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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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Agrobacterium mediated BSMV infection of N. benthamiana, wheat, barley and B. distachyon.(A) Appearance of large necrotic local lesions on leaves of C. amaranticolor at 10 days after mechanical inoculation with either circular (right) or linearized (left) plasmids. CP shows western blots of BSMV CP isolated from two separate leaves. (B) N. benthamiana leaves infiltrated with equal concentrations of Agrobacterium mixtures harboring pCaBS-α, pCaBS-β, and pCaBS-γ. Infiltrated leaves at 10 dpi (left) and upper uninfiltrated leaves developing mild mottling symptoms at 15 dpi (right). CP is as designated panel A. (C) Typical BSMV symptoms on systemically infected leaves of wheat, barley and B. distachyon emerging at 14 dpi after inoculation of plants with infected N. benthamiana leaf sap. The presence of BSMV RNA and CP was confirmed by RT-PCR and Western blots.
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pone-0026468-g002: Agrobacterium mediated BSMV infection of N. benthamiana, wheat, barley and B. distachyon.(A) Appearance of large necrotic local lesions on leaves of C. amaranticolor at 10 days after mechanical inoculation with either circular (right) or linearized (left) plasmids. CP shows western blots of BSMV CP isolated from two separate leaves. (B) N. benthamiana leaves infiltrated with equal concentrations of Agrobacterium mixtures harboring pCaBS-α, pCaBS-β, and pCaBS-γ. Infiltrated leaves at 10 dpi (left) and upper uninfiltrated leaves developing mild mottling symptoms at 15 dpi (right). CP is as designated panel A. (C) Typical BSMV symptoms on systemically infected leaves of wheat, barley and B. distachyon emerging at 14 dpi after inoculation of plants with infected N. benthamiana leaf sap. The presence of BSMV RNA and CP was confirmed by RT-PCR and Western blots.

Mentions: To confirm the infectivity of pCaBS-α, pCaBS-β and pCaBS-γ, the plasmids were mixed and rub-inoculated on Chenopodium amaranticolor leaves. Large necrotic lesions similar to those resulting from BSMV RNAs or purified BSMV inoculum appeared at 7 to 10 days post-inoculation (dpi) only on leaves abraded with either circular or linearized plasmids (Fig. 2A). After infiltration with equal concentrations of A. tumefaciens EHA105 mixtures harboring pCaBS-α, pCaBS-β and pCaBS-γ, the upper uninfiltrated N. benthamiana leaves developed mild mottling symptoms at 7 to 10 dpi. Western blotting revealed high levels of coat protein, and PCR analyses verified the presence of the gRNAs (data not shown), indicating systemic movement from the infiltrated leaves (Fig. 2B).


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)

Agrobacterium mediated BSMV infection of N. benthamiana, wheat, barley and B. distachyon.(A) Appearance of large necrotic local lesions on leaves of C. amaranticolor at 10 days after mechanical inoculation with either circular (right) or linearized (left) plasmids. CP shows western blots of BSMV CP isolated from two separate leaves. (B) N. benthamiana leaves infiltrated with equal concentrations of Agrobacterium mixtures harboring pCaBS-α, pCaBS-β, and pCaBS-γ. Infiltrated leaves at 10 dpi (left) and upper uninfiltrated leaves developing mild mottling symptoms at 15 dpi (right). CP is as designated panel A. (C) Typical BSMV symptoms on systemically infected leaves of wheat, barley and B. distachyon emerging at 14 dpi after inoculation of plants with infected N. benthamiana leaf sap. The presence of BSMV RNA and CP was confirmed by RT-PCR and Western blots.
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Related In: Results  -  Collection

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

pone-0026468-g002: Agrobacterium mediated BSMV infection of N. benthamiana, wheat, barley and B. distachyon.(A) Appearance of large necrotic local lesions on leaves of C. amaranticolor at 10 days after mechanical inoculation with either circular (right) or linearized (left) plasmids. CP shows western blots of BSMV CP isolated from two separate leaves. (B) N. benthamiana leaves infiltrated with equal concentrations of Agrobacterium mixtures harboring pCaBS-α, pCaBS-β, and pCaBS-γ. Infiltrated leaves at 10 dpi (left) and upper uninfiltrated leaves developing mild mottling symptoms at 15 dpi (right). CP is as designated panel A. (C) Typical BSMV symptoms on systemically infected leaves of wheat, barley and B. distachyon emerging at 14 dpi after inoculation of plants with infected N. benthamiana leaf sap. The presence of BSMV RNA and CP was confirmed by RT-PCR and Western blots.
Mentions: To confirm the infectivity of pCaBS-α, pCaBS-β and pCaBS-γ, the plasmids were mixed and rub-inoculated on Chenopodium amaranticolor leaves. Large necrotic lesions similar to those resulting from BSMV RNAs or purified BSMV inoculum appeared at 7 to 10 days post-inoculation (dpi) only on leaves abraded with either circular or linearized plasmids (Fig. 2A). After infiltration with equal concentrations of A. tumefaciens EHA105 mixtures harboring pCaBS-α, pCaBS-β and pCaBS-γ, the upper uninfiltrated N. benthamiana leaves developed mild mottling symptoms at 7 to 10 dpi. Western blotting revealed high levels of coat protein, and PCR analyses verified the presence of the gRNAs (data not shown), indicating systemic movement from the infiltrated leaves (Fig. 2B).

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