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Comparisons of Ribosomal Protein Gene Promoters Indicate Superiority of Heterologous Regulatory Sequences for Expressing Transgenes in Phytophthora infestans.

Poidevin L, Andreeva K, Khachatoorian C, Judelson HS - PLoS ONE (2015)

Bottom Line: This sequence is enriched in genes associated with transcription, translation, and DNA replication, including tRNA and rRNA biogenesis.Five of the six promoters yielded strong expression of a GUS reporter, but the stability of expression was higher using the P. capsici promoters.With the RPS9 and RPL10 promoters of P. infestans, about half of transformants stopped making GUS over two years of culture, while their P. capsici orthologs conferred stable expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Pathology and Microbiology, University of California, Riverside, California, United States of America.

ABSTRACT
Molecular genetics approaches in Phytophthora research can be hampered by the limited number of known constitutive promoters for expressing transgenes and the instability of transgene activity. We have therefore characterized genes encoding the cytoplasmic ribosomal proteins of Phytophthora and studied their suitability for expressing transgenes in P. infestans. Phytophthora spp. encode a standard complement of 79 cytoplasmic ribosomal proteins. Several genes are duplicated, and two appear to be pseudogenes. Half of the genes are expressed at similar levels during all stages of asexual development, and we discovered that the majority share a novel promoter motif named the PhRiboBox. This sequence is enriched in genes associated with transcription, translation, and DNA replication, including tRNA and rRNA biogenesis. Promoters from the three P. infestans genes encoding ribosomal proteins S9, L10, and L23 and their orthologs from P. capsici were tested for their ability to drive transgenes in stable transformants of P. infestans. Five of the six promoters yielded strong expression of a GUS reporter, but the stability of expression was higher using the P. capsici promoters. With the RPS9 and RPL10 promoters of P. infestans, about half of transformants stopped making GUS over two years of culture, while their P. capsici orthologs conferred stable expression. Since cross-talk between native and transgene loci may trigger gene silencing, we encourage the use of heterologous promoters in transformation studies.

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In planta expression of PcRPS9 promoter.Detached tomato leaflets were inoculated with a P. infestans transformant expressing a fusion between GUS and the promoter from PcRPS9, and stained histochemically after 4 days. (A, B) edges of lesions in which P. infestans was growing within the leaflet. The direction of growth is from right to left. Little staining is observed in the older part of the lesion, since the hyphae there have become vacuolated. (C) region of leaflet where sporulation was starting, showing staining of hyphae emerging on the plant surface.
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pone.0145612.g008: In planta expression of PcRPS9 promoter.Detached tomato leaflets were inoculated with a P. infestans transformant expressing a fusion between GUS and the promoter from PcRPS9, and stained histochemically after 4 days. (A, B) edges of lesions in which P. infestans was growing within the leaflet. The direction of growth is from right to left. Little staining is observed in the older part of the lesion, since the hyphae there have become vacuolated. (C) region of leaflet where sporulation was starting, showing staining of hyphae emerging on the plant surface.

Mentions: To confirm that the PcRPS9 promoter was expressed during plant infection, transformants were inoculated on tomato leaflets and stained for GUS. Expression was observed both in hyphae within the plant and on surface hyphae at 4 days post-infection (Fig 8). Similar results were obtained with transformants in which GUS was driven by the PcRPL10 promoter (not shown).


Comparisons of Ribosomal Protein Gene Promoters Indicate Superiority of Heterologous Regulatory Sequences for Expressing Transgenes in Phytophthora infestans.

Poidevin L, Andreeva K, Khachatoorian C, Judelson HS - PLoS ONE (2015)

In planta expression of PcRPS9 promoter.Detached tomato leaflets were inoculated with a P. infestans transformant expressing a fusion between GUS and the promoter from PcRPS9, and stained histochemically after 4 days. (A, B) edges of lesions in which P. infestans was growing within the leaflet. The direction of growth is from right to left. Little staining is observed in the older part of the lesion, since the hyphae there have become vacuolated. (C) region of leaflet where sporulation was starting, showing staining of hyphae emerging on the plant surface.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4696810&req=5

pone.0145612.g008: In planta expression of PcRPS9 promoter.Detached tomato leaflets were inoculated with a P. infestans transformant expressing a fusion between GUS and the promoter from PcRPS9, and stained histochemically after 4 days. (A, B) edges of lesions in which P. infestans was growing within the leaflet. The direction of growth is from right to left. Little staining is observed in the older part of the lesion, since the hyphae there have become vacuolated. (C) region of leaflet where sporulation was starting, showing staining of hyphae emerging on the plant surface.
Mentions: To confirm that the PcRPS9 promoter was expressed during plant infection, transformants were inoculated on tomato leaflets and stained for GUS. Expression was observed both in hyphae within the plant and on surface hyphae at 4 days post-infection (Fig 8). Similar results were obtained with transformants in which GUS was driven by the PcRPL10 promoter (not shown).

Bottom Line: This sequence is enriched in genes associated with transcription, translation, and DNA replication, including tRNA and rRNA biogenesis.Five of the six promoters yielded strong expression of a GUS reporter, but the stability of expression was higher using the P. capsici promoters.With the RPS9 and RPL10 promoters of P. infestans, about half of transformants stopped making GUS over two years of culture, while their P. capsici orthologs conferred stable expression.

View Article: PubMed Central - PubMed

Affiliation: Department of Plant Pathology and Microbiology, University of California, Riverside, California, United States of America.

ABSTRACT
Molecular genetics approaches in Phytophthora research can be hampered by the limited number of known constitutive promoters for expressing transgenes and the instability of transgene activity. We have therefore characterized genes encoding the cytoplasmic ribosomal proteins of Phytophthora and studied their suitability for expressing transgenes in P. infestans. Phytophthora spp. encode a standard complement of 79 cytoplasmic ribosomal proteins. Several genes are duplicated, and two appear to be pseudogenes. Half of the genes are expressed at similar levels during all stages of asexual development, and we discovered that the majority share a novel promoter motif named the PhRiboBox. This sequence is enriched in genes associated with transcription, translation, and DNA replication, including tRNA and rRNA biogenesis. Promoters from the three P. infestans genes encoding ribosomal proteins S9, L10, and L23 and their orthologs from P. capsici were tested for their ability to drive transgenes in stable transformants of P. infestans. Five of the six promoters yielded strong expression of a GUS reporter, but the stability of expression was higher using the P. capsici promoters. With the RPS9 and RPL10 promoters of P. infestans, about half of transformants stopped making GUS over two years of culture, while their P. capsici orthologs conferred stable expression. Since cross-talk between native and transgene loci may trigger gene silencing, we encourage the use of heterologous promoters in transformation studies.

Show MeSH
Related in: MedlinePlus