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 h...

Celý popis

Uloženo v:

Podrobná bibliografie
Vydáno v:	Virology journal Ročník 5; číslo 1; s. 135
Hlavní autoři:	Urbino, Cica, Thébaud, Gael, Granier, Martine, Blanc, Stéphane, Peterschmitt, Michel
Médium:	Journal Article
Jazyk:	angličtina
Vydáno:	London BioMed Central 31.10.2008 BioMed Central Ltd BMC
Témata:	Begomovirus - classification Begomovirus - genetics Biomass Biomedical and Life Sciences Biomedicine Cloning Cloning, Molecular - methods Genetic aspects Genome, Viral Genotype Life Sciences Lycopersicon esculentum - growth & development Lycopersicon esculentum - virology Methodology Methods Mutation Phenotype Plant viruses Sequence Analysis, DNA Vegetal Biology Virology Virus diseases of plants France Virus Accumulation Roll Circle Amplification Tomato Yellow Leaf Curl Virus Tomato Plant Mutation Frequency
ISSN:	1743-422X, 1743-422X
On-line přístup:	Získat plný text
Tagy:	Přidat tag Žádné tagy, Buďte první, kdo vytvoří štítek k tomuto záznamu!

Popis
Shrnutí:	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.
Bibliografie:	ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23
ISSN:	1743-422X 1743-422X
DOI:	10.1186/1743-422X-5-135