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Characterization of female sexual development‐1 ( fsd‐1 ) Transcript Structure, Expression, and Localization in the Fungus Neurospora crassa
Author(s) -
Hurysz Thomas,
Gebhardt Bryce,
Pyatt Mary,
Emmens Kathryn,
Toufexis Christine,
Hutchison Elizabeth
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.648.12
Subject(s) - biology , neurospora crassa , intron , crassa , exon , genetics , chromodomain , gene , microbiology and biotechnology , rna , helicase , mutant
Neurospora crassa is a filamentous ascomycete fungus that can reproduce sexually and asexually. The signaling pathways that control sexual development in N. crassa are not well characterized. FSD‐1 is an N. crassa transcription factor that controls both early and late stages of sexual development. Although we know that fsd‐1 regulates female sexual development (including fertility and spore maturation), little is known about (1) its transcriptional regulation, (2) RNA isoforms, (3) protein expression, and (4) localization. Intron and exon boundaries of fsd‐1 were mapped using reverse transcription (RT)‐PCR and sequencing and we determined that fsd‐1 can be expressed as three different transcripts, which differ in length and intron/exon structure. Quantitative RT‐PCR during a time course of sexual development showed that expression of fsd‐1 transcripts is not detectable until N. crassa initiates sexual development. In addition, one transcript variant is predominantly expressed during sexual development, and expression of this transcript peaks approximately five days after crossing (mating). It is unknown whether all three fsd‐1 transcripts are translated into protein, and thus we will monitor FSD‐1 protein expression using immunoprecipitation and western blotting. The three fsd‐1 transcript variants encode for predicted proteins of slightly different sizes, which can be visualized via western blot. To investigate FSD‐1 localization, we constructed a GFP‐tagged version of FSD‐1 using overlap PCR and targeted this construct to the native locus. FSD‐1‐GFP localization was monitored via fluorescence microscopy throughout a time course of development. We found that FSD‐1‐GFP localized to the nuclei of female reproductive tissues, such as paraphyses, but not to tissues where meiosis occurs. In addition, we confirmed that the FSD‐1‐GFP strain complements the sexual development‐related phenotypes that occur in an fsd‐1 knockout strain, such as sterility and lack of ascospore melanization. Finally, we constructed strains where fsd‐1 has an overexpression promoter, and we will determine whether overexpression of fsd‐1 has a dominant negative effect on wild‐type strains during mating. Little is known about the regulatory pathways that function in non‐meiotic filamentous fungal female sexual tissues, and our work on fsd‐1 provides an important first step in characterizing this aspect of fungal development. Support or Funding Information Geneseo Foundation This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .