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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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PDS and magnesium chelatase subunit H (ChlH) gene silencing phenotypes in barley, wheat and B. distachyon.(A) Barley showing phenotypes typical of suppression of ChlH and PDS by BSMV:HvChlH or BSMV:HvPDS inserts of the indicated lengths. (B) Upper panel: Wheat inoculated with BSMV:TaChlH250 showing more than 90% of inoculated plants developing the chlorotic phenotype associated with suppression of the ChlH gene. Middle panel: PDS and ChlH gene silencing phenotypes on wheat leaves infected with BSMV:TaChlH and BSMV:PDS derivatives with different length inserts. Bottom panel: PCR amplification of transcripts from leaves shown in the middle panel. (C) B. distachyon leaves showing effects of PDS suppression after infection with BSMV:BdPDS inserts. In these experiments, plants were inoculated at the two-leaf stage with infected N. benthamiana sap harboring BSMV derivatives targeting PDS and ChlH cognate genes from each species. Leaf photographs were taken at 14 dpi, and fragment lengths from each source are indicated as subscripts above each leaf. Relative transcript levels of PDS and ChlH genes in the leaves infected with the different BSMV derivatives are shown under the leaf photographs. RNA extracted from the leaves was subjected to semi-quantitative RT-PCR amplification (28 cycles for wheat and barley, 31 cycles for B. distachyon) with the gene-specific oligonucleotide primers shown in Table S1. Amplified species-specific 18S rRNA served as internal controls for each species.
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pone-0026468-g006: PDS and magnesium chelatase subunit H (ChlH) gene silencing phenotypes in barley, wheat and B. distachyon.(A) Barley showing phenotypes typical of suppression of ChlH and PDS by BSMV:HvChlH or BSMV:HvPDS inserts of the indicated lengths. (B) Upper panel: Wheat inoculated with BSMV:TaChlH250 showing more than 90% of inoculated plants developing the chlorotic phenotype associated with suppression of the ChlH gene. Middle panel: PDS and ChlH gene silencing phenotypes on wheat leaves infected with BSMV:TaChlH and BSMV:PDS derivatives with different length inserts. Bottom panel: PCR amplification of transcripts from leaves shown in the middle panel. (C) B. distachyon leaves showing effects of PDS suppression after infection with BSMV:BdPDS inserts. In these experiments, plants were inoculated at the two-leaf stage with infected N. benthamiana sap harboring BSMV derivatives targeting PDS and ChlH cognate genes from each species. Leaf photographs were taken at 14 dpi, and fragment lengths from each source are indicated as subscripts above each leaf. Relative transcript levels of PDS and ChlH genes in the leaves infected with the different BSMV derivatives are shown under the leaf photographs. RNA extracted from the leaves was subjected to semi-quantitative RT-PCR amplification (28 cycles for wheat and barley, 31 cycles for B. distachyon) with the gene-specific oligonucleotide primers shown in Table S1. Amplified species-specific 18S rRNA served as internal controls for each species.

Mentions: Although we were unable to agroinfect cereals directly, N. benthamiana infiltrated leaves provided an excellent source for secondary inoculations to wheat, barley, and the model grass, B. distachyon (Fig. 3; Table S2). Using LIC cloning techniques, a series of PDS or magnesium chelatase subunit H (ChlH) target gene fragments of different lengths from each species to be tested were cloned into the LIC site of pCa-╬│bLIC (Fig. 3). When the first two emerging leaves of wheat, barley, and B. distachyon were inoculated with infected N. benthamiana sap, silencing phenotypes appeared 10 to 14 dpi on upper leaves emerging after infection that corresponded to the cognate PDS or ChlH sequences were used for silencing (Fig. 6; Fig. S1; Table S2). Leaves of all three cereal species targeted for down regulation of PDS developed a longitudinal white streaks that appeared to be devoid of carotenoid pigment. These photobleached segments occupied various portions of the parallel venation and were interspersed within green regions of the leaf blades. In the case of the ChlH phenotype, a more intense yellowing sometimes covered large areas of the leaf, but the yellowing was often interspersed with green sections. In contrast, BSMV or BSMV:00 infected leaves developed typical mosaic symptoms consisting of mild light yellow chlorotic streaks (Fig. 6 and Fig. S1). In all three species, the virus symptoms could be easily distinguished visually from the PDS or ChlH phenotypic effects. In most cases, a high proportion of the plants (>80%) developed the silencing phenotype as illustrated in Fig. 6B, which shows several pots of wheat at 14 dpi, in which >90% of the plants exhibit the chlorotic phenotype associated with suppression of TaChlH transcripts.


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)

PDS and magnesium chelatase subunit H (ChlH) gene silencing phenotypes in barley, wheat and B. distachyon.(A) Barley showing phenotypes typical of suppression of ChlH and PDS by BSMV:HvChlH or BSMV:HvPDS inserts of the indicated lengths. (B) Upper panel: Wheat inoculated with BSMV:TaChlH250 showing more than 90% of inoculated plants developing the chlorotic phenotype associated with suppression of the ChlH gene. Middle panel: PDS and ChlH gene silencing phenotypes on wheat leaves infected with BSMV:TaChlH and BSMV:PDS derivatives with different length inserts. Bottom panel: PCR amplification of transcripts from leaves shown in the middle panel. (C) B. distachyon leaves showing effects of PDS suppression after infection with BSMV:BdPDS inserts. In these experiments, plants were inoculated at the two-leaf stage with infected N. benthamiana sap harboring BSMV derivatives targeting PDS and ChlH cognate genes from each species. Leaf photographs were taken at 14 dpi, and fragment lengths from each source are indicated as subscripts above each leaf. Relative transcript levels of PDS and ChlH genes in the leaves infected with the different BSMV derivatives are shown under the leaf photographs. RNA extracted from the leaves was subjected to semi-quantitative RT-PCR amplification (28 cycles for wheat and barley, 31 cycles for B. distachyon) with the gene-specific oligonucleotide primers shown in Table S1. Amplified species-specific 18S rRNA served as internal controls for each species.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198768&req=5

pone-0026468-g006: PDS and magnesium chelatase subunit H (ChlH) gene silencing phenotypes in barley, wheat and B. distachyon.(A) Barley showing phenotypes typical of suppression of ChlH and PDS by BSMV:HvChlH or BSMV:HvPDS inserts of the indicated lengths. (B) Upper panel: Wheat inoculated with BSMV:TaChlH250 showing more than 90% of inoculated plants developing the chlorotic phenotype associated with suppression of the ChlH gene. Middle panel: PDS and ChlH gene silencing phenotypes on wheat leaves infected with BSMV:TaChlH and BSMV:PDS derivatives with different length inserts. Bottom panel: PCR amplification of transcripts from leaves shown in the middle panel. (C) B. distachyon leaves showing effects of PDS suppression after infection with BSMV:BdPDS inserts. In these experiments, plants were inoculated at the two-leaf stage with infected N. benthamiana sap harboring BSMV derivatives targeting PDS and ChlH cognate genes from each species. Leaf photographs were taken at 14 dpi, and fragment lengths from each source are indicated as subscripts above each leaf. Relative transcript levels of PDS and ChlH genes in the leaves infected with the different BSMV derivatives are shown under the leaf photographs. RNA extracted from the leaves was subjected to semi-quantitative RT-PCR amplification (28 cycles for wheat and barley, 31 cycles for B. distachyon) with the gene-specific oligonucleotide primers shown in Table S1. Amplified species-specific 18S rRNA served as internal controls for each species.
Mentions: Although we were unable to agroinfect cereals directly, N. benthamiana infiltrated leaves provided an excellent source for secondary inoculations to wheat, barley, and the model grass, B. distachyon (Fig. 3; Table S2). Using LIC cloning techniques, a series of PDS or magnesium chelatase subunit H (ChlH) target gene fragments of different lengths from each species to be tested were cloned into the LIC site of pCa-╬│bLIC (Fig. 3). When the first two emerging leaves of wheat, barley, and B. distachyon were inoculated with infected N. benthamiana sap, silencing phenotypes appeared 10 to 14 dpi on upper leaves emerging after infection that corresponded to the cognate PDS or ChlH sequences were used for silencing (Fig. 6; Fig. S1; Table S2). Leaves of all three cereal species targeted for down regulation of PDS developed a longitudinal white streaks that appeared to be devoid of carotenoid pigment. These photobleached segments occupied various portions of the parallel venation and were interspersed within green regions of the leaf blades. In the case of the ChlH phenotype, a more intense yellowing sometimes covered large areas of the leaf, but the yellowing was often interspersed with green sections. In contrast, BSMV or BSMV:00 infected leaves developed typical mosaic symptoms consisting of mild light yellow chlorotic streaks (Fig. 6 and Fig. S1). In all three species, the virus symptoms could be easily distinguished visually from the PDS or ChlH phenotypic effects. In most cases, a high proportion of the plants (>80%) developed the silencing phenotype as illustrated in Fig. 6B, which shows several pots of wheat at 14 dpi, in which >90% of the plants exhibit the chlorotic phenotype associated with suppression of TaChlH transcripts.

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