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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

qRT-PCR analysis of UPR-dependent expression of pit1, pit2 and tin1-1.RNA was prepared from exponentially growing U. maydis strains SG200 (WT) and cib1 deletion (Δcib1) in YNB liquid medium supplemented with 5 μg/ml TM or 3 mM DTT. Expression of pit1 and the effector genes pit2 and tin1-1 was measured in response to UPR induction. Expression values represent the mean of three biological replicates with two technical duplicates each. Error bars represent the standard error (SE). *indicates p-value < 0.05; **< 0.01 and ***< 0.001, respectively.
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pone.0153861.g001: qRT-PCR analysis of UPR-dependent expression of pit1, pit2 and tin1-1.RNA was prepared from exponentially growing U. maydis strains SG200 (WT) and cib1 deletion (Δcib1) in YNB liquid medium supplemented with 5 μg/ml TM or 3 mM DTT. Expression of pit1 and the effector genes pit2 and tin1-1 was measured in response to UPR induction. Expression values represent the mean of three biological replicates with two technical duplicates each. Error bars represent the standard error (SE). *indicates p-value < 0.05; **< 0.01 and ***< 0.001, respectively.

Mentions: tin1-1 is part of the effector gene cluster 19A, which contains 24 predicted effector genes. Deletion of the entire gene cluster almost completely abolished tumor formation [9]. However, deletion of a tin1-1 containing sub-fragment of cluster 19A had only a minor effect on pathogenicity [49]. pit2 encodes a secreted cysteine-protease inhibitor that is genetically linked to pit1 (encoding a trans-membrane protein of unknown function). Both genes are highly upregulated during the infection process and crucial for pathogenic development [11]. Since expression of pit1 and pit2 is driven by a putatively bi-directional promoter, we speculated that pit1 expression might be as well affected by ER stress. By qRT-PCR analysis, UPR-dependent gene expression of tin1-1, pit1 and pit2 was tested (Fig 1). We further included bip1 and actin as positive and negative control to test assay conditions (S1 Fig). This showed significant upregulation of tin1-1, pit1 and pit2 in response to ER stress (Fig 1). Induced expression of all three genes was dependent on the presence of cib1. Interestingly, the predicted UPRE in the pit1/2 promoter region is located in approximately the same distance between pit1 and pit2 ORFs, suggesting that a common regulatory element might mediate induction of both 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)

qRT-PCR analysis of UPR-dependent expression of pit1, pit2 and tin1-1.RNA was prepared from exponentially growing U. maydis strains SG200 (WT) and cib1 deletion (Δcib1) in YNB liquid medium supplemented with 5 μg/ml TM or 3 mM DTT. Expression of pit1 and the effector genes pit2 and tin1-1 was measured in response to UPR induction. Expression values represent the mean of three biological replicates with two technical duplicates each. Error bars represent the standard error (SE). *indicates p-value < 0.05; **< 0.01 and ***< 0.001, respectively.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4836707&req=5

pone.0153861.g001: qRT-PCR analysis of UPR-dependent expression of pit1, pit2 and tin1-1.RNA was prepared from exponentially growing U. maydis strains SG200 (WT) and cib1 deletion (Δcib1) in YNB liquid medium supplemented with 5 μg/ml TM or 3 mM DTT. Expression of pit1 and the effector genes pit2 and tin1-1 was measured in response to UPR induction. Expression values represent the mean of three biological replicates with two technical duplicates each. Error bars represent the standard error (SE). *indicates p-value < 0.05; **< 0.01 and ***< 0.001, respectively.
Mentions: tin1-1 is part of the effector gene cluster 19A, which contains 24 predicted effector genes. Deletion of the entire gene cluster almost completely abolished tumor formation [9]. However, deletion of a tin1-1 containing sub-fragment of cluster 19A had only a minor effect on pathogenicity [49]. pit2 encodes a secreted cysteine-protease inhibitor that is genetically linked to pit1 (encoding a trans-membrane protein of unknown function). Both genes are highly upregulated during the infection process and crucial for pathogenic development [11]. Since expression of pit1 and pit2 is driven by a putatively bi-directional promoter, we speculated that pit1 expression might be as well affected by ER stress. By qRT-PCR analysis, UPR-dependent gene expression of tin1-1, pit1 and pit2 was tested (Fig 1). We further included bip1 and actin as positive and negative control to test assay conditions (S1 Fig). This showed significant upregulation of tin1-1, pit1 and pit2 in response to ER stress (Fig 1). Induced expression of all three genes was dependent on the presence of cib1. Interestingly, the predicted UPRE in the pit1/2 promoter region is located in approximately the same distance between pit1 and pit2 ORFs, suggesting that a common regulatory element might mediate induction of both 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