Abstract
Cryptococcus neoformans is a human fungal pathogen with a defined sexual cycle. Nutrient-limiting conditions and pheromones induce a dimorphic transition from unicellular yeast to multicellular hyphae and the production of infectious spores. Sexual reproduction involves cells of either opposite (bisexual) or one (unisexual) mating type. Bisexual and unisexual reproduction are governed by shared components of the conserved pheromone-sensing Cpk1 MAPK signal transduction cascade and by Mat2, the major transcriptional regulator of the pathway. However, the downstream targets of the pathway are largely unknown, and homology-based approaches have failed to yield downstream transcriptional regulators or other targets. In this study, we applied insertional mutagenesis via Agrobacterium tumefaciens transkingdom DNA delivery to identify mutants with unisexual reproduction defects. In addition to elements known to be involved in sexual development (Crg1, Ste7, Mat2, and Znf2), three key regulators of sexual development were identified by our screen: Znf3, Spo11, and Ubc5. Spo11 and Ubc5 promote sporulation during both bisexual and unisexual reproduction. Genetic and phenotypic analyses provide further evidence implicating both genes in the regulation of meiosis. Phenotypic analysis of sexual development showed that Znf3 is required for hyphal development during unisexual reproduction and also plays a central role during bisexual reproduction. Znf3 promotes cell fusion and pheromone production through a pathway parallel to and independent of the pheromone signaling cascade. Surprisingly, Znf3 participates in transposon silencing during unisexual reproduction and may serve as a link between RNAi silencing and sexual development. Our studies illustrate the power of unbiased genetic screens to reveal both novel and conserved circuits that operate sexual reproduction.
Highlights
Sexual reproduction in eukaryotes facilities genetic diversity and eliminates deleterious mutations leading to better fit progeny
To identify genes encoding novel components governing unisexual reproduction we utilized the ability of A. tumefaciens to transfer and mutagenize its target by randomly introducing a known DNA sequence into the genome
A. tumefaciens-mediated transkingdom DNA delivery has been successfully applied in C. neoformans to identify and characterize novel virulence factors and components of other sensing pathways [26,30,31,34]
Summary
Sexual reproduction in eukaryotes facilities genetic diversity and eliminates deleterious mutations leading to better fit progeny. Sex often involves two cells of opposite mating type (heterothallism) that secrete pheromones in order to induce cell fusion and subsequently nuclear fusion and meiosis generate recombinant progeny. In other fungi, solo incubation of an individual isolate can result in sexual reproduction, and this selfing process is referred to as homothallism. Paradigmatic examples of fungi with both modes of sexual reproduction are Saccharomyces cerevisiae and Cryptococcus neoformans. Nutrients activate cellular receptors and the cAMP-dependent pathway to govern the expression of genes evoking the dimorphic transition [5]. Core components of these pathways are highly conserved throughout the fungal kingdom; the downstream targets are often species-specific
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