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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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Silencing of endogenous phytoene desaturase (PDS) and plastid transketolase (TK) genes in N. benthamiana by Agrobacterium mediated BSMV VIGS.The four-leaf stage of N. benthamiana was infiltrated with an Agrobacterium mixture containing pCaBS-α, pCaBS-β and pCa-γb:NbPDS370 (BSMV:NbPDS370) or pCa-γb:NbTK400 (BSMV:NbTK400). (A) Large areas of photobleaching occurring in leaves infected with BSMV:NbPDS370 40 dpi, and an intense white photobleaching often appearing in stems and petioles (see magnified inset panel). (B) Chlorotic sectors with a distinctive carotenoid coloration interspersed with pale green regions in upper uninfiltrated leaves of plants infected at 23 dpi with BSMV:NbTK400. A leaf of a plant infiltrated with the empty vector (BSMV:00) is shown in the inset panel. (C) Relative transcript levels of NbPDS and NbTK in leaves infected with the BSMV:00, BSMV:NbPDS370 or BSMV:NbTK400. RNA extracted from the leaves was subjected to semi-quantitative RT-PCR amplification (27 cycles) with the gene-specific oligonucleotide primers shown in Table S1. Amplified tobacco 18S rRNA served as an internal control.
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pone-0026468-g004: Silencing of endogenous phytoene desaturase (PDS) and plastid transketolase (TK) genes in N. benthamiana by Agrobacterium mediated BSMV VIGS.The four-leaf stage of N. benthamiana was infiltrated with an Agrobacterium mixture containing pCaBS-α, pCaBS-β and pCa-γb:NbPDS370 (BSMV:NbPDS370) or pCa-γb:NbTK400 (BSMV:NbTK400). (A) Large areas of photobleaching occurring in leaves infected with BSMV:NbPDS370 40 dpi, and an intense white photobleaching often appearing in stems and petioles (see magnified inset panel). (B) Chlorotic sectors with a distinctive carotenoid coloration interspersed with pale green regions in upper uninfiltrated leaves of plants infected at 23 dpi with BSMV:NbTK400. A leaf of a plant infiltrated with the empty vector (BSMV:00) is shown in the inset panel. (C) Relative transcript levels of NbPDS and NbTK in leaves infected with the BSMV:00, BSMV:NbPDS370 or BSMV:NbTK400. RNA extracted from the leaves was subjected to semi-quantitative RT-PCR amplification (27 cycles) with the gene-specific oligonucleotide primers shown in Table S1. Amplified tobacco 18S rRNA served as an internal control.

Mentions: Leaves infiltrated with pCaBS-α, pCaBS-β and pCa-γb:NbPDS370 to elicit PDS silencing developed a mottled photobleaching phenotype in the 5th or 6th leaves at 9 to 10 dpi, and about 5 days later (15 dpi) larger areas of more uniform white PDS silencing were observed at the 6 to 8 leaf stage (data not shown). PDS silencing was most pronounced at 30 to 45 dpi when large areas of white photobleaching were evident in most leaves and, in many cases, on stems and petioles (Fig. 4A). Leaf and stem silencing persisted until the leaves began to senesce, but silencing in younger emerging leaves began to gradually dissipate as green sectors at the tips and margins of the leaves became increasingly evident.


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)

Silencing of endogenous phytoene desaturase (PDS) and plastid transketolase (TK) genes in N. benthamiana by Agrobacterium mediated BSMV VIGS.The four-leaf stage of N. benthamiana was infiltrated with an Agrobacterium mixture containing pCaBS-α, pCaBS-β and pCa-γb:NbPDS370 (BSMV:NbPDS370) or pCa-γb:NbTK400 (BSMV:NbTK400). (A) Large areas of photobleaching occurring in leaves infected with BSMV:NbPDS370 40 dpi, and an intense white photobleaching often appearing in stems and petioles (see magnified inset panel). (B) Chlorotic sectors with a distinctive carotenoid coloration interspersed with pale green regions in upper uninfiltrated leaves of plants infected at 23 dpi with BSMV:NbTK400. A leaf of a plant infiltrated with the empty vector (BSMV:00) is shown in the inset panel. (C) Relative transcript levels of NbPDS and NbTK in leaves infected with the BSMV:00, BSMV:NbPDS370 or BSMV:NbTK400. RNA extracted from the leaves was subjected to semi-quantitative RT-PCR amplification (27 cycles) with the gene-specific oligonucleotide primers shown in Table S1. Amplified tobacco 18S rRNA served as an internal control.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026468-g004: Silencing of endogenous phytoene desaturase (PDS) and plastid transketolase (TK) genes in N. benthamiana by Agrobacterium mediated BSMV VIGS.The four-leaf stage of N. benthamiana was infiltrated with an Agrobacterium mixture containing pCaBS-α, pCaBS-β and pCa-γb:NbPDS370 (BSMV:NbPDS370) or pCa-γb:NbTK400 (BSMV:NbTK400). (A) Large areas of photobleaching occurring in leaves infected with BSMV:NbPDS370 40 dpi, and an intense white photobleaching often appearing in stems and petioles (see magnified inset panel). (B) Chlorotic sectors with a distinctive carotenoid coloration interspersed with pale green regions in upper uninfiltrated leaves of plants infected at 23 dpi with BSMV:NbTK400. A leaf of a plant infiltrated with the empty vector (BSMV:00) is shown in the inset panel. (C) Relative transcript levels of NbPDS and NbTK in leaves infected with the BSMV:00, BSMV:NbPDS370 or BSMV:NbTK400. RNA extracted from the leaves was subjected to semi-quantitative RT-PCR amplification (27 cycles) with the gene-specific oligonucleotide primers shown in Table S1. Amplified tobacco 18S rRNA served as an internal control.
Mentions: Leaves infiltrated with pCaBS-α, pCaBS-β and pCa-γb:NbPDS370 to elicit PDS silencing developed a mottled photobleaching phenotype in the 5th or 6th leaves at 9 to 10 dpi, and about 5 days later (15 dpi) larger areas of more uniform white PDS silencing were observed at the 6 to 8 leaf stage (data not shown). PDS silencing was most pronounced at 30 to 45 dpi when large areas of white photobleaching were evident in most leaves and, in many cases, on stems and petioles (Fig. 4A). Leaf and stem silencing persisted until the leaves began to senesce, but silencing in younger emerging leaves began to gradually dissipate as green sectors at the tips and margins of the leaves became increasingly evident.

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