A novel cloning strategy for isolating, genotyping and phenotyping genetic variants of geminiviruses

被引:24
作者
Urbino, Cica [1 ]
Thebaud, Gael [2 ]
Granier, Martine [1 ]
Blanc, Stephane [2 ]
Peterschmitt, Michel [1 ]
机构
[1] CIRAD UMR BGPI, F-34398 Montpellier, France
[2] INRA UMR BGPI, F-34398 Montpellier, France
关键词
D O I
10.1186/1743-422X-5-135
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
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 x 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.
引用
收藏
页数:10
相关论文
共 29 条
[21]   Biolistic inoculation of plants with Tomato yellow leaf curl virus DNA [J].
Lapidot, Moshe ;
Weil, Galit ;
Cohen, Lidya ;
Segev, Limor ;
Gaba, Victor .
JOURNAL OF VIROLOGICAL METHODS, 2007, 144 (1-2) :143-148
[22]   Real-time PCR for the quantitation of Tomato yellow leaf curl Sardinia virus in tomato plants and in Bemisia tabaci [J].
Mason, Giovanna ;
Caciagli, Piero ;
Accotto, Gian Paolo ;
Noris, Emanuela .
JOURNAL OF VIROLOGICAL METHODS, 2008, 147 (02) :282-289
[23]   The distribution of fitness effects caused by single-nucleotide substitutions in an RNA virus [J].
Sanjuán, R ;
Moya, A ;
Elena, SF .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2004, 101 (22) :8396-8401
[24]   High rate of viral evolution associated with the emergence of carnivore parvovirus [J].
Shackelton, LA ;
Parrish, CR ;
Truyen, U ;
Holmes, EC .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 2005, 102 (02) :379-384
[25]   ANALYSIS OF AFRICAN CASSAVA MOSAIC-VIRUS RECOMBINANTS SUGGESTS STRAND NICKING OCCURS WITHIN THE CONSERVED NONANUCLEOTIDE MOTIF DURING THE INITIATION OF ROLLING CIRCLE DNA-REPLICATION [J].
STANLEY, J .
VIROLOGY, 1995, 206 (01) :707-712
[26]   REPLICATIONAL RELEASE OF GEMINIVIRUS GENOMES FROM TANDEMLY REPEATED COPIES - EVIDENCE FOR ROLLING-CIRCLE REPLICATION OF A PLANT VIRAL-DNA [J].
STENGER, DC ;
REVINGTON, GN ;
STEVENSON, MC ;
BISARO, DM .
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, 1991, 88 (18) :8029-8033
[27]   GENETIC-ANALYSIS OF TOMATO GOLDEN MOSAIC-VIRUS - ORF-AL2 IS REQUIRED FOR COAT PROTEIN ACCUMULATION WHILE ORF-AL3 IS NECESSARY FOR EFFICIENT DNA-REPLICATION [J].
SUNTER, G ;
HARTITZ, MD ;
HORMUZDI, SG ;
BROUGH, CL ;
BISARO, DM .
VIROLOGY, 1990, 179 (01) :69-77
[28]   TRANSACTIVATION OF GEMINIVIRUS-AR1 AND GEMINIVIRUS-BR1 GENE-EXPRESSION BY THE VIRAL-AL2 GENE-PRODUCT OCCURS AT THE LEVEL OF TRANSCRIPTION [J].
SUNTER, G ;
BISARO, DM .
PLANT CELL, 1992, 4 (10) :1321-1331
[29]   A simplified method of constructing infectious clones of begomovirus employing limited restriction enzyme digestion of products of rolling circle amplification [J].
Wu, Chia-Ying ;
Lai, Yi-Chin ;
Lin, Na-Sheng ;
Hsu, Yau-Heiu ;
Tsai, Hsin-Tzu ;
Liao, Jye-Yann ;
Hu, Chung-Chi .
JOURNAL OF VIROLOGICAL METHODS, 2008, 147 (02) :355-359