RNA silencing in fungi

Abstract

RNA silencing is an evolutionarily conserved mechanism in eukaryotic organisms induced by double-stranded RNA (dsRNA) and plays an essential role in regulating gene expression and maintaining genome stability. RNA silencing occurs at both posttranscriptional levels through sequence-specific RNA degradation or translational repression and at transcriptional levels through RNA-directed DNA methylation and/or heterochromatin formation. RNA silencing pathways have been relatively well characterized in plants and animals, and are now also being widely investigated in diverse fungi, some of which are important plant pathogens. This review focuses primarily on the current understanding of the dsRNA-mediated posttranscriptional gene silencing processes in fungi, but also discusses briefly the known gene silencing pathways that appear to be independent of the RNA silencing machineries. We review RNA silencing studies for a variety of fungi and highlight some of the mechanistic differences observed in different fungal organisms. As RNA silencing is being exploited as a technology in gene function studies in fungi as well as in engineering anti-fungal resistance in plants and animals, we also discuss the recent progress towards understanding dsRNA uptake in fungi.

Key words： RNA silencing; gene silencing; heterochromatin formation; fungi; small interfering RNA; micro-RNA

References

1 Ah-Fong A M V, Bormann-Chung C A, Judelson H S (2008). Optimization of transgene-mediated silencing in Phytophthora infestans and its association with small-interfering RNAs. Fungal Genet Biol, 45(8): 1197–1205

2 Barnes S E, Alcocer M J C, Archer D B (2008). siRNA as a molecular tool for use in Aspergillus niger. Biotechnol Lett, 30(5): 885–890

3 Blanco F A, Judelson H S (2005). A bZIP transcription factor from Phytophthora interacts with a protein kinase and is required for zoospore motility and plant infection. Mol Microbiol, 56(3): 638–648

4 Bohse M L, Woods J P (2007). RNA interference-mediated silencing of the YPS3 gene of Histoplasma capsulatum reveals virulence defects. Infect Immun, 75(6): 2811–2817

5 Braumann I, van den Berg M, Kempken F (2008). Repeat induced point mutation in two asexual fungi, Aspergillus niger and Penicillium chrysogenum. Curr Genet, 53(5): 287–297

6 Buchon N, Vaury C (2006). RNAi: a defensive RNA-silencing against viruses and transposable elements. Heredity, 96(2): 195–202

7 Cam H P, Sugiyama T, Chen E S, Chen X, FitzGerald P C, Grewal S I S (2005). Comprehensive analysis of heterochromatin- and RNAi-mediated epigenetic control of the fission yeast genome. Nat Genet, 37(8): 809–819

8 Camblong J, Beyrouthy N, Guffanti E, Schlaepfer G, Steinmetz L M, Stutz F (2009). Trans-acting antisense RNAs mediate transcriptional gene cosuppression in S. cerevisiae. Genes Dev, 23(13): 1534–1545

9 Camblong J, Iglesias N, Fickentscher C, Dieppois G, Stutz F (2007). Antisense RNA stabilization induces transcriptional gene silencing via histone deacetylation in S. cerevisiae. Cell, 131(4): 706–717

10 Cardoza R E, Hermosa M R, Vizcaíno J A, González F J, Llobell A, Monte E, Gutiérrez S (2007). Partial silencing of a hydroxy-methylglutaryl-CoA reductase-encoding gene in Trichoderma harzianum CECT 2413 results in a lower level of resistance to lovastatin and lower antifungal activity. Fungal Genet Biol, 44(4): 269–283

11 Cardoza R E, Vizcaíno J A, Hermosa M R, Sousa S, González F J, Llobell A, Monte E, Gutiérrez S (2006). Cloning and characterization of the erg1 gene of Trichoderma harzianum: effect of the erg1 silencing on ergosterol biosynthesis and resistance to terbinafine. Fungal Genet Biol, 43(3): 164–178

12 Caribé dos Santos A C, Sena J A L, Santos S C, Dias C V, Pirovani C P, Pungartnik C, Valle R R, Cascardo J C M, Vincentz M (2009). dsRNA-induced gene silencing in Moniliophthora perniciosa, the causal agent of witches’ broom disease of cacao. Fungal Genet Biol, 46(11): 825–836

13 Catalanotto C, Azzalin G, Macino G, Cogoni C (2000). Gene silencing in worms and fungi. Nature, 404(6775): 245

14 Catalanotto C, Azzalin G, Macino G, Cogoni C (2002). Involvement of small RNAs and role of the qde genes in the gene silencing pathway in Neurospora. Genes Dev, 16(7): 790–795

15 Catalanotto C, Nolan T, Cogoni C (2006). Homology effects in Neurospora crassa. FEMS Microbiol Lett, 254(2): 182–189

16 Catalanotto C, Pallotta M, ReFalo P, Sachs M S, Vayssie L, Macino G, Cogoni C (2004). Redundancy of the two dicer genes in transgeneinduced posttranscriptional gene silencing in Neurospora crassa. Mol Cell Biol, 24(6): 2536–2545

17 Cecere G, Cogoni C (2009). Quelling targets the rDNA locus and functions in rDNA copy number control. BMC Microbiol, 9(1): 44–54

18 Choudhary S, Lee H C, Maiti M, He Q, Cheng P, Liu Q, Liu Y (2007). A double-stranded-RNA response program important for RNA interference efficiency. Mol Cell Biol, 27(11): 3995–4005

19 Cogoni C, Irelan J T, Schumacher M, Schmidhauser T J, Selker E U, Macino G (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(12): 3153–3163

20 Cogoni C, Macino G (1997). Isolation of quelling-defective (qde) mutants impaired in posttranscriptional transgene-induced gene silencing in Neurospora crassa. Proc Natl Acad Sci USA, 94(19): 10233–10238

21 Cogoni C, Macino G (1999a). Gene silencing in Neurospora crassa requires a protein homologous to RNA-dependent RNA polymerase. Nature, 399(6732): 166–169

22 Cogoni C, Macino G (1999b). Posttranscriptional gene silencing in Neurospora by a RecQ DNA helicase. Science, 286(5448): 2342–2344

23 Cogoni C, Romano N, Macino G (1994). Suppression of gene expression by homologous transgenes. Antonie van Leeuwenhoek, 65(3): 205–209

24 Costa A M S B, Mills P R, Bailey A M, Foster G D, Challen M P (2008). Oligonucleotide sequences forming short self-complimentary hairpins can expedite the down-regulation of Coprinopsis cinerea genes. J Microbiol Methods, 75(2): 205–208

25 Costa A S M B, Thomas D J I, Eastwood D, Cutler S B, Bailey A M, Foster G D, Mills P R, Challen M P (2009). Quantifiable downregulation of endogenous genes in Agaricus bisporus mediated by expression of RNA hairpins. J Microbiol Biotechnol, 19(3): 271–276

26 Davis C A, Ares M Jr (2006). Accumulation of unstable promoter-associated transcripts upon loss of the nuclear exosome subunit Rrp6p in Saccharomyces cerevisiae. Proc Natl Acad Sci USA, 103(9): 3262–3267

27 de Haro J P, Calo S, Cervantes M, Nicolás F E, Torres-Martínez S, Ruiz-Vázquez R M (2009). A single dicer gene is required for efficient gene silencing associated with two classes of small antisense RNAs in Mucor circinelloides. Eukaryot Cell, 8(10): 1486–1497

28 de Jong J F, Deelstra H J, Wösten H A B, Lugones L G (2006). RNA-mediated gene silencing in monokaryons and dikaryons of Schizophyllum commune. Appl Environ Microbiol, 72(2): 1267–1269

29 Der S D, Zhou A, Williams B R G, Silverman R H (1998). Identification of genes differentially regulated by interferon alpha, beta, or gamma using oligonucleotide arrays. Proc Natl Acad Sci USA, 95(26): 15623–15628

30 Disney M D, Haidaris C G, Turner D H (2003). Uptake and antifungal activity of oligonucleotides in Candida albicans. Proc Natl Acad Sci USA, 100(4): 1530–1534

31 Djupedal I, Kos-Braun I C, Mosher R A, Söderholm N, Simmer F, Hardcastle T J, Fender A, Heidrich N, Kagansky A, Bayne E, Wagner E G H, Baulcombe D C, Allshire R C, Ekwall K (2009). Analysis of small RNA in fission yeast; centromeric siRNAs are potentially generated through a structured RNA. EMBO J, 28(24): 3832–3844

32 Drinnenberg I A, Weinberg D E, Xie K T, Mower J P, Wolfe K H, Fink G R, Bartel D P (2009). RNAi in budding yeast. Science, 326(5952): 544–550

33 Duxbury M S, Ashley S W, Whang E E (2005). RNA interference: a mammalian SID-1 homologue enhances siRNA uptake and gene silencing efficacy in human cells. Biochem Biophys Res Commun, 331(2): 459–463

34 Eamens A, Wang M B, Smith N A, Waterhouse P M (2008). RNA silencing in plants: yesterday, today, and tomorrow. Plant Physiol, 147(2): 456–468

35 Eastwood D C, Challen M P, Zhang C, Jenkins H, Henderson J, Burton K S (2008). Hairpin-mediated down-regulation of the urea cycle enzyme argininosuccinate lyase in Agaricus bisporus. Mycol Res, 112: 708–716

36 Fire A, Xu S, Montgomery M K, Kostas S A, Driver S E, Mello C C (1998). Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 391(6669): 806–811

37 Fitzgerald A, Van Kan J A L, Plummer K M (2004). Simultaneous silencing of multiple genes in the apple scab fungus, Venturia inaequalis, by expression of RNA with chimeric inverted repeats. Fungal Genet Biol, 41(10): 963–971

38 Freitag M, Lee D W, Kothe G O, Pratt R J, Aramayo R, Selker E U (2004). DNA methylation is independent of RNA interference in Neurospora. Science, 304(5679): 1939

39 Galagan J E, Selker E U (2004). RIP: the evolutionary cost of genome defense. Trends Genet, 20(9): 417–423

40 Gaulin E, Jauneau A, Villalba F, Rickauer M, Esquerré-Tugayé M T, Bottin A (2002). The CBEL glycoprotein of Phytophthora parasitica var. nicotianae is involved in cell wall deposition and adhesion to cellulosic substrates. J Cell Sci, 115(23): 4565–4575

41 Ghildiyal M, Zamore P D (2009). Small silencing RNAs: an expanding universe. Nat Rev Genet, 10(2): 94–108

42 Goldoni M, Azzalin G, Macino G, Cogoni C (2004). Efficient gene silencing by expression of double stranded RNA in Neurospora crassa. Fungal Genet Biol, 41(11): 1016–1024

43 Goodbourn S, Didcock L, Randall R E (2000). Interferons: cell signalling, immune modulation, antiviral response and virus countermeasures. J Gen Virol, 81(Pt 10): 2341–2364

44 Hamada W, Spanu P D (1998). Co-suppression of the hydrophobin gene HCf-1 is correlated with antisense RNA biosynthesis in Cladosporium fulvum. Mol Gen Genet, 259(6): 630–638

45 Hammond T M, Bok J W, Andrewski M D, Reyes-Domínguez Y, Scazzocchio C, Keller N P (2008). RNA silencing gene truncation in the filamentous fungus Aspergillus nidulans. Eukaryot Cell, 7(2): 339–349

46 Hammond T M, Keller N P (2005). RNA silencing in Aspergillus nidulans is independent of RNA-dependent RNA polymerases. Genetics, 169(2): 607–617

47 Heneghan M N, Costa A M S B, Challen M P, Mills P R, Bailey A, Foster G D (2007). A comparison of methods for successful triggering of gene silencing in Coprinus cinereus. Mol Biotechnol, 35(3): 283–296

48 Himeno M, Maejima K, Komatsu K, Ozeki J, Hashimoto M, Kagiwada S, Yamaji Y, Namba S (2010). Significantly low level of small RNA accumulation derived from an encapsidated mycovirus with dsRNA genome. Virology, 396(1): 69–75

49 Hinas A, Reimegård J, Wagner E G, Nellen W, Ambros V R, Söderbom F (2007). The small RNA repertoire of Dictyostelium discoideum and its regulation by components of the RNAi pathway. Nucleic Acids Res, 35(20): 6714–6726

50 Idnurm A, Howlett B J (2003). Analysis of loss of pathogenicity mutants reveals that repeat-induced point mutations can occur in the Dothideomycete Leptosphaeria maculans. Fungal Genet Biol, 39(1): 31–37

51 Iida T, Nakayama J I, Moazed D (2008). siRNA-mediated heterochromatin establishment requires HP1 and is associated with antisense transcription. Mol Cell, 31: 178–189

52 Irelan J T, Selker E U (1996). Gene silencing in filamentous fungi: RIP, MIP and quelling. J Genet, 75(3): 313–324

53 Jöchl C, Loh E, Ploner A, Haas H, Hüttenhofer A (2009). Development-dependent scavenging of nucleic acids in the filamentous fungus Aspergillus fumigatus. RNA Biol6(2): 178–186

54 Jöchl C, Rederstorff M, Hertel J, Stadler P F, Hofacker I L, Schrettl M, Haas H, Hüttenhofer A (2008). Small ncRNA transcriptome analysis from Aspergillus fumigatus suggests a novel mechanism for regulation of protein synthesis. Nucleic Acids Res, 36(8): 2677–2689

55 Judelson H S, Tani S (2007). Transgene-induced silencing of the zoosporogenesis-specific NIFC gene cluster of Phytophthora infestans involves chromatin alterations. Eukaryot Cell, 6(7): 1200–1209

56 Kadotani N, Murata T, Quoc N B, Adachi Y, Nakayashiki H (2008). Transcriptional control and protein specialization have roles in the functional diversification of two dicer-like proteins in Magnaporthe oryzae. Genetics, 180(2): 1245–1249

57 Kadotani N, Nakayashiki H, Tosa Y, Mayama S (2003). RNA silencing in the phytopathogenic fungus Magnaporthe oryzae. Mol Plant Microbe Interact, 16(9): 769–776

58 Kadotani N, Nakayashiki H, Tosa Y, Mayama S (2004). One of the two Dicer-like proteins in the filamentous fungi Magnaporthe oryzae genome is responsible for hairpin RNA-triggered RNA silencing and related small interfering RNA accumulation. J Biol Chem, 279(43): 44467–44474

59 Kato H, Goto D B, Martienssen R A, Urano T, Furukawa K, Murakami Y (2005). RNA polymerase II is required for RNAi-dependent heterochromatin assembly. Science, 309(5733): 467–469

60 Kemppainen M, Duplessis S, Martin F, Pardo A G (2009). RNA silencing in the model mycorrhizal fungus Laccaria bicolor: gene knock-down of nitrate reductase results in inhibition of symbiosis with Populus. Environ Microbiol, 11(7): 1878–1896

61 Keon J P R, Owen J W, Hargreaves J A (1999). Lack of evidence for antisense suppression in the fungal plant pathogen Ustilago maydis. Antisense Nucleic Acid Drug Dev, 9(1): 101–104

62 Khatri M, Rajam M V (2007). Targeting polyamines of Aspergillus nidulans by siRNA specific to fungal ornithine decarboxylase gene. Med Mycol, 45(3): 211–220

63 Klattenhoff C, Theurkauf W (2008). Biogenesis and germline functions of piRNAs. Development, 135(1): 3–9

64 Lacroix H, Spanu P D (2009). Silencing of six hydrophobins in Cladosporium fulvum: complexities of simultaneously targeting multiple genes. Appl Environ Microbiol, 75(2): 542–546

65 Latijnhouwers M, Govers F (2003). A Phytophthora infestans G-protein β subunit is involved in sporangium formation. Eukaryot Cell, 2(5): 971–977

66 Laurie J D, Linning R, Bakkeren G (2008). Hallmarks of RNA silencing are found in the smut fungus Ustilago hordei but not in its close relative Ustilago maydis. Curr Genet, 53(1): 49–58

67 Lee H C, Chang S S, Choudhary S, Aalto A P, Maiti M, Bamford D H, Liu Y (2009). qiRNA is a new type of small interfering RNA induced by DNA damage. Nature, 459(7244): 274–277

68 Liu H, Cottrell T R, Pierini L M, Goldman W E, Doering T L (2002). RNA interference in the pathogenic fungus Cryptococcus neoformans. Genetics, 160(2): 463–470

69 Malone C D, Hannon G J (2009). Small RNAs as guardians of the genome. Cell, 136(4): 656–668

70 McDonald T, Brown D, Keller N P, Hammond T M (2005). RNA silencing of mycotoxin production in Aspergillus and Fusarium species. Mol Plant Microbe Interact, 18(6): 539–545

71 Molnar A, Bassett A, Thueneman E, Schwach F, Karkare S, Ossowski S, Weigel D, Baulcombe D (2009). Highly specific gene silencing by artificial microRNAs in the unicellular alga Chlamydomonas reinhardtii. Plant J, 58(1): 165–174

72 Molnár A, Schwach F, Studholme D J, Thuenemann E C, Baulcombe D C (2007). miRNAs control gene expression in the single-cell alga Chlamydomonas reinhardtii. Nature, 447(7148): 1126–1129

73 Morin R D, Aksay G, Dolgosheina E, Ebhardt H A, Magrini V, Mardis E R, Sahinalp S C, Unrau P J (2008). Comparative analysis of the small RNA transcriptomes of Pinus contorta and Oryza sativa. Genome Res, 18(4): 571–584

74 Moriwaki A, Ueno M, Arase S, Kihara J (2007). RNA-mediated gene silencing in the phytopathogenic fungus Bipolaris oryzae. FEMS Microbiol Lett, 269(1): 85–89

75 Motamedi M R, Verdel A, Colmenares S U, Gerber S A, Gygi S P, Moazed D (2004). Two RNAi complexes, RITS and RDRC, physically interact and localize to noncoding centromeric RNAs. Cell, 119(6): 789–802

76 Mouyna I, Henry C, Doering T L, Latgé J P (2004). Gene silencing with RNA interference in the human pathogenic fungus Aspergillus fumigatus. FEMS Microbiol Lett, 237(2): 317–324

77 Murata T, Kadotani N, Yamaguchi M, Tosa Y, Mayama S, Nakayashiki H (2007). siRNA-dependent and-independent post-transcriptional cosuppression of the LTR-retrotransposon MAGGY in the phytopathogenic fungus Magnaporthe oryzae. Nucleic Acids Res, 35(18): 5987–5994

78 Nakayashiki H (2005). RNA silencing in fungi: mechanisms and applications. FEBS Lett, 579(26): 5950–5957

79 Nakayashiki H, Hanada S, Nguyen B Q, Kadotani N, Tosa Y, Mayama S (2005). RNA silencing as a tool for exploring gene function in ascomycete fungi. Fungal Genet Biol, 42(4): 275–283

80 Nakayashiki H, Kadotani N, Mayama S (2006). Evolution and diversification of RNA silencing proteins in fungi. J Mol Evol, 63(1): 127–135

81 Nakayashiki H, Nguyen Q B (2008). RNA interference: roles in fungal biology. Curr Opin Microbiol, 11(6): 494–502

82 Namekawa S H, Iwabata K, Sugawara H, Hamada F N, Koshiyama A, Chiku H, Kamada T, Sakaguchi K (2005). Knockdown of LIM15/DMC1 in the mushroom Coprinus cinereus by double-stranded RNA-mediated gene silencing. Microbiology, 151(11): 3669–3678

83 Nicolás F E, de Haro J P, Torres-Martínez S, Ruiz-Vázquez R M (2007). Mutants defective in a Mucor circinelloides dicer-like gene are not compromised in siRNA silencing but display developmental defects. Fungal Genet Biol, 44(6): 504–516

84 Nicolás F E, Torres-Martínez S, Ruiz-Vázquez R M (2003). Two classes of small antisense RNAs in fungal RNA silencing triggered by non-integrative transgenes. EMBO J, 22(15): 3983–3991

85 Nolan T, Cecere G, Mancone C, Alonzi T, Tripodi M, Catalanotto C, Cogoni C (2008). The RNA-dependent RNA polymerase essential for post-transcriptional gene silencing in Neurospora crassa interacts with replication protein A. Nucleic Acids Res, 36(2): 532–538

86 Osborne E A, Dudoit S, Rine J (2009). The establishment of gene silencing at single-cell resolution. Nat Genet, 41(7): 800–806

87 Rappleye C A, Engle J T, Goldman W E (2004). RNA interference in Histoplasma capsulatum demonstrates a role for α-(1,3)-glucan in virulence. Mol Microbiol, 53(1): 153–165

88 Roignant J Y, Carré C, Mugat B, Szymczak D, Lepesant J A, Antoniewski C (2003). Absence of transitive and systemic pathways allows cell-specific and isoform-specific RNAi in Drosophila. RNA, 9(3): 299–308

89 Romano N, Macino G (1992). Quelling: transient inactivation of gene expression in Neurospora crassa by transformation with homologous sequences. Mol Microbiol, 6(22): 3343–3353

90 Saleh M C, van Rij R P, Hekele A, Gillis A, Foley E, O’Farrell P H, Andino R (2006). The endocytic pathway mediates cell entry of dsRNA to induce RNAi silencing. Nat Cell Biol, 8(8): 793–802

91 Schuurs T A, Schaeffer E A M, Wessels J G H (1997). Homology-dependent silencing of the SC3 gene in Schizophyllum commune. Genetics, 147(2): 589–596

92 Segers G C, van Wezel R, Zhang X, Hong Y, Nuss D L (2006). Hypovirus papain-like protease p29 suppresses RNA silencing in the natural fungal host and in a heterologous plant system. Eukaryot Cell, 5(6): 896–904

93 Segers G C, Zhang X, Deng F, Sun Q, Nuss D L (2007). Evidence that RNA silencing functions as an antiviral defense mechanism in fungi. Proc Natl Acad Sci USA, 104(31): 12902–12906

94 Sigova A, Rhind N, Zamore P D (2004). A single Argonaute protein mediates both transcriptional and posttranscriptional silencing in Schizosaccharomyces pombe. Genes Dev, 18(19): 2359–2367

95 Simmer F, Buscaino A, Kos-Braun I C, Kagansky A, Boukaba A, Urano T, Kerr A R W, Allshire R C (2010). Hairpin RNA induces secondary small interfering RNA synthesis and silencing in trans in fission yeast. EMBO Rep, 11(2): 112–118

96 Smith N A, Singh S P, Wang M B, Stoutjesdijk P A, Green A G, Waterhouse P M (2000). Total silencing by intron-spliced hairpin RNAs. Nature, 407(6802): 319–320

97 Sugiyama T, Cam H, Verdel A, Moazed D, Grewal S I S (2005). RNA-dependent RNA polymerase is an essential component of a self-enforcing loop coupling heterochromatin assembly to siRNA production. Proc Natl Acad Sci USA, 102(1): 152–157

98 Sun Q, Choi G H, Nuss D L (2009). A single argonaute gene is required for induction of RNA silencing antiviral defense and promotes viral RNA recombination. Proc Natl Acad Sci USA, 106(42): 17927–17932

99 Timmons L, Fire A (1998). Specific interference by ingested dsRNA. Nature, 395(6705): 854

100 van West P, Kamoun S, van’t Klooster J W, Govers F (1999). Internuclear gene silencing in Phytophthora infestans. Mol Cell, 3(3): 339–348

101 van West P, Shepherd S J, Walker C A, Li S, Appiah A A, Grenville-Briggs L J, Govers F, Gow N A R (2008). Internuclear gene silencing in Phytophthora infestans is established through chromatin remodelling. Microbiology, 154(5): 1482–1490

102 Verdel A, Jia S, Gerber S, Sugiyama T, Gygi S, Grewal S I S, Moazed D (2004). RNAi-mediated targeting of heterochromatin by the RITS complex. Science, 303(5658): 672–676

103 Verdel A, Vavasseur A, Le Gorrec M, Touat-Todeschini L (2009). Common themes in siRNA-mediated epigenetic silencing pathways. Int J Dev Biol, 53(2-3): 245–257

104 Volpe T A, Kidner C, Hall I M, Teng G, Grewal S I S, Martienssen R A (2002). Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi. Science, 297(5588): 1833–1837

105 Wälti M A, Villalba C, Buser R M, Grünler A, Aebi M, Künzler M (2006). Targeted gene silencing in the model mushroom Coprinopsis cinerea (Coprinus cinereus) by expression of homologous hairpin RNAs. Eukaryot Cell, 5(4): 732–744

106 Waterhouse P M, Graham M W, Wang M B (1998). Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proc Natl Acad Sci USA, 95(23): 13959–13964

107 Waterhouse P M, Wang M B, Lough T (2001). Gene silencing as an adaptive defence against viruses. Nature, 411(6839): 834–842

108 Whisson S C, Avrova A O, van West P, Jones J T (2005). A method for double-stranded RNA-mediated transient gene silencing in Phytophthora infestans. Mol Plant Pathol, 6(2): 153–163

109 Winston W M, Molodowitch C, Hunter C P (2002). Systemic RNAi in C. elegans requires the putative transmembrane protein SID-1. Science, 295(5564): 2456–2459

110 Yamada O, Ikeda R, Ohkita Y, Hayashi R, Sakamoto K, Akita O (2007). Gene silencing by RNA interference in the koji mold Aspergillus oryzae. Biosci Biotechnol Biochem, 71(1): 138–144

111 Zhang X, Segers G C, Sun Q, Deng F, Nuss D L (2008). Characterization of hypovirus-derived small RNAs generated in the chestnut blight fungus by an inducible DCL-2-dependent pathway. J Virol, 82(6): 2613–2619

112 Zhao T, Li G, Mi S, Li S, Hannon G J, Wang X J, Qi Y (2007). A complex system of small RNAs in the unicellular green alga Chlamydomonas reinhardtii. Genes Dev, 21(10): 1190–1203

113 Zhao T, Wang W, Bai X, Qi Y (2009). Gene silencing by artificial miRNAs in Chlamydomonas. Plant J, 58: 157–164