scholarly journals Enhancer Trapping Identifies TRI, an Arabidopsis Gene Up-Regulated by Pathogen Infection

2004 ◽  
Vol 17 (10) ◽  
pp. 1086-1094 ◽  
Author(s):  
Ingela Fridborg ◽  
Alan Williams ◽  
Aidong Yang ◽  
Stuart MacFarlane ◽  
Katherine Coutts ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Tobacco Rattle Virus ◽  
Defense Responses ◽  
Gus Activity ◽  
Enhancer Trap ◽  
Cauliflower Mosaic Virus ◽  
35S Promoter ◽  
Resistance Response ◽  
Enhancer Element ◽  
Enhancer Trap Line

Enhancer trap Arabidopsis thaliana plants were screened for genes up-regulated by virus infection. The plants carried T-DNA insertions comprising a minimal -60-bp Cauliflower mosaic virus 35S promoter fused to the β-glucuronidase (GUS) reporter gene. Approximately 12,000 plants were assayed for GUS activity before and after rub-inoculation with Tobacco rattle virus (TRV) tagged with the green fluorescent protein (GFP). One plant and its progeny consistently showed upregulation of GUS activity in response to TRV-GFP infection, indicating that a virus-responsive enhancer element was “tagged” by the T-DNA in this line. Other viruses, bacteria, and oomycetes, but not wounding, up-regulated GUS activity in the enhancer trap line, indicating that the response was not specific to TRV-GFP infection. A pathogen-inducible, alternatively spliced gene was identified, which we have termed TRI for TRV-induced gene. A pathogen-responsive element was localized to a 1.1-kb region upstream of the T-DNA insertion, and two different cis-acting elements, both implicated in defense responses, were found in the sequence upstream of TRI. Sequence analyses revealed that TRI is similar to ACRE169, a gene that is up-regulated in Cf-9-expressing tobacco when treated with Avr-9, the Cladosporium fulvum elicitor of the Cf-9 resistance response.

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.

Chloroplast-dependent and light-independent expression of the tobacco rbcS promoter–GUS chimeric gene in black spruce

1996 ◽  
Vol 26 (6) ◽  
pp. 909-917
Author(s):  
Madoka Gray-Mitsumune ◽  
Bong Y. Yoo ◽  
Pierre J. Charest
Keyword(s):  
Fusion Gene ◽  
Black Spruce ◽  
Gus Activity ◽  
Chimeric Gene ◽  
Cauliflower Mosaic Virus ◽  
35S Promoter ◽  
Zygotic Embryos ◽  
Ribulose Bisphosphate Carboxylase ◽  
Bisphosphate Carboxylase ◽  
Rbcs Promoter

The tobacco rbcS (ribulose bisphosphate carboxylase small subunit) promoter, fused to the β-glucuronidase (GUS) reporter gene, was delivered to black spruce (Piceamariana (Mill.) BSP) tissues via microprojectile DNA bombardment, and its regulation was studied. The expression of the tobacco rbcS promoter–GUS chimeric gene was dependent on the presence of chloroplasts in black spruce tissues, as demonstrated in two ways: (i) there was no GUS activity expressed in zygotic embryos where no chloroplasts were observed, whereas it was expressed in light- and dark-grown seedlings that contained mature or immature chloroplasts; (ii) a herbicide, Norflurazon, destroyed chloroplast structure in seedlings and inhibited the expression of the tobacco rbcS promoter–GUS chimeric gene. A control chimeric gene, the cauliflower mosaic virus (CaMV) 35S promoter–GUS fusion gene was not inhibited by Norflurazon. Unlike in angiosperms, light had no effect on the expression of tobacco rbcS promoter–GUS chimeric gene. Both light- and dark-grown seedlings showed GUS activity, and expression in dark-grown seedlings was not enhanced by light. These results suggest that the tissue-specific regulation of the rbcS promoter may be conserved between angiosperms and conifers, but that the light regulation of this promoter may not be conserved.

scholarly journals The LeATL6-Associated Ubiquitin/Proteasome System May Contribute to Fungal Elicitor-Activated Defense Response via the Jasmonic Acid-Dependent Signaling Pathway in Tomato

2007 ◽  
Vol 20 (1) ◽  
pp. 72-81 ◽  
Author(s):  
Daisuke Hondo ◽  
Shu Hase ◽  
Yoshinori Kanayama ◽  
Nobuyuki Yoshikawa ◽  
Shigehito Takenaka ◽  
...  
Keyword(s):  
Jasmonic Acid ◽  
Signaling Pathway ◽  
Ubiquitin Proteasome System ◽  
Defense Responses ◽  
Cauliflower Mosaic Virus ◽  
35S Promoter ◽  
Pythium Oligandrum ◽  
Wild Tomato ◽  
Ubiquitin Proteasome ◽  
Dependent Signaling Pathway

The expression of LeATL6, an ortholog of Arabidopsis ATL6 that encodes a RING-H2 finger protein, was induced in tomato roots treated with a cell wall protein fraction (CWP) elicitor of the biocontrol agent Pythium oligandrum. The LeATL6 protein was expressed as a fusion protein with a maltose-binding protein (MBP) in Escherichia coli, and it catalyzed the transfer of ubiquitin to the MBP moiety on incubation with ubiquitin, the ubiquitin-activating enzyme E1, and the ubiquitin-conjugating enzyme E2; this indicated that LeATL6 represents ubiquitin ligase E3. LeATL6 expression also was induced by elicitor treatment of jai1-1 mutant tomato cells in which the jasmonic acid (JA)-mediated signaling pathway was impaired; however, JA-dependent expression of the basic PR-6 and TPI-1 genes that encode proteinase inhibitor II and I, respectively, was not induced in elicitor-treated jai1-1 mutants. Furthermore, transient overexpression of LeATL6 under the control of the Cauliflower mosaic virus 35S promoter induced the basic PR6 and TPI-1 expression in wild tomato but not in the jai1-1 mutant. In contrast, LeATL6 overexpression did not activate salicylic acid-responsive acidic PR-1 and PR-2 promoters in wild tomato. These results indicated that elicitor-responsive LeATL6 probably regulates JA-dependent basic PR6 and TPI-1 gene expression in tomato. The LeATL6-associated ubiquitin/proteasome system may contribute to elicitor-activated defense responses via a JA-dependent signaling pathway in plants.

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 MaRING2: A Positive Regulator of an Abscisic Acid-dependent Response to Cold Stress from Banana Fruit

2015 ◽  
Vol 140 (4) ◽  
pp. 373-382
Author(s):  
Jiao Chen ◽  
De-bao Yuan ◽  
Chao-zheng Wang ◽  
Yi-xing Li ◽  
Fen-fang Li ◽  
...  
Keyword(s):  
Abscisic Acid ◽  
Cold Stress ◽  
Expression Profiles ◽  
Functional Characterization ◽  
Gene Expression Profiles ◽  
Defense Responses ◽  
Cauliflower Mosaic Virus ◽  
35S Promoter ◽  
Banana Fruit ◽  
Positive Regulator

Many reports indicate that an abundance of really interesting new gene (RING) play key roles in regulating defense responses against abiotic and biotic stresses in plants. In this study, the cloning and functional characterization of a RING gene, MaRING2, in banana (Musa acuminata) fruit are reported. MaRING2 belongs to the NEP1-interacting protein (NIP) RING-H2 finger protein family. Gene expression profiles revealed that MaRING2 was cold responsive and induced by abscisic acid (ABA) treatment during cold storage. In this study, the MaRING2 under control of the Cauliflower mosaic virus 35S (CaMV 35S) promoter was transformed to tobacco (Nicotiana benthamiana) using agrobacterium (Agrobacterium tumefaciens)-mediated transformation. The resultant MaRING2-overexpressing transgenic plants (35S:MaRING2) exhibited significantly increased tolerance to low temperatures and were hypersensitive to exogenous ABA in terms of germination and early seedling growth. In addition, overexpression of MaRING2 enhanced the expression of stress-responsive genes under normal (before cold stress) or cold conditions. These results demonstrate the biological role of MaRING2 in conferring cold tolerance. Taken together, these results suggest that MaRING2, a C3H2C3-type RING protein, is a positive regulator of the ABA-dependent stress response.

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 (285) Characterization of a Composite Promoter from Genomic Sequences of Grapevine

HortScience ◽  
2006 ◽  
Vol 41 (4) ◽  
pp. 1053C-1053
Author(s):  
Manjul Dutt ◽  
Zhijian T. Li ◽  
Sadanand Dhekney ◽  
Dennis J. Gray
Keyword(s):  
Mosaic Virus ◽  
Genomic Sequence ◽  
Fusion Gene ◽  
Core Promoter ◽  
Gus Activity ◽  
Cauliflower Mosaic Virus ◽  
35S Promoter ◽  
2S Albumin ◽  
Wide Range ◽  
Constitutive Gene Expression

Genetic transformation of plants necessitates the use of promoters to control transgene expression. Numerous promoters have been isolated from a wide range of organisms for use in plants. However, many of these natural promoters exhibit relatively low activity and/or have limited use. To provide an alternative, we constructed a composite promoter (EP) using a genomic DNA sequence and a 35 bp TATA-containing fragment from the 2S albumin (VvAlb1) gene core promoter of grapevine. The 0.9-kb genomic sequence was identified after TAIL-PCR, based on the presence of several unique cis-acting elements. The sequence showed no homology to any known plant gene, enhancer, and promoter. Two binary vectors, pEP-EGFP/NPT and pEP-GUS, containing a bifunctional EGFP/NPTII fusion gene and a GUS gene, respectively, were constructed to test transcriptional activity of the composite promoter both qualitatively and quantitatively. Transient GFP expression was observed in somatic embryos (SE) of Vitis vinifera `Thompson Seedless' after Agrobacterium-mediated transformation using pEP-EGFP/NPT. Quantitative GUS assay of stably transformed SE containing pEP-GUS indicated that the EP composite promoter was capable of producing GUS activity as high as 12% of that from a doubly enhanced Cauliflower Mosaic Virus 35S promoter or eight times higher than that from a doubly enhanced Cassava Vein Mosaic Virus promoter. In addition, transformation of Arabidopsis with pEP-GUS yielded comparable GUS activity throughout the plant. These data indicate that the EP composite promoter can be used in transformation studies to provide sustained constitutive gene expression in plants.

scholarly journals Nematode-Responsive Activity of the Cauliflower Mosaic Virus 35S Promoter and Its Subdomains

1999 ◽  
Vol 12 (3) ◽  
pp. 189-196 ◽  
Author(s):  
David J. Bertioli ◽  
Matthew Smoker ◽  
Paul R. Burrows
Keyword(s):  
Mosaic Virus ◽  
Giant Cells ◽  
Gus Activity ◽  
Cauliflower Mosaic Virus ◽  
35S Promoter ◽  
Host Gene Expression ◽  
Root Knot Nematode ◽  
Cyst Nematodes ◽  
Feeding Sites ◽  
Cauliflower Mosaic Virus 35S

Root-knot and cyst nematodes are obligate plant parasites that induce complex biotrophic feeding structures in host roots. The mechanisms by which nematodes regulate host gene expression to produce feeding sites are unknown. The cauliflower mosaic virus (CaMV) 35S promoter has been reported to be repressed strongly in the feeding sites of both root-knot and cyst nematodes. In contrast, other work has indicated that this promoter is partially active in some feeding sites. Considering the importance of the 35S promoter in biotechnology, we have defined the nematoderesponsive nature of this promoter in more detail. Transgenic tobacco harboring various 35S-uidA constructs was assayed for β-glucuronidase (GUS) activity after infection by root-knot nematodes (Meloidogyne incognita) and cyst nematodes (Globodera tabacum subsp. tabacum). The entire 35S promoter (-343 to +8) was active in giant cells induced by M. incognita and, to a lesser extent, the syncytia of G. tabacum subsp. tabacum. In the latter case, activity decreased as the feeding sites matured. Subdomains of the 35S promoter were also active in feeding sites, particularly B4 and B5 in giant cells. However, subdomain B3 was strongly down-regulated in gall tissue and syncytia. In total, 14 constructs were studied and nematode-responsive expression was always stronger and more consistent with the root-knot nematode than the cyst nematode.

scholarly journals Evaluation of Constitutive Viral Promoters in Transgenic Soybean Roots and Nodules

2008 ◽  
Vol 21 (8) ◽  
pp. 1027-1035 ◽  
Author(s):  
Manjula Govindarajulu ◽  
James M. Elmore ◽  
Thomas Fester ◽  
Christopher G. Taylor
Keyword(s):  
Mosaic Virus ◽  
Hairy Roots ◽  
Target Genes ◽  
Root Nodules ◽  
Gus Activity ◽  
Cauliflower Mosaic Virus ◽  
35S Promoter ◽  
Symbiotic Interaction ◽  
Strawberry Vein Banding Virus ◽  
Viral Promoters

The efficiency of β-glucuronidase (GUS) expression was evaluated with five viral promoters to identify the most suitable promoter or promoters for use in soybean hairy roots, including applications to study the symbiotic interaction with Bradyrhizobium japonicum. Levels of GUS activity were fluorimetrically and histochemically assayed when the GUS (uidA) gene was driven by the Cauliflower mosaic virus (CaMV) 35S promoter and enhanced 35S (E35S) promoter, the Cassava vein mosaic virus (CsVMV) promoter, the Figwort mosaic virus (FMV) promoter, and the Strawberry vein banding virus (SVBV2) promoter. We demonstrate that GUS activity was highest when driven by the FMV promoter and that the promoter activity of 35S and SVBV2 was significantly lower than that of the CsVMV and E35S promoters when tested in soybean hairy roots. In mature soybean root nodules, strong GUS activity was evident when the FMV, 35S, and CsVMV promoters were used. These results indicate that the FMV promoter facilitates the strong expression of target genes in soybean hairy roots and root nodules.

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.

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