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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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Effect of PcRPS9 promoter size on GUS expression.(A) Expression vectors based on the 500, 420, and 325-nt versions of the PcRPS9 promoter. (B) Expression driven by different versions of the PcRPS9 promoter in P. infestans. Each bar represents values from independent transformants, based on the average of two biological replicates. NC is an empty-vector control.
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pone.0145612.g006: Effect of PcRPS9 promoter size on GUS expression.(A) Expression vectors based on the 500, 420, and 325-nt versions of the PcRPS9 promoter. (B) Expression driven by different versions of the PcRPS9 promoter in P. infestans. Each bar represents values from independent transformants, based on the average of two biological replicates. NC is an empty-vector control.

Mentions: We chose to develop an expression vector using PcRPS9 regulatory sequences since it resulted in higher average GUS levels than the PcRPL10 promoter, although both resulted in durable expression. First, we exchanged the PcRPS9 promoter for the B. lactucae ham34 promoter in pTOR, a vector that is used widely by the oomycete community (Fig 6A). We also inserted a GUS gene into the plasmid to allow us to test 500, 420, and 325-nt versions of the PcRPS9 promoter. The smaller PcRPS9 promoters were tested in an attempt to reduce vector size and minimize unneeded sequences that might affect stability. PcRPS9 transcription is predicted to start about 30-nt upstream of the 3' end of the promoter.


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)

Effect of PcRPS9 promoter size on GUS expression.(A) Expression vectors based on the 500, 420, and 325-nt versions of the PcRPS9 promoter. (B) Expression driven by different versions of the PcRPS9 promoter in P. infestans. Each bar represents values from independent transformants, based on the average of two biological replicates. NC is an empty-vector control.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0145612.g006: Effect of PcRPS9 promoter size on GUS expression.(A) Expression vectors based on the 500, 420, and 325-nt versions of the PcRPS9 promoter. (B) Expression driven by different versions of the PcRPS9 promoter in P. infestans. Each bar represents values from independent transformants, based on the average of two biological replicates. NC is an empty-vector control.
Mentions: We chose to develop an expression vector using PcRPS9 regulatory sequences since it resulted in higher average GUS levels than the PcRPL10 promoter, although both resulted in durable expression. First, we exchanged the PcRPS9 promoter for the B. lactucae ham34 promoter in pTOR, a vector that is used widely by the oomycete community (Fig 6A). We also inserted a GUS gene into the plasmid to allow us to test 500, 420, and 325-nt versions of the PcRPS9 promoter. The smaller PcRPS9 promoters were tested in an attempt to reduce vector size and minimize unneeded sequences that might affect stability. PcRPS9 transcription is predicted to start about 30-nt upstream of the 3' end of the promoter.

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