Transgene Induced Co-Suppression during Vegetative Growth in
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.
Vyšlo v časopise:
Transgene Induced Co-Suppression during Vegetative Growth in. PLoS Genet 8(8): e32767. doi:10.1371/journal.pgen.1002885
Kategorie:
Research Article
prolekare.web.journal.doi_sk:
https://doi.org/10.1371/journal.pgen.1002885
Souhrn
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.
Zdroje
1. CogoniC, MacinoG (1999) Homology-dependent gene silencing in plants and fungi: a number of variations on the same theme. Curr Opin Microbiol 2: 657–662.
2. KooterJM, MatzkeMA, MeyerP (1999) Listening to the silent genes: transgene silencing, gene regulation and pathogen control. Trends Plant Sci 4: 340–347.
3. StamM, MolJNM, KooterJM (1997) The silence of genes in transgenic plants. Ann Bot 79: 3–12.
4. CatalanottoC, AzzalinG, MacinoG, CogoniC (2002) Involvement of small RNAs and role of the qde genes in the gene silencing pathway in Neurospora. Genes Dev 16: 790–795.
5. CatalanottoC, PallottaM, ReFaloP, SachsMS, VayssieL, et al. (2004) Redundancy of the two dicer genes in transgene-induced posttranscriptional gene silencing in Neurospora crassa. Mol Cell Biol 24: 2536–2545.
6. CogoniC, IrelanJT, SchumacherM, SchmidhauserTJ, SelkerEU, et al. (1996) Transgene silencing of the al-1 gene in vegetative cells of Neurospora is mediated by a cytoplasmic effector and does not depend on DNA-DNA interactions or DNA methylation. EMBO J 15: 3153–3163.
7. RomanoN, MacinoG (1992) Quelling: transient inactivation of gene expression in Neurospora crassa by transformation with homologous sequences. Mol Microbiol 6: 3343–3353.
8. SelkerEU, CambareriEB, JensenBC, HaackKR (1987) Rearrangement of duplicated DNA in specialized cells of Neurospora. Cell 51: 741–752.
9. RossignolJL, FaugeronG (1994) Gene inactivation triggered by recognition between DNA repeats. Experientia 15: 307–317.
10. FreedmanT, PukkilaPJ (1993) De novo methylation of repeated sequences in Coprinus cinereus. Genetics 135: 357–366.
11. BarryC, FaugeronG, RossignolJL (1993) Methylation induced premeiotically in Ascobolus: coextension with DNA repeat lengths and effect on transcript elongation. Proc Natl Acad Sci U S A 90: 4557–4561.
12. CambareriE, JensenB, SchabtachE, SelkerE (1989) Repeat-induced G-C to A-T mutations in Neurospora. Science 244: 1571–1575.
13. CogoniC, MacinoG (1999) Gene silencing in Neurospora crassa requires a protein homologous to RNA-dependent RNA polymerase. Nature 399: 166–169.
14. LeeDW, PrattRJ, McLaughlinM, AramayoR (2003) An argonaute-like protein is required for meiotic silencing. Genetics 164: 821–828.
15. FulciV, MacinoG (2007) Quelling: post-transcriptional gene silencing guided by small RNAs in Neurospora crassa. Curr Opin Microbiol 10: 199–203.
16. Pal-BhadraM, BhadraU, BirchlerJA (1997) Cosuppression in Drosophila: gene silencing of alcohol dehydrogenase by white-Adh transgenes is polycomb dependent. Cell 90: 479–490.
17. VaucheretH, BeclinC, FagardM (2001) Post-transcriptional gene silencing in plants. J Cell Sci 114: 3083–3091.
18. DernburgAF, ZalevskyJ, ColaiacovoMP, VilleneuveAM (2000) Transgene-mediated cosuppression in the C. elegans germ line. Genes Dev 14: 1578–1583.
19. DalmayT, HamiltonA, RuddS, AngellS, BaulcombeDC (2000) An RNA-dependent RNA polymerase gene in Arabidopsis is required for posttranscriptional gene silencing mediated by a transgene but not by a virus. Cell 101: 543–553.
20. BrenneckeJ, AravinAA, StarkA, DusM, KellisM, et al. (2007) Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila. Cell 128: 1089–1103.
21. KlattenhoffC, TheurkaufW (2008) Biogenesis and germline functions of piRNAs. Development 135: 3–9.
22. RuizMT, VoinnetO, BaulcombeCD (1998) Initiation and maintenance of virus-induced gene silencing. Plant Cell 10: 937–946.
23. JonesL, HamiltonAJ, VoinnetO, ThomasCL, MauleAJ, et al. (1999) RNA-DNA interactions and DNA methylation in post-transcriptional gene silencing. Plant Cell 1: 2291–2301.
24. WangX, HsuehY-P, LiW, FloydA, SkalskyR, et al. (2010) Sex-induced silencing defends the genome of Cryptococcus neoformans via RNAi. Genes Dev 24: 2566–2582.
25. WangP, CardenasME, CoxGM, PerfectJR, HeitmanJ (2001) Two cyclophilin A homologs with shared and distinct functions important for growth and virulence of Cryptococcus neoformans. EMBO Rep 2: 511–518.
26. LoftusBJ, FungE, RoncagliaP, RowleyD, AmedeoP, et al. (2005) The genome of the basidiomycetous yeast and human pathogen Cryptococcus neoformans. Science 307: 1321–1324.
27. AlspaughJA, PerfectJR, HeitmanJ (1997) Cryptococcus neoformans mating and virulence are regulated by the G-protein alpha subunit GPA1 and cAMP. Genes Dev 11: 3206–3217.
28. PanditNN, RussoVEA (1992) Reversible inactivation of a foreign gene, hph, during the asexual cycle in Neurospora crassa transformants. Mol Gen Genet 234: 412–422.
29. CatalanottoC, NolanT, CogoniC (2006) Homology effects in Neurospora crassa. FEMS Microbiol Lett 254: 182–189.
30. JanbonG, MaengS, YangDH, KoYJ, JungKW, et al. (2010) Characterizing the role of RNA silencing components in Cryptococcus neoformans. Fungal Genet Biol 47: 1070–1080.
31. LeeHC, AaltoAP, YangQ, ChangSS, HuangG, et al. (2010) The DNA/RNA-dependent RNA polymerase QDE-1 generates aberrant RNA and dsRNA for RNAi in a process requiring replication protein A and a DNA helicase. PLoS Biology 8: e1000496 doi:10.1371/journal.pbio.1000496.
32. NolanT, CecereG, ManconeC, AlonziT, TripodiM, et al. (2008) The RNA-dependent RNA polymerase essential for post-transcriptional gene silencing in Neurospora crassa interacts with replication protein A. Nucleic Acids Research 36: 532–538.
33. IftodeC, DanielyY, BorowiecJA (1999) Replication protein A (RPA): the eukaryotic SSB. Crit Rev Biochem Mol Biol 34: 141–180.
34. WoldMS (1997) Replication protein A: a heterotrimeric, single-stranded DNA-binding protein required for eukaryotic DNA metabolism. Annu Rev Biochem 66: 61–92.
35. NakayashikiH, KadotaniN, MayamaS (2006) Evolution and diversification of RNA silencing proteins in fungi. J Mol Evol 63: 127–135.
36. LiuH, CottrellTR, PieriniLM, GoldmanWE, DoeringTL (2002) RNA interference in the pathogenic fungus Cryptococcus neoformans. Genetics 160: 463–470.
37. BoseI, DoeringTL (2011) Efficient implementation of RNA interference in the pathogenic yeast Cryptococcus neoformans. J Microbiol Methods 86: 156–159.
38. StamM, De BruinR, KenterS, Van Der HoornRAL, Van BloklandR, et al. (1997) Post-transcriptional silencing of chalcone synthase in Petunia by inverted transgene repeats. Plant J 12: 63–82.
39. EnglishJJ, MuellerE, BaulcombeDC (1996) Suppression of virus accumulation in transgenic plants exhibiting silencing of nuclear genes. Plant Cell 8: 179–188.
40. LeeHC, ChangSS, ChoudharyS, AaltoAP, MaitiM, et al. (2009) qiRNA is a new type of small interfering RNA induced by DNA damage. Nature 459: 274–277.
41. ToffalettiDL, RudeTH, JohnstonSA, DurackDT, PerfectJR (1993) Gene transfer in Cryptococcus neoformans by use of biolistic delivery of DNA. J Bacteriol 175: 1405–1411.
42. SudarshanS, DavidsonRC, HeitmanJ, AlspaughJA (1999) Molecular analysis of the Cryptococcus neoformans ADE2 gene, a selectable marker for transformation and gene disruption. Fungal Genet Biol 27: 36–48.
43. XueC, TadaY, DongX, HeitmanJ (2007) The human fungal pathogen Cryptococcus can complete its sexual cycle during a pathogenic association with plants. Cell Host Microbe 1: 263–273.
44. FraserJA, SubaranRL, NicholsCB, HeitmanJ (2003) Recapitulation of the sexual cycle of the primary fungal pathogen Cryptococcus neoformans var. gattii: implications for an outbreak on Vancouver Island, Canada. Eukaryot Cell 2: 1036–1045.
45. DavidsonRC, CruzMC, SiaRA, AllenB, AlspaughJA, et al. (2000) Gene disruption by biolistic transformation in serotype D strains of Cryptococcus neoformans. Fungal Genet Biol 29: 38–48.
46. BreslowDK, CameronDM, CollinsSR, SchuldinerM, Stewart-OrnsteinJ, et al. (2008) A comprehensive strategy enabling high-resolution functional analysis of the yeast genome. Nat Meth 5: 711–718.
47. SchuldinerM, CollinsSR, ThompsonNJ, DenicV, BhamidipatiA, et al. (2005) Exploration of the function and organization of the yeast early secretory pathway through an epistatic miniarray profile. Cell 123: 507–519.
48. BahnYS, KojimaK, CoxGM, HeitmanJ (2005) Specialization of the HOG pathway and its impact on differentiation and virulence of Cryptococcus neoformans. Mol Biol Cell 16: 2285–2300.
Štítky
Genetika Reprodukčná medicínaČlánok vyšiel v časopise
PLOS Genetics
2012 Číslo 8
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