Post-Transcriptional Gene Silencing in the Root System of the Actinorhizal Tree Allocasuarina verticillata

In recent years, RNA interference has been exploited as a tool for investigating gene function in plants. We tested the potential of double-stranded RNA interference technology for silencing a transgene in the actinorhizal tree Allocasuarina verticillata. The approach was undertaken using stably transformed shoots expressing the β-glucuronidase (GUS) gene under the control of the constitutive promoter 35S; the shoots were further transformed with the Agrobacterium rhizogenes A4RS containing hairpin RNA (hpRNA) directed toward the GUS gene, and driven by the 35S promoter. The silencing and control vectors contained the reporter gene of the green fluorescent protein (GFP), thus allowing a screening of GUS-silenced composite plantlets for autofluorescence. With this rapid procedure, histochemical data established that the reporter gene was strongly silenced in both fluorescent roots and actinorhizal nodules. Fluorometric data further established that the level of GUS silencing was usually greater than 90% in the hairy roots containing the hairpin GUS sequences. We found that the silencing process of the reporter gene did not spread to the aerial part of the composite A. verticillata plants. Real-time quantitative polymerase chain reaction showed that GUS mRNAs were substantially reduced in roots and, thereby, confirmed the knock-down of the GUS transgene in the GFP+ hairy roots. The approach described here will provide a versatile tool for the rapid assessment of symbiotically related host genes in actinorhizal plants of the Casuarinaceae family.

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Successful genetic transformation in date palm (Phoenix dactylifera)

Journal of the Bangladesh Agricultural University ◽

10.3329/jbau.v11i2.19841 ◽

2014 ◽

Vol 11 (2) ◽

pp. 171-176 ◽

Cited By ~ 2

Author(s):

L Hassan

Keyword(s):

Agrobacterium Tumefaciens ◽

Agrobacterium Rhizogenes ◽

Reporter Gene ◽

Hairy Roots ◽

Date Palm ◽

Binary Vector ◽

Nptii Gene ◽

Neomycin Phosphotransferase ◽

Gus Gene ◽

Nos Terminator

The introduction of foreign genes into most of the Phoenix spp using recombinant DNA technology is not a straight forward task. In Phoenix spp application of this technology towards successful transformation proved to be a more difficult one so far no report on the successful regeneration of transgenic date palm plants has been published. We developed an efficient and reproducible variety-independent method for producing transgenic date palm (Phoenix spp) via Agrobacterium-mediated transformation. Agrobacterium rhizogenes strains LBA 9402 were used and for cotransformation experiments the strain LBA 9402 with the binary vector pBIN19 with the p35S GUS INT gene was used. Off-shoot segments from different Phoenix spp cultivars were infected with Agrobacterium rhizogenes. The development of hairy roots at a high frequency only on infected tissue pieces showed that transformation is possible. Various parameters like, effect of different genotypes on root initiation, root number and root length have been studied. Regeneration of transformed root cultures to plantlets was also attempted. Histochemical GUS assay and polymerase chain reaction analysis of hairy roots confirmed the presence of GUS gene. Agrobacterium tumifaciensmediated transformation was also performed using the leaves of off-shoot explants. Agrobacterium tumefaciens strains: I) GV3101 with the vir plasmid pMP90 the strain C58C1 ATHV with the vir-plasmid pTiBo542 (=pEHA101; Hood et al. 1986) was used. The nptII gene (neomycin phosphotransferase) was used as a selectable marker gene. The ?-Glucuronidase-gene (GUS-Gene: Jefferson et al. 1987) under control of the Ubi- and 35S-Promotors, with an Intron (Vancanneyt et al. 1990), was used as the reporter gene. We also used the genetically engineered Agrobacterium tumefaciens strain LBA4404 as a vector for infection in the transformation experiment, which contains plasmid pBI121 of 14 KDa (binary vector). This binary vector contains following genes within the right border (RB) and left border (LB) region of the construct: The udiA gene (Jefferson, 1986) predetermining GUS (?-glucuronidase), driven by CaMV promoter and NOS terminator. This reporter gene can be used to assess the efficiency of transformation. The nptII gene (Herrera-Estrella et al., 1983) encoding neomycin phosphotransferase II (nptII) conferring kanamycin resistance, driven by NOS promoter and NOS terminator. The bacterium also contains plasmid pAL4404 which is a disarmed Ti plasmid (132 KDa) containing the virulence genes. For the confirmation of transgenes, calli were taken from the growing callus mass for DNA isolation. PCR- and Southern analysis was performed to determine the integration and the copy number of the transgene. The GUS-test was performed to demonstrate ß-glucuronidase expression. The transgenic plantlets were kept in a hardening room for four weeks and they will be transferred to a growth chamber with controlled environment for further establishment. DOI: http://dx.doi.org/10.3329/jbau.v11i2.19841 J. Bangladesh Agril. Univ. 11(2): 171-176, 2013

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Transformed Hairy Roots of Arachis hypogea: A Tool for Studying Root Nodule Symbiosis in a Non–Infection Thread Legume of the Aeschynomeneae Tribe

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-22-2-0132 ◽

2009 ◽

Vol 22 (2) ◽

pp. 132-142 ◽

Cited By ~ 37

Author(s):

Senjuti Sinharoy ◽

Sudip Saha ◽

Susanta Roy Chaudhury ◽

Maitrayee DasGupta

Keyword(s):

Hairy Roots ◽

Fluorescent Protein ◽

Root Hairs ◽

Infection Thread ◽

Natural Soil ◽

35S Promoter ◽

Wild Type ◽

Cortical Cells ◽

Arachis Hypogea ◽

Green Fluorescent

Arachis hypogea is a non–“infection thread” (IT) legume where rhizobial entry or dissemination in the nodules never involves IT. Rhizobia invade through epidermal “cracks” and directly access the cortical cells to develop the characteristic aeschynomenoid nodules. For investigating these nonclassical nodulation features in Arachis spp., we developed an efficient procedure for Agrobacterium rhizogenes R1000-mediated transformation of this plant. In this study, we optimized the induction of hairy roots and nodulation of composite Arachis hypogea plants in the presence of Bradyrhizobium sp. (Arachis) strain NC92. 35S promoter-driven green fluorescent protein and β-glucuronidase expression indicated transformation frequency to be above 80%. The transformed roots had the characteristic rosette-type root hairs and had normal level of expression of symbiosis-related genes SymRK and CCaMK. The transgenic nodules resembled the wild-type nodules with an exception of 2 to 3%, where they structurally deviated from the wild-type nodules to form nodular roots. A 16S rRNA profile of an infected-zone metagenome indicated that identical populations of bradyrhizobia invaded both composite wild-type plants grown in natural soil. Our results demonstrate that Arachis hairy root is an attractive system for undertaking investigations of the nonclassical features associated with its nitrogen-fixing symbiotic interactions.

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V2 of tomato yellow leaf curl virus can suppress methylation-mediated transcriptional gene silencing in plants

Journal of General Virology ◽

10.1099/vir.0.055798-0 ◽

2014 ◽

Vol 95 (1) ◽

pp. 225-230 ◽

Cited By ~ 53

Author(s):

Bi Wang ◽

Fangfang Li ◽

Changjun Huang ◽

Xiuling Yang ◽

Yajuan Qian ◽

...

Keyword(s):

Gene Silencing ◽

Fluorescent Protein ◽

Methylation Status ◽

Promoter Sequence ◽

Tomato Yellow Leaf ◽

Transcriptional Gene Silencing ◽

35S Promoter ◽

Yellow Leaf ◽

Leaf Curl Virus ◽

Leaf Curl

Tomato yellow leaf curl virus (TYLCV) is a DNA virus belonging to the genus Begomovirus. TYLCV replicates using double-stranded DNA intermediates that can become the target of plant transcriptional gene silencing (TGS). Here, we show that the V2 protein of TYLCV can suppress TGS of a transcriptionally silenced green fluorescent protein (GFP) transgene in Nicotiana benthamiana line 16-TGS. Through bisulfite sequencing and chop-PCR, we demonstrated that the TYLCV V2 can reverse GFP transgene silencing by reducing the methylation levels in the 35S promoter sequence. Both AtSN1 and MEA-ISR loci in Arabidopsis thaliana were previously reported to be strongly methylated, and we show that the methylation status of both loci was significantly reduced in TYLCV V2 transgenic Arabidopsis plants. We conclude that TYLCV can efficiently suppress TGS when it infects plants, and its V2 protein is responsible for the TGS suppression activity.

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Construction and Transient Expression of Chimeric Cassettes Containing CaMV 35S or OsAER1 Promoter and GUS Gene Fusion in Tobacco

Jurnal AgroBiogen ◽

10.21082/jbio.v14n2.2018.p55-64 ◽

2018 ◽

Vol 14 (2) ◽

pp. 55

Author(s):

Anniversari Apriana ◽

Atmitri Sisharmini ◽

Hajrial Awidinnoor ◽

Kurniawan Rudi Trijatmiko ◽

Sudarsono Sudarsono

Keyword(s):

Reporter Gene ◽

Gene Promoter ◽

Camv 35S Promoter ◽

35S Promoter ◽

Transient Transformation ◽

Gus Gene ◽

Promoter Fragment ◽

Camv 35S ◽

Promoter Construct ◽

Tobacco Seedlings

<p>Reporter gene assays are commonly used to study the expression pattern of a gene and the promoter activity. The purpose of this study was to assemble the chimeric gene constructs consisting of CaMV 35S promoter orOsAER1 gene promoter connected to the β-glucuronidase (GUS) reporter gene encoding the β-glucuronidase enzyme and to obtain an efficient method for Agrobacterium tumefaciens-mediated transient transformation of tobacco sprouts. The CaMV 35S promoter fragment reamplified from pCAMBIA1301 binary vector and the OsAER1 gene promoter fragment amplified from rice cv. Awan Kuning were ligated into pCAMBIA1300int::gus::tNOS to produce binary vectors pCAMBIA1300int::p35S::gus::tNOS and pCAMBIA1300int::prOsAER1::gus::tNOS. The vectors were used for transient transformation of 5–day old tobacco seedlings. The transformation was carried out using two bacterial cultures with densities of OD600 = 0.5 or OD600 = 1.0 combined with a vacuum for 15 or 30 minutes. Tobacco seedlings transformed with pCAMBIA1300int::p35S::gus::tNOS showed higher transformation efficiency as compared tothe ones transformed with pCAMBIA1300int::prOsAER1::gus::tNOS. A higher efficiency was obtained from transformation using bacterial culture with density of OD600 = 0.5 in combination with a vacuum for 30 minutes. Expression of GUS gene in the tobacco sprouts transformed with CaMV 35S promoter construct was observed through out the sprouts area (root, hypocotyl, cotyledon, and leaf), where as expression of GUS gene was observed in root, hypocotyl, and cotyledon, but not in leaf on tobacco sprouts transformed with OsAER1 promoter construct. These results indicate that the transient transformation is a quick and simple method for testing a chimeric gene construct.</p>

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Mastrevirus Rep and RepA Proteins Suppress de novo Transcriptional Gene Silencing

International Journal of Molecular Sciences ◽

10.3390/ijms222111462 ◽

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.

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Viroids as a Tool to Study RNA-Directed DNA Methylation in Plants

Cells ◽

10.3390/cells10051187 ◽

2021 ◽

Vol 10 (5) ◽

pp. 1187

Author(s):

Michael Wassenegger ◽

Athanasios Dalakouras

Keyword(s):

Dna Methylation ◽

Rna Interference ◽

Gene Silencing ◽

Plant Cells ◽

Transcriptional Gene Silencing ◽

Rnai Machinery ◽

Double Stranded Rna ◽

Post Transcriptional Gene Silencing ◽

Cumulative Amount

Viroids are plant pathogenic, circular, non-coding, single-stranded RNAs (ssRNAs). Members of the Pospiviroidae family replicate in the nucleus of plant cells through double-stranded RNA (dsRNA) intermediates, thus triggering the host’s RNA interference (RNAi) machinery. In plants, the two RNAi pillars are Post-Transcriptional Gene Silencing (PTGS) and RNA-directed DNA Methylation (RdDM), and the latter has the potential to trigger Transcriptional Gene Silencing (TGS). Over the last three decades, the employment of viroid-based systems has immensely contributed to our understanding of both of these RNAi facets. In this review, we highlight the role of Pospiviroidae in the discovery of RdDM, expound the gradual elucidation through the years of the diverse array of RdDM’s mechanistic details and propose a revised RdDM model based on the cumulative amount of evidence from viroid and non-viroid systems.

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RNA Interference of Genes Encoding the Vacuolar-ATPase in Liriomyza trifolii

Insects ◽

10.3390/insects12010041 ◽

2021 ◽

Vol 12 (1) ◽

pp. 41

Author(s):

Ya-Wen Chang ◽

Yu-Cheng Wang ◽

Xiao-Xiang Zhang ◽

Junaid Iqbal ◽

Yu-Zhou Du

Keyword(s):

Rna Interference ◽

Expression Analysis ◽

Target Genes ◽

Fluorescent Protein ◽

Control Strategies ◽

Vacuolar Atpase ◽

Invasive Pest ◽

Liriomyza Trifolii ◽

Horticultural Crops ◽

Genes Encoding

The leafminer fly, Liriomyza trifolii, is an invasive pest of vegetable and horticultural crops in China. In this study, a microinjection method based on dsRNA was developed for RNA interference (RNAi) in L. trifolii using genes encoding vacuolar-ATPase (V-ATPase). Expression analysis indicated that V-ATPase B and V-ATPase D were more highly expressed in L. trifolii adults than in larvae or pupae. Microinjection experiments with dsV-ATPase B and dsV-ATPase D were conducted to evaluate the efficacy of RNAi in L. trifolii adults. Expression analysis indicated that microinjection with 100 ng dsV-ATPase B or dsV-ATPase led to a significant reduction in V-ATPase transcripts as compared to that of the dsGFP control (dsRNA specific to green fluorescent protein). Furthermore, lower dsRNA concentrations were also effective in reducing the expression of target genes when delivered by microinjection. Mortality was significantly higher in dsV-ATPase B- and dsV-ATPase D-treated insects than in controls injected with dsGFP. The successful deployment of RNAi in L. trifolii will facilitate functional analyses of vital genes in this economically-important pest and may ultimately result in new control strategies.

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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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