Gene silencing in potato: allelic differences and effect of ploidy

Wolters, Anne‐Marie A.; Visser, R.G.F.

doi:10.1007/978-94-011-4183-3_18

Cited by 5 publications

(5 citation statements)

References 41 publications

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“…Silencing of GBSSI was more efficient with antisense constructs than with sense constructs (Wolters and Visser, 2000). For antisense inhibition, a positive correlation between the T-DNA integration number and the effect on silencing was found (Kuipers et al, 1995).…”

Section: Introductionmentioning

confidence: 94%

Post-transcriptional Gene Silencing of GBSSI in Potato: Effects of Size and Sequence of the Inverted Repeats

et al. 2006

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In the past, silencing of granule-bound starch synthase (GBSSI) in potato was achieved by antisense technology, where it was observed that inclusion of the 3' end of the GBSSI coding region increased silencing efficiency. Since higher silencing efficiencies were desired, GBSSI inverted repeat constructs were designed and tested in potato. First, large inverted repeats comprising the 5' and the 3' half of the GBSSI cDNA were tested. The 5' IR construct gave a significantly higher silencing efficiency than the 3' IR construct. Since it was not known whether the observed difference was due to the sequence or the orientation of the inverted repeat, the GBSSI cDNA was divided into three regions, after which each region was tested in small inverted repeats in two orientations. To this end large numbers of independent transformants were produced for each construct. The results suggested that there was no effect of inverted repeat orientation on silencing efficiency. The percentage of transformants showing strong inhibition varied from 48% for a 3'-derived construct to 87% for a 5' as well as a middle region-derived construct. Similar to the large inverted repeats, the 3' sequences induced the least efficient silencing implying that the observed differences in silencing efficiency are caused by sequence differences. The small inverted repeat constructs with a repeat size of 500-600 bp and a spacer of about 150 bp were more efficient silencing inducers than the large inverted repeat constructs where the size of the repeat was 1.1 or 1.3 kb whilst the size of spacer was 1.3 or 1.1 kb. The results presented here show that size and sequence of the inverted repeat influenced silencing efficiency.

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Section: Introductionmentioning

confidence: 94%

Post-transcriptional Gene Silencing of GBSSI in Potato: Effects of Size and Sequence of the Inverted Repeats

et al. 2006

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“…Recent findings indicate that peculiarities of transgene arrangements, most frequently observed with integration of direct or inverted repeats, lead to the generation of aberrant RNA and formation of dsRNA. These RNAs can be processed to siRNAs responsible for the ATP-dependent, sequence-specific degradation of mRNA (Wolters and Visser 2000;Ambros 2001;Elbashir et al 2001;Lipardi et al 2001;Sharp 2001). The detection of small RNAs of about 21-nt by both sense and antisense riboprobes of HsfA1 in CS2 and CS3 tomato and the lack of such siRNAs in WT and OE plants (Fig.…”

Section: Molecular Basis Of Hsfa1 Gene Silencingmentioning

confidence: 99%

In the complex family of heat stress transcription factors, HsfA1 has a unique role as master regulator of thermotolerance in tomato

Mishra¹,

Tripp²,

Winkelhaus³

et al. 2002

Genes Dev.

509

481

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We generated transgenic tomato plants with altered expression of heat stress transcription factor HsfA1. Plants with 10-fold overexpression of HsfA1 (OE plants) were characterized by a single HsfA1 transgene cassette, whereas plants harboring a tandem inverted repeat of the cassette showed cosuppression (CS plants) by posttranscriptional silencing of the HsfA1 gene connected with formation of small interfering RNAs. Under normal growth conditions, major developmental parameters were similar for wild-type (WT), OE, and CS plants. However, CS plants and fruits were extremely sensitive to elevated temperatures, because heat stress-induced synthesis of chaperones and Hsfs was strongly reduced or lacking. Despite the complexity of the plant Hsf family with at least 17 members in tomato, HsfA1 has a unique function as master regulator for induced thermotolerance. Using transient reporter assays with mesophyll protoplasts from WT tomato, we demonstrated that plasmid-encoded HsfA1 and HsfA2 were well expressed. However, in CS protoplasts the cosuppression phenomenon was faithfully reproduced. Only transformation with HsfA2 expression plasmid led to normal expression of the transcription factor and reporter gene activation, whereas even high amounts of HsfA1 expression plasmids were silenced. Thermotolerance in CS protoplasts was restored by plasmid-borne HsfA2, resulting in expression of chaperones, thermoprotection of firefly luciferase, and assembly of heat stress granules. (Pelham 1982;Pelham and Bienz 1982;Nover 1987). Similar to other transcription factors, Hsfs have a modular structure with an N-terminal DNA-binding domain characterized by a central helix-turn-helix motif, an adjacent oligomerization domain with a bipartite heptad pattern of hydrophobic amino acid residues (HR-A/B region), and sequence motifs essential for nuclear import and export (NLS, NES;Wu 1995;Morimoto 1998;Heerklotz et al. 2001;Nover et al. 2001). In many cases, the C-terminal activation domains are characterized by short peptide motifs (AHA motifs) shown to be crucial for the activator function (Döring et al. 2000).Sequencing of the Arabidopsis genome revealed a unique complexity of the Hsf family with 21 members, in contrast to yeast and Drosophila with one Hsf and vertebrates with four Hsfs (Wu 1995;Nover et al. 2001). From analyses of expressed sequence tag (EST) libraries, it is evident that the size of the Hsf family is comparable also in other plants, with 17 Hsfs thus far identified from tomato ESTs . By structural characteristics and phylogenetic comparison, plant Hsfs were assigned to three classes. In Arabidopsis, there are 15

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“…4 and 5). Gene silencing is most likely to happen when its sequence shares the highest homology with the antisense RNA sequence (Wolters and Visser 2000). The reduction in soluble protein content via antisense RNA in the present research is, therefore, mainly the result of the expression of inhibition of the patatin gene that is most similar to SK24-1, which leads to further subsequent influences on the down-stream processes.…”

Section: Influence Of Sk24-1 On Microtuber Formation Of the Transformmentioning

confidence: 73%

Functional analysis of a class I patatin gene SK24-1 in microtuber formation of transgenic potatoes

Si¹,

Liu²,

Huang³

et al. 2008

Can. J. Plant Sci.

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. 2008. Functional analysis of a class I patatin gene SK24-1 in microtuber formation of transgenic potatoes. Can. J. Plant Sci. 88: 593Á598. Expression of a class I patatin cDNA clone, SK24-1, in Escherichia coli revealed that the cDNA clone possessed lipid acyl hydrolase (LAH) activity. Transformed potato plants were obtained via Agrobacterium-mediated transformation using the chimeric constructs containing the sense and antisense cDNA under the control cauliflower mosaic virus 35S (CaMV 35S) promoter. In some sense transformed plants, both sense patatin RNA and LAH activity were increased and further resulted in a significant increase of percentage of plantlets that formed microtubers and numbers of microtubers per plantlet in vitro. All antisense plants displayed a reduction in LAH activity. Both sense and antisense RNA could be detected in antisense plants, but transcripts of antisense RNA resulted in a reduction of endogenous sense RNA. Moreover, expression of antisense cDNA in some antisense transformed plants led to a significant decrease in the number of microtubers formed. These results suggest that SK24-1 was involved in regulating microtuber formation.

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Gene silencing in potato: allelic differences and effect of ploidy

Cited by 5 publications

References 41 publications

Post-transcriptional Gene Silencing of GBSSI in Potato: Effects of Size and Sequence of the Inverted Repeats

Post-transcriptional Gene Silencing of GBSSI in Potato: Effects of Size and Sequence of the Inverted Repeats

In the complex family of heat stress transcription factors, HsfA1 has a unique role as master regulator of thermotolerance in tomato

Functional analysis of a class I patatin gene SK24-1 in microtuber formation of transgenic potatoes

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