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Crude extracts of bacterially expressed dsRNA can be used to protect plants against virus infections.

Tenllado F, Martínez-García B, Vargas M, Díaz-Ruíz JR - BMC Biotechnol. (2003)

Bottom Line: Double-stranded RNA (dsRNA) is a potent initiator of gene silencing in a diverse group of organisms that includes plants, Caenorhabditis elegans, Drosophila and mammals.The approach required the in vitro synthesis of large amounts of RNA involving high cost and considerable labour.The main advantage of this mode of dsRNA production is its simplicity and its extremely low cost compared with the requirements for regenerating transgenic plants.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Biología de Plantas, Centro de Investigaciones Biológicas, CSIC, Velázquez 144, Madrid 28006, Spain. tenllado@cib.csic.es

ABSTRACT

Background: Double-stranded RNA (dsRNA) is a potent initiator of gene silencing in a diverse group of organisms that includes plants, Caenorhabditis elegans, Drosophila and mammals. We have previously shown and patented that mechanical inoculation of in vitro-transcribed dsRNA derived from viral sequences specifically prevents virus infection in plants. The approach required the in vitro synthesis of large amounts of RNA involving high cost and considerable labour.

Results: We have developed an in vivo expression system to produce large amounts of virus-derived dsRNAs in bacteria, with a view to providing a practical control of virus diseases in plants. Partially purified bacterial dsRNAs promoted specific interference with the infection in plants by two viruses belonging to the tobamovirus and potyvirus groups. Furthermore, we have demonstrated that easy to obtain, crude extracts of bacterially expressed dsRNAs are equally effective protecting plants against virus infections when sprayed onto plant surfaces by a simple procedure. Virus infectivity was significantly abolished when plants were sprayed with French Press lysates several days before virus inoculation.

Conclusion: Our approach provides an alternative to genetic transformation of plant species with dsRNA-expressing constructs capable to interfere with plant viruses. The main advantage of this mode of dsRNA production is its simplicity and its extremely low cost compared with the requirements for regenerating transgenic plants. This approach provides a reliable and potential tool, not only for plant protection against virus diseases, but also for the study of gene silencing mechanisms in plant virus infections.

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Bacterially expressed dsRNA interferes with PPV infection. (A) Production of PPV dsRNAs in E. coli. HT115 cells were separately transformed with the L4440 double-T7 vector containing either the HC or the CP genes of PPV. Bacterial cultures were induced with IPTG and processed for total nucleic acid. Samples were resolved by electrophoresis on 1% agarose gel before (lanes 1 to 4) or after treatment with RNase A (lanes 5 to 8), and nucleic acid was visualized by staining with ethidium bromide. Markers, λEcoRI-HindIII molecular weight markers. The positions of bacterially expressed 1492-bp HC and 1081-bp CP dsRNAs are indicated in the margin. (B) Detection of PPV in total RNA extracted from systemic leaves of N. benthamiana by RT-PCR at 14 dpi. Plants were mock inoculated or were inoculated with PPV (0.3 μg/ml) alone (-), or with mixtures of PPV plus French Press preparations derived from HT115 harboring either PPV HC dsRNA, PMMoV IR 54 or the empty vector, as indicated. Markers, λEcoRI-HindIII molecular weight markers. RT-PCR was performed with 1 μg of total RNA using primers corresponding to the CP coding sequence of PPV. The position of the 510-bp amplified fragment is indicated in the margin.
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Figure 4: Bacterially expressed dsRNA interferes with PPV infection. (A) Production of PPV dsRNAs in E. coli. HT115 cells were separately transformed with the L4440 double-T7 vector containing either the HC or the CP genes of PPV. Bacterial cultures were induced with IPTG and processed for total nucleic acid. Samples were resolved by electrophoresis on 1% agarose gel before (lanes 1 to 4) or after treatment with RNase A (lanes 5 to 8), and nucleic acid was visualized by staining with ethidium bromide. Markers, λEcoRI-HindIII molecular weight markers. The positions of bacterially expressed 1492-bp HC and 1081-bp CP dsRNAs are indicated in the margin. (B) Detection of PPV in total RNA extracted from systemic leaves of N. benthamiana by RT-PCR at 14 dpi. Plants were mock inoculated or were inoculated with PPV (0.3 μg/ml) alone (-), or with mixtures of PPV plus French Press preparations derived from HT115 harboring either PPV HC dsRNA, PMMoV IR 54 or the empty vector, as indicated. Markers, λEcoRI-HindIII molecular weight markers. RT-PCR was performed with 1 μg of total RNA using primers corresponding to the CP coding sequence of PPV. The position of the 510-bp amplified fragment is indicated in the margin.

Mentions: To test whether extracts derived from bacteria expressing viral dsRNA could prevent infection by plant viruses other than PMMoV, gene sequences corresponding to PPV were introduced into HT115 strain. For these experiments, we cloned cDNA fragments containing either the helper component (HC) or the coat protein (CP) genes of PPV into L4440, a plasmid vector which has two convergent T7 promoters flanking the multiple cloning site [21]. Upon IPTG induction, prominent bands corresponding in size to full-length HC (1492 bp) and CP (1081 bp) dsRNAs were detected in bacterial nucleic acid extracts by agarose gel analysis and confirmed by resistance to RNase A (Fig. 4A). Other minor bands present in the extracts likely represent partial RNA duplexes.


Crude extracts of bacterially expressed dsRNA can be used to protect plants against virus infections.

Tenllado F, Martínez-García B, Vargas M, Díaz-Ruíz JR - BMC Biotechnol. (2003)

Bacterially expressed dsRNA interferes with PPV infection. (A) Production of PPV dsRNAs in E. coli. HT115 cells were separately transformed with the L4440 double-T7 vector containing either the HC or the CP genes of PPV. Bacterial cultures were induced with IPTG and processed for total nucleic acid. Samples were resolved by electrophoresis on 1% agarose gel before (lanes 1 to 4) or after treatment with RNase A (lanes 5 to 8), and nucleic acid was visualized by staining with ethidium bromide. Markers, λEcoRI-HindIII molecular weight markers. The positions of bacterially expressed 1492-bp HC and 1081-bp CP dsRNAs are indicated in the margin. (B) Detection of PPV in total RNA extracted from systemic leaves of N. benthamiana by RT-PCR at 14 dpi. Plants were mock inoculated or were inoculated with PPV (0.3 μg/ml) alone (-), or with mixtures of PPV plus French Press preparations derived from HT115 harboring either PPV HC dsRNA, PMMoV IR 54 or the empty vector, as indicated. Markers, λEcoRI-HindIII molecular weight markers. RT-PCR was performed with 1 μg of total RNA using primers corresponding to the CP coding sequence of PPV. The position of the 510-bp amplified fragment is indicated in the margin.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC153545&req=5

Figure 4: Bacterially expressed dsRNA interferes with PPV infection. (A) Production of PPV dsRNAs in E. coli. HT115 cells were separately transformed with the L4440 double-T7 vector containing either the HC or the CP genes of PPV. Bacterial cultures were induced with IPTG and processed for total nucleic acid. Samples were resolved by electrophoresis on 1% agarose gel before (lanes 1 to 4) or after treatment with RNase A (lanes 5 to 8), and nucleic acid was visualized by staining with ethidium bromide. Markers, λEcoRI-HindIII molecular weight markers. The positions of bacterially expressed 1492-bp HC and 1081-bp CP dsRNAs are indicated in the margin. (B) Detection of PPV in total RNA extracted from systemic leaves of N. benthamiana by RT-PCR at 14 dpi. Plants were mock inoculated or were inoculated with PPV (0.3 μg/ml) alone (-), or with mixtures of PPV plus French Press preparations derived from HT115 harboring either PPV HC dsRNA, PMMoV IR 54 or the empty vector, as indicated. Markers, λEcoRI-HindIII molecular weight markers. RT-PCR was performed with 1 μg of total RNA using primers corresponding to the CP coding sequence of PPV. The position of the 510-bp amplified fragment is indicated in the margin.
Mentions: To test whether extracts derived from bacteria expressing viral dsRNA could prevent infection by plant viruses other than PMMoV, gene sequences corresponding to PPV were introduced into HT115 strain. For these experiments, we cloned cDNA fragments containing either the helper component (HC) or the coat protein (CP) genes of PPV into L4440, a plasmid vector which has two convergent T7 promoters flanking the multiple cloning site [21]. Upon IPTG induction, prominent bands corresponding in size to full-length HC (1492 bp) and CP (1081 bp) dsRNAs were detected in bacterial nucleic acid extracts by agarose gel analysis and confirmed by resistance to RNase A (Fig. 4A). Other minor bands present in the extracts likely represent partial RNA duplexes.

Bottom Line: Double-stranded RNA (dsRNA) is a potent initiator of gene silencing in a diverse group of organisms that includes plants, Caenorhabditis elegans, Drosophila and mammals.The approach required the in vitro synthesis of large amounts of RNA involving high cost and considerable labour.The main advantage of this mode of dsRNA production is its simplicity and its extremely low cost compared with the requirements for regenerating transgenic plants.

View Article: PubMed Central - HTML - PubMed

Affiliation: Departamento de Biología de Plantas, Centro de Investigaciones Biológicas, CSIC, Velázquez 144, Madrid 28006, Spain. tenllado@cib.csic.es

ABSTRACT

Background: Double-stranded RNA (dsRNA) is a potent initiator of gene silencing in a diverse group of organisms that includes plants, Caenorhabditis elegans, Drosophila and mammals. We have previously shown and patented that mechanical inoculation of in vitro-transcribed dsRNA derived from viral sequences specifically prevents virus infection in plants. The approach required the in vitro synthesis of large amounts of RNA involving high cost and considerable labour.

Results: We have developed an in vivo expression system to produce large amounts of virus-derived dsRNAs in bacteria, with a view to providing a practical control of virus diseases in plants. Partially purified bacterial dsRNAs promoted specific interference with the infection in plants by two viruses belonging to the tobamovirus and potyvirus groups. Furthermore, we have demonstrated that easy to obtain, crude extracts of bacterially expressed dsRNAs are equally effective protecting plants against virus infections when sprayed onto plant surfaces by a simple procedure. Virus infectivity was significantly abolished when plants were sprayed with French Press lysates several days before virus inoculation.

Conclusion: Our approach provides an alternative to genetic transformation of plant species with dsRNA-expressing constructs capable to interfere with plant viruses. The main advantage of this mode of dsRNA production is its simplicity and its extremely low cost compared with the requirements for regenerating transgenic plants. This approach provides a reliable and potential tool, not only for plant protection against virus diseases, but also for the study of gene silencing mechanisms in plant virus infections.

Show MeSH
Related in: MedlinePlus