Transgene Induced Co-Suppression during Vegetative Growth in Cryptococcus neoformans

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.

3 RNAi Function and Diversity in Fungi

“…The diminishing of these siRNAs in the rdp1 mutant supports the fact that SIS is related to RNAi and dependent on dsRNA (Wang et al 2010). Another quelling-like co-suppression phenomenon is present in C. neoformans, named mitotic-induced silencing (Wang et al 2012). Transformation of cpa1 resulted in isolates with decreased mRNA levels of CPA1 and CPA2 genes.…”

“…Transformation of cpa1 resulted in isolates with decreased mRNA levels of CPA1 and CPA2 genes. This silencing is dependent on RNAi components Rdp1, Ago1, and Dcr2 (Wang et al 2012). …”

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

“…C. neoformans undergoes two distinct transgene dependent silencing processes, one during normal mitotic growth, called MIS (mitotic induced silencing) (Wang et al, 2012b), and one during the sexual cycle, called SIS (sex induced silencing) (Wang et al, 2010). Both processes function to repress expression of transposons, either at a basal level during vegetative growth (Dumesic et al, 2014; Wang et al, 2012b), or at a much higher level under mating conditions (Wang et al, 2010). The key difference between these two processes is that SIS occurs at a much higher frequency, which is in line with the increased expression of transposons and elevated levels of RNAi components under mating conditions.…”

GGR (Geranylgeranyl Reductase) Expression Affects the Allelopathic Response to Arabidopsis Allelochemicals

“…Although not intended, it is not unusual that attempts to overexpress a transgene may trigger the suppression or reduction of the expression of the endogenous homologous gene along with the transgene (Meyer & Saedler, 1996;Wang et al, 2012). Homology-dependent gene silencing (HDGS) can occur due to multiple gene copies in a genome.…”