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Transcriptional regulation of Culex pipiens mosquitoes by Wolbachia influences cytoplasmic incompatibility.

Pinto SB, Stainton K, Harris S, Kambris Z, Sutton ER, Bonsall MB, Parkhill J, Sinkins SP - PLoS Pathog. (2013)

Bottom Line: Knockdown analysis of this gene using RNAi showed an effect on hatch rates in a Wolbachia infected Culex molestus line.Furthermore, in later stages of development an effect on developmental progression in CI embryos occurs in bidirectionally incompatible crosses.The genome of a wPip Wolbachia strain variant from Culex molestus was sequenced and compared with the genome of a wPip variant with which it was incompatible.

View Article: PubMed Central - PubMed

Affiliation: Peter Medawar Building for Pathogen Research and Nuffield Department of Medicine (NDM), University of Oxford, Oxford, United Kingdom ; Department of Zoology, University of Oxford, Oxford, United Kingdom.

ABSTRACT
Cytoplasmic incompatibility (CI) induced by the endosymbiont Wolbachia pipientis causes complex patterns of crossing sterility between populations of the Culex pipiens group of mosquitoes. The molecular basis of the phenotype is yet to be defined. In order to investigate what host changes may underlie CI at the molecular level, we examined the transcription of a homolog of the Drosophila melanogaster gene grauzone that encodes a zinc finger protein and acts as a regulator of female meiosis, in which mutations can cause sterility. Upregulation was observed in Wolbachia-infected C. pipiens group individuals relative to Wolbachia-cured lines and the level of upregulation differed between lines that were reproductively incompatible. Knockdown analysis of this gene using RNAi showed an effect on hatch rates in a Wolbachia infected Culex molestus line. Furthermore, in later stages of development an effect on developmental progression in CI embryos occurs in bidirectionally incompatible crosses. The genome of a wPip Wolbachia strain variant from Culex molestus was sequenced and compared with the genome of a wPip variant with which it was incompatible. Three genes in inserted or deleted regions were newly identified in the C. molestus wPip genome, one of which is a transcriptional regulator labelled wtrM. When this gene was transfected into adult Culex mosquitoes, upregulation of the grauzone homolog was observed. These data suggest that Wolbachia-mediated regulation of host gene expression is a component of the mechanism of cytoplasmic incompatibility.

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Analysis of transcription factor wtrM.A. Phylogenetic tree of Wolbachia transcriptional regulator (wtr) genes. An unrooted maximum likelihood phylogenetic tree, constructed using PHYLIP, of transcriptional regulator genes in the wPip and wMel genomes from amino acid alignments. Bootstrap percentages for 100 trees are shown for each node. B. Transcription of wtrM in Culex mosquitoes. Expression of wtrM was validated by RNA extraction and PCR on the cDNA using primers internal to the open reading frame of the gene. There is no transcription of wtrM detectable in control untransfected ovaries (a) or carcasses (b) from Pel. There is a high level of transcription of wtrM in ovaries from untransfected Mol mosquitoes (c) but not in carcasses (d). In Pel mosquitoes transfected with wtrM by injection into the thorax (e, f) there is no transcription detected in ovaries (e) but strong expression in carcasses (f). Transfection of wtrM into the abdomen of Pel mosquitoes produced no detectable transcription in ovaries (g) but transcription readily detected in carcasses (h). An untransfected whole female of Pel (i) and Mol (j) are also shown. C. CPIJ005623 upregulation in transfected Culex females. Transcription of the Culex gene CPIJ005623 in Pel females transfected with the wPipMol gene wtrM, compared to Pel females transfected with the same plasmid but containing no insert. Difference was significant at p<0.01 using a Wilcoxon rank sum test.
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ppat-1003647-g005: Analysis of transcription factor wtrM.A. Phylogenetic tree of Wolbachia transcriptional regulator (wtr) genes. An unrooted maximum likelihood phylogenetic tree, constructed using PHYLIP, of transcriptional regulator genes in the wPip and wMel genomes from amino acid alignments. Bootstrap percentages for 100 trees are shown for each node. B. Transcription of wtrM in Culex mosquitoes. Expression of wtrM was validated by RNA extraction and PCR on the cDNA using primers internal to the open reading frame of the gene. There is no transcription of wtrM detectable in control untransfected ovaries (a) or carcasses (b) from Pel. There is a high level of transcription of wtrM in ovaries from untransfected Mol mosquitoes (c) but not in carcasses (d). In Pel mosquitoes transfected with wtrM by injection into the thorax (e, f) there is no transcription detected in ovaries (e) but strong expression in carcasses (f). Transfection of wtrM into the abdomen of Pel mosquitoes produced no detectable transcription in ovaries (g) but transcription readily detected in carcasses (h). An untransfected whole female of Pel (i) and Mol (j) are also shown. C. CPIJ005623 upregulation in transfected Culex females. Transcription of the Culex gene CPIJ005623 in Pel females transfected with the wPipMol gene wtrM, compared to Pel females transfected with the same plasmid but containing no insert. Difference was significant at p<0.01 using a Wilcoxon rank sum test.

Mentions: As a preliminary guide to which of the inserted or deleted regions most warranted further experimental investigation, we examined patterns of presence or absence in some other C. pipiens group populations from different geographical locations using PCR (Table S4). The Wolbachia transcriptional regulator gene wtrM was the only gene that occurred solely in the two C. molestus lines examined, Mol and Italy, which generate bidirectional CI with a selection of other C. pipiens group line with which they have been crossed (Table S1). This gene is a member of a family of Wolbachia transcriptional regulator genes, with seven genes in the wPipPel genome and six in the D. melanogaster wMel Wolbachia genome (Figure 5A). One of the genes missing in wPipMol but present in wPipPel, WP0457, is also a member of this family of transcriptional regulators. WP0457 is identical to WP1341, located in a highly similar cluster of prophage-associated genes. Both of these loci are located close to patatin family phospholipase genes, which encode bacterial virulence factors that can disrupt cell membranes [38], [39].


Transcriptional regulation of Culex pipiens mosquitoes by Wolbachia influences cytoplasmic incompatibility.

Pinto SB, Stainton K, Harris S, Kambris Z, Sutton ER, Bonsall MB, Parkhill J, Sinkins SP - PLoS Pathog. (2013)

Analysis of transcription factor wtrM.A. Phylogenetic tree of Wolbachia transcriptional regulator (wtr) genes. An unrooted maximum likelihood phylogenetic tree, constructed using PHYLIP, of transcriptional regulator genes in the wPip and wMel genomes from amino acid alignments. Bootstrap percentages for 100 trees are shown for each node. B. Transcription of wtrM in Culex mosquitoes. Expression of wtrM was validated by RNA extraction and PCR on the cDNA using primers internal to the open reading frame of the gene. There is no transcription of wtrM detectable in control untransfected ovaries (a) or carcasses (b) from Pel. There is a high level of transcription of wtrM in ovaries from untransfected Mol mosquitoes (c) but not in carcasses (d). In Pel mosquitoes transfected with wtrM by injection into the thorax (e, f) there is no transcription detected in ovaries (e) but strong expression in carcasses (f). Transfection of wtrM into the abdomen of Pel mosquitoes produced no detectable transcription in ovaries (g) but transcription readily detected in carcasses (h). An untransfected whole female of Pel (i) and Mol (j) are also shown. C. CPIJ005623 upregulation in transfected Culex females. Transcription of the Culex gene CPIJ005623 in Pel females transfected with the wPipMol gene wtrM, compared to Pel females transfected with the same plasmid but containing no insert. Difference was significant at p<0.01 using a Wilcoxon rank sum test.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3814344&req=5

ppat-1003647-g005: Analysis of transcription factor wtrM.A. Phylogenetic tree of Wolbachia transcriptional regulator (wtr) genes. An unrooted maximum likelihood phylogenetic tree, constructed using PHYLIP, of transcriptional regulator genes in the wPip and wMel genomes from amino acid alignments. Bootstrap percentages for 100 trees are shown for each node. B. Transcription of wtrM in Culex mosquitoes. Expression of wtrM was validated by RNA extraction and PCR on the cDNA using primers internal to the open reading frame of the gene. There is no transcription of wtrM detectable in control untransfected ovaries (a) or carcasses (b) from Pel. There is a high level of transcription of wtrM in ovaries from untransfected Mol mosquitoes (c) but not in carcasses (d). In Pel mosquitoes transfected with wtrM by injection into the thorax (e, f) there is no transcription detected in ovaries (e) but strong expression in carcasses (f). Transfection of wtrM into the abdomen of Pel mosquitoes produced no detectable transcription in ovaries (g) but transcription readily detected in carcasses (h). An untransfected whole female of Pel (i) and Mol (j) are also shown. C. CPIJ005623 upregulation in transfected Culex females. Transcription of the Culex gene CPIJ005623 in Pel females transfected with the wPipMol gene wtrM, compared to Pel females transfected with the same plasmid but containing no insert. Difference was significant at p<0.01 using a Wilcoxon rank sum test.
Mentions: As a preliminary guide to which of the inserted or deleted regions most warranted further experimental investigation, we examined patterns of presence or absence in some other C. pipiens group populations from different geographical locations using PCR (Table S4). The Wolbachia transcriptional regulator gene wtrM was the only gene that occurred solely in the two C. molestus lines examined, Mol and Italy, which generate bidirectional CI with a selection of other C. pipiens group line with which they have been crossed (Table S1). This gene is a member of a family of Wolbachia transcriptional regulator genes, with seven genes in the wPipPel genome and six in the D. melanogaster wMel Wolbachia genome (Figure 5A). One of the genes missing in wPipMol but present in wPipPel, WP0457, is also a member of this family of transcriptional regulators. WP0457 is identical to WP1341, located in a highly similar cluster of prophage-associated genes. Both of these loci are located close to patatin family phospholipase genes, which encode bacterial virulence factors that can disrupt cell membranes [38], [39].

Bottom Line: Knockdown analysis of this gene using RNAi showed an effect on hatch rates in a Wolbachia infected Culex molestus line.Furthermore, in later stages of development an effect on developmental progression in CI embryos occurs in bidirectionally incompatible crosses.The genome of a wPip Wolbachia strain variant from Culex molestus was sequenced and compared with the genome of a wPip variant with which it was incompatible.

View Article: PubMed Central - PubMed

Affiliation: Peter Medawar Building for Pathogen Research and Nuffield Department of Medicine (NDM), University of Oxford, Oxford, United Kingdom ; Department of Zoology, University of Oxford, Oxford, United Kingdom.

ABSTRACT
Cytoplasmic incompatibility (CI) induced by the endosymbiont Wolbachia pipientis causes complex patterns of crossing sterility between populations of the Culex pipiens group of mosquitoes. The molecular basis of the phenotype is yet to be defined. In order to investigate what host changes may underlie CI at the molecular level, we examined the transcription of a homolog of the Drosophila melanogaster gene grauzone that encodes a zinc finger protein and acts as a regulator of female meiosis, in which mutations can cause sterility. Upregulation was observed in Wolbachia-infected C. pipiens group individuals relative to Wolbachia-cured lines and the level of upregulation differed between lines that were reproductively incompatible. Knockdown analysis of this gene using RNAi showed an effect on hatch rates in a Wolbachia infected Culex molestus line. Furthermore, in later stages of development an effect on developmental progression in CI embryos occurs in bidirectionally incompatible crosses. The genome of a wPip Wolbachia strain variant from Culex molestus was sequenced and compared with the genome of a wPip variant with which it was incompatible. Three genes in inserted or deleted regions were newly identified in the C. molestus wPip genome, one of which is a transcriptional regulator labelled wtrM. When this gene was transfected into adult Culex mosquitoes, upregulation of the grauzone homolog was observed. These data suggest that Wolbachia-mediated regulation of host gene expression is a component of the mechanism of cytoplasmic incompatibility.

Show MeSH
Related in: MedlinePlus