Characterizing the role of RNA silencing components in Cryptococcus neoformans

Abstract

The RNA interference (RNAi) mediated by homology-dependent degradation of the target mRNA with small RNA molecules plays a key role in controlling transcription and translation processes in a number of eukaryotic organisms. The RNAi machinery is also evolutionarily conserved in a wide variety of fungal species, including pathogenic fungi. To elucidate the physiological functions of the RNAi pathway in Cryptococcus neoformans that causes fungal meningitis, here we performed genetic analyses for genes encoding Argonaute (AGO1 and AGO2), RNA-dependent RNA polymerase (RDP1), and Dicers (DCR1 and DCR2) in both serotype A and D C. neoformans. The present study shows that Ago1, Rdp1, and Dcr2 are the major components of the RNAi process occurring in C. neoformans. However, the RNAi machinery is not involved in regulation of production of two virulence factors (capsule and melanin), sexual differentiation, and diverse stress response. Comparative transcriptome analysis of the serotype A and D RNAi mutants revealed that only modest changes occur in the genome-wide transcriptome profiles when the RNAi process was perturbed. Notably, the serotype D rdp1Δ mutants showed an increase in transcript abundance of active retrotransposons and transposons, such as T2 and T3, the latter of which is a novel serotype D-specific transposon of C. neoformans. In a wild type background both T2 and T3 were found to be weakly active mobile elements, although we found no evidence of Cnl1 retrotransposon mobility. In contrast, all three transposable elements exhibited enhanced mobility in the rdp1Δ mutant strain. In conclusion, the RNAi pathway plays an important role in controlling transposon activity and genome integrity of C. neoformans.

Fig. 1

The role of the argonaute-, RdRP-, and Dicer-like genes in the RNA interference process of C. neoformans. (A) Map of the pRNAi plasmid used to transform C. neoformans. (B) Coloration of the different mutant strains after 4 days on YPD agar medium at 30°C. (C) ADE2-specific mRNA levels as measured by quantitative RT-PCR. An ade2 knockdown strain was constructed by integration of the pRNAi plasmid (GeneBank HM352736, kindly given by T. Doering) in its genome. This strain (control, strain name) produced red colonies on complete medium and the levels of ADE2-specific mRNA were fiftyfold lower in the control strain than in the WT strain. To test the importance of each putative RNAi-component on the silencing of ADE2, the control ADE2-knockdown strain was crossed with different single and double RNAi mutant strains. As shown here, RDP1 was the only gene completely necessary for ADE2-silencing. AGO1 and DCR2 also play a major role whereas the deletion of either DCR1 or AGO2 appeared to have a very limited impact on silencing. * Student t-test P value <0.001.** Student t-test P value<0.05

Fig. 2

The role of the RNAi components in virulence factor production, mating, and stress response of C. neoformans. (A) Capsule synthesis levels of the following strains were quantitatively measured by using hematocrit as previously described (Kim et al., 2010): the serotype A MATa WT strain (KN99) and cac1Δ (YSB79), hog1Δ (YSB81), ago1Δ (YSB300), rdp1Δ (YSB306), and dcr2Δ (YSB305) mutant strains. The Y axis indicates the relative capsule volume, which is percent ratio of length of packed cell volume phase versus length of total loading volume phase. (B) Melanin production of the following strains was visualized on L-DOPA or Niger seed medium: the serotype A MATa WT strain (KN99) and cac1Δ (YSB79), hog1Δ (YSB81), ago1Δ (YSB300), rdp1Δ (YSB306), and dcr2Δ (YSB305) mutant strains. The hog1Δ and cac1Δ mutants were used as positive and negative controls, respectively, in both capsule and melanin production (Bahn et al., 2004; Bahn et al., 2005). (C) Each serotype A α mating type strain [WT H99, ago1Δ (YSB299), rdp1Δ (YSB624), and dcr2Δ (YSB304)] was cocultured with a mating type strains [WT KN99, ago1Δ (YSB300), rdp1Δ (YSB307), and dcr2Δ (YSB305)] on V8 medium (pH 5.0) at room temperature in the dark. Representative edges of the mating patches were photographed at 100x magnification after 21 d incubation. (D) Each C. neoformans strain (the WT KN99 strain and ras1Δ [YSB73], cac1Δ [YSB79], hog1Δ [YSB81], ago1Δ (YSB300), rdp1Δ (YSB306), and dcr2Δ (YSB305)) was grown overnight at 30°C in liquid YPD medium, 10-fold serially diluted (1 10⁻⁴ dilutions), and spotted (3 μl of dilution) on YPD agar medium containing the indicated concentrations of KCl, NaCl, fludioxonil, Congo red, hydrogen peroxide (H₂O₂), amphotericin B, itraconazole, hydroxyurea, methylmethane sulfonate (MMS), or TBZ. Cells were incubated at 30°C for 72 h and photographed.

Fig. 3

Increased transcript levels of transposable elements in the serotype D rdp1Δ mutants. Northern blot analysis showed basal expression levels of T2 and T3 transposons in the serotype D WT [NE519 (MATα) and NE520 (MATa)] and increased levels in rdp1Δ mutant [NE517 (MATα) and NE518 (MATa)]. Diagrams of T3 and T2 (long and short versions) transposons are depicted at the right panel.

Fig. 4

Mobility of T2 and T3 transposons and Cnl1 retrotransposon in the WT (MATa) and rdp1Δ mutant strain backgrounds. Analysis of the transposition events by Southern blot analysis was conducted with genomic DNA isolated after prolonged cultures (10 subcultures) of the serotype D WT strain (NE520) and rdp1Δ mutant (NE518) and compared to the reference strain (C, before subculture). Ten colonies per each culture were analyzed. From each independent culture (10 colonies), only novel transposition events were indicated as arrows. “Hop-in” and “pop-out” events of transposons were indicated as red and blue arrows, respectively. Transposition events occurring at the identical position of multiple colonies from a single culture were counted as a single event since they may represent the same transposition event between siblings during subcultures. Genomic DNA was cleaved with PstI for T2, XhoI for T3, and PstI for Cnl1, separated on 0.8% agarose gel, transferred to Nylon membranes, and hybridized with each specific probe generated by PCR with primers listed in Table S2.