Aberrant promoter methylation occurred from multicopy transgene and SU(VAR)3 - 9 homolog 9 (SUVH9) gene in transgenic Nicotiana benthamiana

2012 ◽  
Vol 39 (9) ◽  
pp. 764 ◽  
Author(s):  
Gi-Ho Lee ◽  
Seong-Han Sohn ◽  
Eun-Young Park ◽  
Young-Doo Park
Keyword(s):  
Gene Silencing ◽  
Nicotiana Benthamiana ◽  
Fluorescent Protein ◽  
Cauliflower Mosaic Virus ◽  
Camv 35S Promoter ◽  
35S Promoter ◽  
Camv 35S ◽  
Histone Deacetylase 1 ◽  
Transgenic Lines ◽  
Green Fluorescent

The chemical modification of DNA by methylation is a heritable trait and can be subsequently reversed without altering the original DNA sequence. Methylation can reduce or silence gene expression and is a component of a host’s defence response to foreign nucleic acids. In our study, we employed a plant transformation strategy using Nicotiana benthamiana Domin to study the heritable stability of the introduced transgenes. Through the introduction of the cauliflower mosaic virus (CaMV) 35S promoter and the green fluorescent protein (GFP) reporter gene, we demonstrated that this introduced promoter often triggers a homology-dependent gene-silencing (HDGS) response. These spontaneous transgene-silencing phenomena are due to methylation of the CaMV 35S promoter CAAT box during transgenic plant growth. This process is catalysed by SU(VAR)3–9 homologue 9 (SUVH9), histone deacetylase 1 (HDA1) and domains rearranged methylase 2 (DRM2). In particular, we showed from our data that SUVH9 is the key regulator of methylation activity in epigenetically silenced GFP transgenic lines; therefore, our findings demonstrate that an introduced viral promoter and transgene can be subject to a homology-dependent gene-silencing mechanism that can downregulate its expression and negatively influence the heritable stability of the transgene.

scholarly journals Functional Analysis of a Viral Promoter From the Strawberry Vein Banding Virus (SVBV) Chinese Isolate

2021 ◽  
Author(s):  
Lei Jiang ◽  
Jing Chen ◽  
You-zhi Yang ◽  
Rui Li ◽  
Shuang Li ◽  
...  
Keyword(s):  
Gene Expression ◽  
Fluorescent Protein ◽  
Full Length ◽  
Camv 35S Promoter ◽  
Chinese Isolate ◽  
35S Promoter ◽  
Deletion Mutants ◽  
Camv 35S ◽  
Strawberry Vein Banding Virus ◽  
Green Fluorescent

Abstract BackgroundPromoter is an important factor during gene expression in cells. In this study, we cloned a full-length promoter from the strawberry vein banding virus (SVBV) Chinese isolate and produced several its deletion mutants.MethodsThe full-length promoter of SVBV (SP1) and its three deletion mutants (SP2, SP3, and SP4) were amplified using polymerase chain reaction (PCR). The expression activities controlled by the SVBV SP1, SP2, SP3, and SP4 were evaluated using β-glucuronidase (GUS) and green fluorescent protein (GFP) reporter genes.ResultsOur transient expression assays showed that the SVBV SP1 promoter as well as its three deletion mutants all expressed the reporter genes, but to very different levels. Interestingly, the expression activity driven by the SP1 promoter was much higher than that shown by the CaMV 35S promoter. After stable transformation of a GUS gene into Nicotiana tabacum plants, the transgene expression level driven by the SVBV SP1 promoter was about 2.6-fold greater than that driven by the CaMV 35S promoter. In addition, the GUS gene expression levels could be enhanced by co-infiltrating the plants with the SP1 promoter-driven vector carrying the GUS gene and the vector expressing the SVBV ORF V or ORF VI.ConclusionsThe SVBV Chinese isolate promoter SP1 is a stronger promoter than the CaMV 35S and FLt-US promoter, may be more useful for production of stable transgenic plants.

Transcriptional silencing of RNAi constructs against nematode genes in Arabidopsis

Nematology ◽  
2013 ◽  
Vol 15 (5) ◽  
pp. 519-528 ◽  
Author(s):  
Tina Kyndt ◽  
Hongli Ji ◽  
Bartel Vanholme ◽  
Godelieve Gheysen
Keyword(s):  
Transcriptional Silencing ◽  
Housekeeping Genes ◽  
Cauliflower Mosaic Virus ◽  
Heterodera Schachtii ◽  
Camv 35S Promoter ◽  
35S Promoter ◽  
Expression Levels ◽  
Camv 35S ◽  
General Decline ◽  
Transgenic Lines

In this research, Arabidopsis thaliana plants were transformed with hairpin constructs targeting cyst nematode (Heterodera schachtii) genes, driven by the cauliflower mosaic virus (CaMV) 35S promoter: two housekeeping genes (the splicing factor Hs-U2AF and the vacuolar Hs-H+ATPase) and one candidate effector gene (the ubiquitin extension protein Hs-ubi). Expression of the dsRNA appeared to be extremely variable between and within homozygous T3 lines and even between tissues. Infection experiments showed up to 50% reduction in nematode infection for some transgenic lines. The results varied not only between lines containing the same construct but also between independent repetitions of the experiment. Further focusing on the Hs-U2AF-RNAi lines revealed large variations and a general decline of construct expression levels over the generations. Bisulphite sequencing of a 197 bp part of the CaMV 35S promoter revealed substantial methylation in this region and a negative correlation between the methylation level and expression of the hairpin construct. Taken together, our results show that host-generated RNAi can suffer from high levels of transcriptional silencing of the construct, leading to varying expression levels within and between transgenic lines.

scholarly journals A simple and accurate PCR method for detection of genetically modified rice

2019 ◽  
Vol 17 (2) ◽  
pp. 847-851 ◽  
Author(s):  
Payam Safaei ◽  
Ebrahim Molaee Aghaee ◽  
Gholamreza Jahed Khaniki ◽  
Setareh Agha Kuchak Afshari ◽  
Sassan Rezaie
Keyword(s):  
Genetically Modified ◽  
Sucrose Phosphate Synthase ◽  
Cauliflower Mosaic Virus ◽  
Camv 35S Promoter ◽  
35S Promoter ◽  
Rice Varieties ◽  
Camv 35S ◽  
Nos Terminator ◽  
Gm Rice ◽  
Pcr Method

Abstract Background Legislation regulating for labeling and use of genetically modified (GM) crops are increased considerably worldwide in order to health and safety assurance of consumers. For this purpose, a polymerase chain reaction (PCR) method has been developed for detection of GM rice in people’s food diet. Methods In this study, eighty-one non-labeled rice samples were collected randomly from different market sites of Tehran, Iran. In order to analysis, rice genomic DNA was extracted using MBST DNA extraction kit and subsequently, sucrose phosphate synthase (SPS) gene was used to confirm the quality of extracted DNA. Then, cauliflower mosaic virus (CaMV) 35S promoter and Agrobacterium nopaline synthase (NOS) terminator were selected as screening targets for detection of GM rice sequences by PCR. Results According to our results, 2 out of 81 (2.4%) samples tested were positive for CaMV 35S promoter while no positive result was detected for NOS terminator. Conclusion The obtained data indicated that this method is capable to identify the GM rice varieties. Furthermore, it can demonstrate the possibility of the presence of GM rice in Tehran’s market, thus putting emphasis on the requirement for developing a precise approach to evaluate this product.

scholarly journals Differential Roles of AC2 and AC4 of Cassava Geminiviruses in Mediating Synergism and Suppression of Posttranscriptional Gene Silencing

2004 ◽  
Vol 78 (17) ◽  
pp. 9487-9498 ◽  
Author(s):  
Ramachandran Vanitharani ◽  
Padmanabhan Chellappan ◽  
Justin S. Pita ◽  
Claude M. Fauquet
Keyword(s):  
Gene Silencing ◽  
Mosaic Virus ◽  
Defense Mechanism ◽  
Fluorescent Protein ◽  
Cauliflower Mosaic Virus ◽  
African Cassava Mosaic Virus ◽  
35S Promoter ◽  
Viral Dna ◽  
Posttranscriptional Gene Silencing ◽  
Cassava Mosaic Virus

ABSTRACT Posttranscriptional gene silencing (PTGS) in plants is a natural defense mechanism against virus infection. In mixed infections, virus synergism is proposed to result from suppression of the host defense mechanism by the viruses. Synergistic severe mosaic disease caused by simultaneous infection with isolates of the Cameroon strain of African cassava mosaic virus (ACMV-[CM]) and East African cassava mosaic Cameroon virus (EACMCV) in cassava and tobacco is characterized by a dramatic increase in symptom severity and a severalfold increase in viral-DNA accumulation by both viruses compared to that in singly infected plants. Here, we report that synergism between ACMV-[CM] and EACMCV is a two-way process, as the presence of the DNA-A component of ACMV-[CM] or EACMCV in trans enhanced the accumulation of viral DNA of EACMCV and ACMV-[CM], respectively, in tobacco BY-2 protoplasts. Furthermore, transient expression of ACMV-[CM] AC4 driven by the Cauliflower mosaic virus 35S promoter (p35S-AC4) enhanced EACMCV DNA accumulation by ∼8-fold in protoplasts, while p35S-AC2 of EACMCV enhanced ACMV-[CM] DNA accumulation, also by ∼8-fold. An Agrobacterium-based leaf infiltration assay determined that ACMV-[CM] AC4 and EACMCV AC2, the putative synergistic genes, were able to suppress PTGS induced by green fluorescent protein (GFP) and eliminated the short interfering RNAs associated with PTGS, with a correlated increase in GFP mRNA accumulation. In addition, we have identified AC4 of Sri Lankan cassava mosaic virus and AC2 of Indian cassava mosaic virus as suppressors of PTGS, indicating that geminiviruses evolved differently in regard to interaction with the host. The specific and different roles played by these AC2 and AC4 proteins of cassava geminiviruses in regulating anti-PTGS activity and their relation to synergism are discussed.

scholarly journals High transgene expression is associated with systemic GFP silencing in Nicotiana benthamiana

2021 ◽  
Author(s):  
Bill Hendrix ◽  
Paul Hoffer ◽  
Rick Sanders ◽  
Steve Schwartz ◽  
Wei Zheng ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Nicotiana Benthamiana ◽  
Transgene Expression ◽  
Fluorescent Protein ◽  
Integration Site ◽  
Crop Protection ◽  
Transgene Silencing ◽  
Expression Level ◽  
Transgenic Lines ◽  
Transgene Expression Level

AbstractGene silencing in plants using topical dsRNA is a new approach that has the potential to be a sustainable component of the agricultural production systems of the future. However, more research is needed to enable this technology as an economical and efficacious supplement to current crop protection practices. Systemic gene silencing is one key enabling aspect. The objective of this research was to better understand systemic transgene silencing in Nicotiana benthamiana. Previous reports details sequencing of the integration site of the Green Fluorescent Protein (GFP) transgene in the well-known N. benthamiana GFP16C event revealed inadvertent co-integration of part of a bacterial transposase. To determine the effect of this transgene configuration on systemic silencing, new GFP transgenic lines with or without the transposase sequences were produced. GFP expression levels in the 19 single-copy events and three hemizygous 16C lines produced for this study ranged from 50-72% of the homozygous 16C line. GFP expression was equivalent to 16C in a two-copy event. Local GFP silencing was observed in all transgenic and 16C hemizygous lines after topical application of delivery formulations with a GFP targeting dsRNA. The 16C-like systemic silencing phenotype was only observed in the two-copy line. The partial transposase had no impact on transgene expression level, local GFP silencing, small RNA abundance and distribution, or systemic GFP silencing in the transgenic lines. We conclude that high transgene expression level is a key enabler of systemic transgene silencing in N. benthamiana.

scholarly journals A single chimeric transgene derived from two distinct viruses confers multi-virus resistance in transgenic plants through homology-dependent gene silencing

2000 ◽  
Vol 81 (8) ◽  
pp. 2103-2109 ◽  
Author(s):  
Fuh-Jyh Jan ◽  
Carmen Fagoaga ◽  
Sheng-Zhi Pang ◽  
Dennis Gonsalves
Keyword(s):  
Gene Silencing ◽  
Virus Resistance ◽  
Fluorescent Protein ◽  
Gene Segment ◽  
N Gene ◽  
Transcriptional Gene Silencing ◽  
Multiple Resistance ◽  
Cp Gene ◽  
Transgenic Lines ◽  
Green Fluorescent

We showed previously that 218 and 110 bp N gene segments of tomato spotted wilt virus (TSWV) that were fused to the non-target green fluorescent protein (GFP) gene were able to confer resistance to TSWV via post-transcriptional gene silencing (PTGS). N gene segments expressed alone did not confer resistance. Apparently, the GFP DNA induced PTGS that targetted N gene segments and the incoming homologous TSWV for degradation, resulting in a resistant phenotype. These observations suggested that multiple resistance could be obtained by replacing the GFP DNA with a viral DNA that induces PTGS. The full-length coat protein (CP) gene of turnip mosaic virus (TuMV) was linked to 218 or 110 bp N gene segments and transformed into Nicotiana benthamiana. A high proportion (4 of 18) of transgenic lines with the 218 bp N gene segment linked to the TuMV CP gene were resistant to both viruses, and resistance was transferred to R2 plants. Nuclear run-on and Northern experiments confirmed that resistance was via PTGS. In contrast, only one of 14 transgenic lines with the TuMV CP linked to a 110 bp N gene segment yielded progeny with multiple resistance. Only a few R1 plants were resistant and resistance was not observed in R2 plants. These results clearly show the applicability of multiple virus resistance through the fusion of viral segments to DNAs that induce PTGS.

Research note: The 35S promoter is not constitutively expressed in the transgenic tropical actinorhizal tree Casuarina glauca

2002 ◽  
Vol 29 (5) ◽  
pp. 649 ◽  
Author(s):  
Aziz Smouni ◽  
Laurent Laplaze ◽  
Didier Bogusz ◽  
Fathia Guermache ◽  
Florence Auguy ◽  
...  
Keyword(s):  
Vascular Tissue ◽  
Gus Expression ◽  
Cauliflower Mosaic Virus ◽  
Camv 35S Promoter ◽  
35S Promoter ◽  
Casuarina Glauca ◽  
Camv 35S ◽  
Highly Active ◽  
One Year

The tropical nitrogen-fixing tree, Casuarina glauca Sieb. ex Spreng. was genetically transformed using Agrobacterium tumefaciens C58C1(pGV2260; pBIN19GUSINT). We report on the expression pattern conferred by the cauliflower mosaic virus (CaMV) 35S promoter in transgenic C. glauca plants grown in vitro, and for one year in a greenhouse. Histochemical assays in shoots from in vitro plants revealed β-glucuronidase (GUS) staining in apical and axillary buds, and in nearly all tissues near the base of the stem. In roots, the CaMV 35S drove strong GUS expression in the apex and vascular tissue. In 1-year old plants grown in a greenhouse, the CaMV 35S promoter was highly active, except in peripheral suberized tissues. Transgenic C. glauca plants were nodulated by the actinomycete Frankia. Histochemical assays on vibratome sections of transgenic nodules demonstrated intense GUS activity in the vascular bundle, the phellogen, and in strands of uninfected cells filled with polyphenols. GUS expression was undetectable in Frankia-infected cells.

scholarly journals Cell-to-Cell Movement of the 25K Protein of Potato virus X Is Regulated by Three Other Viral Proteins

2000 ◽  
Vol 13 (6) ◽  
pp. 599-605 ◽  
Author(s):  
Yang Yang ◽  
Biao Ding ◽  
David C. Baulcombe ◽  
Jeanmarie Verchot
Keyword(s):  
Transgenic Tobacco ◽  
Potato Virus ◽  
Fluorescent Protein ◽  
Cell Movement ◽  
Potato Virus X ◽  
Viral Proteins ◽  
Cauliflower Mosaic Virus ◽  
35S Promoter ◽  
Green Fluorescent ◽  
Potential Interactions

The 25K, 12K, and 8K proteins and coat protein (CP) of Potato virus X (PVX) are required for virus cell-to-cell movement. In this study, experiments were conducted to determine whether the PVX 25K protein moves cell to cell and to explore potential interactions between the PVX 25K, 12K, and 8K proteins and CP. The PVX 25K gene was fused to the green fluorescent protein (GFP) gene and inserted into plasmids adjacent to the cauliflower mosaic virus 35S promoter. These plasmids were introduced by biolistic bombardment to transgenic tobacco expressing the PVX 12K, 8K, and CP genes. The GFP:25K fused proteins moved cell to cell on nontransgenic tobacco and tobacco expressing either the 12K or 8K proteins. However, the GFP:25K proteins did not move on transgenic tobacco expressing the combined 12K/8K genes or the CP gene. Thus, movement of the PVX 25K protein through plas-modesmata may be regulated by interactions with other PVX proteins.

scholarly journals Engineering Resistance to Multiple Prunus Fruit Viruses Through Expression of Chimeric Hairpins

2007 ◽  
Vol 132 (3) ◽  
pp. 407-414 ◽  
Author(s):  
Zongrang Liu ◽  
Ralph Scorza ◽  
Jean-Michel Hily ◽  
Simon W. Scott ◽  
Delano James
Keyword(s):  
Gene Silencing ◽  
Mosaic Virus ◽  
Fruit Production ◽  
Cauliflower Mosaic Virus ◽  
Ringspot Virus ◽  
35S Promoter ◽  
Plum Pox Virus ◽  
In Planta ◽  
Posttranscriptional Gene Silencing ◽  
Transgenic Lines

Prunus L. fruit production is seriously affected by several predominant viruses. The development of new cultivars resistant to these viruses is challenging but highly desired by breeders and growers. We report a posttranscriptional gene silencing-based approach for engineering multivirus resistance in plants. A single chimeric transgene, PTRAP6, was created by the fusion of 400 to 500-base pair (bp) gene fragments from six major Prunus fruit viruses, including american plum line pattern virus, peach mosaic virus, plum pox virus (PPV), prune dwarf virus (PDV), prunus necrotic ringspot virus, and tomato ringspot virus (ToRSV). Both strands of PTRAP6 were found being transcribed as an ≈2.5-kilobp transcript in planta without splicing interruption. To induce gene silencing/virus resistance, we placed two copies of PTRAP6 in an inverted repeat under the control of the cauliflower mosaic virus 35S promoter and separated by an intron spacer fragment to create PTRAP6i. Inoculation of the resulting transgenic Nicotiana benthamiana Domin. plants revealed that 12 of 28 R0 PTRAP6i transgenic lines (43%) were resistant to ToRSV ranging from mild symptoms to symptom-free phenotypes. Detailed analysis of two of three highly resistant homozygous R3 generation lines demonstrated that they were resistant to all three viruses tested, including PDV, PPV, and ToRSV. The remaining three viruses targeted by PTRAP6i were either unavailable for this study or were unable to systemically infect N. benthamiana. Transgene-wide and -specific small interfering RNA species were detected along with disappearance of transgene transcript in the resistant lines, indicating that posttranscriptional gene silencing underlies the mechanism of resistance. This work presents evidence that PTRAP6i is able to confer gene silencing-based resistance to multiple Prunus fruit viruses.

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