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A high throughput barley stripe mosaic virus vector for virus induced gene silencing in monocots and dicots.

Yuan C, Li C, Yan L, Jackson AO, Liu Z, Han C, Yu J, Li D - PLoS ONE (2011)

Bottom Line: Suppression of an Arabidopsis orthologue cloned from wheat (TaPMR5) also interfered with wheat powdery mildew (Blumeria graminis f. sp. tritici) infections in a manner similar to that of the A. thaliana PMR5 loss-of-function allele.These results imply that the PMR5 gene has maintained similar functions across monocot and dicot families.Our BSMV VIGS system provides substantial advantages in expense, cloning efficiency, ease of manipulation and ability to apply VIGS for high throughput genomics studies.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Agro-Biotechnology, China Agricultural University, Beijing, People's Republic of China.

ABSTRACT
Barley stripe mosaic virus (BSMV) is a single-stranded RNA virus with three genome components designated alpha, beta, and gamma. BSMV vectors have previously been shown to be efficient virus induced gene silencing (VIGS) vehicles in barley and wheat and have provided important information about host genes functioning during pathogenesis as well as various aspects of genes functioning in development. To permit more effective use of BSMV VIGS for functional genomics experiments, we have developed an Agrobacterium delivery system for BSMV and have coupled this with a ligation independent cloning (LIC) strategy to mediate efficient cloning of host genes. Infiltrated Nicotiana benthamiana leaves provided excellent sources of virus for secondary BSMV infections and VIGS in cereals. The Agro/LIC BSMV VIGS vectors were able to function in high efficiency down regulation of phytoene desaturase (PDS), magnesium chelatase subunit H (ChlH), and plastid transketolase (TK) gene silencing in N. benthamiana and in the monocots, wheat, barley, and the model grass, Brachypodium distachyon. Suppression of an Arabidopsis orthologue cloned from wheat (TaPMR5) also interfered with wheat powdery mildew (Blumeria graminis f. sp. tritici) infections in a manner similar to that of the A. thaliana PMR5 loss-of-function allele. These results imply that the PMR5 gene has maintained similar functions across monocot and dicot families. Our BSMV VIGS system provides substantial advantages in expense, cloning efficiency, ease of manipulation and ability to apply VIGS for high throughput genomics studies.

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Related in: MedlinePlus

Schematic representation of use of the pCa-γbLIC vector for target gene fragment cloning.Individual steps in LIC cloning and insertion of target fragments to be used for VIGS assessment are illustrated.
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pone-0026468-g003: Schematic representation of use of the pCa-γbLIC vector for target gene fragment cloning.Individual steps in LIC cloning and insertion of target fragments to be used for VIGS assessment are illustrated.

Mentions: One limitation of in vitro transcription and DNA based BSMV VIGS vectors [25], [27] is the relatively inefficient cloning of target genes needed for efficient high throughput VIGS operations. To circumvent this limitation, a LIC site (5′-GAAGGGCCCGGTGGTGGTGGT-3′) with an ApaI site (underlined) was inserted into pCaBS-γ at three positions downstream of the γa (polymerase subunit) gene. The plasmid pCa-γbLIC was engineered by integrating the LIC site immediately upstream of the stop codon of the γb coding region, to provide a functional γb protein with an extension of two C-terminal residues (Glu and Val) after inserting target fragments (Fig. 1B and Fig. 3). For pCa-LICγb, the LIC site was designed to replace the γb AUG start codon and eliminate γb protein expression, and the LIC site was engineered as a replacement for γb to generate pCa-LICΔγb (Fig. 1B).


A high throughput barley stripe mosaic virus vector for virus induced gene silencing in monocots and dicots.

Yuan C, Li C, Yan L, Jackson AO, Liu Z, Han C, Yu J, Li D - PLoS ONE (2011)

Schematic representation of use of the pCa-γbLIC vector for target gene fragment cloning.Individual steps in LIC cloning and insertion of target fragments to be used for VIGS assessment are illustrated.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3198768&req=5

pone-0026468-g003: Schematic representation of use of the pCa-γbLIC vector for target gene fragment cloning.Individual steps in LIC cloning and insertion of target fragments to be used for VIGS assessment are illustrated.
Mentions: One limitation of in vitro transcription and DNA based BSMV VIGS vectors [25], [27] is the relatively inefficient cloning of target genes needed for efficient high throughput VIGS operations. To circumvent this limitation, a LIC site (5′-GAAGGGCCCGGTGGTGGTGGT-3′) with an ApaI site (underlined) was inserted into pCaBS-γ at three positions downstream of the γa (polymerase subunit) gene. The plasmid pCa-γbLIC was engineered by integrating the LIC site immediately upstream of the stop codon of the γb coding region, to provide a functional γb protein with an extension of two C-terminal residues (Glu and Val) after inserting target fragments (Fig. 1B and Fig. 3). For pCa-LICγb, the LIC site was designed to replace the γb AUG start codon and eliminate γb protein expression, and the LIC site was engineered as a replacement for γb to generate pCa-LICΔγb (Fig. 1B).

Bottom Line: Suppression of an Arabidopsis orthologue cloned from wheat (TaPMR5) also interfered with wheat powdery mildew (Blumeria graminis f. sp. tritici) infections in a manner similar to that of the A. thaliana PMR5 loss-of-function allele.These results imply that the PMR5 gene has maintained similar functions across monocot and dicot families.Our BSMV VIGS system provides substantial advantages in expense, cloning efficiency, ease of manipulation and ability to apply VIGS for high throughput genomics studies.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Agro-Biotechnology, China Agricultural University, Beijing, People's Republic of China.

ABSTRACT
Barley stripe mosaic virus (BSMV) is a single-stranded RNA virus with three genome components designated alpha, beta, and gamma. BSMV vectors have previously been shown to be efficient virus induced gene silencing (VIGS) vehicles in barley and wheat and have provided important information about host genes functioning during pathogenesis as well as various aspects of genes functioning in development. To permit more effective use of BSMV VIGS for functional genomics experiments, we have developed an Agrobacterium delivery system for BSMV and have coupled this with a ligation independent cloning (LIC) strategy to mediate efficient cloning of host genes. Infiltrated Nicotiana benthamiana leaves provided excellent sources of virus for secondary BSMV infections and VIGS in cereals. The Agro/LIC BSMV VIGS vectors were able to function in high efficiency down regulation of phytoene desaturase (PDS), magnesium chelatase subunit H (ChlH), and plastid transketolase (TK) gene silencing in N. benthamiana and in the monocots, wheat, barley, and the model grass, Brachypodium distachyon. Suppression of an Arabidopsis orthologue cloned from wheat (TaPMR5) also interfered with wheat powdery mildew (Blumeria graminis f. sp. tritici) infections in a manner similar to that of the A. thaliana PMR5 loss-of-function allele. These results imply that the PMR5 gene has maintained similar functions across monocot and dicot families. Our BSMV VIGS system provides substantial advantages in expense, cloning efficiency, ease of manipulation and ability to apply VIGS for high throughput genomics studies.

Show MeSH
Related in: MedlinePlus