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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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HT115-expressed PMMoV IR 54 specifically interferes with PMMoV infection. (A) Northern blot analysis of total RNA extracted from inoculated (lanes 2 to 5) or uppermost systemic leaves (lanes 6 and 7) of N. benthamiana at 7 dpi. Plants were inoculated with mixtures of PMMoV (5 μg/ml) plus nucleic acid extracts prepared from HT115 harboring either pGEM/IR 54 (IR 54) or the empty vector (HT115). Extract from HT115/IR 54 used in the inoculum was run on lane 1 for comparison. RNA samples (1 μg) were fractionated by 1% agarose gel electrophoresis, and a DIG-labeled PMMoV 54-kDa RNA was used as a probe. (B) N. benthamiana plants were initially inoculated with mixtures of PMMoV plus bacterial nucleic acid extracts as indicated above. In addition, mixtures of PMMoV plus nucleic acid extracts prepared from BL21 carrying pGEM/IR 54 (BL21/IR 54) were included. After 7 days, 1:1000 diluted extracts from systemic leaves were assessed on opposite half-leaves of N. tabacum cv. Xanthi nc as indicated. Similar numbers of local lesions were observed in both halves of the leaf shown at the right. No visible local response was observed in the half-leaf inoculated with plant extracts derived from PMMoV plus HT115/IR 54 shown at the left. (C) Agarose gel analysis of total RNA (3 μg) extracted from systemic leaves of N. benthamiana plants that were mock inoculated or were inoculated with mixtures of TMV (5 μg/ml) plus nucleic acid extracts prepared from HT115 harboring either pGEM/IR 54 (IR 54) or the empty vector (HT115), as indicated. M, λEcoRI-HindIII molecular weight markers. TMV, purified TMV RNA (100 ng) was loaded as a control. The positions of PMMoV RNA, TMV RNA, and RNA species derived from partially denatured, input dsRNA are indicated.
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Figure 2: HT115-expressed PMMoV IR 54 specifically interferes with PMMoV infection. (A) Northern blot analysis of total RNA extracted from inoculated (lanes 2 to 5) or uppermost systemic leaves (lanes 6 and 7) of N. benthamiana at 7 dpi. Plants were inoculated with mixtures of PMMoV (5 μg/ml) plus nucleic acid extracts prepared from HT115 harboring either pGEM/IR 54 (IR 54) or the empty vector (HT115). Extract from HT115/IR 54 used in the inoculum was run on lane 1 for comparison. RNA samples (1 μg) were fractionated by 1% agarose gel electrophoresis, and a DIG-labeled PMMoV 54-kDa RNA was used as a probe. (B) N. benthamiana plants were initially inoculated with mixtures of PMMoV plus bacterial nucleic acid extracts as indicated above. In addition, mixtures of PMMoV plus nucleic acid extracts prepared from BL21 carrying pGEM/IR 54 (BL21/IR 54) were included. After 7 days, 1:1000 diluted extracts from systemic leaves were assessed on opposite half-leaves of N. tabacum cv. Xanthi nc as indicated. Similar numbers of local lesions were observed in both halves of the leaf shown at the right. No visible local response was observed in the half-leaf inoculated with plant extracts derived from PMMoV plus HT115/IR 54 shown at the left. (C) Agarose gel analysis of total RNA (3 μg) extracted from systemic leaves of N. benthamiana plants that were mock inoculated or were inoculated with mixtures of TMV (5 μg/ml) plus nucleic acid extracts prepared from HT115 harboring either pGEM/IR 54 (IR 54) or the empty vector (HT115), as indicated. M, λEcoRI-HindIII molecular weight markers. TMV, purified TMV RNA (100 ng) was loaded as a control. The positions of PMMoV RNA, TMV RNA, and RNA species derived from partially denatured, input dsRNA are indicated.

Mentions: To evaluate the capability of the PMMoV IR 54 produced in bacteria to interfere with PMMoV infection, Nicotiana benthamiana plants were inoculated with mixtures of PMMoV (5 μg/ml) and phenol-extracted, nucleic acid extracts prepared from HT115 harboring either pGEM/IR 54 (HT115/IR 54) or the empty vector (HT115). Total nucleic acid extracted from BL21 carrying pGEM/IR 54 (BL21/IR 54) was used as control. Plants inoculated with PMMoV plus bacterial extracts derived from HT115 or BL21/IR 54 displayed typical disease symptoms in upper leaves at 6 days post inoculation (dpi). In contrast, all the plants (35 plants in 6 independent experiments) that were inoculated with PMMoV plus extracts derived from HT115/IR 54 were free of symptoms until they flower, typically after 10 weeks post-inoculation. The failure of nucleic acid extracts derived from BL21/IR 54 to interfere with PMMoV infection precludes any effect concerning plasmid DNA homologous to the virus on the interference observed with extracts derived from HT115/IR 54. Northern blot analysis of total RNA showed that PMMoV RNA accumulated in both the inoculated and the upper leaf tissue of HT115- and BL21/IR 54-treated plants at 7 dpi (Fig. 2A and data not shown). In contrast, viral RNA levels were below the limit of Northern blot detection in plants coinoculated with the virus and the PMMoV IR 54-containing HT115 extract. Instead, two faster migrating signals that hybridised with the PMMoV-specific probe were consistently detected in the inoculated leaves of these plants. These hybridization bands have been previously reported in plants inoculated with in vitro-transcribed PMMoV 54 dsRNA [19] and in mosquitoes injected with dsRNA corresponding to the Defensin gene [25]. We interpret these bands as denatured and non-denatured input dsRNA, as they are also present in the bacterial extract used as inoculum (Fig. 2A, lane 1). A corollary is that the loop region present in the hairpin structure encoded by pGEM/IR 54, is probably cleaved by nucleases in the course of bacterial induction rendering non-covalently linked dsRNA.


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)

HT115-expressed PMMoV IR 54 specifically interferes with PMMoV infection. (A) Northern blot analysis of total RNA extracted from inoculated (lanes 2 to 5) or uppermost systemic leaves (lanes 6 and 7) of N. benthamiana at 7 dpi. Plants were inoculated with mixtures of PMMoV (5 μg/ml) plus nucleic acid extracts prepared from HT115 harboring either pGEM/IR 54 (IR 54) or the empty vector (HT115). Extract from HT115/IR 54 used in the inoculum was run on lane 1 for comparison. RNA samples (1 μg) were fractionated by 1% agarose gel electrophoresis, and a DIG-labeled PMMoV 54-kDa RNA was used as a probe. (B) N. benthamiana plants were initially inoculated with mixtures of PMMoV plus bacterial nucleic acid extracts as indicated above. In addition, mixtures of PMMoV plus nucleic acid extracts prepared from BL21 carrying pGEM/IR 54 (BL21/IR 54) were included. After 7 days, 1:1000 diluted extracts from systemic leaves were assessed on opposite half-leaves of N. tabacum cv. Xanthi nc as indicated. Similar numbers of local lesions were observed in both halves of the leaf shown at the right. No visible local response was observed in the half-leaf inoculated with plant extracts derived from PMMoV plus HT115/IR 54 shown at the left. (C) Agarose gel analysis of total RNA (3 μg) extracted from systemic leaves of N. benthamiana plants that were mock inoculated or were inoculated with mixtures of TMV (5 μg/ml) plus nucleic acid extracts prepared from HT115 harboring either pGEM/IR 54 (IR 54) or the empty vector (HT115), as indicated. M, λEcoRI-HindIII molecular weight markers. TMV, purified TMV RNA (100 ng) was loaded as a control. The positions of PMMoV RNA, TMV RNA, and RNA species derived from partially denatured, input dsRNA are indicated.
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Related In: Results  -  Collection

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Figure 2: HT115-expressed PMMoV IR 54 specifically interferes with PMMoV infection. (A) Northern blot analysis of total RNA extracted from inoculated (lanes 2 to 5) or uppermost systemic leaves (lanes 6 and 7) of N. benthamiana at 7 dpi. Plants were inoculated with mixtures of PMMoV (5 μg/ml) plus nucleic acid extracts prepared from HT115 harboring either pGEM/IR 54 (IR 54) or the empty vector (HT115). Extract from HT115/IR 54 used in the inoculum was run on lane 1 for comparison. RNA samples (1 μg) were fractionated by 1% agarose gel electrophoresis, and a DIG-labeled PMMoV 54-kDa RNA was used as a probe. (B) N. benthamiana plants were initially inoculated with mixtures of PMMoV plus bacterial nucleic acid extracts as indicated above. In addition, mixtures of PMMoV plus nucleic acid extracts prepared from BL21 carrying pGEM/IR 54 (BL21/IR 54) were included. After 7 days, 1:1000 diluted extracts from systemic leaves were assessed on opposite half-leaves of N. tabacum cv. Xanthi nc as indicated. Similar numbers of local lesions were observed in both halves of the leaf shown at the right. No visible local response was observed in the half-leaf inoculated with plant extracts derived from PMMoV plus HT115/IR 54 shown at the left. (C) Agarose gel analysis of total RNA (3 μg) extracted from systemic leaves of N. benthamiana plants that were mock inoculated or were inoculated with mixtures of TMV (5 μg/ml) plus nucleic acid extracts prepared from HT115 harboring either pGEM/IR 54 (IR 54) or the empty vector (HT115), as indicated. M, λEcoRI-HindIII molecular weight markers. TMV, purified TMV RNA (100 ng) was loaded as a control. The positions of PMMoV RNA, TMV RNA, and RNA species derived from partially denatured, input dsRNA are indicated.
Mentions: To evaluate the capability of the PMMoV IR 54 produced in bacteria to interfere with PMMoV infection, Nicotiana benthamiana plants were inoculated with mixtures of PMMoV (5 μg/ml) and phenol-extracted, nucleic acid extracts prepared from HT115 harboring either pGEM/IR 54 (HT115/IR 54) or the empty vector (HT115). Total nucleic acid extracted from BL21 carrying pGEM/IR 54 (BL21/IR 54) was used as control. Plants inoculated with PMMoV plus bacterial extracts derived from HT115 or BL21/IR 54 displayed typical disease symptoms in upper leaves at 6 days post inoculation (dpi). In contrast, all the plants (35 plants in 6 independent experiments) that were inoculated with PMMoV plus extracts derived from HT115/IR 54 were free of symptoms until they flower, typically after 10 weeks post-inoculation. The failure of nucleic acid extracts derived from BL21/IR 54 to interfere with PMMoV infection precludes any effect concerning plasmid DNA homologous to the virus on the interference observed with extracts derived from HT115/IR 54. Northern blot analysis of total RNA showed that PMMoV RNA accumulated in both the inoculated and the upper leaf tissue of HT115- and BL21/IR 54-treated plants at 7 dpi (Fig. 2A and data not shown). In contrast, viral RNA levels were below the limit of Northern blot detection in plants coinoculated with the virus and the PMMoV IR 54-containing HT115 extract. Instead, two faster migrating signals that hybridised with the PMMoV-specific probe were consistently detected in the inoculated leaves of these plants. These hybridization bands have been previously reported in plants inoculated with in vitro-transcribed PMMoV 54 dsRNA [19] and in mosquitoes injected with dsRNA corresponding to the Defensin gene [25]. We interpret these bands as denatured and non-denatured input dsRNA, as they are also present in the bacterial extract used as inoculum (Fig. 2A, lane 1). A corollary is that the loop region present in the hairpin structure encoded by pGEM/IR 54, is probably cleaved by nucleases in the course of bacterial induction rendering non-covalently linked dsRNA.

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