scholarly journals T4 Lysozyme and Attacin Genes Enhance Resistance of Transgenic `Galaxy' Apple against Erwinia amylovora

2002 ◽  
Vol 127 (4) ◽  
pp. 515-519 ◽  
Author(s):  
Kisung Ko ◽  
John L. Norelli ◽  
Jean-Paul Reynoird ◽  
Herb S. Aldwinckle ◽  
Susan K. Brown
Keyword(s):  
Mosaic Virus ◽  
Erwinia Amylovora ◽  
Alfalfa Mosaic Virus ◽  
Cauliflower Mosaic Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
35S Promoter ◽  
In Planta ◽  
Mechanical Wounding ◽  
Genes Encoding ◽  
Transgenic Lines

Genes encoding lysozyme (T4L) from T4 bacteriophage and attacin E (attE) from Hyalophora cecropia were used, either singly or in combination, to construct plant binary vectors, pLDB15, p35SAMVT4, and pPin2Att35SAMVT4, respectively, for Agrobacterium-mediated transformation of `Galaxy' apple, to enhance resistance to Erwinia amylovora. In these plasmids, the T4L gene was controlled by the cauliflower mosaic virus 35S promoter with duplicated upstream domain and the untranslated leader sequence of alfalfa mosaic virus RNA 4, and the attE gene was controlled by the potato proteinase inhibitor II (Pin2) promoter. All transgenic lines were screened by polymerase chain reaction (PCR) for T4L and attE genes, and a double-antibody sandwich enzyme-linked immunosorbent assay for neomycin phosphotransferase II. Amplification of T4L and attE genes was observed in reverse transcriptase-PCR, indicating that these genes were transcribed in all tested transgenic lines containing each gene. The attacin protein was detected in all attE transgenic lines. The expression of attE under the Pin2 promoter was constitutive but higher levels of expression were observed after mechanical wounding. Some T4L or attE transgenic lines had significant disease reduction compared to nontransgenic `Galaxy'. However, transgenic lines containing both attE and T4L genes were not significantly more resistant than nontransgenic `Galaxy', indicating that there was no in planta synergy between attE and T4L with respect to resistance to E. amylovora.

scholarly journals Engineering Resistance to Multiple Prunus Fruit Viruses Through Expression of Chimeric Hairpins

2007 ◽  
Vol 132 (3) ◽  
pp. 407-414 ◽  
Author(s):  
Zongrang Liu ◽  
Ralph Scorza ◽  
Jean-Michel Hily ◽  
Simon W. Scott ◽  
Delano James
Keyword(s):  
Gene Silencing ◽  
Mosaic Virus ◽  
Fruit Production ◽  
Cauliflower Mosaic Virus ◽  
Ringspot Virus ◽  
35S Promoter ◽  
Plum Pox Virus ◽  
In Planta ◽  
Posttranscriptional Gene Silencing ◽  
Transgenic Lines

Prunus L. fruit production is seriously affected by several predominant viruses. The development of new cultivars resistant to these viruses is challenging but highly desired by breeders and growers. We report a posttranscriptional gene silencing-based approach for engineering multivirus resistance in plants. A single chimeric transgene, PTRAP6, was created by the fusion of 400 to 500-base pair (bp) gene fragments from six major Prunus fruit viruses, including american plum line pattern virus, peach mosaic virus, plum pox virus (PPV), prune dwarf virus (PDV), prunus necrotic ringspot virus, and tomato ringspot virus (ToRSV). Both strands of PTRAP6 were found being transcribed as an ≈2.5-kilobp transcript in planta without splicing interruption. To induce gene silencing/virus resistance, we placed two copies of PTRAP6 in an inverted repeat under the control of the cauliflower mosaic virus 35S promoter and separated by an intron spacer fragment to create PTRAP6i. Inoculation of the resulting transgenic Nicotiana benthamiana Domin. plants revealed that 12 of 28 R0 PTRAP6i transgenic lines (43%) were resistant to ToRSV ranging from mild symptoms to symptom-free phenotypes. Detailed analysis of two of three highly resistant homozygous R3 generation lines demonstrated that they were resistant to all three viruses tested, including PDV, PPV, and ToRSV. The remaining three viruses targeted by PTRAP6i were either unavailable for this study or were unable to systemically infect N. benthamiana. Transgene-wide and -specific small interfering RNA species were detected along with disappearance of transgene transcript in the resistant lines, indicating that posttranscriptional gene silencing underlies the mechanism of resistance. This work presents evidence that PTRAP6i is able to confer gene silencing-based resistance to multiple Prunus fruit viruses.

scholarly journals Robust RNAi-Based Resistance to Mixed Infection of Three Viruses in Soybean Plants Expressing Separate Short Hairpins from a Single Transgene

2011 ◽  
Vol 101 (11) ◽  
pp. 1264-1269 ◽  
Author(s):  
Xiuchun Zhang ◽  
Shirley Sato ◽  
Xiaohong Ye ◽  
Anne E. Dorrance ◽  
T. Jack Morris ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Virus Resistance ◽  
Soybean Mosaic Virus ◽  
Alfalfa Mosaic Virus ◽  
Cauliflower Mosaic Virus ◽  
Systemic Resistance ◽  
35S Promoter ◽  
Virus Infections ◽  
Bean Pod Mottle Virus ◽  
Soybean Plants

Transgenic plants expressing double-stranded RNA (dsRNA) of virus origin have been previously shown to confer resistance to virus infections through the highly conserved RNA-targeting process termed RNA silencing or RNA interference (RNAi). In this study we applied this strategy to soybean plants and achieved robust resistance to multiple viruses with a single dsRNA-expressing transgene. Unlike previous reports that relied on the expression of one long inverted repeat (IR) combining sequences of several viruses, our improved strategy utilized a transgene designed to express several shorter IRs. Each of these short IRs contains highly conserved sequences of one virus, forming dsRNA of less than 150 bp. These short dsRNA stems were interspersed with single-stranded sequences to prevent homologous recombination during the transgene assembly process. Three such short IRs with sequences of unrelated soybean-infecting viruses (Alfalfa mosaic virus, Bean pod mottle virus, and Soybean mosaic virus) were assembled into a single transgene under control of the 35S promoter and terminator of Cauliflower mosaic virus. Three independent transgenic lines were obtained and all of them exhibited strong systemic resistance to the simultaneous infection of the three viruses. These results demonstrate the effectiveness of this very straight forward strategy for engineering RNAi-based virus resistance in a major crop plant. More importantly, our strategy of construct assembly makes it easy to incorporate additional short IRs in the transgene, thus expanding the spectrum of virus resistance. Finally, this strategy could be easily adapted to control virus problems of other crop plants.

scholarly journals Identification, Characterization, and Molecular Detection of Alfalfa mosaic virus in Potato

2006 ◽  
Vol 96 (11) ◽  
pp. 1237-1242 ◽  
Author(s):  
H. Xu ◽  
J. Nie
Keyword(s):  
Mosaic Virus ◽  
Gene Sequence ◽  
Alfalfa Mosaic Virus ◽  
Enzyme Linked Immunosorbent Assay ◽  
Polymorphism Analysis ◽  
Rt Pcr ◽  
Potato Leaf ◽  
Simple Method ◽  
Cp Gene ◽  
Nucleotide Sequence Alignment

Alfalfa mosaic virus (AMV) was detected in potato fields in several provinces in Canada and characterized by bioassay, enzyme-linked immunosorbent assay, and reverse-transcription polymerase chain reaction (RT-PCR). The identity of eight Canadian potato AMV isolates was confirmed by sequence analysis of their coat protein (CP) gene. Sequence and phylogenetic analysis indicated that these eight AMV potato isolates fell into one strain group, whereas a slight difference between Ca175 and the other Canadian AMV isolates was revealed. The Canadian AMV isolates, except Ca175, clustered together among other strains based on alignment of the CP gene sequence. To detect the virus, a pair of primers, AMV-F and AMV-R, specific to the AMV CP gene, was designed based on the nucleotide sequence alignment of known AMV strains. Evaluations showed that RT-PCR using this primer set was specific and sensitive for detecting AMV in potato leaf and tuber samples. AMV RNAs were easily detected in composite samples of 400 to 800 potato leaves or 200 to 400 tubers. Restriction analysis of PCR amplicons with SacI was a simple method for the confirmation of PCR tests. Thus, RT-PCR followed by restriction fragment length polymorphism analysis may be a useful approach for screening potato samples on a large scale for the presence of AMV.

Advances in alfalfa mosaic virus-mediated expression of anthrax antigen in planta

2005 ◽  
Vol 338 (2) ◽  
pp. 717-722 ◽  
Author(s):  
R. Brodzik ◽  
K. Bandurska ◽  
D. Deka ◽  
M. Golovkin ◽  
H. Koprowski
Keyword(s):  
Mosaic Virus ◽  
Alfalfa Mosaic Virus ◽  
In Planta

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 Expression of thaumatin, a new type of alternative sweetener in rice

2020 ◽  
Vol 48 (3) ◽  
pp. 1276-1291
Author(s):  
Shahina AKTER ◽  
Md. Amdadul HUQ ◽  
Yu-Jin JUNG ◽  
Kwon-Kyoo KANG
Keyword(s):  
Transgenic Rice ◽  
Oryza Sativa L ◽  
Plasmid Vector ◽  
Single Copy ◽  
Cauliflower Mosaic Virus ◽  
Pcr Analysis ◽  
35S Promoter ◽  
Alternative Sweetener ◽  
Sweet Proteins ◽  
Transgenic Lines

  Sweet proteins are the natural alternative to the artificial sweeteners as well as flavor enhancers. Among other sweet protein, thaumatin protein was isolated from Thaumatococcus daniellii Benth plant fruit. In this study, pinII Ti plasmid vector was constructed with thaumatin gene, where thaumatin was placed under the control of the duel cauliflower mosaic virus 35S promoter into rice (Oryza sativa L. var. japonica cv. ‘Dongjinbyeo’) by Agrobacterium-mediated transformation to generate transgenic plants. Thirteen plant lines were regenerated and the transgenic rice lines were confirmed by different molecular analysis. The genomic PCR result revealed that all of the plant lines were transgenic. The single copy and intergenic plant lines were selected by Taqman PCR analysis and FST analysis, respectively. Expression of thaumatin gene in transgenic rice resulted in the accumulation of thaumatin protein in the leave. Thaumatin protein was also accumulated in leave of T1 generation. Sensory analysis result suggested that the thaumatin protein expressing transgenic lines exerted sweet tasting activity. These results demonstrated that thaumatin was expressed in transgenic rice plants.

scholarly journals Quantification of the 35S Promoter in DNA Extracts from Genetically Modified Organisms Using Real-Time Polymerase Chain Reaction and Specificity Assessment on Various Genetically Modified Organisms, Part I: Operating Procedure

2005 ◽  
Vol 88 (2) ◽  
pp. 547-557 ◽  
Author(s):  
Sophie Fernandez ◽  
Chrystèle Charles-Delobel ◽  
Angèle Geldreich ◽  
Georges Berthier ◽  
Francine Boyer ◽  
...  
Keyword(s):  
Mosaic Virus ◽  
Real Time ◽  
Genetically Modified Organisms ◽  
Limit Of Detection ◽  
Genetically Modified ◽  
Collaborative Study ◽  
Cauliflower Mosaic Virus ◽  
Performance Criteria ◽  
35S Promoter ◽  
Conserved Sequence

Abstract A highly sensitive quantitative real-time assay targeted on the 35S promoter of a commercial genetically modified organism (GMO) was characterized (sF/sR primers) and developed for an ABI Prism® 7700 Sequence Detection System and TaqMan® chemistry. The specificity assessment and performance criteria of sF/sR assay were compared to other P35S-targeted published assays. sF/sR primers amplified a 79 base pair DNA sequence located in a part of P35S that is highly conserved among many caulimovirus strains, i.e., this consensus part of CaMV P35S is likely to be present in many GM events. According to the experimental conditions, the absolute limit of detection for Bt176 corn was estimated between 0.2 and 2 copies of equivalent genome (CEG). The limit of quantification was reached below 0.1% Bt176 content. A Cauliflower Mosaic Virus control (CaMV) qualitative assay targeted on the ORF III of the viral genome was also used as a control (primers 3F/3R) to assess the presence of CaMV in plant-derived products. The specificity of this test was assessed on various CaMV strains, including the Figwort Mosaic Virus (FMV) and solanaceous CaMV strains. Considering the performance of sF/sR quantification test, the highly conserved sequence, and the small size of the amplicon, this assay was tested in a collaborative study in order to be proposed as an international standard.

Sign in / Sign up

Export Citation Format

Share Document