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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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Multiple alignments (top) and phylogenetic analyses (bottom) of the 337 bp TaPMR5 gene sequence with similar cDNA or EST sequences in wheat, barley, rice, maize, sorghum, B. distachyon, a species of fescue (Festuca arundinacea) and an interspecific hybrid of two wild rye species (Leymus cinereus x Leymus triticoides).This region has 90.3% identity in these cereal plants. A. thaliana PMR5 gene and its Chinese cabbage orthologue were used as control outgroups. Nucleotides with white lettering in black boxes are 100% conserved, turquoise shading with white lettering indicates 75% or greater conservation, and unshaded nucleotides with black letters are less than 75% conserved. A phylogenetic tree of the PMR5 gene fragments from different species was obtained by analysis with DNAMAN software (Version 5.2.10, Lynnon corporation, Quebec, Canada). Ta  =  Triticum aestivum (GenBank accession: CV759357); Le  =  Leymus cinereus x Leymus triticoides (EG378784); Bd  =  B. distachyon (ADDN01000398); Fa  =  Festuca arundinacea (DT707549); Os  =  Oryza sativa (NM_001056332); Zm  =  Zea mays (NM_001148816); Sb  =  Sorghum bicolor (XM_002467995); Hv  =  H. vulgare (BE413258); Br  =  Brassica rapa subsp. Pekinensis (EX125594); At  =  A. thaliana (AK117382).
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pone-0026468-g009: Multiple alignments (top) and phylogenetic analyses (bottom) of the 337 bp TaPMR5 gene sequence with similar cDNA or EST sequences in wheat, barley, rice, maize, sorghum, B. distachyon, a species of fescue (Festuca arundinacea) and an interspecific hybrid of two wild rye species (Leymus cinereus x Leymus triticoides).This region has 90.3% identity in these cereal plants. A. thaliana PMR5 gene and its Chinese cabbage orthologue were used as control outgroups. Nucleotides with white lettering in black boxes are 100% conserved, turquoise shading with white lettering indicates 75% or greater conservation, and unshaded nucleotides with black letters are less than 75% conserved. A phylogenetic tree of the PMR5 gene fragments from different species was obtained by analysis with DNAMAN software (Version 5.2.10, Lynnon corporation, Quebec, Canada). Ta  =  Triticum aestivum (GenBank accession: CV759357); Le  =  Leymus cinereus x Leymus triticoides (EG378784); Bd  =  B. distachyon (ADDN01000398); Fa  =  Festuca arundinacea (DT707549); Os  =  Oryza sativa (NM_001056332); Zm  =  Zea mays (NM_001148816); Sb  =  Sorghum bicolor (XM_002467995); Hv  =  H. vulgare (BE413258); Br  =  Brassica rapa subsp. Pekinensis (EX125594); At  =  A. thaliana (AK117382).

Mentions: To identify potential PMR5 cereal orthologous, we conducted a BLAST search with the 337 bp TaPMR5 sequence, which revealed the presence of related cDNA or EST sequences in wheat, barley, rice, maize, sorghum, B. distachyon, fescue species (Festuca arundinacea), and a wild rye interspecific hybrid (Leymus cinereus x Leymus triticoides). Multiple alignments of the 337 bp sequence revealed >90% sequence identity in these cereals when the A. thaliana PMR5 gene and the Chinese cabbage orthologue provided a control outgroup (Fig. 9). These results support previous suggestions that PMR5, like other PMR genes involved in powdery mildew invasion [78], represents an ancient lineage whose functions have been conserved across the plant kingdom over ∼200 million years since divergence of the monocots and dicots [79].


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)

Multiple alignments (top) and phylogenetic analyses (bottom) of the 337 bp TaPMR5 gene sequence with similar cDNA or EST sequences in wheat, barley, rice, maize, sorghum, B. distachyon, a species of fescue (Festuca arundinacea) and an interspecific hybrid of two wild rye species (Leymus cinereus x Leymus triticoides).This region has 90.3% identity in these cereal plants. A. thaliana PMR5 gene and its Chinese cabbage orthologue were used as control outgroups. Nucleotides with white lettering in black boxes are 100% conserved, turquoise shading with white lettering indicates 75% or greater conservation, and unshaded nucleotides with black letters are less than 75% conserved. A phylogenetic tree of the PMR5 gene fragments from different species was obtained by analysis with DNAMAN software (Version 5.2.10, Lynnon corporation, Quebec, Canada). Ta  =  Triticum aestivum (GenBank accession: CV759357); Le  =  Leymus cinereus x Leymus triticoides (EG378784); Bd  =  B. distachyon (ADDN01000398); Fa  =  Festuca arundinacea (DT707549); Os  =  Oryza sativa (NM_001056332); Zm  =  Zea mays (NM_001148816); Sb  =  Sorghum bicolor (XM_002467995); Hv  =  H. vulgare (BE413258); Br  =  Brassica rapa subsp. Pekinensis (EX125594); At  =  A. thaliana (AK117382).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3198768&req=5

pone-0026468-g009: Multiple alignments (top) and phylogenetic analyses (bottom) of the 337 bp TaPMR5 gene sequence with similar cDNA or EST sequences in wheat, barley, rice, maize, sorghum, B. distachyon, a species of fescue (Festuca arundinacea) and an interspecific hybrid of two wild rye species (Leymus cinereus x Leymus triticoides).This region has 90.3% identity in these cereal plants. A. thaliana PMR5 gene and its Chinese cabbage orthologue were used as control outgroups. Nucleotides with white lettering in black boxes are 100% conserved, turquoise shading with white lettering indicates 75% or greater conservation, and unshaded nucleotides with black letters are less than 75% conserved. A phylogenetic tree of the PMR5 gene fragments from different species was obtained by analysis with DNAMAN software (Version 5.2.10, Lynnon corporation, Quebec, Canada). Ta  =  Triticum aestivum (GenBank accession: CV759357); Le  =  Leymus cinereus x Leymus triticoides (EG378784); Bd  =  B. distachyon (ADDN01000398); Fa  =  Festuca arundinacea (DT707549); Os  =  Oryza sativa (NM_001056332); Zm  =  Zea mays (NM_001148816); Sb  =  Sorghum bicolor (XM_002467995); Hv  =  H. vulgare (BE413258); Br  =  Brassica rapa subsp. Pekinensis (EX125594); At  =  A. thaliana (AK117382).
Mentions: To identify potential PMR5 cereal orthologous, we conducted a BLAST search with the 337 bp TaPMR5 sequence, which revealed the presence of related cDNA or EST sequences in wheat, barley, rice, maize, sorghum, B. distachyon, fescue species (Festuca arundinacea), and a wild rye interspecific hybrid (Leymus cinereus x Leymus triticoides). Multiple alignments of the 337 bp sequence revealed >90% sequence identity in these cereals when the A. thaliana PMR5 gene and the Chinese cabbage orthologue provided a control outgroup (Fig. 9). These results support previous suggestions that PMR5, like other PMR genes involved in powdery mildew invasion [78], represents an ancient lineage whose functions have been conserved across the plant kingdom over ∼200 million years since divergence of the monocots and dicots [79].

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