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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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Bwc1 and Bwc2 Physically InteractThe coding regions of the BWC1 and BWC2 genes were fused adjacent to the AD or BD of S. cerevisiae GAL4. Plasmids were cotransformed into a S. cerevisiae strain in which Gal4 regulates the ADE2, HIS3, and lacZ genes. Growth of strains in the absence of adenine (−ade) or histidine (−his) and increased β-galactosidase activity (β-gal, ± one standard error, Miller units) indicate protein-protein interactions.
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pbio-0030095-g006: Bwc1 and Bwc2 Physically InteractThe coding regions of the BWC1 and BWC2 genes were fused adjacent to the AD or BD of S. cerevisiae GAL4. Plasmids were cotransformed into a S. cerevisiae strain in which Gal4 regulates the ADE2, HIS3, and lacZ genes. Growth of strains in the absence of adenine (−ade) or histidine (−his) and increased β-galactosidase activity (β-gal, ± one standard error, Miller units) indicate protein-protein interactions.

Mentions: A yeast two-hybrid analysis was conducted to test whether Bwc1 and Bwc2 physically interact. cDNA clones were fused to the S. cerevisiae Gal4 transcription factor activation (AD) or DNA binding (BD) domains and expressed in a S. cerevisiae strain in which the GAL promoter regulates ADE2, HIS3, and lacZ reporter genes. In S. cerevisiae strains expressing AD-Bwc1 and BD-Bwc2, or AD-Bwc2 and BD-Bwc1, the ADE2, HIS3, and lacZ reporter genes were all induced and the cells grew in the absence of adenine or histidine and expressed increased levels of β-galactosidase activity (Figure 6). In contrast, S. cerevisiae strains containing single Gal4-Bwc1/2 fusions and the corresponding Gal4 domain did not. These observations indicate that Bwc1 and Bwc2 can interact with one another in vivo. There was no evidence for homodimer formation for either Bwc1 or Bwc2, and no effects of light on the reporter gene-dependent growth of the strains were observed. Attempts to demonstrate Bwc1-Bwc2 interaction in C. neoformans itself via coimmunoprecipitation of epitope-tagged forms of Bwc1 and Bwc2 have been unsuccessful so far, due to the low abundance of the proteins, cross-reactivity of the antibodies with endogenous fungal proteins, and loss-of-function of tagged proteins in strains overexpressing these proteins (unpublished data). These findings demonstrate that Bwc1 and Bwc2 can physically interact when expressed in the nucleus of another fungal species, and that they can do so in a light-independent manner.


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

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

Bwc1 and Bwc2 Physically InteractThe coding regions of the BWC1 and BWC2 genes were fused adjacent to the AD or BD of S. cerevisiae GAL4. Plasmids were cotransformed into a S. cerevisiae strain in which Gal4 regulates the ADE2, HIS3, and lacZ genes. Growth of strains in the absence of adenine (−ade) or histidine (−his) and increased β-galactosidase activity (β-gal, ± one standard error, Miller units) indicate protein-protein interactions.
© Copyright Policy
Related In: Results  -  Collection

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

pbio-0030095-g006: Bwc1 and Bwc2 Physically InteractThe coding regions of the BWC1 and BWC2 genes were fused adjacent to the AD or BD of S. cerevisiae GAL4. Plasmids were cotransformed into a S. cerevisiae strain in which Gal4 regulates the ADE2, HIS3, and lacZ genes. Growth of strains in the absence of adenine (−ade) or histidine (−his) and increased β-galactosidase activity (β-gal, ± one standard error, Miller units) indicate protein-protein interactions.
Mentions: A yeast two-hybrid analysis was conducted to test whether Bwc1 and Bwc2 physically interact. cDNA clones were fused to the S. cerevisiae Gal4 transcription factor activation (AD) or DNA binding (BD) domains and expressed in a S. cerevisiae strain in which the GAL promoter regulates ADE2, HIS3, and lacZ reporter genes. In S. cerevisiae strains expressing AD-Bwc1 and BD-Bwc2, or AD-Bwc2 and BD-Bwc1, the ADE2, HIS3, and lacZ reporter genes were all induced and the cells grew in the absence of adenine or histidine and expressed increased levels of β-galactosidase activity (Figure 6). In contrast, S. cerevisiae strains containing single Gal4-Bwc1/2 fusions and the corresponding Gal4 domain did not. These observations indicate that Bwc1 and Bwc2 can interact with one another in vivo. There was no evidence for homodimer formation for either Bwc1 or Bwc2, and no effects of light on the reporter gene-dependent growth of the strains were observed. Attempts to demonstrate Bwc1-Bwc2 interaction in C. neoformans itself via coimmunoprecipitation of epitope-tagged forms of Bwc1 and Bwc2 have been unsuccessful so far, due to the low abundance of the proteins, cross-reactivity of the antibodies with endogenous fungal proteins, and loss-of-function of tagged proteins in strains overexpressing these proteins (unpublished data). These findings demonstrate that Bwc1 and Bwc2 can physically interact when expressed in the nucleus of another fungal species, and that they can do so in a light-independent manner.

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