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Unfolded Protein Response (UPR) Regulator Cib1 Controls Expression of Genes Encoding Secreted Virulence Factors in Ustilago maydis.

Hampel M, Jakobi M, Schmitz L, Meyer U, Finkernagel F, Doehlemann G, Heimel K - PLoS ONE (2016)

Bottom Line: While a role of the UPR in effector secretion has been described previously, we investigated a potential UPR-dependent regulation of genes encoding secreted effector proteins.Targeted deletion of the UPRE abolished Cib1-dependent expression of pit2 and significantly affected virulence.This study expands the role of the UPR as a signal hub in fungal virulence and illustrates, how biotrophic fungi can coordinate cellular physiology, development and regulation of secreted virulence factors.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Genetics, Institute for Microbiology and Genetics, Göttingen, Germany.

ABSTRACT
The unfolded protein response (UPR), a conserved eukaryotic signaling pathway to ensure protein homeostasis in the endoplasmic reticulum (ER), coordinates biotrophic development in the corn smut fungus Ustilago maydis. Exact timing of UPR activation is required for virulence and presumably connected to the elevated expression of secreted effector proteins during infection of the host plant Zea mays. In the baker's yeast Saccharomyces cerevisiae, expression of UPR target genes is induced upon binding of the central regulator Hac1 to unfolded protein response elements (UPREs) in their promoters. While a role of the UPR in effector secretion has been described previously, we investigated a potential UPR-dependent regulation of genes encoding secreted effector proteins. In silico prediction of UPREs in promoter regions identified the previously characterized effector genes pit2 and tin1-1, as bona fide UPR target genes. Furthermore, direct binding of the Hac1-homolog Cib1 to the UPRE containing promoter fragments of both genes was confirmed by quantitative chromatin immunoprecipitation (qChIP) analysis. Targeted deletion of the UPRE abolished Cib1-dependent expression of pit2 and significantly affected virulence. Furthermore, ER stress strongly increased Pit2 expression and secretion. This study expands the role of the UPR as a signal hub in fungal virulence and illustrates, how biotrophic fungi can coordinate cellular physiology, development and regulation of secreted virulence factors.

No MeSH data available.


Related in: MedlinePlus

qChIP analysis of Cib1 binding to the pit1/2- and tin1-1-promoter.(A) Schematic overview of promoter organization and probe regions used for qChIP experiments. Sequence of the predicted Cib1 binding sites (UPRE) in the pit1/2 and tin1-1 promoter region is given in bold in case of identical nucleotides in pit1/2 and tin1-1 UPREs. (B) qChIP analysis of Cib1 binding to the pit1/2 and tin1-1 promoter in strain SG200cib1-3xHA 3 h after DTT (3 mM) treatment. The HA-tagged Cib1 protein was immunoprecipitated with anti HA-antibody coupled agarose beads (Sigma). Enrichment of immunoprecipitated DNA is shown relative to the input control. PCR-amplicons corresponding to the pit1/2 and tin1-1 promoter are significantly enriched compared to ORF controls. No significant enrichment was observed for PCR-amplicons corresponding to pit1, pit2 and tin1-1 ORFs in comparison to the eIF2b control. Given are the mean values of four independent experiments. Error bars represent the standard error (SE). Statistical significance was tested using Student's t-test. *indicates p-value < 0.05; **< 0.01 and ***< 0.001, respectively.
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pone.0153861.g002: qChIP analysis of Cib1 binding to the pit1/2- and tin1-1-promoter.(A) Schematic overview of promoter organization and probe regions used for qChIP experiments. Sequence of the predicted Cib1 binding sites (UPRE) in the pit1/2 and tin1-1 promoter region is given in bold in case of identical nucleotides in pit1/2 and tin1-1 UPREs. (B) qChIP analysis of Cib1 binding to the pit1/2 and tin1-1 promoter in strain SG200cib1-3xHA 3 h after DTT (3 mM) treatment. The HA-tagged Cib1 protein was immunoprecipitated with anti HA-antibody coupled agarose beads (Sigma). Enrichment of immunoprecipitated DNA is shown relative to the input control. PCR-amplicons corresponding to the pit1/2 and tin1-1 promoter are significantly enriched compared to ORF controls. No significant enrichment was observed for PCR-amplicons corresponding to pit1, pit2 and tin1-1 ORFs in comparison to the eIF2b control. Given are the mean values of four independent experiments. Error bars represent the standard error (SE). Statistical significance was tested using Student's t-test. *indicates p-value < 0.05; **< 0.01 and ***< 0.001, respectively.

Mentions: To address whether Cib1 directly regulates expression of pit1, pit2 and tin1-1, we investigated binding of Cib1 to the predicted UPREs in respective promoter regions by quantitative chromatin immunoprecipitation (qChIP) analysis. To this end, strain SG200cib1-3xHA was generated for expression of a Cib1-3xHA fusion protein under the control of its endogenous promoter. To facilitate expression from the native genomic locus, the construct was integrated by homologous recombination, replacing the endogenous cib1 gene. ER stress sensitivity of SG200cib1-3xHA was indistinguishable from the SG200 progenitor strain, demonstrating functionality of the fusion protein (S2 Fig). Enrichment was quantified relative to the input control for UPRE containing promoter regions, as well as for fragments corresponding to the ORF regions of pit1, pit2, tin1-1, and of eIF2b (Fig 2A) that is not regulated by the UPR. We observed a significant enrichment of fragments corresponding to UPRE containing promoter regions of pit1/2 and tin1-1, whereas no enrichment was observed for regions corresponding to the respective ORFs (Fig 2B). In comparison to the eIF2b control, UPRE containing promoter fragments of pit1/2 and tin1-1 were 74.9 fold (+/- 7.8) and 95.1 fold (+/- 11.7) enriched, respectively. The predicted UPREs in the promoter of pit1/2 and tin1-1 share a consensus of TGCCACGT followed by CG or GT, respectively. This indicates that Cib1 regulates expression of pit1, pit2 and tin1-1 by direct binding to the promoter of these genes.


Unfolded Protein Response (UPR) Regulator Cib1 Controls Expression of Genes Encoding Secreted Virulence Factors in Ustilago maydis.

Hampel M, Jakobi M, Schmitz L, Meyer U, Finkernagel F, Doehlemann G, Heimel K - PLoS ONE (2016)

qChIP analysis of Cib1 binding to the pit1/2- and tin1-1-promoter.(A) Schematic overview of promoter organization and probe regions used for qChIP experiments. Sequence of the predicted Cib1 binding sites (UPRE) in the pit1/2 and tin1-1 promoter region is given in bold in case of identical nucleotides in pit1/2 and tin1-1 UPREs. (B) qChIP analysis of Cib1 binding to the pit1/2 and tin1-1 promoter in strain SG200cib1-3xHA 3 h after DTT (3 mM) treatment. The HA-tagged Cib1 protein was immunoprecipitated with anti HA-antibody coupled agarose beads (Sigma). Enrichment of immunoprecipitated DNA is shown relative to the input control. PCR-amplicons corresponding to the pit1/2 and tin1-1 promoter are significantly enriched compared to ORF controls. No significant enrichment was observed for PCR-amplicons corresponding to pit1, pit2 and tin1-1 ORFs in comparison to the eIF2b control. Given are the mean values of four independent experiments. Error bars represent the standard error (SE). Statistical significance was tested using Student's t-test. *indicates p-value < 0.05; **< 0.01 and ***< 0.001, respectively.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4836707&req=5

pone.0153861.g002: qChIP analysis of Cib1 binding to the pit1/2- and tin1-1-promoter.(A) Schematic overview of promoter organization and probe regions used for qChIP experiments. Sequence of the predicted Cib1 binding sites (UPRE) in the pit1/2 and tin1-1 promoter region is given in bold in case of identical nucleotides in pit1/2 and tin1-1 UPREs. (B) qChIP analysis of Cib1 binding to the pit1/2 and tin1-1 promoter in strain SG200cib1-3xHA 3 h after DTT (3 mM) treatment. The HA-tagged Cib1 protein was immunoprecipitated with anti HA-antibody coupled agarose beads (Sigma). Enrichment of immunoprecipitated DNA is shown relative to the input control. PCR-amplicons corresponding to the pit1/2 and tin1-1 promoter are significantly enriched compared to ORF controls. No significant enrichment was observed for PCR-amplicons corresponding to pit1, pit2 and tin1-1 ORFs in comparison to the eIF2b control. Given are the mean values of four independent experiments. Error bars represent the standard error (SE). Statistical significance was tested using Student's t-test. *indicates p-value < 0.05; **< 0.01 and ***< 0.001, respectively.
Mentions: To address whether Cib1 directly regulates expression of pit1, pit2 and tin1-1, we investigated binding of Cib1 to the predicted UPREs in respective promoter regions by quantitative chromatin immunoprecipitation (qChIP) analysis. To this end, strain SG200cib1-3xHA was generated for expression of a Cib1-3xHA fusion protein under the control of its endogenous promoter. To facilitate expression from the native genomic locus, the construct was integrated by homologous recombination, replacing the endogenous cib1 gene. ER stress sensitivity of SG200cib1-3xHA was indistinguishable from the SG200 progenitor strain, demonstrating functionality of the fusion protein (S2 Fig). Enrichment was quantified relative to the input control for UPRE containing promoter regions, as well as for fragments corresponding to the ORF regions of pit1, pit2, tin1-1, and of eIF2b (Fig 2A) that is not regulated by the UPR. We observed a significant enrichment of fragments corresponding to UPRE containing promoter regions of pit1/2 and tin1-1, whereas no enrichment was observed for regions corresponding to the respective ORFs (Fig 2B). In comparison to the eIF2b control, UPRE containing promoter fragments of pit1/2 and tin1-1 were 74.9 fold (+/- 7.8) and 95.1 fold (+/- 11.7) enriched, respectively. The predicted UPREs in the promoter of pit1/2 and tin1-1 share a consensus of TGCCACGT followed by CG or GT, respectively. This indicates that Cib1 regulates expression of pit1, pit2 and tin1-1 by direct binding to the promoter of these genes.

Bottom Line: While a role of the UPR in effector secretion has been described previously, we investigated a potential UPR-dependent regulation of genes encoding secreted effector proteins.Targeted deletion of the UPRE abolished Cib1-dependent expression of pit2 and significantly affected virulence.This study expands the role of the UPR as a signal hub in fungal virulence and illustrates, how biotrophic fungi can coordinate cellular physiology, development and regulation of secreted virulence factors.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Microbiology and Genetics, Institute for Microbiology and Genetics, Göttingen, Germany.

ABSTRACT
The unfolded protein response (UPR), a conserved eukaryotic signaling pathway to ensure protein homeostasis in the endoplasmic reticulum (ER), coordinates biotrophic development in the corn smut fungus Ustilago maydis. Exact timing of UPR activation is required for virulence and presumably connected to the elevated expression of secreted effector proteins during infection of the host plant Zea mays. In the baker's yeast Saccharomyces cerevisiae, expression of UPR target genes is induced upon binding of the central regulator Hac1 to unfolded protein response elements (UPREs) in their promoters. While a role of the UPR in effector secretion has been described previously, we investigated a potential UPR-dependent regulation of genes encoding secreted effector proteins. In silico prediction of UPREs in promoter regions identified the previously characterized effector genes pit2 and tin1-1, as bona fide UPR target genes. Furthermore, direct binding of the Hac1-homolog Cib1 to the UPRE containing promoter fragments of both genes was confirmed by quantitative chromatin immunoprecipitation (qChIP) analysis. Targeted deletion of the UPRE abolished Cib1-dependent expression of pit2 and significantly affected virulence. Furthermore, ER stress strongly increased Pit2 expression and secretion. This study expands the role of the UPR as a signal hub in fungal virulence and illustrates, how biotrophic fungi can coordinate cellular physiology, development and regulation of secreted virulence factors.

No MeSH data available.


Related in: MedlinePlus