Transgene Inactivation: Plants Fight Back!

Finnegan, Jean; McElroy, David

doi:10.1038/nbt0994-883

Cited by 323 publications

(178 citation statements)

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“…It has also been associated with paramutation (a natural example of trans-inactivation; Brink, 1973;Martienssen, 1996;Matzke et al, 1996) in maize. Methylation has often been associated with transgene inactivation in dicots (Finnegan and McElroy, 1994), and there is mounting evidence that de novo methylation is involved in gene silencing (Flavell, 1994).…”

Section: Transgene Silencing and Methylationmentioning

confidence: 99%

“…Cytosine methylation has been correlated with many instances of gene silencing in dicotyledonous plants (Finnegan et al, 1993;Finnegan and McElroy, 1994;Flavell, 1994). Therefore, the methylation status of the bar gene in bialaphos-resistant and bialaphos-sensitive plants was investigated by genomic analysis using the restriction endonuclease isoschizomers Hpall and Mspl (Fig.…”

Section: Silencing Of the Bar Genementioning

confidence: 99%

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Epigenetic Transcriptional Silencing and 5-Azacytidine-Mediated Reactivation of a Complex Transgene in Rice

et al. 1997

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Despite a growing number of reports indicating non-Mendelian inheritance of transgene expression in monocots, no detailed description of the structure and stability of the transgene exists for transformants generated by direct DNA-transfer techniques, making the cause for these observations difficult to determine. In this paper we describe the complex organization of Btf crylllA and bar transgenes in rice (Oryza safiva 1.) that displayed aberrant segregation in R, progeny. Silencing rather than rearrangement of the bar gene was implicated because the herbicide-sensitive R, plants had a DNA hybridization profile identical to that of the resistant R, parent and R, siblings. Cenomic DNA analysis revealed substantial methylation of the Ubil/bar sequences in silenced plants and, to a lesser degree, in herbicide-resistant plants, suggesting that the transgene locus was potentiated for silencing. Nuclease protection and nuclear run-on assays confirmed that silencing was due to transcriptional inactivation. Treatment of R, progeny of silenced plants with 5-azacytidine resulted in demethylation of the Ubil promoter and reactivation of bar gene expression, demonstrating a functional relationship for methylation in gene silencing. These findings indicate that methylation-based silencing may be frequent in cereals transformed by direct DNA protocols that insert multiple, often rearranged sequences.

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Section: Transgene Silencing and Methylationmentioning

confidence: 99%

Section: Silencing Of the Bar Genementioning

confidence: 99%

Epigenetic Transcriptional Silencing and 5-Azacytidine-Mediated Reactivation of a Complex Transgene in Rice

et al. 1997

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“…Data sets that indicate a change in pollination phenotype due to high-level expression of the transgene are underlined, nd, no data. (Finnegan and McElroy, 1994). Plants with relatively low expression (or unsuppressed plants in the antisense experiments) served as additional negative controls.…”

Section: S Rnase Expression In Transgenic Plantsmentioning

confidence: 99%

S RNase and Interspecific Pollen Rejection in the Genus Nicotiana: Multiple Pollen-Rejection Pathways Contribute to Unilateral Incompatibility between Self-Incompatible and Self-Compatible Species.

et al. 1996

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In self-incompatible (SI) plants, the S locus acts to prevent growth of self-pollen and thus promotes outcrossing within the species. lnterspecific crosses between SI and self-compatible (SC) species often show unilateral lncompatibility that follows the S I x SC rule: S I specles reject pollen from SC specles, but the reciproca1 crosses are usually compatible. The general validity of the S I x SC rule suggests a link between S I and interspeclflc pollen rejectlon; however, this link has been questioned because of a number of exceptlons to the rule. To clarlfy the role of the S locus in interspecific pollen rejection, we transformed severa1 Nicotlana species and hybrids with genes encodlng S A~ or SC,O RNase from S I N. alata. Compatibillty phenotypes in the transgenlc plants were tested using pollen from three SC specles showing unilateral incompatibility with N. alata. S RNase was lmpllcated ln rejecting pollen from all three specles. Rejection of N. plumbaginifolia pollen was similar to S allele-speclfic pollen rejection, showing a requirement for both S RNase and other genetic factors from N. alata. In contrast, S RNase-dependent rejectlon of N. glutinosa and N. tabacum pollen proceeded without these additional factors. N. alata also rejects pollen fmm the latter two specles through an S RNaseindependent mechanism. Our results lmplicate the S locus in all three systems, but lt 1s clear that multlple mechanisms contribute to interspecific pollen rejectlon. INTRODUCTIONMany plants have evolved genetically controlled selfincompatibility (SI) systems that promote outcrossing by restricting pollination between closely related individuals of the same species (de Nettancourt, 1977). Mechanisms also exist to restrict pollination between different species, but comparatively little is known about the control of interspecific pollination.As in other solanaceous plants, SI in the genus Nicotiana is controlled by a single multiallelic locus, the S locus (Newbigin et al., 1993). These plants employ a gametophytic SI system in which pollen is rejected if the S allele in the haploid pollen is the same as either S allele in the diploid pistil. S allele-specific pollen rejection occurs as pollen tubes grow through the extracellular matrix of the stylar transmitting tract (Newbigin et al., 1993). The products of the S locus in the style are the To whom correspondence should be addressed.S RNases (McClure et al., 1989). These glycoproteins are very abundant in the extracellular matrix of the transmitting tract and are also expressed in the stigma and in the epidermis of the placenta (Cornish et Anderson et al., 1989;McClure et al., 1993). S RNases are essential for S allele-specific pollen rejection Murfett et al., 1994), and their ribonuclease activity is required for this function . Following incompatible pollinations, RNA in selfpollen tubes is degraded, and this degradation is consistent with a cytotoxic model for pollen rejection (McClure et al., 1990;Gray et al., 1991; Dickinson, 1994). SI is therefore an active process i...

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“…In the latter respect, the phenotypic stability of the hpRNA gene silencing over many generations is of crucial importance for the reliable application of gene silencing for crop improvement. The possibility of inactivation of transgenes through promoter methylation (Finnegan and McElroy, 1994) has been raised as a potential cause of phenotypic reversion of the transgeneencoded trait, a phenomenon that may gradually emerge over a number of generations. Although studies in Arabidopsis have shown that dsRNAmediated gene silencing can be inherited in simple Mendelian fashion in the T 2 generation when targeted against either the endogenous CLV3 and API genes (Chuang and Meyerowitz, 2000) or a stably integrated ␤-glucuronidase reporter transgene , there are no studies reporting on the long-term phenotypic stability of such changes over several generations.…”

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confidence: 99%

hpRNA-Mediated Targeting of the Arabidopsis FAD2 Gene Gives Highly Efficient and Stable Silencing

et al. 2002

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The endogenous ⌬12-desaturase gene (FAD2) in Arabidopsis was targeted for silencing using seed-specific cosuppression (CS), hairpin (HP) RNA (hpRNA), and intron-spliced HP (iHP) constructs. The iHP construct, incorporating the 120-bp 3Ј-untranslated region of the FAD2 gene, gave the highest degree of silencing. In some iHP lines ⌬12-desaturase activity was reduced to levels as low as those in the null fad2-1 mutant, and every primary transformant showed a pronounced reduction in FAD2 activity. One highly silenced iHP line was propagated for five generations and showed no reversion or diminution in its degree of silencing. About 75% of plants transformed with the HP construct, targeting the FAD2 coding region, gave dramatically reduced ⌬12-desaturase activity, whereas approximately 50% of plants transformed with the CS construct, containing the same coding region sequence, showed silencing at a much less profound level. In all three types of constructs, the degree of silencing was increased when the transgenes were homozygous, but this was much more pronounced for the CS constructs. All three types of construct could give a single locus that was capable of effective silencing, but in the one such CS line where this was the case, the locus had a complex insertion pattern. This is consistent with the concept that posttranscriptional gene silencing is induced by double-stranded, or self-complementary, RNA that is formed in cases of CS by complex insertion patterns at a single locus and that the most effective way of generating profoundly silenced plants is by the use of constructs that encode hpRNAs. Furthermore, these results demonstrate for the first time, to our knowledge, that iHP constructs targeted against an endogenous seed-expressed gene are clearly able to generate phenotypic changes that are inherited stably over several generations, making this approach a reliable technique for genetic modification of seed quality and possibly other traits in agricultural plants.

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Transgene Inactivation: Plants Fight Back!

Cited by 323 publications

References 66 publications

Epigenetic Transcriptional Silencing and 5-Azacytidine-Mediated Reactivation of a Complex Transgene in Rice

Epigenetic Transcriptional Silencing and 5-Azacytidine-Mediated Reactivation of a Complex Transgene in Rice

S RNase and Interspecific Pollen Rejection in the Genus Nicotiana: Multiple Pollen-Rejection Pathways Contribute to Unilateral Incompatibility between Self-Incompatible and Self-Compatible Species.

hpRNA-Mediated Targeting of the Arabidopsis FAD2 Gene Gives Highly Efficient and Stable Silencing

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