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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

Secretion of Pit2-mCherry is strongly increased upon ER stress and dependent on Cib1.(A) The influence of DTT-mediated UPR activation on levels of Pit2-mCherry was investigated by immunoblot analysis using anti-mCherry antibodies in strains SG200Δpit2-pit2-mCherry (WT) and the Δcib1 derivative were grown in CM liquid medium and treated with 3 mM DTT (+) to induce ER stress, or left untreated under otherwise identical conditions (-). After 4 h, cell pellets and supernatant were separated by centrifugation and proteins were subjected to SDS-PAGE analysis followed by immunodetection using anti-mCherry antibodies. Coomassie stained bands served as loading control (LC). (B) Posttranscriptional effects of an active UPR on Pit2-mCherry expression and secretion were monitored in strain SG200Δpit2-Potef:pit2-mCherry (WT) and the Δcib1 derivative. Expression of pit2-mcherry is under control of the constitutively active otef-promoter. The experiment was performed as described for (A).
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pone.0153861.g005: Secretion of Pit2-mCherry is strongly increased upon ER stress and dependent on Cib1.(A) The influence of DTT-mediated UPR activation on levels of Pit2-mCherry was investigated by immunoblot analysis using anti-mCherry antibodies in strains SG200Δpit2-pit2-mCherry (WT) and the Δcib1 derivative were grown in CM liquid medium and treated with 3 mM DTT (+) to induce ER stress, or left untreated under otherwise identical conditions (-). After 4 h, cell pellets and supernatant were separated by centrifugation and proteins were subjected to SDS-PAGE analysis followed by immunodetection using anti-mCherry antibodies. Coomassie stained bands served as loading control (LC). (B) Posttranscriptional effects of an active UPR on Pit2-mCherry expression and secretion were monitored in strain SG200Δpit2-Potef:pit2-mCherry (WT) and the Δcib1 derivative. Expression of pit2-mcherry is under control of the constitutively active otef-promoter. The experiment was performed as described for (A).

Mentions: The unfolded protein response is supposed to be required for efficient secretion of effector proteins during biotrophic growth of U. maydis [31,36]. To determine the cumulative result of transcriptional and posttranscriptional effects of UPR activation on secretion of Pit2, we analyzed the amount of secreted Pit2-mCherry fusion protein in strain SG200Δpit2-pit2-mCherry [11] and its Δcib1 derivative by immunoblot analysis. In these strains expression of Pit2-mCherry fusion protein is under control of its native promoter. Induction of DTT-mediated ER stress resulted in strongly increased levels of secreted Pit2-mCherry in the supernatant, compared to the untreated WT. In contrast, the level of secreted Pit2-mCherry was not affect by DTT treatment in the Δcib1 background (Fig 5A). Moreover, analysis of the pellet fraction revealed increased levels of Pit2-mCherry after DTT treatment in WT and to a lesser extent, also in Δcib1 strains (Fig 5A), which is in line with pit2 expression levels under these conditions (Fig 1). Interestingly, in case of the Δcib1 strain, DTT-induced ER stress led to accumulation of a slightly higher migrating band that was not observed in the WT background or in the supernatant. Thus, our data indicates that UPR activation facilitates increased expression and secretion of Pit2-mCherry. However, assuming that the higher migrating band corresponds to the unprocessed Pit2-mCherry fusion protein including the 25 amino acid signal peptide (predicted molecular weight 39 kDa) and the lower migrating band to the processed form (predicted molecular weight 36 kDa), UPR might also be important for efficient processing and cleavage of the Pit2 signal peptide during ER stress.


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)

Secretion of Pit2-mCherry is strongly increased upon ER stress and dependent on Cib1.(A) The influence of DTT-mediated UPR activation on levels of Pit2-mCherry was investigated by immunoblot analysis using anti-mCherry antibodies in strains SG200Δpit2-pit2-mCherry (WT) and the Δcib1 derivative were grown in CM liquid medium and treated with 3 mM DTT (+) to induce ER stress, or left untreated under otherwise identical conditions (-). After 4 h, cell pellets and supernatant were separated by centrifugation and proteins were subjected to SDS-PAGE analysis followed by immunodetection using anti-mCherry antibodies. Coomassie stained bands served as loading control (LC). (B) Posttranscriptional effects of an active UPR on Pit2-mCherry expression and secretion were monitored in strain SG200Δpit2-Potef:pit2-mCherry (WT) and the Δcib1 derivative. Expression of pit2-mcherry is under control of the constitutively active otef-promoter. The experiment was performed as described for (A).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4836707&req=5

pone.0153861.g005: Secretion of Pit2-mCherry is strongly increased upon ER stress and dependent on Cib1.(A) The influence of DTT-mediated UPR activation on levels of Pit2-mCherry was investigated by immunoblot analysis using anti-mCherry antibodies in strains SG200Δpit2-pit2-mCherry (WT) and the Δcib1 derivative were grown in CM liquid medium and treated with 3 mM DTT (+) to induce ER stress, or left untreated under otherwise identical conditions (-). After 4 h, cell pellets and supernatant were separated by centrifugation and proteins were subjected to SDS-PAGE analysis followed by immunodetection using anti-mCherry antibodies. Coomassie stained bands served as loading control (LC). (B) Posttranscriptional effects of an active UPR on Pit2-mCherry expression and secretion were monitored in strain SG200Δpit2-Potef:pit2-mCherry (WT) and the Δcib1 derivative. Expression of pit2-mcherry is under control of the constitutively active otef-promoter. The experiment was performed as described for (A).
Mentions: The unfolded protein response is supposed to be required for efficient secretion of effector proteins during biotrophic growth of U. maydis [31,36]. To determine the cumulative result of transcriptional and posttranscriptional effects of UPR activation on secretion of Pit2, we analyzed the amount of secreted Pit2-mCherry fusion protein in strain SG200Δpit2-pit2-mCherry [11] and its Δcib1 derivative by immunoblot analysis. In these strains expression of Pit2-mCherry fusion protein is under control of its native promoter. Induction of DTT-mediated ER stress resulted in strongly increased levels of secreted Pit2-mCherry in the supernatant, compared to the untreated WT. In contrast, the level of secreted Pit2-mCherry was not affect by DTT treatment in the Δcib1 background (Fig 5A). Moreover, analysis of the pellet fraction revealed increased levels of Pit2-mCherry after DTT treatment in WT and to a lesser extent, also in Δcib1 strains (Fig 5A), which is in line with pit2 expression levels under these conditions (Fig 1). Interestingly, in case of the Δcib1 strain, DTT-induced ER stress led to accumulation of a slightly higher migrating band that was not observed in the WT background or in the supernatant. Thus, our data indicates that UPR activation facilitates increased expression and secretion of Pit2-mCherry. However, assuming that the higher migrating band corresponds to the unprocessed Pit2-mCherry fusion protein including the 25 amino acid signal peptide (predicted molecular weight 39 kDa) and the lower migrating band to the processed form (predicted molecular weight 36 kDa), UPR might also be important for efficient processing and cleavage of the Pit2 signal peptide during ER stress.

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