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Light controls growth and development via a conserved pathway in the fungal kingdom.

Idnurm A, Heitman J - PLoS Biol. (2005)

Bottom Line: One UV-sensitive mutant that filaments equally well in the light and the dark was identified and found to have an insertion in the BWC2 gene, whose product is structurally similar to N. crassa WC-2.Deletion of BWC1 or BWC2 reduces the virulence of C. neoformans in a murine model of infection; the Bwc1-Bwc2 system thus represents a novel protein complex that influences both development and virulence in a pathogenic fungus.These results demonstrate that a role for blue/UV light in controlling development is an ancient process that predates the divergence of the fungi into the ascomycete and basidiomycete phyla.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Microbiology, Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina, USA.

ABSTRACT
Light inhibits mating and haploid fruiting of the human fungal pathogen Cryptococcus neoformans, but the mechanisms involved were unknown. Two genes controlling light responses were discovered through candidate gene and insertional mutagenesis approaches. Deletion of candidate genes encoding a predicted opsin or phytochrome had no effect on mating, while strains mutated in the white collar 1 homolog gene BWC1 mated equally well in the light or the dark. The predicted Bwc1 protein shares identity with Neurospora crassa WC-1, but lacks the zinc finger DNA binding domain. BWC1 regulates cell fusion and repression of hyphal development after fusion in response to blue light. In addition, bwc1 mutant strains are hypersensitive to ultraviolet light. To identify other components required for responses to light, a novel self-fertile haploid strain was created and subjected to Agrobacterium-mediated insertional mutagenesis. One UV-sensitive mutant that filaments equally well in the light and the dark was identified and found to have an insertion in the BWC2 gene, whose product is structurally similar to N. crassa WC-2. The C. neoformans Bwc1 and Bwc2 proteins interact in the yeast two-hybrid assay. Deletion of BWC1 or BWC2 reduces the virulence of C. neoformans in a murine model of infection; the Bwc1-Bwc2 system thus represents a novel protein complex that influences both development and virulence in a pathogenic fungus. These results demonstrate that a role for blue/UV light in controlling development is an ancient process that predates the divergence of the fungi into the ascomycete and basidiomycete phyla.

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The BWC2 Gene also Mediates UV/Blue Light Responses in C. neoformans(A) A self-filamentous haploid strain (MATa +SXI1α) exhibits light-repressed filamentation. This strain was mutated by Agrobacterium-mediated T-DNA insertion, and insertional mutant strain 25F8 filaments equally well in the light and the dark.(B) Comparison of the structures of Bwc1 and Bwc2. The Bwc1 predicted protein (1,097 amino acids) has a LOV domain, two additional PAS domains and a nuclear localization signal. The Bwc2 predicted protein (392 amino acids) has a PAS domain and zinc finger DNA binding domain.(C) Bilateral mating between wild-type (WT), bwcl, bwc2, or bwc1 bwc2 double (bwc1,2) mutant strains on V8 medium in the dark and the light (48 h). Filamentation is repressed in wild-type crosses in the light, but not in crosses between mutants or in the dark.(D) Fusion efficiency of strains under different wavelengths of light. Matings were between wild-type (+) partners, bwc2 mutant (Δ) partners, or one wild-type and one mutant partner (bwc2 α × WT a, or WT α × bwc2 a). Mutation of bwc2 in either or both mating partners relieves inhibition of fusion by white or blue light. Bars indicate the standard error of the mean of three replicates.(E) Filament formation in wild-type or bwc2/bwc2 mutant diploid strains (24 h). Light does not repress filament formation in bwc2/bwc2 diploids.(F) The bwc2 and double bwc1 bwc2 (bwc1,2) mutants are as hypersensitive to UV light as bwc1 mutants. Ten-fold serial dilutions of yeast cells were plated in duplicate onto YPD medium, and one set was UV irradiated (~48 mJ/cm2).
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pbio-0030095-g005: The BWC2 Gene also Mediates UV/Blue Light Responses in C. neoformans(A) A self-filamentous haploid strain (MATa +SXI1α) exhibits light-repressed filamentation. This strain was mutated by Agrobacterium-mediated T-DNA insertion, and insertional mutant strain 25F8 filaments equally well in the light and the dark.(B) Comparison of the structures of Bwc1 and Bwc2. The Bwc1 predicted protein (1,097 amino acids) has a LOV domain, two additional PAS domains and a nuclear localization signal. The Bwc2 predicted protein (392 amino acids) has a PAS domain and zinc finger DNA binding domain.(C) Bilateral mating between wild-type (WT), bwcl, bwc2, or bwc1 bwc2 double (bwc1,2) mutant strains on V8 medium in the dark and the light (48 h). Filamentation is repressed in wild-type crosses in the light, but not in crosses between mutants or in the dark.(D) Fusion efficiency of strains under different wavelengths of light. Matings were between wild-type (+) partners, bwc2 mutant (Δ) partners, or one wild-type and one mutant partner (bwc2 α × WT a, or WT α × bwc2 a). Mutation of bwc2 in either or both mating partners relieves inhibition of fusion by white or blue light. Bars indicate the standard error of the mean of three replicates.(E) Filament formation in wild-type or bwc2/bwc2 mutant diploid strains (24 h). Light does not repress filament formation in bwc2/bwc2 diploids.(F) The bwc2 and double bwc1 bwc2 (bwc1,2) mutants are as hypersensitive to UV light as bwc1 mutants. Ten-fold serial dilutions of yeast cells were plated in duplicate onto YPD medium, and one set was UV irradiated (~48 mJ/cm2).

Mentions: We hypothesized that Bwc1 binds to an interacting DNA binding protein, because Bwc1 would be unable to act as a transcription factor on its own. Systematic deletion of all of the C. neoformans transcription factors, assuming these were annotated, is not technically feasible at this stage. Similarly, standard insertional mutagenesis poses a problem because the filamentation phenotype requires that both the MATa and MATα mating partners bear the same mutation. However, overexpression of a mating type-specific homeodomain protein in a haploid strain of the opposite mating type confers a self-filamentous morphology [27]. We reasoned that such a self-filametous strain could be employed to perform random insertional mutagenesis, and devised a screen to identify the hypothetical protein interacting with Bwc1. The SXI1α gene, which encodes the MATα-specific homeodomain protein [27], was introduced into the genome of a MATa haploid strain. The resulting MATa +SXI1α strain (AI49) exhibited self-filamentous growth that was regulated by temperature, light, and nutrients. The strain grew as a budding yeast at 37 °C and filamented at 25 °C, and, like MATa/MATα diploids, filamentation was inhibited by light, and was most robust on V8 mating medium (Figure 5A).


Light controls growth and development via a conserved pathway in the fungal kingdom.

Idnurm A, Heitman J - PLoS Biol. (2005)

The BWC2 Gene also Mediates UV/Blue Light Responses in C. neoformans(A) A self-filamentous haploid strain (MATa +SXI1α) exhibits light-repressed filamentation. This strain was mutated by Agrobacterium-mediated T-DNA insertion, and insertional mutant strain 25F8 filaments equally well in the light and the dark.(B) Comparison of the structures of Bwc1 and Bwc2. The Bwc1 predicted protein (1,097 amino acids) has a LOV domain, two additional PAS domains and a nuclear localization signal. The Bwc2 predicted protein (392 amino acids) has a PAS domain and zinc finger DNA binding domain.(C) Bilateral mating between wild-type (WT), bwcl, bwc2, or bwc1 bwc2 double (bwc1,2) mutant strains on V8 medium in the dark and the light (48 h). Filamentation is repressed in wild-type crosses in the light, but not in crosses between mutants or in the dark.(D) Fusion efficiency of strains under different wavelengths of light. Matings were between wild-type (+) partners, bwc2 mutant (Δ) partners, or one wild-type and one mutant partner (bwc2 α × WT a, or WT α × bwc2 a). Mutation of bwc2 in either or both mating partners relieves inhibition of fusion by white or blue light. Bars indicate the standard error of the mean of three replicates.(E) Filament formation in wild-type or bwc2/bwc2 mutant diploid strains (24 h). Light does not repress filament formation in bwc2/bwc2 diploids.(F) The bwc2 and double bwc1 bwc2 (bwc1,2) mutants are as hypersensitive to UV light as bwc1 mutants. Ten-fold serial dilutions of yeast cells were plated in duplicate onto YPD medium, and one set was UV irradiated (~48 mJ/cm2).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1064852&req=5

pbio-0030095-g005: The BWC2 Gene also Mediates UV/Blue Light Responses in C. neoformans(A) A self-filamentous haploid strain (MATa +SXI1α) exhibits light-repressed filamentation. This strain was mutated by Agrobacterium-mediated T-DNA insertion, and insertional mutant strain 25F8 filaments equally well in the light and the dark.(B) Comparison of the structures of Bwc1 and Bwc2. The Bwc1 predicted protein (1,097 amino acids) has a LOV domain, two additional PAS domains and a nuclear localization signal. The Bwc2 predicted protein (392 amino acids) has a PAS domain and zinc finger DNA binding domain.(C) Bilateral mating between wild-type (WT), bwcl, bwc2, or bwc1 bwc2 double (bwc1,2) mutant strains on V8 medium in the dark and the light (48 h). Filamentation is repressed in wild-type crosses in the light, but not in crosses between mutants or in the dark.(D) Fusion efficiency of strains under different wavelengths of light. Matings were between wild-type (+) partners, bwc2 mutant (Δ) partners, or one wild-type and one mutant partner (bwc2 α × WT a, or WT α × bwc2 a). Mutation of bwc2 in either or both mating partners relieves inhibition of fusion by white or blue light. Bars indicate the standard error of the mean of three replicates.(E) Filament formation in wild-type or bwc2/bwc2 mutant diploid strains (24 h). Light does not repress filament formation in bwc2/bwc2 diploids.(F) The bwc2 and double bwc1 bwc2 (bwc1,2) mutants are as hypersensitive to UV light as bwc1 mutants. Ten-fold serial dilutions of yeast cells were plated in duplicate onto YPD medium, and one set was UV irradiated (~48 mJ/cm2).
Mentions: We hypothesized that Bwc1 binds to an interacting DNA binding protein, because Bwc1 would be unable to act as a transcription factor on its own. Systematic deletion of all of the C. neoformans transcription factors, assuming these were annotated, is not technically feasible at this stage. Similarly, standard insertional mutagenesis poses a problem because the filamentation phenotype requires that both the MATa and MATα mating partners bear the same mutation. However, overexpression of a mating type-specific homeodomain protein in a haploid strain of the opposite mating type confers a self-filamentous morphology [27]. We reasoned that such a self-filametous strain could be employed to perform random insertional mutagenesis, and devised a screen to identify the hypothetical protein interacting with Bwc1. The SXI1α gene, which encodes the MATα-specific homeodomain protein [27], was introduced into the genome of a MATa haploid strain. The resulting MATa +SXI1α strain (AI49) exhibited self-filamentous growth that was regulated by temperature, light, and nutrients. The strain grew as a budding yeast at 37 °C and filamented at 25 °C, and, like MATa/MATα diploids, filamentation was inhibited by light, and was most robust on V8 mating medium (Figure 5A).

Bottom Line: One UV-sensitive mutant that filaments equally well in the light and the dark was identified and found to have an insertion in the BWC2 gene, whose product is structurally similar to N. crassa WC-2.Deletion of BWC1 or BWC2 reduces the virulence of C. neoformans in a murine model of infection; the Bwc1-Bwc2 system thus represents a novel protein complex that influences both development and virulence in a pathogenic fungus.These results demonstrate that a role for blue/UV light in controlling development is an ancient process that predates the divergence of the fungi into the ascomycete and basidiomycete phyla.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Microbiology, Howard Hughes Medical Institute, Duke University Medical Center, Durham, North Carolina, USA.

ABSTRACT
Light inhibits mating and haploid fruiting of the human fungal pathogen Cryptococcus neoformans, but the mechanisms involved were unknown. Two genes controlling light responses were discovered through candidate gene and insertional mutagenesis approaches. Deletion of candidate genes encoding a predicted opsin or phytochrome had no effect on mating, while strains mutated in the white collar 1 homolog gene BWC1 mated equally well in the light or the dark. The predicted Bwc1 protein shares identity with Neurospora crassa WC-1, but lacks the zinc finger DNA binding domain. BWC1 regulates cell fusion and repression of hyphal development after fusion in response to blue light. In addition, bwc1 mutant strains are hypersensitive to ultraviolet light. To identify other components required for responses to light, a novel self-fertile haploid strain was created and subjected to Agrobacterium-mediated insertional mutagenesis. One UV-sensitive mutant that filaments equally well in the light and the dark was identified and found to have an insertion in the BWC2 gene, whose product is structurally similar to N. crassa WC-2. The C. neoformans Bwc1 and Bwc2 proteins interact in the yeast two-hybrid assay. Deletion of BWC1 or BWC2 reduces the virulence of C. neoformans in a murine model of infection; the Bwc1-Bwc2 system thus represents a novel protein complex that influences both development and virulence in a pathogenic fungus. These results demonstrate that a role for blue/UV light in controlling development is an ancient process that predates the divergence of the fungi into the ascomycete and basidiomycete phyla.

Show MeSH
Related in: MedlinePlus