the expression of viral silencing suppressors. Out of approximately 25,000 mRNAs tested 362 were found to be differentially expressed due to the activity of silencing suppressors. Interestingly a large majority of genes (80%) was down-regulated. The fact that quite a number of genes were differentially expressed was to be expected since RNA silencing also plays a role in regulating certain mRNA levels via the activity of so-called micro-RNAs (miRNAs). Any disturbances in this processes will produce complex transcriptional changes for instance by directly inhibiting the activity of miRNAs (as it has been shown for HC-Pro for instance).
To conclude it can be stated that the interactions between antiviral RNA silencing and the countermeasures viruses have evolved to frustrate such process are on one hand a very important topic in virology and on the other hand a strong starting point for breakthroughs in other fields of research such as functional genomics and development. In an application environment, RNA silencing has allowed us to develop efficient and broad virus resistance in plants.
The influenza A virus NS1 protein binds small interfering RNAs and suppresses RNA silencing in plants
Bucher, E.C. ; Hemmes, J.C. ; Haan, P. de; Goldbach, R.W. ; Prins, M.W. - \ 2004
Journal of General Virology 85 (2004)4. - ISSN 0022-1317 - p. 983 - 991.
double-stranded-rna - translation initiation-factor - nicotiana-benthamiana - messenger-rna - transgenic plants - gene-expression - coat protein - human-cells - c-elegans - targets
RNA silencing comprises a set of sequence-specific RNA degradation pathways that occur in a wide range of eukaryotes, including animals, fungi and plants. A hallmark of RNA silencing is the presence of small interfering RNA molecules (siRNAs). The siRNAs are generated by cleavage of larger double-stranded RNAs (dsRNAs) and provide the sequence specificity for degradation of cognate RNA molecules. In plants, RNA silencing plays a key role in developmental processes and in control of virus replication. It has been shown that many plant viruses encode proteins, denoted RNA silencing suppressors, that interfere with this antiviral response. Although RNA silencing has been shown to occur in vertebrates, no relationship with inhibition of virus replication has been demonstrated to date. Here we show that the NS1 protein of human influenza A virus has an RNA silencing suppression activity in plants, similar to established RNA silencing suppressor proteins of plant viruses. In addition, NS1 was shown to be capable of binding siRNAs. The data presented here fit with a potential role for NS1 in counteracting innate antiviral responses in vertebrates by sequestering siRNAs.
|Molecular pathology of tomato spotted wilt virus, a plant-infecting bunyavirus
Goldbach, R.W. ; Knippenberg, I.C. van; Kormelink, R.J.M. ; Bucher, E.C. ; Prins, M.W. - \ 2003
In: EMBO Workshop Genomci Apporaches in Plant Virology, May 28-31, Keszthely, Hungary Keszthely, Hungary : - p. 42 - 42.
|Investigating the interaction between negative strand viruses and natural RNA silencing defense and exploiting it for tospoviurus resistance
Bucher, E.C. ; Goldbach, R.W. ; Prins, M.W. - \ 2003
In: EMBO workshop Genomic Apporaches in Plant Virology, May 28-31, Keszthely, Hungary Keszthely, Hungary : - p. 8 - 8.
Negative -strand tospoviruses and tenuiviruses carry a gene for a suppressor of gene silencing at analogous genomic positions
Bucher, E.C. ; Sijen, T. ; Haan, P. de; Goldbach, R.W. ; Prins, M.W. - \ 2003
Journal of Virology 77 (2003). - ISSN 0022-538X - p. 1329 - 1336.
spotted wilt virus - nonstructural protein nss - s-rna segment - nicotiana-benthamiana - nucleotide-sequence - transgenic plants - expression - interference - determinants - bunyavirus
Posttranscriptional silencing of a green fluorescent protein (GFP) transgene in Nicotiana benthamiana plants was suppressed when these plants were infected with Tomato spotted wilt virus (TSWV), a plant-infecting member of the Bunyaviridae. Infection with TSWV resulted in complete reactivation of GFP expression, similar to the case for Potato virus Y, but distinct from that for Cucumber mosaic virus, two viruses known to carry genes encoding silencing suppressor proteins. Agrobacterium-based leaf injections with individual TSWV genes identified the NSS gene to be responsible for the RNA silencing-suppressing activity displayed by this virus. The absence of short interfering RNAs in NSS-expressing leaf sectors suggests that the tospoviral NSS protein interferes with the intrinsic RNA silencing present in plants. Suppression of RNA silencing was also observed when the NS3 protein of the Rice hoja blanca tenuivirus, A nonenveloped negative-strand virus, was expressed. These results indicate that plant-infecting negative-strand RNA viruses carry a gene for a suppressor of RNA silencing.
Resistance mechanisms to plant viruses: an overview
Goldbach, R.W. ; Bucher, E.C. ; Prins, A.H. - \ 2003
Virus Research 92 (2003). - ISSN 0168-1702 - p. 207 - 212.
ribosome-inactivating proteins - replicase-mediated resistance - pathogen-derived resistance - transgenic tobacco plants - cucumber mosaic-virus - nicotiana-benthamiana - genetic interference - confers resistance - movement protein - immune-system
To obtain virus-resistant host plants, a range of operational strategies can be followed nowadays. While for decades plant breeders have been able to introduce natural resistance genes in susceptible genotypes without knowing precisely what these resistance traits were, currently a growing number of (mostly) dominant resistance genes have been cloned and analyzed. This has led not only to a better understanding of the plant's natural defence systems, but also opened the way to use these genes beyond species borders. Besides using natural resistance traits, also several novel, "engineered" forms of virus resistance have been developed over the past 15 years. The first successes were obtained embarking from the principle of pathogen-derived resistance (PDR) by transforming host plants with viral genes or sequences with the purpose to block a specific step during virus multiplication in the plant. As an unforeseen spin-off of these investments, the phenomenon of post-translational gene silencing (PTGS) was discovered, which to date is by far the most successful way to engineer resistance. It is generally believed that PTGS reflects a natural defence system of the plant, and part of the hypothesized components required for PTGS have been identified. As counteracting strategy, and confirming PTGS to be a natural phenomenon, a considerable number of viruses have acquired gene functions by which they can suppress PTGS. In addition to PDR and PTGS, further strategies for engineered virus resistance have been explored, including the use of pokeweed antiviral protein (PAP), 2',5'-oligoadenylate synthetase and "plantibodies". This paper will give a brief overview of the major strategies that have become operational during the past 10 years. (C) 2003 Elsevier Science B.V. All rights reserved.