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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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Flow chart showing the protocol used for analysis of the effects of TaPMR5 silencing on powdery mildew invasion.The timing of N. benthamiana agroinfiltrations and use of infiltrated leaves for secondary inoculation of wheat in coordination with applications of powdery mildew conidia are illustrated.
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pone-0026468-g007: Flow chart showing the protocol used for analysis of the effects of TaPMR5 silencing on powdery mildew invasion.The timing of N. benthamiana agroinfiltrations and use of infiltrated leaves for secondary inoculation of wheat in coordination with applications of powdery mildew conidia are illustrated.

Mentions: To determine the effects of TaPMR5 on wheat powdery mildew (B. graminis f. sp. triticum) infections, a 170 bp fragment was amplified from wheat DNA with the primer pair PMR5-8/PMR5-10 (Table S1) and inserted into pCa-γbLIC in the sense orientation to generate pCa-γb:TaPMR5170. This derivative was infiltrated into N. benthamiana, and sap from the infected leaves was inoculated to the first two emerging leaves of the highly susceptible wheat line Xuezao to assess the effects on mildew pathogenesis (Fig. 7). BSMV:00 (pCa-γbLIC) was tested as a vector alone control, and BSMV:TaPDS200 was used to confirm viral infection and spread.


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)

Flow chart showing the protocol used for analysis of the effects of TaPMR5 silencing on powdery mildew invasion.The timing of N. benthamiana agroinfiltrations and use of infiltrated leaves for secondary inoculation of wheat in coordination with applications of powdery mildew conidia are illustrated.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026468-g007: Flow chart showing the protocol used for analysis of the effects of TaPMR5 silencing on powdery mildew invasion.The timing of N. benthamiana agroinfiltrations and use of infiltrated leaves for secondary inoculation of wheat in coordination with applications of powdery mildew conidia are illustrated.
Mentions: To determine the effects of TaPMR5 on wheat powdery mildew (B. graminis f. sp. triticum) infections, a 170 bp fragment was amplified from wheat DNA with the primer pair PMR5-8/PMR5-10 (Table S1) and inserted into pCa-γbLIC in the sense orientation to generate pCa-γb:TaPMR5170. This derivative was infiltrated into N. benthamiana, and sap from the infected leaves was inoculated to the first two emerging leaves of the highly susceptible wheat line Xuezao to assess the effects on mildew pathogenesis (Fig. 7). BSMV:00 (pCa-γbLIC) was tested as a vector alone control, and BSMV:TaPDS200 was used to confirm viral infection and spread.

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