Abstract
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.
Highlights
Introduction ofDNA sequences into the genome often results in homology-dependent gene silencing in organisms as diverse as plants, fungi, flies, nematodes, and mammals
Transgenes can provoke silencing of endogenous genes with which they share sequence homology
Compared with the sex induced silencing (SIS) phenomenon previously discovered in C. neoformans, which efficiently silences genes during mating (,50%) but not during vegetative growth (,0.2%), asexual co-suppression operates efficiently during vegetative growth to suppress transgene expression and may silence transposons and other repetitive sequences
Summary
Introduction ofDNA sequences into the genome often results in homology-dependent gene silencing in organisms as diverse as plants, fungi, flies, nematodes, and mammals. 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. In genetically modified plants and fungi, introduced transgenes are in some cases silenced and can cause silencing of endogenous genes if they share sufficient homology [1]. This phenomenon has been termed co-suppression or homologydependent gene silencing (HDGS), and usually occurs when multiple copies of particular sequences are present in the genome [2,3]. An RNAi degradation process is central to PTGS, in which a dsRNA intermediate homologous to the target gene or transgene is generated and processed into siRNAs of 21–25 nucleotides. siRNAs subsequently guide gene silencing in a sequence-specific manner [4,5]
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