Virus‐induced gene silencing in transgenic plants: transgene silencing and reactivation associate with two patterns of transgene body methylation

We used bisulfite sequencing to study the methylation of a viral transgene whose expression was silenced upon plum pox virus infection of the transgenic plant and its subsequent recovery as a consequence of so‐called virus‐induced gene silencing (VIGS). VIGS was associated with a general increase in the accumulation of small RNAs corresponding to the coding region of the viral transgene. After VIGS, the transgene promoter was not methylated and the coding region showed uneven methylation, with the 5′ end being mostly unmethylated in the recovered tissue or mainly methylated at CG sites in regenerated silenced plants. The methylation increased towards the 3′ end, which showed dense methylation in all three contexts (CG, CHG and CHH). This methylation pattern and the corresponding silenced status were maintained after plant regeneration from recovered silenced tissue and did not spread into the promoter region, but were not inherited in the sexual offspring. Instead, a new pattern of methylation was observed in the progeny plants consisting of disappearance of the CHH methylation, similar CHG methylation at the 3′ end, and an overall increase in CG methylation in the 5′ end. The latter epigenetic state was inherited over several generations and did not correlate with transgene silencing and hence virus resistance. These results suggest that the widespread CG methylation pattern found in body gene bodies located in euchromatic regions of plant genomes may reflect an older silencing event, and most likely these genes are no longer silenced.     

Gene silencing results in instability of antibody production in transgenic plants

The stability of antibody and F_ab expression was assessed in five different homozygous transgenic Arabidopsis lines. Each of these lines showed silencing of the transgenes that encode the antibody polypeptides, leading to instability of antibody production. However, each line had a different and specific instability profile. The characteristic variation in the level of antibody accumulation in each line as a function of developmental stage indicated that the T-DNA integration pattern played a role in triggering silencing, and also that the history and the integration position of simple transgene loci can influence the susceptibility to epigenetic silencing. In different lines with low antibody accumulation levels, methylation was found either in the promoter alone, in both the promoter and the transcribed region, in the transcribed region only, or in the transcribed region and downstream sequences. In conclusion, our data suggest that epigenetic effects result in different transgene expression profiles in each of the five Arabidopsis lines analyzed. Received: 27 July 1998 / Accepted: 12 October 1998     

Isolation of soybean plants with stable transgene expression by visual selection based on green fluorescent protein

Particle bombardment is a common platform for soybean transformation but tends to cause transgene silencing due to the integration of rearranged or multiple copies of transgenes. We now describe the isolation of a total of 44 independent transgenic soybean plants after transformation by particle bombardment with one of two gene constructs, pHV and pHVS. Both constructs contain the hygromycin phosphotransferase gene (hpt) as a selectable marker and a modified glycinin gene (V3-1) for evaluation of homology-dependent silencing of endogenous glycinin genes; pHVS also contains sGFP(S65T), which encodes a modified form of green fluorescent protein (GFP), as a reporter gene in the flanking region of V3-1. Fluorescence microscopy revealed that the leaves of 8 of the 25 independent transgenic plants obtained with pHVS expressed GFP; most of these GFP-positive plants also contained V3-1 mRNA and an increased glycinin content in their seeds, and they exhibited simple banding patterns on Southern blots that were indicative of a low copy number of each of the three transgenes. In contrast, most of the transgenic plants obtained with pHVS that did not express GFP, as well as most of those obtained with pHV, lacked endogenous glycinin in their seeds and exhibited more complex patterns of transgene integration. The use of a reporter gene such as sGFP(S65T) in addition to an antibiotic resistance gene may thus help to reduce the problem of gene silencing associated with direct DNA transformation systems and facilitate the recovery of transgenic plants that stably express the gene of interest.     

Chemically defined polyethylene glycol siRNA conjugates with enhanced gene silencing effect

The therapeutic application of siRNA suffers from poor bioavailability caused by rapid degradation and elimination. The covalent attachment of PEG is a universal concept to increase molecular size and enhance the pharmacokinetic properties of biomacromolecules. We devised a facile approach for attachment of PEG molecules with a defined molecular weight, and successful purification of the resulting conjugates. We directly conjugated structurally defined PEG chains with twelve ethylene glycol units to the 3′-terminal hydroxyl group of both sense and antisense strands via an aminoalkyl linker. The conjugates were easily purified by HPLC and successful PEGylation and molecule integrity were confirmed by ESI-MS. The evaluation of in vitro gene knockdown of two different targets in MCF-7 breast cancer cells showed stable pharmacologic activity when combined with a standard transfection reagent. Sense strand PEGylation even increased the silencing potency of a CRCX4-siRNA which had modest activity in its wild-type form. The results indicate that PEG chains at the 3′-terminus of both strands of siRNA are well tolerated by the RNAi effector. The attachment of short, chemically defined PEG chains is a feasible approach to improve the pharmacokinetic properties of siRNA, and can be combined with other targeted and untargeted delivery vehicles.     

The influence of matrix attachment regions on transgene expression in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> wild type and gene silencing mutants

Many studies in both animal and plant systems have shown that matrix attachment regions (MARs) can increase the expression of flanking transgenes. However, our previous studies revealed no effect of the chicken lysozyme MARs (chiMARs) on transgene expression in the first generation transgenic Arabidopsis thaliana plants transformed with a β-glucuronidase gene (uidA) unless gene silencing mutants were used as genetic background for transformation. In the present study, we investigated why chiMARs do not influence transgene expression in transgenic wild-type Arabidopsis plants. We first studied the effect of chiMARs on transgene expression in the progeny of primary transformants harboring chiMAR-flanked T-DNAs. Our data indicate that chiMARs do not affect transgene expression in consecutive generations of wild-type A. thaliana plants. Next, we examined whether these observed results in A. thaliana transformants are influenced by the applied transformation method. The results from in vitro transformed A. thaliana plants are in accordance with those from in planta transformed A. thaliana plants and again reveal no influence of chiMARs on transgene expression in A. thaliana wild-type transformants. The effect of chiMARs on transgene expression is also examined in in vitro transformed Nicotiana tabacum plants, but as for A. thaliana, the transgene expression in tobacco transformants is not altered by the presence of chiMARs. Taken together, our results show that the applied method or the plant species used for transformation does not influence whether and how chiMARs have an effect on transgene expression. Finally, we studied the effect of MARs (tabMARs) of plant origin (tobacco) on the transgene expression in A. thaliana wild-type plants and suppressed gene silencing (sgs2) mutants. Our results clearly show that similar to chiMARs, the tobacco-derived MARs do not enhance transgene expression in a wild-type background but can be used to enhance transgene expression in a mutant impaired in gene silencing. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. Miguel F.C. De Bolle, Katleen M.J. Butaye Contributed equally to this work     

Characterization of two rice DNA methyltransferase genes and RNAi-mediated reactivation of a silenced transgene in rice callus

Two genomic clones ( OsMET1-1, AF 462029 and OsMET1-2, TPA BK001405), each encoding a cytosine-5 DNA methyltransferase (MTase), were isolated from rice ( Oryza sativa L.) BAC libraries. OsMET1-1 has an open reading frame of 4,566 nucleotides with 12 exons and 11 introns while OsMET1-2 has an open reading frame of 4,491 nucleotides with 11 exons and 10 introns. Although OsMET1-1 and OsMET1-2 have high sequence similarity overall, they share only 24% identity in exon 1, and intron 3 of OsMET1-1 is absent from OsMET1-2. As for other eukaryotic DNA MTases of the Dnmt1/MET l class, the derived amino acid sequences of OsMET1-1 and OsMET1-2 suggest that they are comprised of two-thirds regulatory domain and one-third catalytic domain. Most functional domains identified for other MTases were present in the rice MET1 sequences. Amino acid sequence comparison indicated high similarity (56-75% identity) of rice MET1 proteins to other plant MET1 sequences but limited similarity (approx. 24% identity) to animal Dnmt1 proteins. Genomic blot and database analysis indicated the presence of a single copy of OsMET1-1 (on chromosome 3) and single copy of OsMET1-2 (on chromosome 7). Ribonuclease protection assays revealed expression of both OsMET1-1 and OsMET1-2 in highly dividing cells, but the steady-state level of OsMET1-2 was 7- to 12-fold higher than that for OsMET1-1 in callus, root and inflorescence. The functional involvement of the rice DNA MTases in gene silencing was investigated using an RNAi strategy. Inverted repeat constructs of either the N- or C-terminal regions of OsMET1-1 were supertransformed into calli derived from a rice line bearing a silenced 35S-uidA-nos transgene. Restoration of uidA expression in the bombarded calli was consistent with the inactivation of maintenance methylation and with previous evidence for the involvement of methylation in silencing of this line.     

转基因植物中外源基因沉默机制及防止对策

系统论述了诱发转基因沉默的因素、植物转基因沉默发生水平并提出了基因沉默的防止对策,基因沉默现象是导致外源基因不能在转化植物中正常表达的重要原因。甲基化、重复序列、反式失活和共抑制是基因沉默的主要诱因。其作用水平主要有三种：位置效应、转录水平的基因沉默和转录后水平的基因沉默,避免基因间的同源性、避免重复序列的出现、消除甲基化的影响、使用MAR以及使用诱导型启动子,可以提高外源基因的表达水平,有效防止基因沉默。     

RETRACTED ARTICLE: Virus-induced gene silencing for functional analysis of selected genes

Virus-induced gene silencing (VIGS) is a technology that exploits an RNA-mediated antiviral defense mechanism and has been shown to be of great potential in plant reverse genetics. Circumvention of plant transformation, methodological simplicity, robustness, and speedy results makes VIGS an attractive alternative instrument in functional genomics, even in a high throughput fashion. The system is well established in Nicotiana benthamiana, and efforts are being made to improve VIGS in other species, including monocots. Here, we discuss the issues specific to the application of VIGS technology to determine gene function, which has revealed the roles of a variety of genes in disease resistance, abiotic stress, cellular signaling and secondary metabolite biosynthesis. M. R. Godge and A. Purkayastha made equal contributions and hence should be treated as joint first authors for this paper.     

Expression of phytoene synthase gene (Psy) is enhanced during fruit ripening of Cara Cara navel orange (Citrus sinensis Osbeck)

Citrus is an important fruit crop as regards accumulation of carotenoids. In plant carotenoid biosynthesis, phytoene synthase gene (Psy) plays a key role in catalyzing the head-to-head condensation of geranylgeranyl diphosphate molecules to produce colorless phytoene. In the present paper, we reported the phytoene contents determination and characterization of Psy during fruit ripening of “Washington” navel orange and its red-fleshed mutant “Cara Cara”. Results showed that phytoene was exclusively accumulated in peel and pulp of “Cara Cara”. Although phytoene was observed accumulating with fruit ripening of “Cara Cara”, the contents in pulp were 10 times higher than those in peel. The isolated two Psy cDNAs were both 1520 bp in full length, containing 436 deduced amino acid residues, with a different amino acid at 412th. Genomic hybridization results showed that one or two copies might be present in “Cara Cara” and “Washington” genomes. During “Cara Cara” and “Washington” fruit coloration, expression of Psy was observed to be up-regulated, as revealed by tissue specific profiles in the flavedo, albedo, segment membrane and juice sacs. However, Psy expression in albedo of “Cara Cara” was higher than that in “Washington”, as evidenced by phytoene accumulation in the peel.     

The effects of thiophosphate substitutions on native siRNA gene silencing

RNA mediated interference has emerged as a powerful tool in controlling gene expression in mammalian cells. We investigated the gene silencing properties of six thiophosphate substituted siRNAs (all based on a commercial luciferase medium silencer) compared to that of unmodified siRNA. We also examined the cytotoxicity and dose-response using several thiophosphate modified siRNAs with unmodified siRNA. Our results show that two thiophosphate siRNA sequences convert from medium to high silencers with the addition of four randomly placed thiophosphates. Both thiophosphate siRNAs have a statistically significant difference in luciferase gene silencing (5% and 6% activity) relative to the unmodified native medium silencer referred to as siRNA-2 (18% activity) and four other thiophosphate siRNAs that maintain their medium silencing capability. This indicates that specific thiophosphate substitutions may alter native siRNA function. Further, this shows that thiophosphate siRNAs with the same nucleotide sequence but with different sulfur modification positions have different silencing effects. Both the native siRNA and the thio siRNAs showed a concentration dependent relationship, i.e., with concentration increase, the luciferase gene silencing effect also increased. Confirming cytotoxicity experiments showed no significant changes when HeLa cells were treated with 10nM thiophosphate siRNAs over the course of several days. These results suggest that specific placement of thiophosphates could play an important role in the development of siRNAs as therapeutics by engineering in properties such as strength of binding, nuclease sensitivity, and ultimately efficacy.     

Long time-course monitoring of ZFP809-mediated gene silencing in transgene expression driven by promoters containing MLV-derived PBS

Expression of Moloney murine leukemia virus (MoMLV)-typed retroviral vectors is strictly suppressed in immature cells such as embryonic stem cells. The phenomenon known as gene silencing is primed by the sequence-specific binding of the zinc finger protein 809 (ZFP809) to the primer-binding site of the vectors. However, it has yet to be determined whether the ZFP809-mediated gene silencing is maintained over a long period. In this study, we established an experimental system that can monitor gene silencing during a long-term cell culture using flow cytometry technology combined with fluorescent reporters for the expression of ZFP809 and the transgene expression driven by the promoters of interest. Time-course analysis using our system revealed that ZFP809 maintains gene silencing effect even at a longtime period. Furthermore, our system was useful for the monitoring of ZFP809-mediated gene silencing regardless of the types of vectors and cell lines.     

The capacity of transgenic tobacco to send a systemic RNA silencing signal depends on the nature of the inducing transgene locus

RNA silencing is a conserved eukaryotic pathway in which double-stranded RNA (dsRNA) triggers destruction of homologous target RNA via production of short-interfering RNA (siRNA). In plants, at least some cases of RNA silencing can spread systemically. The signal responsible for systemic spread is expected to include an RNA component to account for the sequence specificity of the process, and transient silencing assays have shown that the capacity for systemic silencing correlates with the accumulation of a particular class of small RNA. Here, we report the results of grafting experiments to study transmission of silencing from stably transformed tobacco lines in the presence or absence of helper component-proteinase (HC-Pro), a viral suppressor of silencing. The studied lines carry either a tail-to-tail inverted repeat, the T4-IR transgene locus, or one of two different amplicon transgene loci encoding replication-competent viral RNA. We find that the T4-IR locus, like many sense-transgene-silenced loci, can send a systemic silencing signal, and this ability is not detectably altered by HC-Pro. Paradoxically, neither amplicon locus effectively triggers systemic silencing except when suppressed for silencing by HC-Pro. In contrast to results from transient assays, these grafting experiments reveal no consistent correlation between capacity for systemic silencing and accumulation of any particular class of small RNA. In addition, although all transgenic lines used to transmit systemic silencing signals were methylated at specific sites within the transgene locus, silencing in grafted scions occurred without detectable methylation at those sites in the target locus of the scion.