scholarly journals Tombusvirus P19-Mediated Suppression of Virus-Induced Gene Silencing Is Controlled by Genetic and Dosage Features That Influence Pathogenicity

2002 ◽  
Vol 15 (3) ◽  
pp. 269-280 ◽  
Author(s):  
Wenping Qiu ◽  
Jong-Won Park ◽  
Herman B. Scholthof
Keyword(s):  
Gene Silencing ◽  
Fluorescent Protein ◽  
Biological Activities ◽  
Potato Virus X ◽  
Virus Spread ◽  
Virus Induced Gene Silencing ◽  
Suppressor Activity ◽  
Gfp Gene ◽  
Pathogenicity Determinant ◽  
Green Fluorescent

The p19 protein (P19) of Tomato bushy stunt virus (TBSV) is a pathogenicity determinant with host-dependent effects on virus spread and symptom induction. In addition, results in this study confirm that Potato virus X-mediated delivery of P19 suppresses posttranscriptional gene silencing (PTGS). To study the relevance of this activity for TBSV biology, we evaluated whether TBSV activates virus-induced gene silencing (VIGS) and if this process is suppressed by P19. TBSV vectors with the green fluorescent protein (GFP) gene, either active or inactive for P19 expression, were inoculated onto GFP-transgenic Nicotiana bentha-miana plants. In the absence of P19 expression, VIGS was activated, as evidenced by the disappearance of GFP mRNA and green fluorescence. Coexpression of GFP and P19 from the TBSV vector suppressed VIGS, except in the newly emerging leaves. The suppressor activity required a central P19 region that is also known to be essential for host-dependent virus spread and symptom induction. Defective interfering RNAs (DIs) that contained the 3′ end of the GFP gene induced silencing very effectively. The concomitant DI-instigated reduction in P19 accumulation failed to suppress this process, analogous to the known P19 dosage effects for other biological activities. In conclusion, (i) TBSV and its DIs are very effective inducers of VIGS, (ii) P19 is a strong suppressor of PTGS, (iii) P19 is a moderate suppressor of VIGS, and (iv) the suppressor activity is influenced by genetic and dosage features that are also important for P19-associated pathogenesis.

scholarly journals Tobacco mosaic virus 126-kDa Protein Increases the Susceptibility of Nicotiana tabacum to Other Viruses and Its Dosage Affects Virus-Induced Gene Silencing

2008 ◽  
Vol 21 (12) ◽  
pp. 1539-1548 ◽  
Author(s):  
Phillip A. Harries ◽  
Karuppaiah Palanichelvam ◽  
Sumana Bhat ◽  
Richard S. Nelson
Keyword(s):  
Nicotiana Tabacum ◽  
Gene Silencing ◽  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
Fluorescent Protein ◽  
Tobacco Rattle Virus ◽  
Potato Virus X ◽  
Brome Mosaic Virus ◽  
Virus Induced Gene Silencing ◽  
Suppressor Activity

The Tobacco mosaic virus (TMV) 126-kDa protein is a suppressor of RNA silencing previously shown to delay the silencing of transgenes in Nicotiana tabacum and N. benthamiana. Here, we demonstrate that expression of a 126-kDa protein–green fluorescent protein (GFP) fusion (126-GFP) in N. tabacum increases susceptibility to a broad assortment of viruses, including Alfalfa mosaic virus, Brome mosaic virus, Tobacco rattle virus (TRV), and Potato virus X. Given its ability to enhance TRV infection in tobacco, we tested the effect of 126-GFP expression on TRV-mediated virus-induced gene silencing (VIGS) and demonstrate that this protein can enhance silencing phenotypes. To explain these results, we examined the poorly understood effect of suppressor dosage on the VIGS response and demonstrated that enhanced VIGS corresponds to the presence of low levels of suppressor protein. A mutant version of the 126-kDa protein, inhibited in its ability to suppress silencing, had a minimal effect on VIGS, suggesting that the suppressor activity of the 126-kDa protein is indeed responsible for the observed dosage effects. These findings illustrate the sensitivity of host plants to relatively small changes in suppressor dosage and have implications for those interested in enhancing silencing phenotypes in tobacco and other species through VIGS.

scholarly journals Satellite Panicum Mosaic Virus Capsid Protein Elicits Symptoms on a Nonhost Plant and Interferes with a Suppressor of Virus-Induced Gene Silencing

2004 ◽  
Vol 17 (3) ◽  
pp. 263-271 ◽  
Author(s):  
Wenping Qiu ◽  
Karen-Beth G. Scholthof
Keyword(s):  
Gene Silencing ◽  
Mosaic Virus ◽  
Capsid Protein ◽  
Fluorescent Protein ◽  
Potato Virus X ◽  
Helper Virus ◽  
Gene Vector ◽  
Virus Induced Gene Silencing ◽  
Suppressor Activity ◽  
Pathogenicity Factor

The capsid protein (CP) of satellite panicum mosaic virus (SPMV) has been implicated as a pathogenicity factor, inducing severe chlorosis on millet plants co-infected with SPMV and its helper virus, Panicum mosaic virus (PMV). In this study, we tested the effects of SPMV CP on Nicotiana benthamiana, a plant that does not support PMV+SPMV infections. SPMV CP expressed from a Potato virus X (PVX) gene vector elicited necrotic lesions on N. benthamiana. Pathogenicity factors often have the additional feature of acting as suppressors of gene silencing; therefore, several assays were developed to test if SPMV CP could act in such a capacity. The results showed that SPMV CP failed to act as a suppressor of posttranscriptional gene silencing when such tests were performed with transgenic N. benthamiana plants silenced for green fluorescent protein (GFP) expression by agroinfiltration or plant virus vectors. However SPMV CP expressed from the PVX gene vector did interfere with suppressor activity associated with PVX p25. This included a rebounded level of GFP silencing along the vascular tissues, including the veins on upper noninoculated leaves. Therefore, the roles of the SPMV CP now include encapsidation of the SPMV RNA, activity as a pathogenicity factor in both host and nonhost plants, and the enigmatic feature of interfering with suppression of gene silencing.

scholarly journals Potyvirus-induced gene silencing: the dynamic process of systemic silencing and silencing suppression

2007 ◽  
Vol 88 (8) ◽  
pp. 2337-2346 ◽  
Author(s):  
Elin Gammelgård ◽  
Maradumane Mohan ◽  
Jari P. T. Valkonen
Keyword(s):  
Gene Silencing ◽  
Fluorescent Protein ◽  
Uv Light ◽  
Deletion Mutants ◽  
Virus Induced Gene Silencing ◽  
Potato Virus A ◽  
Silencing Suppression ◽  
Dark Green ◽  
Green Fluorescent ◽  
Interfering Rna

Potato virus A (PVA; genus Potyvirus) was used for virus-induced gene silencing in a model system that included transgenic Nicotiana benthamiana (line 16c) expressing the gfp transgene for green fluorescent protein (GFP) and chimeric PVA (PVA–GFP) carrying gfp in the P1-encoding region. Infection of the 16c plants with PVA–GFP in five experiments resulted in a reproducible pattern of systemic gfp transgene silencing, despite the presence of the strong silencing-suppressor protein, HC-Pro, produced by the virus. PVA–GFP was also targeted by silencing, and virus-specific short interfering RNA accumulated from the length of the viral genome. Viral deletion mutants lacking the gfp insert appeared in systemically infected leaves and reversed silencing of the gfp transgene in limited areas. However, systemic gfp silencing continued in newly emerging leaves in the absence of the gfp-carrying virus, which implicated a systemic silencing signal that moved from lower leaves without interference by HC-Pro. Use of GFP as a visual marker revealed a novel, mosaic-like recovery phenotype in the top leaves. The leaf areas appearing red or purple under UV light (no GFP expression) contained little PVA and gfp mRNA, and corresponded to the dark-green islands observed under visible light. The surrounding green fluorescent tissues contained actively replicating viral deletion mutants that suppressed GFP silencing. Taken together, systemic progression of gene silencing and antiviral defence (RNA silencing) and circumvention of the silencing by the virus could be visualized and analysed in a novel manner.

147 GENE KNOCKDOWN OF MATERNAL- AND EMBRYONIC-EXPRESSED GREEN FLUORESCENT PROTEIN IN MURINE EMBRYOS BY THE INJECTION OF A SHORT INTERFERING RNA (siRNA)

2007 ◽  
Vol 19 (1) ◽  
pp. 191
Author(s):  
K. Iqbal ◽  
W. A. Kues ◽  
J. W. Carnwath ◽  
H. Niemann
Keyword(s):  
Green Fluorescent Protein ◽  
Gene Silencing ◽  
Fluorescent Protein ◽  
Mrna Levels ◽  
Knock Down ◽  
Gfp Gene ◽  
Mammalian Embryos ◽  
Murine Embryos ◽  
Green Fluorescent

RNA interference (RNAi) is now widely used for gene silencing in various biological systems. Injection of long double-stranded RNAs has been shown to specifically knock down gene expression in mammalian embryos. The utilization of short interfering RNAs (siRNA) to target specific embryonic genes would make this approach flexible and efficient enough for studying physiological functions in development. To demonstrate the feasibility of a single class of siRNA molecules for achieving long-lasting effects after injection into mammalian zygotes, we used siRNAs to knock down expression of the green fluorescent protein (GFP) in transgenic murine embryos of the OG2 transgenic line. Homozygous OG2-animals, carrying the Oct4-GFP transgene, were mated with NMRI animals to produce OG2 hemizygous zygotes, which show a parentally dependent expression pattern of the marker gene. Hemizygous zygotes with a maternally inherited Oct4-GFP gene continuously express the GFP marker; hemizygous zygotes with a paternally inherited Oct4-GFP start transcription of the GFP gene at the 4–8 cell stage, i.e., after onset of embryonic genomic activation. Thus efficacy and duration of gene silencing could be tested under 2 different conditions where (i) GFP mRNA was already present at the time point of injection, and (ii) GFP transcription started 2–3 cell cycles after siRNA injection. Zygotes were microinjected with either a 22-basepair GFP-siRNA or a control siRNA and then cultured in vitro. The siRNAs were conjugated with the fluorochome rhodamine to allow monitoring of injection and subsequent degradation of siRNAs. At the end of the in vitro culture, the developmental stage, the number of nuclei, GFP fluorescence (Table 1), and the GFP mRNA levels were determined by RT-PCR. In conclusion, results demonstrate that injection of a synthetic siRNA is sufficient to knock down a target gene transcript with either maternal or embryonic expression in mammalian embryos. Importantly, maternally derived GFP proteins showed a delayed functional knockdown of GFP for 2 days. The advantages of this approach are that (i) off-target effects of long double-stranded RNAs can be avoided, (ii) siRNAs against any known transcript can be rapidly designed and synthesized, and (iii) developmentally important genes can be silenced in embryos for at least 5 days following injection. Table 1.siRNA microinjection into zygotes with maternally and paternally inherited GFP

scholarly journals Gene C2 of the Monopartite Geminivirus Tomato yellow leaf curl virus-China Encodes a Pathogenicity Determinant That Is Localized in the Nucleus

2001 ◽  
Vol 14 (9) ◽  
pp. 1125-1128 ◽  
Author(s):  
Rene van Wezel ◽  
Huangting Liu ◽  
Po Tien ◽  
John Stanley ◽  
Yiguo Hong
Keyword(s):  
Fluorescent Protein ◽  
Potato Virus X ◽  
Tomato Yellow Leaf ◽  
Yellow Leaf ◽  
In Planta ◽  
Cell Nuclei ◽  
Pathogenicity Determinant ◽  
Green Fluorescent ◽  
Leaf Curl Virus ◽  
Leaf Curl

Expression of the Tomato yellow leaf curl virus-China (TYLCV-C) C2 protein and green fluorescent protein (GFP) fused to the C2 protein (C2-GFP) in Nicotiana benthamiana from a Potato virus X (PVX) vector induced necrotic ringspots on inoculated leaves as well as necrotic vein banding and severe necrosis on systemically infected leaves. The localization of GFP fluorescence in plant cells infected with PVX/C2-GFP and in insect cells transfected with Baculovirus expressing C2-GFP indicates that the TYLCV-C C2 protein is capable of shuttling GFP into plant and insect cell nuclei. Our data demonstrate that the TYLCV-C C2 protein may contribute to viral pathogenicity in planta and is nuclear localized.

scholarly journals Mastrevirus Rep and RepA Proteins Suppress de novo Transcriptional Gene Silencing

2021 ◽  
Vol 22 (21) ◽  
pp. 11462
Author(s):  
Kikyo Watanabe ◽  
Masashi Ugaki
Keyword(s):  
Gene Silencing ◽  
Defense Mechanism ◽  
Fluorescent Protein ◽  
De Novo ◽  
Transcriptional Gene Silencing ◽  
35S Promoter ◽  
Gene Encoding ◽  
Suppressor Activity ◽  
Yellow Dwarf Virus ◽  
Green Fluorescent

Transcriptional gene silencing (TGS) in plants is a defense mechanism against DNA virus infection. The genomes of viruses in the Geminiviridae family encode several TGS suppressors. In this study, we induced de novo TGS against the transgenic GFP gene encoding green fluorescent protein by expressing a hairpin-shaped self-complementary RNA corresponding to the enhancer region of the 35S promoter (hpE35S). In addition, we examined the TGS suppression activity of proteins encoded in the genome of Tobacco yellow dwarf virus (TYDV, genus Mastrevirus). The results show that the replication-associated protein (Rep) and RepA encoded by TYDV have TGS suppressor activity and lead to decreased accumulation of 24-nt siRNAs. These results suggest that Rep and RepA can block the steps before the loading of siRNAs into Argonaute (AGO) proteins. This is the first report of TGS suppressors in the genus Mastrevirus.

scholarly journals The Reporter System for GPCR Assay with the Fission YeastSchizosaccharomyces pombe

Scientifica ◽  
2012 ◽  
Vol 2012 ◽  
pp. 1-11 ◽  
Author(s):  
Shintaro Sasuga ◽  
Toshiya Osada
Keyword(s):  
Fluorescent Protein ◽  
Signal To Noise Ratio ◽  
Biological Activities ◽  
Inducible Promoter ◽  
Upstream Region ◽  
Reporter Plasmid ◽  
Reporter System ◽  
High Background ◽  
Downstream Region ◽  
Green Fluorescent

G protein-coupled receptors (GPCRs) are associated with a great variety of biological activities. Yeasts are often utilized as a host for heterologous GPCR assay. We engineered the intense reporter plasmids for fission yeast to produce green fluorescent protein (GFP) through its endogenous GPCR pathway. As a control region of GFP expression on the reporter plasmid, we focused on seven endogenous genes specifically activated through the pathway. When upstream regions of these genes were used as an inducible promoter in combination with LPI terminator, themam2upstream region produced GFP most rapidly and intensely despite the high background. Subsequently, LPI terminator was replaced with the corresponding downstream regions. The SPBC4.01 downstream region enhanced the response with the low background. Furthermore, combining SPBC4.01 downstream region with the sxa2 upstream region, the signal to noise ratio was obviously better than those of other regions. We also evaluated the time- and dose-dependent GFP productions of the strains transformed with the reporter plasmids. Finally, we exhibited a model of simplified GPCR assay with the reporter plasmid by expressing endogenous GPCR under the control of the foreign promoter.

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 Molecular biology of potexviruses: recent advances

2007 ◽  
Vol 88 (6) ◽  
pp. 1643-1655 ◽  
Author(s):  
Jeanmarie Verchot-Lubicz ◽  
Chang-Ming Ye ◽  
Devinka Bamunusinghe
Keyword(s):  
Gene Silencing ◽  
Rna Synthesis ◽  
Triple Gene Block ◽  
Potato Virus X ◽  
Future Research ◽  
Virus Movement ◽  
Suppressor Activity ◽  
Gene Block ◽  
Recent Advances

Recent advances in potexvirus research have produced new models describing virus replication, cell-to-cell movement, encapsidation, R gene-mediated resistance and gene silencing. Interactions between distant RNA elements are a central theme in potexvirus replication. The 5′ non-translated region (NTR) regulates genomic and subgenomic RNA synthesis and encapsidation, as well as virus plasmodesmal transport. The 3′ NTR regulates both plus- and minus-strand RNA synthesis. How the triple gene-block proteins interact for virus movement is still elusive. As the potato virus X (PVX) TGBp1 protein gates plasmodesmata, regulates virus translation and is a suppressor of RNA silencing, further research is needed to determine how these properties contribute to propelling virus through the plasmodesmata. Specifically, TGBp1 suppressor activity is required for virus movement, but how the silencing machinery relates to plasmodesmata is not known. The TGBp2 and TGBp3 proteins are endoplasmic reticulum (ER)-associated proteins required for virus movement. TGBp2 associates with ER-derived vesicles that traffic along the actin network. Future research will determine whether the virus-induced vesicles are cytopathic structures regulating events along the ER or are vehicles carrying virus to the plasmodesmata for transfer into neighbouring cells. Efforts to assemble virions in vitro identified a single-tailed particle (STP) comprising RNA, coat protein (CP) and TGBp1. It has been proposed that TGBp1 aids in transport of virions or STP between cells and ensures translation of RNA in the receiving cells. PVX is also a tool for studying Avr–R gene interactions and gene silencing in plants. The PVX CP is the elicitor for the Rx gene. Recent reports of the PVX CP reveal how CP interacts with the Rx gene product.

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