scholarly journals The Occurrence of CMV-Specific Short RNAs in Transgenic Tobacco Expressing Virus-Derived Double-Stranded RNA is Indicative of Resistance to the Virus

2002 ◽  
Vol 15 (8) ◽  
pp. 826-833 ◽  
Author(s):  
Kriton Kalantidis ◽  
Stavros Psaradakis ◽  
Martin Tabler ◽  
Mina Tsagris
Keyword(s):  
Transgenic Plants ◽  
Gene Silencing ◽  
Transgenic Tobacco ◽  
Virus Resistance ◽  
Northern Analysis ◽  
Specific Gene ◽  
Posttranscriptional Gene Silencing ◽  
Short Rnas ◽  
Transgenic Lines ◽  
Viral Sequences

Expression or introduction of double-stranded (ds)RNA in eukaryotic cells can trigger sequence-specific gene silencing of transgenes, endogenes, and viruses. Transgenic plants producing dsRNAs with homology to viral sequences are likely to exhibit pathogen-derived resistance to the virus. Cucumber mosaic virus (CMV), a very widespread virus with over 1,000 host species, has the natural ability to suppress silencing in order to establish infection. Here, we report the generation of transgenic tobacco lines, where a DNA transgene containing an inverted repeat of CMV cDNA had been introduced. Expression of this DNA construct delivered an RNA transcript that is able to form an intramolecular double strand. Transgenic plants were challenged with CMV. Three categories of plants could be discriminated: susceptible plants, which typically reacted with milder symptoms than the wild-type control; a “recovery” phenotype, in which newly emerging leaves were free of symptoms; and plants that showed complete resistance. Northern analysis showed that the expression of CMV dsRNA caused, in some transgenic lines, the generation of short RNAs characteristic of posttranscriptional gene silencing. Those lines were CMV resistant. The correlation between the detection of short RNAs and virus resistance provides a molecular marker that makes it possible to predict success in attempts to engineer virus resistance by dsRNA.

scholarly journals Chitin Biosynthesis Inhibition of Meloidogyne incognita by RNAi-Mediated Gene Silencing Increases Resistance to Transgenic Tobacco Plants

2020 ◽  
Vol 21 (18) ◽  
pp. 6626
Author(s):  
Vimalraj Mani ◽  
Chinreddy Subramanyam Reddy ◽  
Seon-Kyeong Lee ◽  
Soyoung Park ◽  
Hyoung-Rai Ko ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Transgenic Tobacco ◽  
Meloidogyne Incognita ◽  
Chitin Synthase ◽  
Egg Mass ◽  
Transgenic Tobacco Plants ◽  
Tobacco Plants ◽  
Wide Range ◽  
Transgenic Lines ◽  
Plant Parasitic

Meloidogyne incognita is a devastating plant parasitic nematode that causes root knot disease in a wide range of plants. In the present study, we investigated host-induced RNA interference (RNAi) gene silencing of chitin biosynthesis pathway genes (chitin synthase, glucose-6-phosphate isomerase, and trehalase) in transgenic tobacco plants. To develop an RNAi vector, ubiquitin (UBQ1) promoter was directly cloned, and to generate an RNAi construct, expression of three genes was suppressed using the GATEWAY system. Further, transgenic Nicotiana benthamiana lines expressing dsRNA for chitin synthase (CS), glucose-6-phosphate isomerase (GPI), and trehalase 1 (TH1) were generated. Quantitative PCR analysis confirmed endogenous mRNA expression of root knot nematode (RKN) and revealed that all three genes were more highly expressed in the female stage than in eggs and in the parasitic stage. In vivo, transformed roots were challenged with M. incognita. The number of eggs and root knots were significantly decreased by 60–90% in RNAi transgenic lines. As evident, root galls obtained from transgenic RNAi lines exhibited 0.01- to 0.70-fold downregulation of transcript levels of targeted genes compared with galls isolated from control plants. Furthermore, phenotypic characteristics such as female size and width were also marginally altered, while effect of egg mass per egg number in RNAi transgenic lines was reduced. These results indicate the relevance and significance of targeting chitin biosynthesis genes during the nematode lifespan. Overall, our results suggest that further developments in RNAi efficiency in commercially valued crops can be applied to employ RNAi against other plant parasitic nematodes.

scholarly journals The Arabidopsis expansin gene (AtEXPA18) is capable to ameliorate drought stress tolerance in transgenic tobacco plants

2021 ◽  
Author(s):  
Alireza Abbasi ◽  
Meysam Malekpour ◽  
Sajjad Sobhanverdi
Keyword(s):  
Drought Stress ◽  
Transgenic Plants ◽  
Transgenic Tobacco ◽  
Dry Weight ◽  
Conventional Polymerase Chain Reaction ◽  
Specific Gene ◽  
Severe Drought ◽  
Transgenic Tobacco Plants ◽  
Tobacco Plants ◽  
Reverse Transcription Pcr

Abstract Expansins are cell wall proteins that, due to changes in pH, causing the expansion of the cell walls. In this study, a previously gene construct designed based on a root-specific gene, AtEXPA18, was utilized to assess its potential roles on different morphological, physiological, and cellular levels of generated transgenic tobacco plants in response to moderate and severe drought stress. AtEXPA18 gene was successfully transferred to the tobacco plants through an agrobacterium-mediate transformation system. Upon obtaining the second generation, tobacco transgenic plants were confirmed by conventional polymerase chain reaction (PCR) technique alongside reverse transcription PCR (RT-PCR) using specific primers. Under drought stress, the transgenic lines showed remarkable growth and significantly improved based on morphological traits such as height and stem diameter, leaf area, leaf number, root dry weight, and Abscisic acid (ABA) levels of leaves compared control plants. As a result, the Cytokinin content of transgenic plants has increased under severe stress levels. Notably, the area's expansion for abaxial epidermal cells under the microscope confirmed in transgene cells compared with the -transgene cells. These results, altogether, could support the AtEXPA18 gene implication in cell expansion and improving tolerance capacity of transgenic crops under drought stress.

scholarly journals A single chimeric transgene derived from two distinct viruses confers multi-virus resistance in transgenic plants through homology-dependent gene silencing

2000 ◽  
Vol 81 (8) ◽  
pp. 2103-2109 ◽  
Author(s):  
Fuh-Jyh Jan ◽  
Carmen Fagoaga ◽  
Sheng-Zhi Pang ◽  
Dennis Gonsalves
Keyword(s):  
Gene Silencing ◽  
Virus Resistance ◽  
Fluorescent Protein ◽  
Gene Segment ◽  
N Gene ◽  
Transcriptional Gene Silencing ◽  
Multiple Resistance ◽  
Cp Gene ◽  
Transgenic Lines ◽  
Green Fluorescent

We showed previously that 218 and 110 bp N gene segments of tomato spotted wilt virus (TSWV) that were fused to the non-target green fluorescent protein (GFP) gene were able to confer resistance to TSWV via post-transcriptional gene silencing (PTGS). N gene segments expressed alone did not confer resistance. Apparently, the GFP DNA induced PTGS that targetted N gene segments and the incoming homologous TSWV for degradation, resulting in a resistant phenotype. These observations suggested that multiple resistance could be obtained by replacing the GFP DNA with a viral DNA that induces PTGS. The full-length coat protein (CP) gene of turnip mosaic virus (TuMV) was linked to 218 or 110 bp N gene segments and transformed into Nicotiana benthamiana. A high proportion (4 of 18) of transgenic lines with the 218 bp N gene segment linked to the TuMV CP gene were resistant to both viruses, and resistance was transferred to R2 plants. Nuclear run-on and Northern experiments confirmed that resistance was via PTGS. In contrast, only one of 14 transgenic lines with the TuMV CP linked to a 110 bp N gene segment yielded progeny with multiple resistance. Only a few R1 plants were resistant and resistance was not observed in R2 plants. These results clearly show the applicability of multiple virus resistance through the fusion of viral segments to DNAs that induce PTGS.

scholarly journals Analysis of recombination between viral RNAs and transgene mRNA under conditions of high selection pressure in favour of recombinants

2009 ◽  
Vol 90 (11) ◽  
pp. 2798-2807 ◽  
Author(s):  
Marco Morroni ◽  
Jeremy R. Thompson ◽  
Mark Tepfer
Keyword(s):  
Transgenic Plants ◽  
Selection Pressure ◽  
In Planta ◽  
Coding Region ◽  
Tobacco Plants ◽  
Viral Rnas ◽  
Transgenic Lines ◽  
High Selection ◽  
The One ◽  
Viral Sequences

One possible environmental risk related to the utilization of virus-resistant transgenic plants expressing viral sequences is the emergence of new viruses generated by recombination between the viral transgene mRNA and the RNA of an infecting virus. This hypothesis has been tested recently for cucumber mosaic virus (CMV) by comparing the recombinant populations in transgenic and non-transgenic plants under conditions of minimal selection pressure in favour of the recombinants. Equivalent populations were observed in transgenic and non-transgenic plants but, in both, there was a strongly dominant hotspot recombinant which was shown recently to be nonviable alone in planta, suggesting that its predominance could be reduced by applying an increased selection pressure in favour of viable recombinants. Partially disabled I17F-CMV mutants were created by engineering 6 nt deletions in five sites in the RNA3 3′-non-coding region (3′-NCR). One mutant was used to inoculate transgenic tobacco plants expressing the coat protein and 3′-NCR of R-CMV. A total of 22 different recombinant types were identified, of which 12 were, as expected, between the transgene mRNA and the mutated I17F-CMV RNA3, while 10 resulted from recombination between the mutated RNA3 and I17F-CMV RNA1. Twenty recombinants were of the aberrant type, while two, including the dominant one detected previously under conditions of minimal selection pressure, were homologous recombinants. All recombinants detected were very similar to ones observed in nature, suggesting that the deployment of transgenic lines similar to the one studied here would not lead to the emergence of new viruses.

scholarly journals Cloning of TaPRP from Wheat and Its Cold Tolerance Analysis in Transgenic Tobacco

2016 ◽  
Vol 8 (5) ◽  
pp. 61
Author(s):  
Zhang Baolei ◽  
Li Da ◽  
Zhang Weidong ◽  
Liu Dasheng ◽  
Gao Qingrong ◽  
...  
Keyword(s):  
Transgenic Plants ◽  
Cold Tolerance ◽  
Transgenic Tobacco ◽  
Tolerance Analysis ◽  
Treatment Temperature ◽  
Nucleotide Sequence Analysis ◽  
Amino Acid Residues ◽  
Rt Pcr ◽  
Sequence Homologies ◽  
Transgenic Lines

<p><em>TaPRP</em>, a proline rich protein (PRP) cDNA, was cloned by RT-PCR from winter wheat. Nucleotide sequence analysis showed <em>TaPRP</em> is composed of 1137 bp (378 amino acid residues with a Mr of 42.19 kD).<em> TaPRP</em> shows 92.6%, 89.3%, 73.0%, and 73.3% sequence homologies with <em>PRP</em> genes from wheat, sorghum, rice, and maize, respectively. The deduced protein includes 170 prolines, presenting a normal <em>PRP</em> primary structure. Expression vector pBI<em>-TaPRP</em> was constructed, in which <em>TaPRP</em> was driven by CaMV35S promoter and stopped by NospolyA. Tobaccos were transformed by<em> Agrobacterium</em> containing the constructed vectors. Three transgenic lines were confirmed by PCR detection and Southern blot. Under the same low temperature stress conditions, transgenic plants had lower conductivity rate compared with the non-transgenic plants, suggesting that cold tolerance in transgenic tobacco plants was improved. However, the different transgenic plants showed significant differences in cold resistant, and there also existed significant interactions between plant and treatment temperature. <em>TaPRP </em>might have an important role in wheat in cold adaptation process.</p>

scholarly journals Arabidopsis SGS2 and SGS3 Genes Are Required for Posttranscriptional Gene Silencing and Natural Virus Resistance

Cell ◽  
2000 ◽  
Vol 101 (5) ◽  
pp. 533-542 ◽  
Author(s):  
Philippe Mourrain ◽  
Christophe Béclin ◽  
Taline Elmayan ◽  
Frank Feuerbach ◽  
Christian Godon ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Virus Resistance ◽  
Posttranscriptional Gene Silencing ◽  
Natural Virus

scholarly journals Geminivirus Resistance in Transgenic Tobacco Expressing Mutated BC1 Protein

1997 ◽  
Vol 10 (5) ◽  
pp. 617-623 ◽  
Author(s):  
Y.-P. Duan ◽  
C. A. Powell ◽  
S. E. Webb ◽  
D. E. Purcifull ◽  
E. Hiebert
Keyword(s):  
Transgenic Plants ◽  
Transgenic Tobacco ◽  
Movement Protein ◽  
Enzyme Linked Immunosorbent Assay ◽  
Continuous Exposure ◽  
Symptom Development ◽  
Dot Blot ◽  
Disease Symptoms ◽  
Transgenic Lines ◽  
Antisense Constructs

Tobacco explants were transformed by Agrobacterium-mediated transformation with sense and antisense constructs of the movement protein genes (BC1 and BV1) of tomato mottle geminivirus (TMoV). Transgenic plants were tested for virus resistance either by exposure to viruliferous whiteflies carrying TMoV or cabbage leaf curl geminivirus (CabLCV) for a 72-h inoculation period or by continuous exposure to TMoV during the life of the plants. The transgenic lines were scored for disease symptoms, and virus replication and distribution were determined by enzyme-linked immunosorbent assay and dot blot hybridizations. Transgenic plants which expressed a mutated form (identified in a previous study) of the BC1 gene showed TMoV and CabLCV resistance. Three resistant phenotypes were observed: a delay in symptom development, a recovery from early symptoms, and an absence of virus symptoms at all stages. Geminivirus was detected in inoculated leaves but was not readily detected in leaves beyond the inoculation sites in the highly resistant plants. The geminivirus resistance conferred by expression of the spontaneously mutated BC1 gene (detected after transformation) in transgenic tobacco may involve transdominant negative interference.

scholarly journals Can We Explain RNA-Mediated Virus Resistance by Homology-Dependent Gene Silencing?

1998 ◽  
Vol 11 (7) ◽  
pp. 717-723 ◽  
Author(s):  
Tom van den Boogaart ◽  
George P. Lomonossoff ◽  
Jeffrey W. Davies
Keyword(s):  
Gene Silencing ◽  
Virus Resistance ◽  
Viral Protein ◽  
Transgene Expression ◽  
Standard Technique ◽  
Protein S ◽  
The Other ◽  
Critical View ◽  
Homologous Sequences ◽  
Viral Sequences

The use of viral sequences to produce virus-resistant plants is now almost a standard technique. A variety of sequences from a large number of viruses have been used but the mechanisms remain largely unknown. There are probably at least two distinct types of mechanisms operating: one requiring the expression of the viral protein(s) and the other dependent only on the presence of transgene-derived RNA. In this review, we will discuss this RNA-mediated resistance and its similarities with cosuppression, a recently described phenomenon leading to suppression of transgene expression and homologous sequences. We present a critical view of the current models available to explain this type of resistance.

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