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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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Effects of down regulation of TaPMR5 on powdery mildew pathogenesis.(A) Highly susceptible wheat line (Xuezao) plants with visible symptoms of pCa-γbLIC (BSMV:00) or pCa-γb:TaPMR5170 (BSMV:TaPMR5170) challenged by dusting with B. graminis f. sp. triticum conidia. The photographs depict colonization of the 4th leaf emerging after inoculation with BSMV:00 or BSMV:TaPMR5170, and show the extent of mycelial development at 4 and 5 days after conidial dusting. (B) Relative transcript levels of TaPMR5 and TaPDS in the 4th leaves emerging after infection with the BSMV derivatives (BSMV:00, BSMV:TaPDS200 or BSMV:TaPMR5170). RNA extracted from the leaves was subjected to semi-quantitative RT-PCR amplification (35 cycles) with the gene-specific oligonucleotide primers shown in Table S1. Amplified wheat 18S rRNA served as an internal control. (C) Higher resolution Olympus IX71 microscope observations of powdery mildew colonies appearing on leaf segments at 3 and 4 days after infection with BSMV:00 or BSMV:TaPMR5170. Leaf tissue was fixed in 75% ethanol/25% glacial acetic acid, and stained with 0.05% Coomassie blue R250. The bars represent 500 µm (40X) or 200 µm (100X). (D) Statistical analyses of colony formation and mycelia growth in wheat after TaPMR5 suppression. The numbers of colonies per visual field were counted and the longest axis of the colonies was measured at 3 and 4 days after conidial applications. The ±SD analyses and error bars reflect the means of three independent experiments.
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pone-0026468-g008: Effects of down regulation of TaPMR5 on powdery mildew pathogenesis.(A) Highly susceptible wheat line (Xuezao) plants with visible symptoms of pCa-γbLIC (BSMV:00) or pCa-γb:TaPMR5170 (BSMV:TaPMR5170) challenged by dusting with B. graminis f. sp. triticum conidia. The photographs depict colonization of the 4th leaf emerging after inoculation with BSMV:00 or BSMV:TaPMR5170, and show the extent of mycelial development at 4 and 5 days after conidial dusting. (B) Relative transcript levels of TaPMR5 and TaPDS in the 4th leaves emerging after infection with the BSMV derivatives (BSMV:00, BSMV:TaPDS200 or BSMV:TaPMR5170). RNA extracted from the leaves was subjected to semi-quantitative RT-PCR amplification (35 cycles) with the gene-specific oligonucleotide primers shown in Table S1. Amplified wheat 18S rRNA served as an internal control. (C) Higher resolution Olympus IX71 microscope observations of powdery mildew colonies appearing on leaf segments at 3 and 4 days after infection with BSMV:00 or BSMV:TaPMR5170. Leaf tissue was fixed in 75% ethanol/25% glacial acetic acid, and stained with 0.05% Coomassie blue R250. The bars represent 500 µm (40X) or 200 µm (100X). (D) Statistical analyses of colony formation and mycelia growth in wheat after TaPMR5 suppression. The numbers of colonies per visual field were counted and the longest axis of the colonies was measured at 3 and 4 days after conidial applications. The ±SD analyses and error bars reflect the means of three independent experiments.

Mentions: The potential effects of TaPMR5 silencing on powdery mildew infections were assessed at 3 weeks after BSMV:00 or BSMV:TaPMR5170 inoculation of Xuezao plants. Colonies emerged on all plants by 4 days after dusting with conidia, but the number of colonies on BSMV:TaPMR5170 inoculated leaves were obviously lower than those on the BSMV:00 treated leaves at 4 and 5 days after mildew infection (Fig. 8A). By 11 days after conidia applications, most BSMV:00 treated plants had wilted and displayed extensive mildew colonization and growth, whereas the BSMV:TaPMR5170 treated plants were more robust and had much lower levels of mildew development (data not shown).


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)

Effects of down regulation of TaPMR5 on powdery mildew pathogenesis.(A) Highly susceptible wheat line (Xuezao) plants with visible symptoms of pCa-γbLIC (BSMV:00) or pCa-γb:TaPMR5170 (BSMV:TaPMR5170) challenged by dusting with B. graminis f. sp. triticum conidia. The photographs depict colonization of the 4th leaf emerging after inoculation with BSMV:00 or BSMV:TaPMR5170, and show the extent of mycelial development at 4 and 5 days after conidial dusting. (B) Relative transcript levels of TaPMR5 and TaPDS in the 4th leaves emerging after infection with the BSMV derivatives (BSMV:00, BSMV:TaPDS200 or BSMV:TaPMR5170). RNA extracted from the leaves was subjected to semi-quantitative RT-PCR amplification (35 cycles) with the gene-specific oligonucleotide primers shown in Table S1. Amplified wheat 18S rRNA served as an internal control. (C) Higher resolution Olympus IX71 microscope observations of powdery mildew colonies appearing on leaf segments at 3 and 4 days after infection with BSMV:00 or BSMV:TaPMR5170. Leaf tissue was fixed in 75% ethanol/25% glacial acetic acid, and stained with 0.05% Coomassie blue R250. The bars represent 500 µm (40X) or 200 µm (100X). (D) Statistical analyses of colony formation and mycelia growth in wheat after TaPMR5 suppression. The numbers of colonies per visual field were counted and the longest axis of the colonies was measured at 3 and 4 days after conidial applications. The ±SD analyses and error bars reflect the means of three independent experiments.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198768&req=5

pone-0026468-g008: Effects of down regulation of TaPMR5 on powdery mildew pathogenesis.(A) Highly susceptible wheat line (Xuezao) plants with visible symptoms of pCa-γbLIC (BSMV:00) or pCa-γb:TaPMR5170 (BSMV:TaPMR5170) challenged by dusting with B. graminis f. sp. triticum conidia. The photographs depict colonization of the 4th leaf emerging after inoculation with BSMV:00 or BSMV:TaPMR5170, and show the extent of mycelial development at 4 and 5 days after conidial dusting. (B) Relative transcript levels of TaPMR5 and TaPDS in the 4th leaves emerging after infection with the BSMV derivatives (BSMV:00, BSMV:TaPDS200 or BSMV:TaPMR5170). RNA extracted from the leaves was subjected to semi-quantitative RT-PCR amplification (35 cycles) with the gene-specific oligonucleotide primers shown in Table S1. Amplified wheat 18S rRNA served as an internal control. (C) Higher resolution Olympus IX71 microscope observations of powdery mildew colonies appearing on leaf segments at 3 and 4 days after infection with BSMV:00 or BSMV:TaPMR5170. Leaf tissue was fixed in 75% ethanol/25% glacial acetic acid, and stained with 0.05% Coomassie blue R250. The bars represent 500 µm (40X) or 200 µm (100X). (D) Statistical analyses of colony formation and mycelia growth in wheat after TaPMR5 suppression. The numbers of colonies per visual field were counted and the longest axis of the colonies was measured at 3 and 4 days after conidial applications. The ±SD analyses and error bars reflect the means of three independent experiments.
Mentions: The potential effects of TaPMR5 silencing on powdery mildew infections were assessed at 3 weeks after BSMV:00 or BSMV:TaPMR5170 inoculation of Xuezao plants. Colonies emerged on all plants by 4 days after dusting with conidia, but the number of colonies on BSMV:TaPMR5170 inoculated leaves were obviously lower than those on the BSMV:00 treated leaves at 4 and 5 days after mildew infection (Fig. 8A). By 11 days after conidia applications, most BSMV:00 treated plants had wilted and displayed extensive mildew colonization and growth, whereas the BSMV:TaPMR5170 treated plants were more robust and had much lower levels of mildew development (data not shown).

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