Sex-Induced Silencing Operates During Opposite-Sex and Unisexual Reproduction in Cryptococcus neoformans

Abstract

Cryptococcus neoformans is a human fungal pathogen that undergoes a dimorphic transition from yeast to hyphae during a-a opposite-sex mating and a-a unisexual reproduction (same-sex mating). Infectious spores are generated during both processes. We previously identified a sex-induced silencing (SIS) pathway in the C. neoformans serotype A var. grubii lineage, in which tandem transgene arrays trigger RNAi-dependent gene silencing at a high frequency during a-a opposite-sex mating, but at an 250-fold lower frequency during asexual mitotic vegetative growth. Here we report that SIS also operates during a-a unisexual reproduction. A self-fertile strain containing either SXI2a-URA5 or NEO-URA5 transgene arrays exhibited an elevated silencing frequency during solo and unisexual mating compared with mitotic vegetative growth. We also found that SIS operates at a similar efficiency on transgene arrays of the same copy number during either a-a unisexual reproduction or a-a opposite-sex mating. URA5-derived small RNAs were detected in the silenced progeny of a-a unisexual reproduction and RNAi core components were required, providing evidence that SIS induced by same-sex mating is also mediated by RNAi via sequence-specific small RNAs. In addition, our data show that the SIS RNAi pathway also operates to defend the genome via squelching transposon activity during same-sex mating as it does during opposite-sex mating. Taken together, our results confirm that SIS is conserved between the divergent C. neoformans serotype A and serotype D cryptic sibling species. COMPLEX genomes have evolved sophisticated mechanisms to sense the presence, and to control the behavior of genomic invaders such as transposable elements and viruses. Among a panoply of diverse strategies, homologydependent gene silencing (HDGS) is a ubiquitous phenomenon among fungi, plants, and animals (Cogoni 2001). HDGS, also known as cosuppression, was first found to be triggered by transgenes, viruses, or unusual duplications of endogenous genes in both plants and fungi (Cogoni et al. 1996;English et al. 1996;Bingham 1997;Cogoni and Macino 1999a). Although a broad variety of HDGS phenomena are known, all of these gene silencing processes are based on the recognition of nucleic acid sequence homology, followed by inactivation of homologous sequences. Gene silencing can occur at a transcriptional or post-transcriptional level involving sequence-specific mRNA degradation. The most studied and best-characterized HDGS phenomenon in fungi is a process known as quelling, which occurs in Neurospora crassa vegetative tissue (Cogoni et al. 1996). Quelling is an RNAi-mediated silencing mechanism that post-transcriptionally inactivates duplicated sequences, in many cases resulting from introduction of transgenes. In quelling, the silenced loci can act in trans, leading to silencing of all homologous genes throughout the genome. The core RNAi components, including Argonaute, Dicer-like proteins, and RNA-dependent RNA polymerase (RdRP), are all required for quellin...

Genetic Circuits that Govern Bisexual and Unisexual Reproduction in Cryptococcus neoformans

“…Surprisingly, we also found a significant increase in the expression of the T1 and T3 DNA transposons (Table 2). Previous studies have shown that transposable elements are highly expressed during bisexual and unisexual reproduction; however, the RNAi pathway inhibits transposon activity via Sex-Induced Silencing (SIS) and protects the genome against transposition and mutations [44], [45], [46]. Deletion of RDP1 , a component of the RNAi pathway (the RNA-dependent RNA polymerase), induces the expression of retrotransposons during sexual development [44], [46].…”

Unisexual versus bisexual mating in Cryptococcus neoformans: Consequences and biological impacts

“…Deletion of RNAi components allows increased expression and movement of transposable elements, which can have direct functional consequences, such as development of FK506 resistance (Wang et al, 2010). Notably, SIS occurs during both bisexual and unisexual mating (Wang et al, 2013b). In addition, the VGII subtype of C. gattii has conspicuously lost its RNAi pathway (Billmyre et al, 2013; D’Souza et al, 2011; Wang et al, 2010).…”

RNAi function, diversity, and loss in the fungal kingdom

“…For example, silencing of a tandem insertion of a triplicated SXI2 a - URA5 transgene during a -α opposite-sex reproduction occurs at 250-fold higher frequency than in vegetative mitotic asexual growth. Because C. neoformans can produce spores via two distinct mating pathways: a -α opposite sex and α- α unisexual reproduction, a subsequent study tested and demonstrated that SIS is not limited to a -α opposite-sex but also operates during α- α unisexual reproduction (Wang et al, 2013). More importantly, the silencing frequency observed during opposite-sex and unisexual reproduction is comparable.…”

“…In rdp1 mutant strains, siRNAs generated from these repetitive loci were abolished, supporting a role for RNAi in transposon control in C. neoformans . Furthermore, two lines of evidence may explain why C. neoformans evolved a more robust sex-related silencing mechanism (Wang et al, 2010, Wang et al, 2013). First, transposons and retrotransposons are highly expressed during sexual reproduction of RNAi mutant strains.…”