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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

Loss of UPR-dependent pit1/2 expression leads to significantly impaired virulence.The haploid pathogenic strain SG200 (WT) and derivatives were inoculated into seven-day-old VA35 maize seedlings. Comparison between WT and Δpit1/2 complemented strains harboring (WT-CP) or lacking the UPRE (ΔUPRE). Disease symptoms were rated eight days after inoculation and grouped into categories depicted on the right. n represents the number of inoculated plants. Statistical significance of alteration in disease rating was calculated using the Wilcoxon-rank-sum test. *P value < 0.05, **<0.01.
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pone.0153861.g004: Loss of UPR-dependent pit1/2 expression leads to significantly impaired virulence.The haploid pathogenic strain SG200 (WT) and derivatives were inoculated into seven-day-old VA35 maize seedlings. Comparison between WT and Δpit1/2 complemented strains harboring (WT-CP) or lacking the UPRE (ΔUPRE). Disease symptoms were rated eight days after inoculation and grouped into categories depicted on the right. n represents the number of inoculated plants. Statistical significance of alteration in disease rating was calculated using the Wilcoxon-rank-sum test. *P value < 0.05, **<0.01.

Mentions: We next addressed expression levels of pit1 and pit2 in WT-CP and ΔUPRE strains during plant colonization. Quantification by qRT-PCR revealed reduced expression of pit1/2 in ΔUPRE strains in comparison to the WT-CP control at 2 dpi, whereas at 4 and 8 dpi expression levels were almost identical to the control (Fig 3B). To test the impact of the pit1/2 UPRE deletion on virulence plant infection experiments were performed. As expected deletion of pit1/2 completely abolished virulence (S3 Fig). Importantly, virulence was fully restored in the SG200-pit1/2 (WT-CP) complemented strain. By contrast, strain SG200-pit1/2ΔUPRE (ΔUPRE), lacking the UPRE motif showed significantly reduced virulence when compared to SG200 (WT) and SG200-pit1/2 (WT-CP) (Fig 4A). This suggests, that UPR-dependent expression of pit1/2 is required for full virulence of U. maydis.


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)

Loss of UPR-dependent pit1/2 expression leads to significantly impaired virulence.The haploid pathogenic strain SG200 (WT) and derivatives were inoculated into seven-day-old VA35 maize seedlings. Comparison between WT and Δpit1/2 complemented strains harboring (WT-CP) or lacking the UPRE (ΔUPRE). Disease symptoms were rated eight days after inoculation and grouped into categories depicted on the right. n represents the number of inoculated plants. Statistical significance of alteration in disease rating was calculated using the Wilcoxon-rank-sum test. *P value < 0.05, **<0.01.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0153861.g004: Loss of UPR-dependent pit1/2 expression leads to significantly impaired virulence.The haploid pathogenic strain SG200 (WT) and derivatives were inoculated into seven-day-old VA35 maize seedlings. Comparison between WT and Δpit1/2 complemented strains harboring (WT-CP) or lacking the UPRE (ΔUPRE). Disease symptoms were rated eight days after inoculation and grouped into categories depicted on the right. n represents the number of inoculated plants. Statistical significance of alteration in disease rating was calculated using the Wilcoxon-rank-sum test. *P value < 0.05, **<0.01.
Mentions: We next addressed expression levels of pit1 and pit2 in WT-CP and ΔUPRE strains during plant colonization. Quantification by qRT-PCR revealed reduced expression of pit1/2 in ΔUPRE strains in comparison to the WT-CP control at 2 dpi, whereas at 4 and 8 dpi expression levels were almost identical to the control (Fig 3B). To test the impact of the pit1/2 UPRE deletion on virulence plant infection experiments were performed. As expected deletion of pit1/2 completely abolished virulence (S3 Fig). Importantly, virulence was fully restored in the SG200-pit1/2 (WT-CP) complemented strain. By contrast, strain SG200-pit1/2ΔUPRE (ΔUPRE), lacking the UPRE motif showed significantly reduced virulence when compared to SG200 (WT) and SG200-pit1/2 (WT-CP) (Fig 4A). This suggests, that UPR-dependent expression of pit1/2 is required for full virulence of U. maydis.

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