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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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GUS expression driven by five ribosomal protein promoters in stable transformants.The box plots for each gene reflect the distribution of activities from a minimum of ten independent transformants for each construct, which employed 500-nt promoter fragments from P. infestans (Pi) or P. capsici (Pc) genes. Expression driven by the PiRPL23 promoter (PiL23) is not shown due to its instability.
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pone.0145612.g003: GUS expression driven by five ribosomal protein promoters in stable transformants.The box plots for each gene reflect the distribution of activities from a minimum of ten independent transformants for each construct, which employed 500-nt promoter fragments from P. infestans (Pi) or P. capsici (Pc) genes. Expression driven by the PiRPL23 promoter (PiL23) is not shown due to its instability.

Mentions: The six promoters were tested in stable transformants of P. infestans using the GUS reporter (Fig 3). This involved placing 500-nt of sequences upstream of their start codons in front of a promoter-less GUS reporter gene, in a plasmid backbone that confers resistance to G418. This size was chosen since the median intergenic distance in P. infestans is 430-nt [44]. The overall fraction of G418-resistant transformants that initially stained positive for GUS was 42%, and we did not observe a significant difference between plasmids or promoters except for the PiRPL23 construct. Less than 10% of primary transformants with the PiRPL23 promoter exhibited GUS activity, and most stopped growing after one or two transfers. The PiRPL23 promoter sequences may have been lethal, perhaps by triggering silencing of an essential native gene.


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)

GUS expression driven by five ribosomal protein promoters in stable transformants.The box plots for each gene reflect the distribution of activities from a minimum of ten independent transformants for each construct, which employed 500-nt promoter fragments from P. infestans (Pi) or P. capsici (Pc) genes. Expression driven by the PiRPL23 promoter (PiL23) is not shown due to its instability.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0145612.g003: GUS expression driven by five ribosomal protein promoters in stable transformants.The box plots for each gene reflect the distribution of activities from a minimum of ten independent transformants for each construct, which employed 500-nt promoter fragments from P. infestans (Pi) or P. capsici (Pc) genes. Expression driven by the PiRPL23 promoter (PiL23) is not shown due to its instability.
Mentions: The six promoters were tested in stable transformants of P. infestans using the GUS reporter (Fig 3). This involved placing 500-nt of sequences upstream of their start codons in front of a promoter-less GUS reporter gene, in a plasmid backbone that confers resistance to G418. This size was chosen since the median intergenic distance in P. infestans is 430-nt [44]. The overall fraction of G418-resistant transformants that initially stained positive for GUS was 42%, and we did not observe a significant difference between plasmids or promoters except for the PiRPL23 construct. Less than 10% of primary transformants with the PiRPL23 promoter exhibited GUS activity, and most stopped growing after one or two transfers. The PiRPL23 promoter sequences may have been lethal, perhaps by triggering silencing of an essential native gene.

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