Introduction of DNA sequences into the genome often results in homology-dependent gene silencing in organisms as diverse as plants, fungi, flies, nematodes, and mammals. We previously showed in Cryptococcus neoformans that a repeat transgene array can induce gene silencing at a high frequency during mating (∼50%), but at a much lower frequency during vegetative growth (∼0.2%). Here we report a robust asexual co-suppression phenomenon triggered by the introduction of a cpa1::ADE2 transgene. Multiple copies of the cpa1::ADE2 transgene were ectopically integrated into the genome, leading to silencing of the endogenous CPA1 and CPA2 genes encoding the cyclosporine A target protein cyclophilin A. Given that CPA1-derived antisense siRNAs were detected in the silenced isolates, and that RNAi components (Rdp1, Ago1, and Dcr2) are required for silencing, we hypothesize that an RNAi pathway is involved, in which siRNAs function as trans factors to silence both the CPA1 and the CPA2 genes. The silencing efficiency of the CPA1 and CPA2 genes is correlated with the transgene copy number and reached ∼90% in the presence of >25 copies of the transgene. We term this transgene silencing phenomenon asexual co-suppression to distinguish it from the related sex-induced silencing (SIS) process. We further show that replication protein A (RPA), a single-stranded DNA binding complex, is required for transgene silencing, suggesting that RPA might play a similar role in aberrant RNA production as observed for quelling in Neurospora crassa. Interestingly, we also observed that silencing of the ADE2 gene occurred at a much lower frequency than the CPA1/2 genes even though it is present in the same transgene array, suggesting that factors in addition to copy number influence silencing. Taken together, our results illustrate that a transgene induced co-suppression process operates during C. neoformans vegetative growth that shares mechanistic features with quelling.

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...