A novel cloning strategy for isolating, genotyping and phenotyping genetic variants of geminiviruses
Background: Viruses of the genus Begomovirus (Geminiviridae) are emerging economically important plant viruses with a circular, single-stranded DNA genome. Previous studies have shown that geminiviruses and RNA viruses exhibit similar mutation frequencies, although geminiviruses are replicated by host DNA polymerases and RNA viruses by their own virus-encoded error-prone RNA-dependent RNA-polymerase. However, the phenotypic effects of naturally occurring mutations have never been extensively investigated in geminiviruses, particularly because, to be infectious, cloned viral genomes usually require sub-cloning as complete or partial tandem repeats into a binary vector from Agrobacterium tumefaciens. Results: Using Tomato yellow leaf curl virus (TYLCV), we show here that infectivity can be obtained when only a 41-nucleotide region containing a highly conserved stem-loop is repeated. A binary vector containing this 41-nt region and a unique restriction site was created, allowing direct cloning of infectious monomeric viral genomes provided that they harbour the same restriction site at the corresponding nucleotide position. This experimental system, which can be transferable to other geminiviruses, was validated by analysis of the phenotypic effect of mutations appearing in TYLCV genomes in a single tomato host plant originally inoculated with a unique viral sequence. Fourteen full-length infectious genomes extracted from this plant were directly cloned and sequenced. The mutation frequency was 1.38 × 10-4 mutation per nucleotide sequenced, similar to that found previously for another begomovirus by sequencing PCR-amplified partial sequences. Interestingly, even in this minimal pool of analysed genomes, mutants with altered properties were readily identified, one of them being fitter and reducing plant biomass more drastically than the parental clone. Conclusion: The cloning strategy presented here is useful for any extensive phenotyping of geminivirus variants and particularly of artificially generated mutants or recombinants.
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Format: | article biblioteca |
Language: | eng |
Subjects: | H20 - Maladies des plantes, Geminiviridae, phénotype, identification, pouvoir pathogène, variation génétique, Clonage moléculaire, mutation, génie génétique, géminivirus enroulement jaune tomat, begomovirus, http://aims.fao.org/aos/agrovoc/c_32859, http://aims.fao.org/aos/agrovoc/c_5776, http://aims.fao.org/aos/agrovoc/c_3791, http://aims.fao.org/aos/agrovoc/c_5629, http://aims.fao.org/aos/agrovoc/c_15975, http://aims.fao.org/aos/agrovoc/c_27503, http://aims.fao.org/aos/agrovoc/c_5014, http://aims.fao.org/aos/agrovoc/c_15974, http://aims.fao.org/aos/agrovoc/c_32611, http://aims.fao.org/aos/agrovoc/c_61d49fca, |
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dig-cirad-fr-5493922024-01-28T17:05:51Z http://agritrop.cirad.fr/549392/ http://agritrop.cirad.fr/549392/ A novel cloning strategy for isolating, genotyping and phenotyping genetic variants of geminiviruses. Urbino Cica, Thébaud Gaël, Granier Martine, Blanc Stéphane, Peterschmitt Michel. 2008. Virology Journal, 5 (135), 10 p.https://doi.org/10.1186/1743-422X-5-135 <https://doi.org/10.1186/1743-422X-5-135> A novel cloning strategy for isolating, genotyping and phenotyping genetic variants of geminiviruses Urbino, Cica Thébaud, Gaël Granier, Martine Blanc, Stéphane Peterschmitt, Michel eng 2008 Virology Journal H20 - Maladies des plantes Geminiviridae phénotype identification pouvoir pathogène variation génétique Clonage moléculaire mutation génie génétique géminivirus enroulement jaune tomat begomovirus http://aims.fao.org/aos/agrovoc/c_32859 http://aims.fao.org/aos/agrovoc/c_5776 http://aims.fao.org/aos/agrovoc/c_3791 http://aims.fao.org/aos/agrovoc/c_5629 http://aims.fao.org/aos/agrovoc/c_15975 http://aims.fao.org/aos/agrovoc/c_27503 http://aims.fao.org/aos/agrovoc/c_5014 http://aims.fao.org/aos/agrovoc/c_15974 http://aims.fao.org/aos/agrovoc/c_32611 http://aims.fao.org/aos/agrovoc/c_61d49fca Background: Viruses of the genus Begomovirus (Geminiviridae) are emerging economically important plant viruses with a circular, single-stranded DNA genome. Previous studies have shown that geminiviruses and RNA viruses exhibit similar mutation frequencies, although geminiviruses are replicated by host DNA polymerases and RNA viruses by their own virus-encoded error-prone RNA-dependent RNA-polymerase. However, the phenotypic effects of naturally occurring mutations have never been extensively investigated in geminiviruses, particularly because, to be infectious, cloned viral genomes usually require sub-cloning as complete or partial tandem repeats into a binary vector from Agrobacterium tumefaciens. Results: Using Tomato yellow leaf curl virus (TYLCV), we show here that infectivity can be obtained when only a 41-nucleotide region containing a highly conserved stem-loop is repeated. A binary vector containing this 41-nt region and a unique restriction site was created, allowing direct cloning of infectious monomeric viral genomes provided that they harbour the same restriction site at the corresponding nucleotide position. This experimental system, which can be transferable to other geminiviruses, was validated by analysis of the phenotypic effect of mutations appearing in TYLCV genomes in a single tomato host plant originally inoculated with a unique viral sequence. Fourteen full-length infectious genomes extracted from this plant were directly cloned and sequenced. The mutation frequency was 1.38 × 10-4 mutation per nucleotide sequenced, similar to that found previously for another begomovirus by sequencing PCR-amplified partial sequences. Interestingly, even in this minimal pool of analysed genomes, mutants with altered properties were readily identified, one of them being fitter and reducing plant biomass more drastically than the parental clone. Conclusion: The cloning strategy presented here is useful for any extensive phenotyping of geminivirus variants and particularly of artificially generated mutants or recombinants. article info:eu-repo/semantics/article Journal Article info:eu-repo/semantics/publishedVersion http://agritrop.cirad.fr/549392/1/document_549392.pdf application/pdf Cirad license info:eu-repo/semantics/openAccess https://agritrop.cirad.fr/mention_legale.html https://doi.org/10.1186/1743-422X-5-135 10.1186/1743-422X-5-135 info:eu-repo/semantics/altIdentifier/doi/10.1186/1743-422X-5-135 info:eu-repo/semantics/altIdentifier/purl/https://doi.org/10.1186/1743-422X-5-135 |
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H20 - Maladies des plantes Geminiviridae phénotype identification pouvoir pathogène variation génétique Clonage moléculaire mutation génie génétique géminivirus enroulement jaune tomat begomovirus http://aims.fao.org/aos/agrovoc/c_32859 http://aims.fao.org/aos/agrovoc/c_5776 http://aims.fao.org/aos/agrovoc/c_3791 http://aims.fao.org/aos/agrovoc/c_5629 http://aims.fao.org/aos/agrovoc/c_15975 http://aims.fao.org/aos/agrovoc/c_27503 http://aims.fao.org/aos/agrovoc/c_5014 http://aims.fao.org/aos/agrovoc/c_15974 http://aims.fao.org/aos/agrovoc/c_32611 http://aims.fao.org/aos/agrovoc/c_61d49fca H20 - Maladies des plantes Geminiviridae phénotype identification pouvoir pathogène variation génétique Clonage moléculaire mutation génie génétique géminivirus enroulement jaune tomat begomovirus http://aims.fao.org/aos/agrovoc/c_32859 http://aims.fao.org/aos/agrovoc/c_5776 http://aims.fao.org/aos/agrovoc/c_3791 http://aims.fao.org/aos/agrovoc/c_5629 http://aims.fao.org/aos/agrovoc/c_15975 http://aims.fao.org/aos/agrovoc/c_27503 http://aims.fao.org/aos/agrovoc/c_5014 http://aims.fao.org/aos/agrovoc/c_15974 http://aims.fao.org/aos/agrovoc/c_32611 http://aims.fao.org/aos/agrovoc/c_61d49fca |
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H20 - Maladies des plantes Geminiviridae phénotype identification pouvoir pathogène variation génétique Clonage moléculaire mutation génie génétique géminivirus enroulement jaune tomat begomovirus http://aims.fao.org/aos/agrovoc/c_32859 http://aims.fao.org/aos/agrovoc/c_5776 http://aims.fao.org/aos/agrovoc/c_3791 http://aims.fao.org/aos/agrovoc/c_5629 http://aims.fao.org/aos/agrovoc/c_15975 http://aims.fao.org/aos/agrovoc/c_27503 http://aims.fao.org/aos/agrovoc/c_5014 http://aims.fao.org/aos/agrovoc/c_15974 http://aims.fao.org/aos/agrovoc/c_32611 http://aims.fao.org/aos/agrovoc/c_61d49fca H20 - Maladies des plantes Geminiviridae phénotype identification pouvoir pathogène variation génétique Clonage moléculaire mutation génie génétique géminivirus enroulement jaune tomat begomovirus http://aims.fao.org/aos/agrovoc/c_32859 http://aims.fao.org/aos/agrovoc/c_5776 http://aims.fao.org/aos/agrovoc/c_3791 http://aims.fao.org/aos/agrovoc/c_5629 http://aims.fao.org/aos/agrovoc/c_15975 http://aims.fao.org/aos/agrovoc/c_27503 http://aims.fao.org/aos/agrovoc/c_5014 http://aims.fao.org/aos/agrovoc/c_15974 http://aims.fao.org/aos/agrovoc/c_32611 http://aims.fao.org/aos/agrovoc/c_61d49fca Urbino, Cica Thébaud, Gaël Granier, Martine Blanc, Stéphane Peterschmitt, Michel A novel cloning strategy for isolating, genotyping and phenotyping genetic variants of geminiviruses |
description |
Background: Viruses of the genus Begomovirus (Geminiviridae) are emerging economically important plant viruses with a circular, single-stranded DNA genome. Previous studies have shown that geminiviruses and RNA viruses exhibit similar mutation frequencies, although geminiviruses are replicated by host DNA polymerases and RNA viruses by their own virus-encoded error-prone RNA-dependent RNA-polymerase. However, the phenotypic effects of naturally occurring mutations have never been extensively investigated in geminiviruses, particularly because, to be infectious, cloned viral genomes usually require sub-cloning as complete or partial tandem repeats into a binary vector from Agrobacterium tumefaciens. Results: Using Tomato yellow leaf curl virus (TYLCV), we show here that infectivity can be obtained when only a 41-nucleotide region containing a highly conserved stem-loop is repeated. A binary vector containing this 41-nt region and a unique restriction site was created, allowing direct cloning of infectious monomeric viral genomes provided that they harbour the same restriction site at the corresponding nucleotide position. This experimental system, which can be transferable to other geminiviruses, was validated by analysis of the phenotypic effect of mutations appearing in TYLCV genomes in a single tomato host plant originally inoculated with a unique viral sequence. Fourteen full-length infectious genomes extracted from this plant were directly cloned and sequenced. The mutation frequency was 1.38 × 10-4 mutation per nucleotide sequenced, similar to that found previously for another begomovirus by sequencing PCR-amplified partial sequences. Interestingly, even in this minimal pool of analysed genomes, mutants with altered properties were readily identified, one of them being fitter and reducing plant biomass more drastically than the parental clone. Conclusion: The cloning strategy presented here is useful for any extensive phenotyping of geminivirus variants and particularly of artificially generated mutants or recombinants. |
format |
article |
topic_facet |
H20 - Maladies des plantes Geminiviridae phénotype identification pouvoir pathogène variation génétique Clonage moléculaire mutation génie génétique géminivirus enroulement jaune tomat begomovirus http://aims.fao.org/aos/agrovoc/c_32859 http://aims.fao.org/aos/agrovoc/c_5776 http://aims.fao.org/aos/agrovoc/c_3791 http://aims.fao.org/aos/agrovoc/c_5629 http://aims.fao.org/aos/agrovoc/c_15975 http://aims.fao.org/aos/agrovoc/c_27503 http://aims.fao.org/aos/agrovoc/c_5014 http://aims.fao.org/aos/agrovoc/c_15974 http://aims.fao.org/aos/agrovoc/c_32611 http://aims.fao.org/aos/agrovoc/c_61d49fca |
author |
Urbino, Cica Thébaud, Gaël Granier, Martine Blanc, Stéphane Peterschmitt, Michel |
author_facet |
Urbino, Cica Thébaud, Gaël Granier, Martine Blanc, Stéphane Peterschmitt, Michel |
author_sort |
Urbino, Cica |
title |
A novel cloning strategy for isolating, genotyping and phenotyping genetic variants of geminiviruses |
title_short |
A novel cloning strategy for isolating, genotyping and phenotyping genetic variants of geminiviruses |
title_full |
A novel cloning strategy for isolating, genotyping and phenotyping genetic variants of geminiviruses |
title_fullStr |
A novel cloning strategy for isolating, genotyping and phenotyping genetic variants of geminiviruses |
title_full_unstemmed |
A novel cloning strategy for isolating, genotyping and phenotyping genetic variants of geminiviruses |
title_sort |
novel cloning strategy for isolating, genotyping and phenotyping genetic variants of geminiviruses |
url |
http://agritrop.cirad.fr/549392/ http://agritrop.cirad.fr/549392/1/document_549392.pdf |
work_keys_str_mv |
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