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—Silencing of the luciferase (luc) gene in 2-month-old transgenic tobacco plants. (A) Luminescence of Luc in an overproducer of the transgene in the dark, line B117. (B) Reduced Luc luminescence in progeny of selfed B117 plants around the main and small veins in a lower leaf. (C) Progeny of selfed B117 with no detectable Luc activity. (D and E) Fully expanded upper leaf of 60 backcrossed progeny of B117. (E) Strong luminescence in the leaves of all 60 seedlings. (F) luc gene silencing in the leaf of selfed B117 progeny. The region around the veins and some leaves exhibited reduced or no detectable luminescence . Photographs were taken under a fluorescent lamp (D) or in the dark after feeding of luciferin (A–C, E, and F).
Source publication
Transgenic tobacco plants that overproduce luciferase (Luc) frequently exhibit post-transcriptional gene silencing (PTGS) of luc. The silencing was observed over five generations and found not to be inherited but acquired by the next generation at a certain frequency. Luc imaging analysis of silenced plants revealed Luc activity only in proliferati...
Citations
... suppression of post-translational gene silencing in meristems and reproductive tissues, 331 which may decrease the function of the RNAi machinery(Mitsuhara et al., 2002). Using maize 332 cenH3 RNAi lines complemented by the AcGREEN-tail swap-CENH3,Kelliher et al. (2016) 333 have demonstrated that in crosses with wild-type these lines can generate 0.24% maternal and 334 0.07% paternal haploids. ...
Double haploid production is the most effective way of creating true-breeding lines in a single generation. In Arabidopsis, haploid induction via mutation of the centromere-specific histone H3 (cenH3) has been shown when outcrossed to wild-type, whereas the wild-type genome remains in the haploid progeny. However, factors that affect haploid induction are still poorly understood. Here, we report that a mutant of the cenH3 assembly factor, Kinetochore Null2 (KNL2) can be used as a haploid inducer when pollinated by wild-type. We discovered that short temperature stress of the knl2 mutant increased the efficiency of haploid induction by 10-fold. We have also demonstrated that a point mutation in the CENPC-k motif of KNL2 is sufficient to generate haploid inducing lines, suggesting that haploid inducing lines in crops can be identified in a naturally occurring or chemically induced mutant population, avoiding the GMO approach at any stage. Furthermore, a cenh3-4 mutant functions as a haploid inducer in response to short heat stress even though it did not induce haploids under standard conditions. Thus, we identified KNL2 as a new target gene for the generation of haploid inducer lines and showed that exposure of mutants of centromeric proteins to high temperature strongly increases their haploid induction efficiency.
... Although transgenic RNAi techniques are relatively established in crop improvement, the generation and deployment of Genetically Modified (GM) RNAi crops necessitates extensive time and intensive resource commitment. The challenges to the development of transgenic RNAi crops include the absence of reliable methods for plant transformation and regeneration system (Altpeter et al. 2016), difficulties to achieve stable inheritance of silencing genes (Mitsuhara et al. 2002), stringent regulatory rules (Palli 2014), and public unease with GM technology (Scott et al. 2018). While a transgenic RNAi approach appears to be expensive for practical use in agriculture, though perhaps relatively less expensive than the true cost of using pesticides (Bourguet and Guillemaud 2016), non-transformative RNAi techniques have emerged as a promising alternative to GM plants, having applications including biocontrol to protect crops in open fields (see Table 1), and as a physical agent that is able to alter the phenotypic appearance of flowering buds (Lau et al. 2015). ...
Recent developments in modern biotechnology such as the use of RNA interference (RNAi) have broadened the scope of crop genetic modification. RNAi strategies have led to significant achievements in crop protection against biotic and abiotic stresses, modification of plant traits, and yield improvement. As RNAi-derived varieties of crops become more useful in the field, it is important to examine the capacity of current regulatory systems to deal with such varieties, and to determine if changes are needed to improve the existing frameworks. We review the biosafety frameworks from the perspective of developing countries that are increasingly involved in modern biotechnology research, including RNAi applications, and make some recommendations. Malaysia and India have approved laws regulating living modified organisms and products thereof, highlighting that the use of any genetically modified step requires regulatory scrutiny. In view of production methods for exogenously applied double-stranded RNAs and potential risks from the resulting double-stranded RNA-based products, we argue that a process-based system may be inappropriate for the non-transformative RNAi technology. We here propose that the current legislation needs rewording to take account of the non-transgenic RNAi technology, and discuss the best alternative for regulatory systems in India and Malaysia in comparison with the existing frameworks in other countries.
... A dominant gene could be under control of a native promoter, or the promoter could be improved, for example, to increase the expression level and to narrow the tissue specificity. In contrast, the use of a recessive gene must be combined with RNA interference technology or similar techniques; however, it is widely recognized that it is difficult to perfectly suppress the gene activity by these techniques, and the effectiveness of these techniques tends to be unstable in long-term applications (Mitsuhara et al. 2002). The availability of a dominant cassette to increase rice outcrossing fertility will make RSUTMS more efficient and applicable to large-scale rice breeding. ...
Stigma exsertion can enhance the outcrossing fertility in rice. Dominant genes for this trait are expected to be essential for the effective application of a novel outcrossing-based breeding system that uses male sterility in rice, which is normally autogamous. Because reduction of stigma exsertion is a domestication trait, we screened wild rice species as possible donors of genes or QTLs for stigma exsertion. We used in silico image-based screening and selected the Oryza rufipogon accession ‘W0120’. A single F1 individual derived from a cross between the japonica rice cultivar ‘Akidawara’ and ‘W0120’ was used to generate F2 and BC1F1 populations. QTL analysis performed using 114 F2 individuals detected QTLs on chromosomes 2, 3, 4, 8, and 11. Only two major QTLs on chromosomes 3 and 8 showed higher degrees of dominance. On the other hand, there were no QTLs near GS3, which is well known as a gene for stigma exsertion. Validation of these QTLs using 188 BC1F1 individuals provided clear evidence for their dominance. Genotypes of the markers nearest to the two QTLs were also related to grain length. We expect the genes responsible for these QTLs to be promising tools for improving outcrossing-based breeding in rice.
... Transgene silencing has often been observed when transgene dosage was doubled (de Carvalho et al., 1992;Dorlhac de Borne et al., 1994;Hart et al., 1992;Mitsuhara et al., 2002;Palauqui et al., 1996;Velten et al., 2012), suggesting silencing is based on increased transcript levels. Our work may well represent the first report that gene silencing can be triggered by ATFs. ...
Previously, we have shown transcriptional activation of endogenous genes in plants using dCas9-VP64. Here, we develop a second generation of vector systems for enhanced transcriptional activation in plants. We tested multiple strategies for CRISPR-Cas9 based transcriptional activation. Simultaneous recruitment of VP64 by dCas9 and a modified guide RNA scaffold gRNA2.0 (designated CRISPR-Act2.0) yielded stronger transcriptional activation than our first generation dCas9-VP64 activators. In addition, we have built a multiplex transcription activator-like effector activation (mTALE-Act) system for simultaneous activation of up to four genes in plants. Our results suggest that mTALE-Act is even more effective than CRISPR-Act2.0. Moreover, we explored tissue specific gene activation using positive feedback loops. Interestingly, our study revealed that certain endogenous genes are more amenable than others to transcriptional activation, and tightly regulated genes may cause target gene silencing when perturbed by activation probes. Hence, these new tools may be used to investigate gene regulatory networks and their control mechanisms. Assembly of multiplex CRISPR-Act2.0 and mTALE-Act systems are both based on streamlined and PCR-independent Golden Gate and Gateway cloning strategies. The systems will enable transcriptional activation applications in both dicots and monocots, and the vectors in this new toolbox are publicly available to the research community through Addgene.
... Induction of transgene silencing can be affected by various factors . These factors include the manner of T-DNA integration in the genome (i.e., single-copy, dispersed-repeat, directrepeat or inverted-repeat forms; Cluster et al. 1996;Jorgensen et al. 1996;Stam et al. 1998), copy number (Mitsuhara et al. 2002;Lechtenberg et al. 2003;Schubert et al. 2004;Wang et al. 2005), integration site (Vaucheret et al. 1995;Day et al. 2000;van Leeuwen et al. 2001;Kim et al. 2007), transcription rate (Que et al. 1997;Vaucheret et al. 1997;Schubert et al. 2004;Kanazawa et al. 2007), promoter types (Marjanac et al. 2009), or allelic state (i.e., homozygous or hemizygous; de Carvalho et al. 1992;Dehio and Schell 1994;Kalantidis et al. 2006) of the transgene, or read-through transcription of the neighboring transgene . Induction of transgene silencing can also be affected by factors other than the characteristics of the transgene: i.e., the transcription rate or mRNA level of an endogenous gene that has sequence homology with the transgene (Kunz et al. 1996;Han et al. 2004;Kalantidis et al. 2006); environmental conditions and/or developmental stages of the plant (de Carvalho Niebel et al. 1995;Meza et al. 2001;Szittya et al. 2003;Majewski et al. 2009;Marjanac et al. 2009). ...
... Understanding the mechanisms of transgene silencing provides information useful for engineering plants through either stable expression of transgene or stable silencing of a gene by transgene. Early studies in plants of N. tabacum or Nicotiana sylvestris indicated that RNA silencing is released during sexual reproduction (Hart et al. 1992;Dorlhac de Borne et al. 1994;Balandin and Castresana 1997;Mitsuhara et al. 2002). However, attempts to understand the entire picture of transgene silencing over the life cycle of plants and the transmission of the silenced state across generations are very limited. ...
... However, attempts to understand the entire picture of transgene silencing over the life cycle of plants and the transmission of the silenced state across generations are very limited. In particular, whether spread of silencing is inhibited in a particular plant tissue has not been known except for meristems in Nicotiana (Voinnet et al. 1998;Mitsuhara et al. 2002) and a hypocotyl-epicotyl junction in Arabidopsis (Kobayashi and Zambryski 2007;Liang et al. 2012). ...
The expression of transgenes introduced into a plant genome is sometimes suppressed by RNA silencing. Although local and systemic spread of RNA silencing have been studied, little is known about the mechanisms underlying spatial and temporal variation in transgene silencing between individual plants or between plants of different generations, which occurs seemingly stochastically. Here, we analyzed the occurrence, spread, and transmission of RNA silencing of the green fluorescent protein (GFP) gene over multiple generations of the progeny of a single soybean transformant. Observation of GFP fluorescence in entire plants of the T3–T5 generations indicated that the initiation and subsequent spread of GFP silencing varied between individuals, although this GFP silencing most frequently began in the primary leaves. In addition, GFP silencing could spread into the outer layer of seed coat tissues but was hardly detectable in the embryos. These results are consistent with the notion that transgene silencing involves its reset during reproductive phase, initiation after germination, and systemic spread in each generation. GFP silencing was absent in the pulvinus, suggesting that its cortical cells inhibit cell-to-cell spread or induction of RNA silencing. The extent of GFP silencing could differ between the stem and a petiole or between petiolules, which have limited vascular bundles connecting them and thus deter long-distant movement of silencing. Taken together, these observations indicate that the initiation and/or spread of RNA silencing depend on specific features of the architecture of the plant in addition to the mechanisms that can be conserved in higher plants.
... Laboratory studies have shown that the silenced state cannot be inherited directly, meaning that a parent cell will most likely be unable to produce daughter cells with the anti-viral components needed to deal with the viral infection. It is, therefore, believed that undifferentiated and proliferating cells, e.g meristematic tissue, need to mature or be released from cellular reproduction before they can acquire an antiviral state [25]. Hence, we introduce P (t) as the population of proliferating cells that are responsible for promoting new plant growth. ...
... the system of equations (24) can be re-written as follows g 1 (τ 1 ) sin(wτ 1 ) − g 2 (τ 1 ) cos(wτ 1 ) = L 1 (τ 1 ), g 1 (τ 1 ) cos(wτ 1 ) + g 2 (τ 1 ) sin(wτ 1 ) = L 2 (τ 1 ). (25) Solving this system yields ...
In the studies of plant infections, the plant immune response is known to
play an essential role. In this paper we derive and analyse a new mathematical
model of plant immune response with particular account for post-transcriptional
gene silencing (PTGS). Besides biologically accurate representation of the PTGS
dynamics, the model explicitly includes two time delays to represent the
maturation time of the growing plant tissue and the non-instantaneous nature of
the PTGS. Through analytical and numerical analysis of stability of the steady
states of the model we identify parameter regions associated with recovery and
resistant phenotypes, as well as possible chronic infections. Dynamics of the
system in these regimes is illustrated by numerical simulations of the model.
... To confirm the anti-silencing activities of the ASR candidates on different transgenes, we established a TGS-inducing system based on an increasing copy number of P35S regions (an inverse relationship between transgene copy number and expression level is often observed in homology-dependent TGS [3,4,9]). We had previously produced a transgenic tobacco plant (Figure 2A, NW7-24-4) harboring an enhanced P35S::LUC (luciferase gene) that exhibits a markedly high level of LUC expression [15]. Since this transgenic plant is a single-copy-inserted homozygote of the enhanced P35S::LUC construct, which itself carries two copies of the 35S enhancer region and one copy of the 35S promoter region, this plant has four copies of the 35S enhancer region and two copies of the 35S promoter region per diploid (Figure 2A). ...
... We found a considerable number of supertransformed plants showing low levels of LUC expression ( Figure 2D, left). In supertransformants without ASR602 ( Figure 2D, top left, CST/ Total), there was a significant positive correlation between P35S::GUS and P35S::LUC expression levels ( Table S3); most of the supertransformants with low GUS expression also showed extremely low LUC expression, indicating that supertransformation with P35S::GUS induced trans-TGS of P35S::LUC ( Figure 2D Plants harboring multiple copies of a transgene tend to have complicated transgenic loci [18] and/or undergo PTGS [2,15]. ...
... Plants and cultured cells of wild type (Nicotiana tabacum cv. Samsun NN) and transgenic tobacco were grown as described [15]. To select a plant showing trans-TGS activity, plants from transgenic tobacco lines M65 and M66 were used [14]. ...
Transcriptional gene silencing (TGS)-a phenomenon observed in endogenous genes/transgenes in eukaryotes-is a huge hindrance to transgenic technology and occurs mainly when the genes involved share sequence homology in their promoter regions. TGS depends on chromosomal position, suggesting the existence of genomic elements that suppress TGS. However, no systematic approach to identify such DNA elements has yet been reported. Here, we developed a successful novel screening strategy to identify such elements (anti-silencing regions-ASRs), based on their ability to protect a flanked transgene from TGS. A silenced transgenic tobacco plant in which a subsequently introduced transgene undergoes obligatory promoter-homology dependent TGS in trans allowed the ability of DNA elements to prevent TGS to be used as the screening criterion. We also identified ASRs in a genomic library from a different plant species (Lotus japonicus: a perennial legume); the ASRs include portions of Ty1/copia retrotransposon-like and pararetrovirus-like sequences; the retrotransposon-like sequences also showed interspecies anti-TGS activity in a TGS-induction system in Arabidopsis. Anti-TGS elements could provide effective tools to reduce TGS and ensure proper regulation of transgene expression. Furthermore, the screening strategy described here will also facilitate the efficient identification of new classes of anti-TGS elements.
... Due to the stochastic nature of most transgene silencing it has proved difficult to directly address the nature of molecular factors associated with the initiation, spread and maintenance of transgene-targeted silencing. There have been a few reports where transgene silencing was found to start in response to the doubling of transgene dosage occurring within plants made homozygous for a single copy transgene [24,25,26,27,28,29,30,31,32] . These examples provide experimental systems that are theoretically amenable to a molecular examination of early silencing processes, but are often complicated by inconsistent or incomplete transgene silencing. ...
... Resulting de-differentiated tobacco cells displayed strong anthocyanin production, indicating a reversal of transgene silencing (Additional file Fig. S2). The observed reversal of silencing in propagating cells is similar to that seen previously with a silenced green fluorescent protein (GFP) transgene in Nicotiana benthamiana [26,39]. ...
... Duplication, rearrangement and/or read-through transcription of introduced DNA has been found to be strongly associated with transgene silencing and likely reflect an unintended production of dsRNA. Silencing of single copy transgenes also occurs in a subset of plant transformation events and has occasionally been found to be triggered by the doubling of transgene copy number associated with plants made homozygous for the introduced DNA [24,25,26,27,28,29,30,31, 32]. These transgenic lines present an opportunity to examine initiation of silencing in a controlled and otherwise isogenic environment. ...
Transgenic tobacco (Nicotiana tabacum) lines were engineered to ectopically over-express AtMYB90 (PAP2), an R2-R3 Myb gene associated with regulation of anthocyanin production in Arabidopsis thaliana. Independently transformed transgenic lines, Myb27 and Myb237, accumulated large quantities of anthocyanin, generating a dark purple phenotype in nearly all tissues. After self-fertilization, some progeny of the Myb27 line displayed an unexpected pigmentation pattern, with most leaves displaying large sectors of dramatically reduced anthocyanin production. The green-sectored 27Hmo plants were all found to be homozygous for the transgene and, despite a doubled transgene dosage, to have reduced levels of AtMYB90 mRNA. The observed reduction in anthocyanin pigmentation and AtMYB90 mRNA was phenotypically identical to the patterns seen in leaves systemically silenced for the AtMYB90 transgene, and was associated with the presence of AtMYB90-derived siRNA homologous to both strands of a portion of the AtMYB90 transcribed region. Activation of transgene silencing in the Myb27 line was triggered when the 35S::AtMYB90 transgene dosage was doubled, in both Myb27 homozygotes, and in plants containing one copy of each of the independently segregating Myb27 and Myb237 transgene loci. Mapping of sequenced siRNA molecules to the Myb27 TDNA (including flanking tobacco sequences) indicated that the 3' half of the AtMYB90 transcript is the primary target for siRNA associated silencing in both homozygous Myb27 plants and in systemically silenced tissues. The transgene within the Myb27 line was found to consist of a single, fully intact, copy of the AtMYB90 construct. Silencing appears to initiate in response to elevated levels of transgene mRNA (or an aberrant product thereof) present within a subset of leaf cells, followed by spread of the resulting small RNA to adjacent leaf tissues and subsequent amplification of siRNA production.
... Future prospects of the use of RNA silencing in soy- bean 1. Stability and heritability of RNA silencing Induction of transgene-mediated RNA silencing can be affected by various factors such as structure, copy number, or expression level of the transgene, environmental conditions or developmental stages of the plant (Majewski et al. 2009, and references therein). In addition, induction of transgenemediated RNA silencing can be destabilized during cell proliferation and appears to be re-initiated in each generation (Furutani et al. 2007, Mitsuhara et al. 2002). However, transgene-mediated RNA silencing can induce a strong, tissue-specific or ubiquitous silencing and is suitable for producing plants in which one or more genes are stably silenced in the presence of the transgene as far as the transgene is capable of inducing the silencing. ...
RNA silencing refers collectively to diverse RNA-mediated pathways of nucleotide-sequence-specific inhibition of gene expression. It has been used to analyze gene function and engineer novel traits in various organisms. Here, we review the application of RNA silencing in soybean. To produce soybean lines, in which a particular gene is stably silenced, researchers have frequently used a transgene that transcribes inverted repeats of a target gene segment. Suppression of gene expression in developing soybean embryos has been one of the main focuses of metabolic engineering using transgene-induced silencing. Plants that have enhanced resistance against diseases caused by viruses or cyst nematode have also been produced. Meanwhile, Agrobacterium rhizogenes-mediated transformation has been used to induce RNA silencing in roots, which enabled analysis of the roles of gene products in nodulation or disease resistance. RNA silencing has also been induced using viral vectors, which is particularly useful for gene function analysis. So far, three viral vectors for virus-induced gene silencing have been developed for soybean. One of the features of the soybean genome is the presence of a large number of duplicated genes. Potential use of RNA silencing technology in combination with forward genetic approaches for analyzing duplicated genes is discussed.
... The significantly weaker reduction of CENH3 in root tips than in cotyledons and leaves of transformants may be due to lower expression of the interfering RNA controlled by the CaMV 35S promoter, which is about three times less active in roots than in leaves (Holtorf et al., 1995). Another possible explanation may be suppression of post-transcriptional gene silencing in meristems, as reported for floral, shoot and root apices in tobacco (Nicotiana tabacum) (Voinnet et al., 1998;Mitsuhara et al., 2002) as well as for the root apex, young leaves and calli of A. thaliana (Marjanac et al., 2009). Nevertheless, for essential single-copy genes such as the CENH3 encoding gene, for which no T-DNA insertion lines are available, RNAi knockdown of gene expression is still the method of choice to test whether reduction of gene expression in non-meristematic tissues has an impact on growth and development as observed for the CENH3 RNAi transformants. ...
The histone H3 variant (CENH3) of centromeric nucleosomes is essential for kinetochore assembly and thus for chromosome segregation in eukaryotes. The mechanism(s) that determine centromere identity, assembly and maintenance of kinetochores are still poorly understood. Although the role of CENH3 during mitosis has been studied in several organisms, little is known about its meiotic function. We show that RNAi-mediated CENH3 knockdown in Arabidopsis thaliana caused dwarfism as the result of a reduced number of mitotic divisions. The remaining mitotic divisions appeared to be error-free. CENH3 RNAi transformants had reduced fertility because of frequently disturbed meiotic chromosome segregation. N-terminally truncated EYFP–CENH3(C) is deposited to and functional within Arabidopsis centromeres of mitotic chromosomes, but cannot be loaded onto centromeres of meiotic nuclei. Thus the N-terminal part is apparently required for CENH3 loading during meiosis. EYFP–CENH3(C) expression reduces the amount of endogenous CENH3, thus mimicking the effect of RNAi. The consequences of reduced endogenous CENH3 and lack of meiotic incorporation of EYFP–CENH3(C) are reduced fertility caused by insufficient CENH3 loading to the centromeres of meiotic chromosomes, subsequent lagging of chromosomes and formation of micronuclei.
