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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

UPR-dependent pit1 and pit2 expression requires UPRE.(A) qRT-PCR analysis of UPR-dependent pit1, pit2 and bip1 expression. RNA was prepared from exponentially growing U. maydis strains SG200 (WT) and derivatives in YNB liquid medium supplemented with 5 μg/ml TM or 3 mM DTT. We tested two different complementation strains with (WT-CP) or without (ΔUPRE) the predicted UPR element in the pit1/2-promoter. Expression values represent the mean of three biological replicates with two technical duplicates each. Error bars represent the standard error (SE). Statistical significance was calculated using Students t-test. *indicates p-value < 0.05; **< 0.01 and ***< 0.001, respectively. (B) qRT-PCR analysis of infected maize leaves at 2, 4 and 8 days post inoculation (dpi). Maize seedlings were inoculated with equal cell numbers of indicated strains. RNA was prepared from three independent samples. Statistical significance was calculated using Students t-test. * indicates a p-value < 0.05.
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pone.0153861.g003: UPR-dependent pit1 and pit2 expression requires UPRE.(A) qRT-PCR analysis of UPR-dependent pit1, pit2 and bip1 expression. RNA was prepared from exponentially growing U. maydis strains SG200 (WT) and derivatives in YNB liquid medium supplemented with 5 μg/ml TM or 3 mM DTT. We tested two different complementation strains with (WT-CP) or without (ΔUPRE) the predicted UPR element in the pit1/2-promoter. Expression values represent the mean of three biological replicates with two technical duplicates each. Error bars represent the standard error (SE). Statistical significance was calculated using Students t-test. *indicates p-value < 0.05; **< 0.01 and ***< 0.001, respectively. (B) qRT-PCR analysis of infected maize leaves at 2, 4 and 8 days post inoculation (dpi). Maize seedlings were inoculated with equal cell numbers of indicated strains. RNA was prepared from three independent samples. Statistical significance was calculated using Students t-test. * indicates a p-value < 0.05.

Mentions: We focused on the functional analysis of the pit1/2 UPRE, since deletion of pit1/2 leads to almost complete loss of U. maydis virulence, which allows functional readout in infection experiments. To test whether the predicted UPRE is required for UPR-dependent expression of pit1/2 genes, the predicted UPRE motif (TGCCACGTCG) in the pit1/2 promoter was deleted. To this end we first replaced the pit1/2 locus with a nourseothricin resistance cassette, generating strain SG200Δpit1/2. Deletion of the pit1/2 locus had no effect on ER stress resistance (S2 Fig), indicating that pit1/2 function is not related to the ER stress response in general. Subsequently the full pit1/2 locus (including pit1 and pit2 ORFs and promoter), with or without the predicted UPRE, was re-integrated into the native locus (for details see material and methods). The resulting strains SG200-pit1/2 (WT-CP) and SG200-pit1/2ΔUPRE (ΔUPRE) did not show alterations in basal expression levels of pit1 and pit2 in comparison to the SG200 (WT) progenitor strain. Moreover, when tested under DTT- or TM-induced ER stress conditions, we observed a robust induction of the conserved UPR target gene bip1 in all three strains, indicating successful activation of the UPR. By contrast, ER stress induced pit1 and pit2 expression only in WT and WT-CP strains, whereas no induction was observed in the ΔUPRE strain (Fig 3A). This suggests that deletion of the UPRE in the pit1/2 promoter abolished UPR-dependent induction of pit1 and pit2 expression.


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)

UPR-dependent pit1 and pit2 expression requires UPRE.(A) qRT-PCR analysis of UPR-dependent pit1, pit2 and bip1 expression. RNA was prepared from exponentially growing U. maydis strains SG200 (WT) and derivatives in YNB liquid medium supplemented with 5 μg/ml TM or 3 mM DTT. We tested two different complementation strains with (WT-CP) or without (ΔUPRE) the predicted UPR element in the pit1/2-promoter. Expression values represent the mean of three biological replicates with two technical duplicates each. Error bars represent the standard error (SE). Statistical significance was calculated using Students t-test. *indicates p-value < 0.05; **< 0.01 and ***< 0.001, respectively. (B) qRT-PCR analysis of infected maize leaves at 2, 4 and 8 days post inoculation (dpi). Maize seedlings were inoculated with equal cell numbers of indicated strains. RNA was prepared from three independent samples. Statistical significance was calculated using Students t-test. * indicates a p-value < 0.05.
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Related In: Results  -  Collection

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pone.0153861.g003: UPR-dependent pit1 and pit2 expression requires UPRE.(A) qRT-PCR analysis of UPR-dependent pit1, pit2 and bip1 expression. RNA was prepared from exponentially growing U. maydis strains SG200 (WT) and derivatives in YNB liquid medium supplemented with 5 μg/ml TM or 3 mM DTT. We tested two different complementation strains with (WT-CP) or without (ΔUPRE) the predicted UPR element in the pit1/2-promoter. Expression values represent the mean of three biological replicates with two technical duplicates each. Error bars represent the standard error (SE). Statistical significance was calculated using Students t-test. *indicates p-value < 0.05; **< 0.01 and ***< 0.001, respectively. (B) qRT-PCR analysis of infected maize leaves at 2, 4 and 8 days post inoculation (dpi). Maize seedlings were inoculated with equal cell numbers of indicated strains. RNA was prepared from three independent samples. Statistical significance was calculated using Students t-test. * indicates a p-value < 0.05.
Mentions: We focused on the functional analysis of the pit1/2 UPRE, since deletion of pit1/2 leads to almost complete loss of U. maydis virulence, which allows functional readout in infection experiments. To test whether the predicted UPRE is required for UPR-dependent expression of pit1/2 genes, the predicted UPRE motif (TGCCACGTCG) in the pit1/2 promoter was deleted. To this end we first replaced the pit1/2 locus with a nourseothricin resistance cassette, generating strain SG200Δpit1/2. Deletion of the pit1/2 locus had no effect on ER stress resistance (S2 Fig), indicating that pit1/2 function is not related to the ER stress response in general. Subsequently the full pit1/2 locus (including pit1 and pit2 ORFs and promoter), with or without the predicted UPRE, was re-integrated into the native locus (for details see material and methods). The resulting strains SG200-pit1/2 (WT-CP) and SG200-pit1/2ΔUPRE (ΔUPRE) did not show alterations in basal expression levels of pit1 and pit2 in comparison to the SG200 (WT) progenitor strain. Moreover, when tested under DTT- or TM-induced ER stress conditions, we observed a robust induction of the conserved UPR target gene bip1 in all three strains, indicating successful activation of the UPR. By contrast, ER stress induced pit1 and pit2 expression only in WT and WT-CP strains, whereas no induction was observed in the ΔUPRE strain (Fig 3A). This suggests that deletion of the UPRE in the pit1/2 promoter abolished UPR-dependent induction of pit1 and pit2 expression.

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