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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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Stability of inserted fragments in N. benthamiana leaves after infiltration with BSMV:TaPDS400.(A) Time course of BSMV accumulation in infiltrated N. benthamiana leaves. Infiltrated leaves were harvested at 4, 6, 8, 10 and 12 days post infiltration. The two curves in the graph represent ELISA analyses of 1∶40 and 1∶160 sample dilutions. The western blot provides an independent assessment of BSMV coat protein accumulation in the infiltrated leaves. The lower band shows a Rubisco loading control. (B) Leaves infiltrated with BSMV:TaPDS400, and the first 5 leaves above the infiltrated leaves were harvested at 12 dpi, and amplified by RT-PCR with the primer pair BS-10/BS-32 (Fig. 1B; Table S1) to assess insert stability during systemic movement. The third and fourth leaves above the leaves infiltrated with the pCa-γbLIC (BSMV:00) were amplified as controls. Note: A distinct shadow progressively appears below the main 1350 bp band in systemically infected leaves 2 to 4, and an obvious smaller band of ∼950 bp corresponding to the empty LIC site appears in the 5th upper leaves. (C) The first 7 leaves above the infiltrated leaves infiltrated with BSMV:TaPDS400, and the upper 5th and 6th leaves infiltrated with BSMV:00 were harvested for RT-PCR at 20 dpi. In this case, the deletion products corresponding in size to the BSMV:00 fragment were more evident in the 6th and 7th leaves and represent a substantial proportion of the PCR population.
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pone-0026468-g005: Stability of inserted fragments in N. benthamiana leaves after infiltration with BSMV:TaPDS400.(A) Time course of BSMV accumulation in infiltrated N. benthamiana leaves. Infiltrated leaves were harvested at 4, 6, 8, 10 and 12 days post infiltration. The two curves in the graph represent ELISA analyses of 1∶40 and 1∶160 sample dilutions. The western blot provides an independent assessment of BSMV coat protein accumulation in the infiltrated leaves. The lower band shows a Rubisco loading control. (B) Leaves infiltrated with BSMV:TaPDS400, and the first 5 leaves above the infiltrated leaves were harvested at 12 dpi, and amplified by RT-PCR with the primer pair BS-10/BS-32 (Fig. 1B; Table S1) to assess insert stability during systemic movement. The third and fourth leaves above the leaves infiltrated with the pCa-γbLIC (BSMV:00) were amplified as controls. Note: A distinct shadow progressively appears below the main 1350 bp band in systemically infected leaves 2 to 4, and an obvious smaller band of ∼950 bp corresponding to the empty LIC site appears in the 5th upper leaves. (C) The first 7 leaves above the infiltrated leaves infiltrated with BSMV:TaPDS400, and the upper 5th and 6th leaves infiltrated with BSMV:00 were harvested for RT-PCR at 20 dpi. In this case, the deletion products corresponding in size to the BSMV:00 fragment were more evident in the 6th and 7th leaves and represent a substantial proportion of the PCR population.

Mentions: Because a high percentage of plants consistently developed gene silencing phenotypes after agroinfiltration of N. benthamiana, we wished to determine the accumulation of BSMV to provide a basis for efficient transfer of BSMV vectors suitable for VIGS elicitation in secondary plants. When the first four to eight leaves above the N. benthamiana cotyledons were infiltrated, the levels of BSMV coat protein increased for the first 6 days and then leveled off and remained relatively constant for the next six days (Fig. 5A). Moreover, several grams of tissue were recovered from the infiltrated leaves and extracts from this tissue were able to provide >80% infections of cereals used for VIGS tests (data not shown). These results demonstrate that N. benthamiana amplification of BSMV from primary infiltrated leaves provides an inexpensive inoculum source for VIGS trials of a large number of plants.


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)

Stability of inserted fragments in N. benthamiana leaves after infiltration with BSMV:TaPDS400.(A) Time course of BSMV accumulation in infiltrated N. benthamiana leaves. Infiltrated leaves were harvested at 4, 6, 8, 10 and 12 days post infiltration. The two curves in the graph represent ELISA analyses of 1∶40 and 1∶160 sample dilutions. The western blot provides an independent assessment of BSMV coat protein accumulation in the infiltrated leaves. The lower band shows a Rubisco loading control. (B) Leaves infiltrated with BSMV:TaPDS400, and the first 5 leaves above the infiltrated leaves were harvested at 12 dpi, and amplified by RT-PCR with the primer pair BS-10/BS-32 (Fig. 1B; Table S1) to assess insert stability during systemic movement. The third and fourth leaves above the leaves infiltrated with the pCa-γbLIC (BSMV:00) were amplified as controls. Note: A distinct shadow progressively appears below the main 1350 bp band in systemically infected leaves 2 to 4, and an obvious smaller band of ∼950 bp corresponding to the empty LIC site appears in the 5th upper leaves. (C) The first 7 leaves above the infiltrated leaves infiltrated with BSMV:TaPDS400, and the upper 5th and 6th leaves infiltrated with BSMV:00 were harvested for RT-PCR at 20 dpi. In this case, the deletion products corresponding in size to the BSMV:00 fragment were more evident in the 6th and 7th leaves and represent a substantial proportion of the PCR population.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0026468-g005: Stability of inserted fragments in N. benthamiana leaves after infiltration with BSMV:TaPDS400.(A) Time course of BSMV accumulation in infiltrated N. benthamiana leaves. Infiltrated leaves were harvested at 4, 6, 8, 10 and 12 days post infiltration. The two curves in the graph represent ELISA analyses of 1∶40 and 1∶160 sample dilutions. The western blot provides an independent assessment of BSMV coat protein accumulation in the infiltrated leaves. The lower band shows a Rubisco loading control. (B) Leaves infiltrated with BSMV:TaPDS400, and the first 5 leaves above the infiltrated leaves were harvested at 12 dpi, and amplified by RT-PCR with the primer pair BS-10/BS-32 (Fig. 1B; Table S1) to assess insert stability during systemic movement. The third and fourth leaves above the leaves infiltrated with the pCa-γbLIC (BSMV:00) were amplified as controls. Note: A distinct shadow progressively appears below the main 1350 bp band in systemically infected leaves 2 to 4, and an obvious smaller band of ∼950 bp corresponding to the empty LIC site appears in the 5th upper leaves. (C) The first 7 leaves above the infiltrated leaves infiltrated with BSMV:TaPDS400, and the upper 5th and 6th leaves infiltrated with BSMV:00 were harvested for RT-PCR at 20 dpi. In this case, the deletion products corresponding in size to the BSMV:00 fragment were more evident in the 6th and 7th leaves and represent a substantial proportion of the PCR population.
Mentions: Because a high percentage of plants consistently developed gene silencing phenotypes after agroinfiltration of N. benthamiana, we wished to determine the accumulation of BSMV to provide a basis for efficient transfer of BSMV vectors suitable for VIGS elicitation in secondary plants. When the first four to eight leaves above the N. benthamiana cotyledons were infiltrated, the levels of BSMV coat protein increased for the first 6 days and then leveled off and remained relatively constant for the next six days (Fig. 5A). Moreover, several grams of tissue were recovered from the infiltrated leaves and extracts from this tissue were able to provide >80% infections of cereals used for VIGS tests (data not shown). These results demonstrate that N. benthamiana amplification of BSMV from primary infiltrated leaves provides an inexpensive inoculum source for VIGS trials of a large number of plants.

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