Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    BNYVV-derived dsRNA confers resistance to rhizomania disease of sugar beet as evidenced by a novel transgenic hairy root approach
    Pavli, R. ; Panopoulos, N.J. ; Goldbach, R.W. ; Skaracis, G.N. - \ 2010
    Transgenic Research 19 (2010)5. - ISSN 0962-8819 - p. 915 - 922.
    double-stranded-rna - yellow-vein-virus - beta-vulgaris l - gene-expression - mediated resistance - antisense rna - interference - plants - suppression - transformation
    Agrobacterium rhizogenes-transformed sugar beet hairy roots, expressing dsRNA from the Beet necrotic yellow vein virus replicase gene, were used as a novel approach to assess the efficacy of three intron-hairpin constructs at conferring resistance to rhizomania disease. Genetically engineered roots were similar in morphology to wild type roots but were characterized by a profound abundancy, rapid growth rate and, in some cases, plagiotropic development. Upon challenge inoculation, seedlings showed a considerable delay in symptom development compared to untransformed or vector-transformed seedlings, expressing dsRNA from an unrelated source. The transgenic root system of almost all seedlings contained no or very low virus titer while the non-transformed aerial parts of the same plants were found infected, leading to the conclusion that the hairy roots studied were effectively protected against the virus. This readily applicable novel method forms a plausible approach to preliminarily evaluate transgenic rhizomania resistance before proceeding in transformation and whole plant regeneration of sugar beet, a tedious and time consuming process for such a recalcitrant crop species
    Tomato chlorotic mottle virus is a target of RNA silencing but the presence of specific short interfering RNAs does not guarantee resistance in transgenic plants
    Ribeiro, S.G. ; Lohuis, H. ; Goldbach, R.W. ; Prins, M. - \ 2007
    Journal of Virology 81 (2007)4. - ISSN 0022-538X - p. 1563 - 1573.
    golden mosaic-virus - whitefly-transmitted geminiviruses - broad-spectrum resistance - curl-sardinia-virus - double-stranded-rna - dna virus - antisense rna - coat protein - genetic-analysis - replication
    Tomato chlorotic mottle virus (ToCMoV) is a begomovirus found widespread in tomato fields in Brazil. ToCMoV isolate BA-Se1 (ToCMoV-[BA-Se1]) was shown to trigger the plant RNA silencing surveillance in different host plants and, coinciding with a decrease in viral DNA levels, small interfering RNAs (siRNAs) specific to ToCMoV-[BA-Se1] accumulated in infected plants. Although not homogeneously distributed, the siRNA population in both infected Nicotiana benthamiana and tomato plants represented the entire DNA-A and DNA-B genomes. We determined that in N. benthamiana, the primary targets corresponded to the 5' end of AC1 and the embedded AC4, the intergenic region and 5' end of AV1 and overlapping central part of AC5. Subsequently, transgenic N. benthamiana plants were generated that were preprogrammed to express double-stranded RNA corresponding to this most targeted portion of the virus genome by using an intron-hairpin construct. These plants were shown to indeed produce ToCMoV specific siRNAs. When challenge inoculated, most transgenic lines showed significant delays in symptom development, and two lines had immune plants. Interestingly, the levels of transgene- produced siRNAs were similar in resistant and susceptible siblings of the same line. This indicates that, in contrast to RNA viruses, the mere presence of transgene siRNAs corresponding to DNA virus sequences does not guarantee virus resistance and that other factors may play a role in determining RNA-mediated resistance to DNA viruses.
    Presence of an intron in inverted repeat constructs does not necessarily have an effect on efficiency of post-transcriptional gene silencing
    Heilersig, H.J.B. ; Loonen, A.E.H.M. ; Wolters, A.M.A. ; Visser, R.G.F. - \ 2006
    Molecular Breeding 17 (2006)4. - ISSN 1380-3743 - p. 307 - 316.
    bound starch synthase - double-stranded-rna - solanum-tuberosum-l - mediated enhancement - antisense rna - maize cells - expression - potato - granule - interference
    The effect of introns on silencing efficiency was tested in inverted repeat constructs of Granule-Bound Starch Synthase (GBSSI) cDNA by comparing the silencing efficiencies induced by inverted repeat constructs with and without introns. No effect could be attributed to the presence of introns indicating that the introns neither enhance nor inhibit post-transcriptional gene silencing. The effect of a spliceable intron in the spacer was studied by comparing constructs harbouring a spliceable or a non-spliceable intron in the spacer. As opposed to the general belief that splicing of an intron increases silencing efficiency, the use of a spliceable intron in the spacer did not result in enhancement of silencing in our experimental system
    Factors influencing gene silencing of granule-bound starch synthase in potato
    Heilersig, H.J.B. - \ 2005
    Wageningen University. Promotor(en): Richard Visser, co-promotor(en): Anne-Marie Wolters. - [S.l.] : S.n. - ISBN 9789085042471 - 107
    solanum tuberosum - aardappelen - genen - genetische modificatie - genexpressie - transgene planten - genetische transformatie - antisense rna - solanum tuberosum - potatoes - genes - genetic transformation - genetic engineering - gene expression - antisense rna - transgenic plants
    In the past, antisense RNA technology was used to modify the composition of potato tuber starch. Potato starch comprises amylose and amylopectin, polymers of glucose. Amylose production in potato is completely dependent on the presence of granule-bound starch synthase I (GBSSI). Inhibition of GBSSI has been achieved by transformation with antisense and sense GBSSI constructs. However, the percentages of transformants showing strong silencing were relatively low which implicated that large numbers of transformants needed to be generated in order to obtain sufficient transformants showing strong silencing. This was an undesirable situation since in practical breeding many transformants would have to be made, making the effort too time consuming and costly. Therefore, an approach that would enhance the silencing efficiency was desired.

    This thesis describes the application of inverted repeat constructs for the modification of potato tuber starch. Transcription of inverted repeat constructs results in the formation of double stranded RNA (dsRNA). These dsRNA molecules are cleaved in small interfering RNAs (siRNAs) by a dsRNA-specific nuclease named Dicer. Subsequently, one strand of the siRNA is incorporated into the RNA Induced Silencing Complex (RISC) which is guided to the target mRNA through conventional base-pairing interactions. The target mRNA is then cleaved opposite the centre of the guide siRNA and finally, the cleaved mRNA is degraded.

    The antisense strand of the siRNA can also act as a primer on target mRNA after which new dsRNA is synthesized by the action of a cellular RNA-directed RNA polymerase (RdRP). The RdRP-synthesized dsRNA will be recognized by Dicer and degraded to secondary siRNAs. These secondary siRNAs can be derived from regions upstream of the targeted regions and can thus induce RNA silencing of sequences that were not initially targeted. This phenomenon is called transitive RNA silencing.

    First, an extensive study on the Post-Transcriptional Gene Silencing (PTGS) effects of eight different cDNA inverted repeat constructs was performed. These cDNA inverted repeat constructs were designed in such a way that the effect of size, orientation and sequence could be determined. The orientation of the inverted repeat did not affect silencing efficiency but the size and sequence did. Small inverted repeat constructs with a repeat size of 500-600 bp were more efficient silencing inducers than the large inverted repeat constructs with a repeat size of 1.1 or 1.3 kb. The two large inverted repeat constructs comprised the5'and the3'half of the GBSSI cDNA whilst for the construction of the small inverted repeat constructs the GBSSI cDNA was divided in three regions; the5', the middle and the3'region. In both instances, the3'sequences induced the least efficient silencing implying that sequence does influence silencing efficiency. The transformants showing the highest percentages of strong silencing were obtained with two small inverted repeat constructs derived from the5'or middle region of the GBSSI cDNA. In both cases, 87 % of transformants showed strong silencing. Silencing was accompanied by a reduction in GBSSI mRNA levels but no relation between the amount of transcript and the level of silencing could be demonstrated. A similar phenomenon was observed for the accumulation of siRNAs. Although there was a relation between the presence of siRNAs and silencing, no relation between the level of siRNAs and the level of silencing was found.

    Another factor we tested was the influence of introns in the cDNA sequences represented in the small inverted repeat constructs. To test the effect of GBSSI intron number two, two constructs were made; one with the intron in the cDNA and one without intron number two in the cDNA. The same approach was used to address the importance of GBSSI intron number nine, which was tested in two inverted repeat constructs harbouring sequences from the middle region of the GBSSI cDNA. For both introns, no effect on silencing efficiency was found. This indicates that the introns neither enhance nor inhibit silencing.

    Many general silencing vectors are designed with a spliceable intron in the spacer because of a general belief that the splicing of this intron enhances gene silencing. To address whether this would apply to our experimental system, we compared constructs that contained intron number nine in a spliceable or a non-spliceable orientation in the spacer sequence. Both constructs gave rise to similar silencing efficiencies. It is likely that intron number nine was spliced in the construct in which it was in the spliceable orientation, because we demonstrated that this intron was spliced in another construct where it was located in exactly the same surrounding sequences.

    Since we found no differences in silencing efficiency, we postulate that it is not a general rule that inclusion of a spliceable intron in the spacer of an inverted repeat construct enhances silencing efficiency. We think that the extent to which the presence of an intron in the spacer sequence enhances silencing efficiency might be different for each intron.

    In plants and C.elegans examples of transitive silencing, i.e. silencing of sequences that were not initially targeted, have been described. Since it was shown that potato has an RdRP homolog, we investigated whether transitive silencing occurred in potato. Two approaches were used to induce transitive silencing. A construct harbouringa NOSterminator inverted repeat preceded by the middle region of the GBSSI cDNA and transcribed by a GBSSI promoter, was tested for its ability to induce silencing of GBSSI. Silencing of GBSSI could be induced indicating that transitivity occurs in potato in a3'to5'direction. To address whether the endogenous GBSSI mRNA can function as a template for the synthesis of new dsRNA, transformants harbouring GBSSI cDNA IR constructs were analysed for the accumulation of secondary siRNAs derived from regions adjacent to the targeted regions. Secondary siRNAs upstream of the targeted region were detected indicating that, in potato, the endogenous GBSSI transcript can function as a template for the synthesis of new dsRNA. No transitivity in the5'to3'direction could be detected. In plants, transitive silencing has been observed in several transgenic systems but very few examples of transitive silencing of endogenous genes exist. In these examples, it was not investigated whether the endogenous or transgene mRNA was used as a template. To our knowledge, we are first to show that endogenous mRNA is used as a template in transitive silencing of endogenous genes in plants. We also showed that the transitivity could extend over a distance of 761 bp, which is the largest distance reported until now.

    When an inverted repeat construct contains promoter sequences, transcriptional gene silencing (TGS) can be induced. In this case, alterations at the DNA or chromatin level prevent transcription of the targeted genes. An alteration at the DNA level is methylation of cytosine residues. This can be induced by RNA signals and is described as RNA-directed DNA methylation. Even though transformants obtained through the use of GBSSI cDNA inverted repeat constructs show strong silencing of GBSSI, there is still production of GBSSI mRNA. To obtain transformants in which the production of GBSSI mRNA was completely inhibited, we induced TGS of GBSSI by constructing GBSSI promoter inverted repeats. Three different regions of the GBSSI promoter were tested in inverted repeat constructs.

    In potato, four classes of GBSSI alleles are known. These four classes of alleles are highly homologous in the coding region, but vary in promoter sequences. The variation in promoter sequences was exploited to make an 'allele-specific' promoter inverted repeat construct. This construct contained the sequences from -531 to -330 bp relative to the transcription initiation site (TIS) in the GBSSI A2 allele. It was hypothesized that this construct would inhibit the expression of the A2, A3 and A4 GBSSI alleles, but not of the A1 GBSSI allele. However, the percentages of weakly silenced transformants obtained with this construct were too low to distinguish the effect between two potato cultivars that varied in allele composition. Therefore, this approach could not be used to induce allele-specific silencing.

    Weak silencing effects were also observed in 57-60 % of the transformants harbouring the inverted repeat construct with the promoter sequences from -766 to -168 bp relative to the TIS. We demonstrated that within this sequence, CG methylation had occurred, but apparently this was not sufficient to obtain strong silencing.

    The most efficient silencing efficiency was induced with the inverted repeat construct containing the promoter sequences from -766 to +194 bp, relative to the TIS. Very strongly silenced transformants were obtained in which the transcription of GBSSI was completely or almost completely inhibited. This was accompanied by the accumulation of promoter-specific siRNAs and CG and CNN methylation.

    Here, we showed that transcriptional silencing of the endogenous GBSSI promoter can be induced through the use of promoter inverted repeats. However, in order to obtain strong silencing, it is important to include sequences in the vicinity of the TIS. As compared to the PTGS approach, the TGS approach could be a good alternative to obtain very strongly silenced transformants that show a complete or almost complete inhibition of GBSSI transcription. Therefore, we think it is worthwhile to study whether the silencing effect in TGS transformants is stable during storage of tubers and in field experiments.
    High frequency induction of RNA-mediated resistance against Cucumber mosaic virus using inverted repeat constructs
    Chen, Y.K. ; Lohuis, H. ; Goldbach, R.W. ; Prins, M.W. - \ 2004
    Molecular Breeding 14 (2004)3. - ISSN 1380-3743 - p. 215 - 226.
    coat protein gene - double-stranded-rna - pathogen-derived resistance - transgenic tobacco - messenger-rna - antisense rna - cmv strains - silenced tobacco - plants - expression
    The application of RNA-mediated resistance against Cucumber mosaic virus (CMV) by using single transgene constructs generally results in only a small portion of resistant individuals. Inverted repeat constructs encoding self-complementary double-stranded RNA have been demonstrated a potential way to obtain RNA-mediated resistance at high efficiency. To test this observation as a possible method for high frequency induction of CMV resistance, Nicotiana benthamiana plants were transformed with transgenes designed to produce double strand RNA molecules of CMV RNA 2 or coat protein (CP) gene sequences. Seventy-five percent of the tested R0 plants transformed with an RNA 2-derived inverted repeat construct (1534 nt CMV sequence) showed extreme resistance to CMV, while a lower percentage of resistance (30%) was observed in R0 lines transformed with a similar construct of a shorter viral RNA 2 sequence (490 nt). The resistance level conferred by CP sequences was also efficient by using a dsRNA construct, reaching a level of 50%. Self-pollinated (S1) progenies obtained from most resistant R0 plants all showed resistance levels of 100%, perfectly correlating with the expression of transgenic siRNAs. The results indicate that the use of inverted repeat viral transgenes is a highly efficient approach to obtain CMV resistant transgenic plants. Consequently, only a handful of transgenic plants will have to be generated using such constructs for successful resistance, which enables the implementation of this protocol for crops that are difficult to transform, such as ornamental plants in which CMV is an important pathogen
    Antisense RNA mediated inhibition of granule - bound starch synthase gene expression in potato
    Kuipers, A. - \ 1994
    Agricultural University. Promotor(en): E. Jacobsen; W.J. Feenstra; R.G.F. Visser. - S.l. : Kuipers - ISBN 9789054852513 - 125
    plantenveredeling - chemische samenstelling - solanum tuberosum - aardappelen - genetische modificatie - genexpressie - antisense rna - plant breeding - chemical composition - solanum tuberosum - potatoes - genetic engineering - gene expression - antisense rna

    Potato starch and its derivatives are widely used in several fields of application. The manufacturing of most products requires the modification of native starch with respect to, for example, viscosity and physical stability. In addition to the currently used physical, chemical and biochemical derivatization and gelatinization of extracted starch, modification of the starch biosynthetic pathway in planta is regarded as a valuable approach for altering the quality and quantity of potato starch.

    This thesis describes the application of antisense RNA technology for the modification of the composition of potato tuber starch. Starch consists of amylose and arnylopectin, and is predominantly synthesized in amyloplasts of tubers and seeds, where it is deposited in starch granules. Several enzymes are involved in the biosynthesis of starch, of which granule-bound starch synthase (GBSS) catalyses the formation of amylose. Generally, starch contains 20% of amylose and 80% of arnylopectin. However, mutants are known in which the ratio between amylose and arnylopectin has changed. An example is the amylose-free ( amf ) potato mutant, of which the starch exclusively contains arnylopectin due to a recessive mutation in the GBSS-gene. The suppression of the expression of specific genes can also be achieved by means of antisense inhibition, which is supposed to be based on the formation of an RNA-duplex between the target mRNA and the antisense RNA, thus blocking the transport of the mRNA from the nucleus.

    To inhibit the (GBSS) gene expression, several antisense genes were introduced into the potato genome via Agrobacterium -mediated transformation. Among transgenic clones in which inhibition of (GBSS) gene expression was observed. two types could be discerned:
    A. Transgenic clones with completely inhibited (GBSS) gene expression. These clones lacked the (GBSS) protein, (GBSS) activity and amylose. The complete inhibition was demonstrated in microtubers, greenhouse grown tubers and field grown tubers, and was maintained in several tuber generations. The composition of tuber starch from these clones and from the amf -mutant was similar.
    B. Transgenic clones with incompletely inhibited (GBSS) gene expression. In these clones the (GBSS) protein content, (GBSS) activity and amylose content were reduced as compared to non-transformed clones. With respect to the extent of inhibition, variation was observed between transgenic clones which was supposed to result from position effects. Variation was also observed between tubers of individual plants and within individual tubers. This type of variation was suggested to be caused by factors related to tuber development.

    Microscopic analysis of iodine stained tuber starch granules from transgenic clones showed that the reduced amount of amylose was not equally distributed throughout the entire granule, but was confined to the central core of the granule. Iodine-staining of starch granules yielded a blue core, in which the amylose content equalled that of wildtype starch, and a red, amylose-free, outer part. The size of the blue core was shown to be positively related to the amylose content, as determined in starch isolated from the transgenic clones. This finding led to the hypothesis that at a certain point in starch granule development the available amount of (GBSS) protein will become insufficient for the formation of starch with a normal amylose content. From this point onwards, amylose-free starch will be produced throughout further starch granule development.

    With respect to the effectiveness of antisense inhibition, the influence of the composition of the antisense gene on the percentage of transgenic clones with complete or incomplete inhibition was studied. To this end, eleven antisense genes were constructed, based on the full length (GBSS) cDNA, the genomic (GBSS) coding region, and some cDNA- and genomic fragments. Furthermore, a comparison was made between the 35S CaMV promoter and the potato (GBSS) promoter. The extent of inhibition of GBSS gene expression was studied in at least 30 transgenic clones per antisense gene construct. In addition to construct-related differences, the number of T- DNA insertions was found to be positively related to the extent of antisense inhibition. It was concluded that the chance for complete inhibition of (GBSS) gene expression was highest in transgenic clones with three or more copies of the antisense gene based on the full length (GBSS) cDNA driven by the (GBSS) promoter.

    In order to evaluate the applicability of the antisense (GBSS) system, two antisense genes based on the full length (GBSS) cDNA and driven by the (GBSS) or the 35S CaMV promoter were introduced into several potato cultivars via transformation with A . tumefaciens. The trangenic clones of one of the cultivars were analysed for the extent of antisense inhibition and its stability in a field trial based on in vitro plantlets. Complete inhibiton of (GBSS) gene expression, resulting in the formation of amylose-free starch, was demonstrated in mature tubers of four of these clones. Other characteristics, such as the total starch- and sugar content, the total yield and the dry matter content, did not deviate significantly as compared to the non-transformed cultivar. This led to the conclusion that antisense inhibition has good perspectives for the formation of amylose-free starch in potato tubers. In 1993, three of the transgenic clones with amylose-free tuber starch that are described in this thesis, were submitted to the Board for Plant Breeder's Rights for registration.

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