scholarly journals A Core35S Promoter of Cauliflower Mosaic Virus Drives More Efficient Replication of Turnip Crinkle Virus

Plants ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1700
Author(s):  
Md Emran Ali ◽  
Sumyya Waliullah
Keyword(s):  
Virus Replication ◽  
Fluorescent Protein ◽  
Rna Virus ◽  
Cauliflower Mosaic Virus ◽  
35S Promoter ◽  
Cdna Clones ◽  
Post Inoculation ◽  
Turnip Crinkle Virus ◽  
Green Fluorescent ◽  
High Level

The 35S promoter with a duplicated enhancer (frequently referred to as 2X35S) is a strong dicotyledonous plant-specific promoter commonly used in generating transgenic plants to enable high-level expression of genes of interest. It is also used to drive the initiation of RNA virus replication from viral cDNA, with the consensus understanding that high levels of viral RNA production powered by 2X35S permit a more efficient initiation of virus replication. Here, we showed that the exact opposite is true. We found that, compared to the Core35S promoter, the 2X35S promoter-driven initiation of turnip crinkle virus (TCV) infection was delayed by at least 24 h. We first compared three versions of 35S promoter, namely 2X35S, 1X35S, and Core35S, for their ability to power the expression of a non-replicating green fluorescent protein (GFP) gene, and confirmed that 2X35S and Core35S correlated with the highest and lowest GFP expression, respectively. However, when inserted upstream of TCV cDNA, 2X35S-driven replication was not detected until 72 h post-inoculation (72 hpi) in inoculated leaves. By contrast, Core35S-driven replication was detected earlier at 48 hpi. A similar delay was also observed in systemically infected leaves (six versus four days post-inoculation). Combining our results, we hypothesized that the stronger 2X35S promoter might enable a higher accumulation of a TCV protein that became a repressor of TCV replication at higher cellular concentration. Extending from these results, we propose that the Core35S (or mini35S) promoter is likely a better choice for generating infectious cDNA clones of TCV.

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 RNA1-Independent Replication and GFP Expression from Tomato marchitez virus Isolate M Cloned cDNA

2016 ◽  
Vol 106 (5) ◽  
pp. 500-509 ◽  
Author(s):  
I. Ferriol ◽  
M. Turina ◽  
E. J. Zamora-Macorra ◽  
B. W. Falk
Keyword(s):  
Fluorescent Protein ◽  
Rna Virus ◽  
Cell Movement ◽  
Full Length ◽  
Wild Type Virus ◽  
Cdna Clones ◽  
Genomic Rna ◽  
Positive Strand Rna Virus ◽  
Green Fluorescent ◽  
Positive Strand Rna

Tomato marchitez virus (ToMarV; synonymous with Tomato apex necrosis virus) is a positive-strand RNA virus in the genus Torradovirus within the family Secoviridae. ToMarV is an emergent whitefly-transmitted virus that causes important diseases in tomato (Solanum lycopersicum) in Mexico. Here, the genome sequence of the ToMarV isolate M (ToMarV-M) was determined. We engineered full-length cDNA clones of the ToMarV-M genomic RNA (RNA1 and RNA2), separately, into a binary vector. Coinfiltration of both triggered systemic infections in Nicotiana benthamiana, tomato, and tomatillo (Physalis philadelphica) plants and recapitulated the biological activity of the wild-type virus. The viral progeny generated from tomato and tomatillo plants were transmissible by the whitefly Bemisia tabaci biotype B. Also, we assessed whether these infectious clones could be used for screening tomato cultivars for resistance to ToMarV and our results allowed us to differentiate resistant and susceptible tomato lines. We demonstrated that RNA1 of ToMarV-M is required for the replication of RNA2, and it can replicate independently of RNA2. From this, ToMarV-M RNA2 was used to express the green fluorescent protein in N. benthamiana plants, which allowed us to track cell-to-cell movement. The construction of full-length infectious cDNA clones of ToMarV-M provides an excellent tool to investigate virus–host–vector interactions and elucidate the functions of torradovirus-encoded proteins or the mechanisms of replication of torradovirus genomic RNA.

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 Grapevine Fanleaf Virus Replication Occurs on Endoplasmic Reticulum-Derived Membranes

2002 ◽  
Vol 76 (17) ◽  
pp. 8808-8819 ◽  
Author(s):  
C. Ritzenthaler ◽  
C. Laporte ◽  
F. Gaire ◽  
P. Dunoyer ◽  
C. Schmitt ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Virus Replication ◽  
Fluorescent Protein ◽  
De Novo ◽  
Rna Virus ◽  
Lipid Synthesis ◽  
Cell Movement ◽  
Brefeldin A ◽  
Cell Periphery ◽  
Green Fluorescent

ABSTRACT Infection by Grapevine fanleaf nepovirus (GFLV), a bipartite RNA virus of positive polarity belonging to the Comoviridae family, causes extensive cytopathic modifications of the host endomembrane system that eventually culminate in the formation of a perinuclear “viral compartment.” We identified by immunoconfocal microscopy this compartment as the site of virus replication since it contained the RNA1-encoded proteins necessary for replication, newly synthesized viral RNA, and double-stranded replicative forms. In addition, by using transgenic T-BY2 protoplasts expressing green fluorescent protein in the endoplasmic reticulum (ER) or in the Golgi apparatus (GA), we could directly show that GFLV replication induced a depletion of the cortical ER, together with a condensation and redistribution of ER-derived membranes, to generate the viral compartment. Brefeldin A, a drug known to inhibit vesicle trafficking between the GA and the ER, was found to inhibit GFLV replication. Cerulenin, a drug inhibiting de novo synthesis of phospholipids, also inhibited GFLV replication. These observations imply that GFLV replication depends both on ER-derived membrane recruitment and on de novo lipid synthesis. In contrast to proteins involved in viral replication, the 2B movement protein and, to a lesser extent, the 2C coat protein were not confined to the viral compartment but were transported toward the cell periphery, a finding consistent with their role in cell-to-cell movement of virus particles.

scholarly journals Generation of siRNAs by T-DNA Sequences Does Not Require Active Transcription or Homology to Sequences in the Plant

2002 ◽  
Vol 15 (11) ◽  
pp. 1137-1146 ◽  
Author(s):  
Tomas Canto ◽  
Fabrizio Cillo ◽  
Peter Palukaitis
Keyword(s):  
Dna Sequences ◽  
Fluorescent Protein ◽  
Cauliflower Mosaic Virus ◽  
35S Promoter ◽  
Small Interfering Rnas ◽  
Promoter Sequences ◽  
Upstream Promoter ◽  
Active Transcription ◽  
Green Fluorescent ◽  
Transcribed Sequences

Delivery into plants of T-DNAs containing promoter, terminator, or coding sequences generated small interfering RNAs (siRNAs) specific to each type of sequence. When both promoter and transcribed sequences were simultaneously present in the T-DNA, accumulation of siRNAs to transcribed sequences was favored over accumulation of siRNAs to the nontranscribed upstream promoter sequences. The generation of specific siRNA sequences occurred even in the absence of T-DNA homology to sequences in the plant. Delivery of T-DNA, with homology to the transgene limited to the nontranscribed cauliflower mosaic virus 35S promoter (35SP) and the transcribed nopaline synthase transcription termination (NosT)signal sequences, into transgenic plants expressing the green fluorescent protein (GFP), generated siRNAs in infiltrated tissues to both 35SP (35SsiRNAs) and NosT (NosTsiRNAs), but not to the GFP sequence (GFPsiRNAs). In infiltrated tissues, the 35SsiRNAs failed to trigger the transcriptional silencing of the transgene, accumulation of 35SsiRNAs could be prevented by the potyviral HC-Pro, and the NosTsiRNAs required an initial amplification to trigger efficient transgene silencing, which is mediated by transcripts from the exogenous T-DNA, and not from the transgene. In upper leaves, silencing correlated with the presence of GFPsiRNAs and the absence of 35SsiRNAs, confirming that its spread was posttranscriptionally mediated by the transgene mRNA.

scholarly journals Continual Green-Fluorescent Protein Monitoring of Cauliflower Mosaic Virus 35S Promoter Activity in Nematode-Induced Feeding Cells in Arabidopsis thaliana

1997 ◽  
Vol 10 (3) ◽  
pp. 394-400 ◽  
Author(s):  
Peter E. Urwin ◽  
Simon G. Møller ◽  
Catherine J. Lilley ◽  
Michael J. McPherson ◽  
Howard J. Atkinson
Keyword(s):  
Arabidopsis Thaliana ◽  
Green Fluorescent Protein ◽  
Mosaic Virus ◽  
Promoter Activity ◽  
Fluorescent Protein ◽  
Cauliflower Mosaic Virus ◽  
35S Promoter ◽  
Feeding Sites ◽  
Green Fluorescent ◽  
Cauliflower Mosaic Virus 35S

The responsiveness of the cauliflower mosaic virus 35S promoter in feeding sites developed by both sexes of Heterodera schachtii and female Meloidogyne incognita has been studied. The objective was to establish the value of green-fluorescent protein (GFP) as a nondestructive reporter gene system for characterizing promoter activity at nematode feeding sites in vivo. Growth units were devised that allowed individual feeding sites in roots of Arabidopsis thaliana to be observed by both bright-field and epifluorescent illumination. Changes in GFP expression were visually observed under experimental conditions that resulted in chloroplast formation in syncytia but not other root cells. Changes in GFP levels altered the extent of quenching, by this protein, of red light emitted by chlorophyll within the chloroplasts under violet excitation. Image analysis provided a semiquantitative basis for simultaneous measurement of changes in GFP fluorescence and the unquenched emission by chlorophyll. GFP levels were constant in cells surrounding the syncytium induced by H. schachtii, but they fell progressively from 10 to 35 days postinfection within this structure. Significant reduction in GFP levels was not limited to the early part of the time course but also occurred between 27 and 35 days postinfection. GFP was detected by immunoblotting in females of M. incognita but not in H. schachtii parasitizing similar GFP-expressing roots.

scholarly journals Nodulation Studies in the Model Legume Medicago truncatula: Advantages of Using the Constitutive EF1α Promoter and Limitations in Detecting Fluorescent Reporter Proteins in Nodule Tissues

2007 ◽  
Vol 20 (9) ◽  
pp. 1040-1047 ◽  
Author(s):  
Marie-Christine Auriac ◽  
Antonius C. J. Timmers
Keyword(s):  
Medicago Truncatula ◽  
Fluorescent Protein ◽  
Cauliflower Mosaic Virus ◽  
Nodule Development ◽  
35S Promoter ◽  
Fluorescent Reporter ◽  
Model Legume ◽  
Fluorescent Reporters ◽  
Fluorescent Markers ◽  
Green Fluorescent

The Cauliflower mosaic virus 35S promoter currently is being used in RNAi-based approaches for attenuating host gene expression during legume root nodule development and also for the expression of fluorescent reporters in nodule tissues. In this study, we have evaluated the expression of this promoter in the indeterminate nodules of the model plant Medicago truncatula. Our results clearly show that the 35S promoter is inactive in both the nodule meristem and in bacteroid-containing cells of the nodules. On the other hand, the Arabidopsis thaliana EF1α promoter was found to be strongly expressed both in the nodule meristem and in all nodule-invaded cells. Therefore, we conclude that the constitutive EF1α promoter is far superior for mRNAi or overexpression studies in nodule tissues compared with the commonly used 35S promoter. In addition, our experiments have revealed that the intensity of fluorescent markers such as green fluorescent protein is severely attenuated within invaded cells in the nitrogen-fixation zone of the nodule, most likely by fluorescence quenching. This phenomenon may hinder the use of these tools for live-cell imaging in nodule tissue.

scholarly journals Analysis of the Bacillus subtilis spoIIIJ Gene and Its Paralogue Gene, yqjG

2002 ◽  
Vol 184 (7) ◽  
pp. 1998-2004 ◽  
Author(s):  
Takako Murakami ◽  
Koki Haga ◽  
Michio Takeuchi ◽  
Tsutomu Sato
Keyword(s):  
Bacillus Subtilis ◽  
Membrane Proteins ◽  
Vegetative Growth ◽  
Fluorescent Protein ◽  
Specific Expression ◽  
Rich Media ◽  
Green Fluorescent ◽  
High Level ◽  
Fluorescent Protein Reporter

ABSTRACT The Bacillus subtilis spoIIIJ gene, which has been proven to be vegetatively expressed, has also been implicated as a sporulation gene. Recent genome sequencing information in many organisms reveals that spoIIIJ and its paralogous gene, yqjG, are conserved from prokaryotes to humans. A homologue of SpoIIIJ/YqjG, the Escherichia coli YidC is involved in the insertion of membrane proteins into the lipid bilayer. On the basis of this similarity, it was proposed that the two homologues act as translocase for the membrane proteins. We studied the requirements for spoIIIJ and yqjG during vegetative growth and sporulation. In rich media, the growth of spoIIIJ and yqjG single mutants were the same as that of the wild type, whereas spoIIIJ yqjG double inactivation was lethal, indicating that together these B. subtilis translocase homologues play an important role in maintaining the viability of the cell. This result also suggests that SpoIIIJ and YqjG probably control significantly overlapping functions during vegetative growth. spoIIIJ mutations have already been established to block sporulation at stage III. In contrast, disruption of yqjG did not interfere with sporulation. We further show that high level expression of spoIIIJ during vegetative phase is dispensable for spore formation, but the sporulation-specific expression of spoIIIJ is necessary for efficient sporulation even at the basal level. Using green fluorescent protein reporter to monitor SpoIIIJ and YqjG localization, we found that the proteins localize at the cell membrane in vegetative cells and at the polar and engulfment septa in sporulating cells. This localization of SpoIIIJ at the sporulation-specific septa may be important for the role of spoIIIJ during sporulation.

High level Purkinje cell specific expression of green fluorescent protein in transgenic mice

2001 ◽  
Vol 115 (6) ◽  
pp. 455-464 ◽  
Author(s):  
Xulun Zhang ◽  
Stephan L. Baader ◽  
Feng Bian ◽  
Wolfgang Müller ◽  
John Oberdick
Keyword(s):  
Green Fluorescent Protein ◽  
Transgenic Mice ◽  
Purkinje Cell ◽  
Fluorescent Protein ◽  
Specific Expression ◽  
Green Fluorescent ◽  
High Level

Limitations on the use of fused green fluorescent protein to investigate structure–function relationships for the cauliflower mosaic virus movement protein

Microbiology ◽  
2000 ◽  
Vol 81 (7) ◽  
pp. 1851-1855 ◽  
Author(s):  
Carole L. Thomas ◽  
Andrew J. Maule
Keyword(s):  
Green Fluorescent Protein ◽  
Mosaic Virus ◽  
Fluorescent Protein ◽  
Movement Protein ◽  
Cauliflower Mosaic Virus ◽  
Virus Movement ◽  
Wild Type ◽  
Tubule Formation ◽  
Mouth Disease Virus ◽  
Green Fluorescent

To investigate the process of tubule formation for the cauliflower mosaic virus movement protein (CaMV MP), the green fluorescent protein (GFP) was fused to the MP to provide a vital marker for MP location after expression in insect cells. In contrast to the long tubular structures seen previously following baculovirus-based expression of the wild-type MP, the fusion protein produced only aggregates of fluorescing material in the cytoplasm. However, by co-expressing wild-type MP and GFP–MP, or by engineering their co-accumulation by introducing a foot-and-mouth disease virus 2A cleavage sequence between GFP and MP, long GFP-fluorescing tubules were formed. The experiments suggest that the presence of GFP at the N or C terminus of the tubule-forming domain of the CaMV MP places steric constraints upon the aggregation of the MP into a tubule but that this can be overcome by providing wild-type protein for inclusion in the aggregate.

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