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

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.

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

Functional Plant Biology ◽

10.1071/fp12051 ◽

2012 ◽

Vol 39 (9) ◽

pp. 764 ◽

Cited By ~ 6

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.

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

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2000.13.6.599 ◽

2000 ◽

Vol 13 (6) ◽

pp. 599-605 ◽

Cited By ~ 63

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.

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

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.1997.10.3.394 ◽

1997 ◽

Vol 10 (3) ◽

pp. 394-400 ◽

Cited By ~ 76

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.

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

Plants ◽

10.3390/plants10081700 ◽

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.

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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

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-20-9-1040 ◽

2007 ◽

Vol 20 (9) ◽

pp. 1040-1047 ◽

Cited By ~ 34

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.

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Negative modulation of the chicken infectious anemia virus promoter by COUP-TF1 and an E box-like element at the transcription start site binding δEF1

Journal of General Virology ◽

10.1099/vir.0.2008/003103-0 ◽

2008 ◽

Vol 89 (12) ◽

pp. 2998-3003 ◽

Cited By ~ 12

Author(s):

Myrna M. Miller ◽

Keith W. Jarosinski ◽

Karel A. Schat

Keyword(s):

Fluorescent Protein ◽

Transcription Start ◽

Upstream Promoter ◽

Chicken Infectious Anemia Virus ◽

E Box ◽

Promoter Construct ◽

Chicken Infectious Anemia ◽

Lmh Cells ◽

Green Fluorescent ◽

Anemia Virus

Expression of enhanced green fluorescent protein (EGFP) under control of the promoter-enhancer of chicken infectious anemia virus (CAV) is increased in an oestrogen receptor-enhanced cell line when treated with oestrogen and the promoter-enhancer binds unidentified proteins that recognize a consensus oestrogen response element (ERE). Co-transfection assays with the CAV promoter and the nuclear receptor chicken ovalbumin upstream promoter transcription factor 1 (COUP-TF1) showed that expression of EGFP was decreased by 50 to 60 % in DF-1 and LMH cells. The CAV promoter that included sequences at and downstream of the transcription start point had less expression than a short promoter construct. Mutation of a putative E box at this site restored expression levels. Electromobility shift assays showed that the transcription regulator delta-EF1 (δEF1) binds to this E box region. These findings indicate that the CAV promoter activity can be affected directly or indirectly by COUP-TF1 and δEF1.

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Localization of Cold Shock Proteins to Cytosolic Spaces Surrounding Nucleoids in Bacillus subtilis Depends on Active Transcription

Journal of Bacteriology ◽

10.1128/jb.183.21.6435-6443.2001 ◽

2001 ◽

Vol 183 (21) ◽

pp. 6435-6443 ◽

Cited By ~ 32

Author(s):

Michael H. W. Weber ◽

Arsen V. Volkov ◽

Ingo Fricke ◽

Mohamed A. Marahiel ◽

Peter L. Graumann

Keyword(s):

Bacillus Subtilis ◽

Cold Shock ◽

Fluorescent Protein ◽

Cold Shock Protein ◽

Initiation Of Translation ◽

Specific Localization ◽

Active Transcription ◽

Green Fluorescent ◽

Shock Proteins ◽

Mutant Cells

ABSTRACT Using immunofluorescence microscopy and a fusion of a cold shock protein (CSP), CspB, to green fluorescent protein (GFP), we showed that in growing cells Bacillus subtilis CSPs specifically localize to cytosolic regions surrounding the nucleoid. The subcellular localization of CSPs is influenced by the structure of the nucleoid. Decondensed chromosomes in smc mutant cells reduced the sizes of the regions in which CSPs localized, while cold shock-induced chromosome compaction was accompanied by an expansion of the space in which CSPs were present. As a control, histone-like protein HBsu localized to the nucleoids, while β-galactosidase and GFP were detectable throughout the cell. After inhibition of translation, CspB-GFP was still present around the nucleoids in a manner similar to that in cold-shocked cells. However, in stationary-phase cells and after inhibition of transcription, CspB was distributed throughout the cell, indicating that specific localization of CspB depends on active transcription and is not due to simple exclusion from the nucleoid. Furthermore, we observed that nucleoids are more condensed and frequently abnormal incspB cspC and cspB cspDdouble-mutant cells. This suggests that the function of CSPs affects chromosome structure, probably through coupling of transcription to translation, which is thought to decondense nucleoids. In addition, we found that cspB cspD and cspB cspC double mutants are defective in sporulation, with a block at or before stage 0. Interestingly, CspB and CspC are depleted from the forespore compartment but not from the mother cell. In toto, our findings suggest that CSPs localize to zones of newly synthesized RNA, coupling transcription with initiation of translation.

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

Microbiology ◽

10.1099/0022-1317-81-7-1851 ◽

2000 ◽

Vol 81 (7) ◽

pp. 1851-1855 ◽

Cited By ~ 41

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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Optimization of particle bombardment parameters for DNA delivery into the male flowers of banana

Biologia ◽

10.2478/s11756-014-0391-7 ◽

2014 ◽

Vol 69 (7) ◽

Cited By ~ 1

Author(s):

Fatemeh Mahdavi ◽

Maziah Mahmood ◽

Normah Noor

Keyword(s):

Particle Bombardment ◽

Fluorescent Protein ◽

Constitutive Expression ◽

Vacuum Pressure ◽

35S Promoter ◽

Helium Pressure ◽

Gfp Expression ◽

Gfp Gene ◽

Male Flowers ◽

Green Fluorescent

AbstractThe possibility of increasing the efficiency of banana transformation was investigated by particle bombardment of the male flowers of banana plants for constitutive expression of gfp gene. The effects of particle bombardment parameters, such as acceleration pressure, bombardment distance, chamber vacuum pressure, gold microcarrier size, gold quantity, DNA quantity, number of bombardments and pre-culture were examined. Single cauliflower-like bodies (CLBs) clusters, induced from meristemic parts of Musa sapientum cv. Nangka (AAB) male flowers, were bombarded by pCambia1304 plasmid carrying gfp gene driven by the CaMV 35S promoter. Optimal transient expression of green-fluorescent protein (GFP) was obtained when the three-day old cultured tissues were bombarded two times at 1100 psi helium pressure. However, the highest GFP expression was observed when 9 cm was applied as bombardment distance with 28 mmHg chamber vacuum pressure. Gold particle with 1 μm diameter at 60 μg/μL concentrations coated with 1.5 μg/μL of DNA have been used as the optimum bombardment parameter since GFP expression was significantly different compared to other conditions. Application of optimized condition proved effective for the generation of stable transgenic banana plants. PCR and southern blot analyses confirmed the presence and integration of gfp gene in genomic DNA of transformed plants. Transformation frequency achieved with the optimized protocol was 7.5% which was significantly higher than the conventional protocol.

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Effects of Movement Protein Mutations on the Formation of Tubules in Plant Protoplasts Expressing a Fusion Between the Green Fluorescent Protein and Cauliflower mosaic virus Movement Protein

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2001.14.8.1026 ◽

2001 ◽

Vol 14 (8) ◽

pp. 1026-1031 ◽

Cited By ~ 9

Author(s):

Zhong Huang ◽

Yu Han ◽

Stephen H. Howell

Keyword(s):

Green Fluorescent Protein ◽

Mosaic Virus ◽

Fluorescent Protein ◽

Movement Protein ◽

Cauliflower Mosaic Virus ◽

Virus Movement ◽

Plant Protoplasts ◽

Tubule Formation ◽

Leaf Mesophyll ◽

Green Fluorescent

Fusions between the green fluorescent protein (GFP) and the Cauliflower mosaic virus (CaMV) movement protein (MP) induce the formation of fluorescent foci and surface tubules in Arabidopsis thaliana leaf mesophyll protoplasts. Tubules elongate coordinately and progressively in an assembly process approximately 6 to 12 h following transfection of protoplasts with GFP-MP constructs. Tubules are not formed in protoplasts transfected by GFP-MPER2A, a MP mutation that renders CaMV noninfectious. A small number of short tubules are formed on protoplasts transfected by GFP-MPN6 and GFP-MPN13, two second-site revertants of ER2A that partially restore infectivity. Protoplasts cotransfected with cyan fluorescent protein (CFP)-MPWT and GFP-MPER2A form tubules containing both MP fusions, indicating that although the GFP-MPER2A cannot induce tubule formation, GFP-MPER2A can coassemble or colocalize with CFP-MPWT in tubules. Thus, CaMV MP-induced tubule formation in protoplasts correlates closely with the infectivity of mutation ER2A and its revertants, suggesting that tubule-forming capacity in plant protoplasts reflects a process required for virus infection or movement.

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