#PAGE_PARAMS# #ADS_HEAD_SCRIPTS# #MICRODATA#

Epigenetic Control of Effector Gene Expression in the Plant Pathogenic Fungus


Effectors are key players in pathogenicity of microbes toward plants. Effector genes usually show concerted expression during plant infection but how this concerted expression is generated remains a largely unexplored research topic. Epigenetic mechanisms are involved in genome maintenance and integrity but are increasingly considered as important for regulation of gene expression in numerous and diverse organisms. Here we show that the genomic environment has impact on expression of Leptosphaeria maculans effector genes, and that an epigenetic mechanism that relies on two proteins involved in heterochromatin formation and maintenance, HP1 and DIM-5, modulates this expression, leading to repression during growth in axenic culture. Chromatin decondensation by removal of histone H3 lysine 9 methylation and/or HP1 is presumably a prerequisite for effector gene expression during primary infection of oilseed rape. Thus we show chromatin-based transcriptional regulation that can act on effector gene expression in fungi. Our study highlights the importance of heterochromatic landscapes, not only for genome maintenance but also in rapid and efficient adaptation of organisms to changing environmental situations.


Vyšlo v časopise: Epigenetic Control of Effector Gene Expression in the Plant Pathogenic Fungus. PLoS Genet 10(3): e32767. doi:10.1371/journal.pgen.1004227
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1004227

Souhrn

Effectors are key players in pathogenicity of microbes toward plants. Effector genes usually show concerted expression during plant infection but how this concerted expression is generated remains a largely unexplored research topic. Epigenetic mechanisms are involved in genome maintenance and integrity but are increasingly considered as important for regulation of gene expression in numerous and diverse organisms. Here we show that the genomic environment has impact on expression of Leptosphaeria maculans effector genes, and that an epigenetic mechanism that relies on two proteins involved in heterochromatin formation and maintenance, HP1 and DIM-5, modulates this expression, leading to repression during growth in axenic culture. Chromatin decondensation by removal of histone H3 lysine 9 methylation and/or HP1 is presumably a prerequisite for effector gene expression during primary infection of oilseed rape. Thus we show chromatin-based transcriptional regulation that can act on effector gene expression in fungi. Our study highlights the importance of heterochromatic landscapes, not only for genome maintenance but also in rapid and efficient adaptation of organisms to changing environmental situations.


Zdroje

1. OlivaR, WinJ, RaffaeleS, BoutemyL, BozkurtTO, et al. (2010) Recent developments in effector biology of filamentous plant pathogens. Cell Microbiol 12: 705–715.

2. Tyler BM, Rouxel T (2013) Effectors of fungi and oomycetes: their virulence and avirulence functions and translocation from pathogen to host. In: Molecular Plant Immunity, Guido Sessa (Ed.) John Wiley & Sons, Inc. pp. 123–167.

3. StergiopoulosI, de WitPJGM (2009) Fungal effector proteins. Annu Rev Phytopathol 47: 233–263.

4. KamounS (2006) A catalogue of the effector secretome of plant pathogenic oomycetes. Annu Rev Phytopathol 44: 41–60.

5. HaasBJ, KamounS, ZodyMC, JiangRH, HandsakerRE, et al. (2009) Genome sequence and analysis of the Irish potato famine pathogen Phytophthora infestans. Nature 461: 393–398.

6. KämperJ, KahmannR, BölkerM, MaLJ, BrefortT, et al. (2006) Insights from the genome of the biotrophic fungal plant pathogen Ustilago maydis. Nature 444: 97–101.

7. RouxelT, GrandaubertJ, HaneJK, HoedeC, van de WouwAP, et al. (2011) Effector diversification within compartments of the Leptosphaeria maculans genome affected by Repeat-Induced Point mutations. Nat Commun 2: 202.

8. SaundersDGO, WinJ, CanoLM, SzaboLJ, KamounS, et al. (2012) Using hierarchical clustering of secreted protein families to classify and rank candidate effectors of rust fungi. PLoS One 7: e29847.

9. WangQ, HanC, FerreiraAO, YuX, YeW, et al. (2011) Transcriptional programming and functional interactions within the Phytophthora sojae RXLR effector repertoire. Plant Cell 23: 2064–2086.

10. HacquardS, JolyDL, LinYC, TisserantE, FeauN, et al. (2012) A comprehensive analysis of genes encoding small secreted proteins identifies candidate effectors in Melampsora larici-populina (poplar leaf rust). Mol Plant Microbe Interact 25: 279–293.

11. Giraldo MC, Dagdas YF, Gupta YK, Mentlak TA, Yi M, et al. (2013) Two distinct secretion systems facilitate tissue invasion by the rice blast fungus Magnaporthe oryzae. Nat Commun 4: doi:10.1038/ncomms2996.

12. SchornackS, HuitemaE, CanoLM, BozkurtTO, OlivaR, et al. (2009) Ten things to know about oomycete effectors. Mol Plant Pathol 10: 795–803.

13. SchmidtSM, PanstrugaR (2011) Pathogenomics of fungal plant parasites: what have we learnt about pathogenesis? Curr Opin Plant Biol 14: 392–399.

14. BalesdentMH, FudalI, OllivierB, BallyP, GrandaubertJ, et al. (2013) The dispensable chromosome of Leptosphaeria maculans shelters an effector gene conferring avirulence towards Brassica rapa. New Phytol 198: 887–898.

15. OrbachMJ, FarrallL, SweigardJA, ChumleyFG, ValentB (2000) A telomeric avirulence gene determines efficacy for the rice blast resistance gene Pi-ta. Plant Cell 12: 2019–2032.

16. MaLJ, van der DoesHC, BorkovichKA, ColemanJJ, DaboussiMJ, et al. (2010) Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium. Nature 464: 367–373.

17. RaffaeleS, FarrerRA, CanoLM, StudholmeDJ, MacLeanD, et al. (2010) Genome evolution following host jumps in the Irish potato famine pathogen lineage. Science 330: 1540–1543.

18. KasugaT, KozanitasM, BuiM, HüberliD, RizzoDM, et al. (2012) Phenotypic diversification is associated with host-induced transposon derepression in the sudden oak death pathogen Phytophthora ramorum. PLoS One 7: e34728.

19. GoutL, FudalI, KuhnML, BlaiseF, EckertM, et al. (2006) Lost in the middle of nowhere: the AvrLm1 avirulence gene of the Dothideomycete Leptosphaeria maculans. Mol Microbiol 60: 67–80.

20. FudalI, RossS, GoutL, BlaiseF, KuhnML, et al. (2007) Heterochromatin-like regions as ecological niches for avirulence genes in the Leptosphaeria maculans genome: map-based cloning of AvrLm6. Mol Plant Microbe Interact 20: 459–470.

21. ParlangeF, DaverdinG, FudalI, KuhnML, BalesdentMH, et al. (2009) Leptosphaeria maculans avirulence gene AvrLm4-7 confers a dual recognition specificity by the Rlm4 and Rlm7 resistance genes of oilseed rape, and circumvents Rlm4-mediated recognition through a single amino acid change. Mol Microbiol 71: 851–863.

22. GoutL, KuhnML, VincenotL, Bernard-SamainS, CattolicoL, et al. (2007) Genome structure impacts molecular evolution at the AvrLm1 avirulence locus of the plant pathogen Leptosphaeria maculans. Environ Microbiol 9: 2978–2992.

23. FudalI, RossS, BrunH, BesnardAL, ErmelM, et al. (2009) Repeat-Induced Point mutation (RIP) as an alternative mechanism of evolution toward virulence in Leptosphaeria maculans. Mol Plant Microbe Interact 22: 932–941.

24. GrewalSI, JiaS (2007) Heterochromatin revisited. Nat Rev Genet 8: 35–46.

25. TurnerBM (2000) Histone acetylation and an epigenetic code. Bioessays 22: 836–845.

26. JenuweinT, AllisCD (2001) Translating the histone code. Science 293: 1074–1080.

27. JamiesonK, RountreeMR, LewisZA, StajichJE, SelkerEU (2013) Regional control of histone H3 lysine 27 methylation in Neurospora. Proc Natl Acad Sci U S A 110: 6027–6032.

28. TamaruH, SelkerEU (2001) A histone H3 methyltransferase controls DNA methylation in Neurospora crassa. Nature 414: 277–283.

29. TamaruH, ZhangX, McMillenD, SinghPB, NakayamaJ, et al. (2003) Trimethylated lysine 9 of histone H3 is a mark for DNA methylation in Neurospora crassa. Nat Genet 34: 75–79.

30. FreitagM, HickeyPC, KhlafallahTK, ReadND, SelkerEU (2004) HP1 is essential for DNA methylation in Neurospora. Mol Cell 13: 427–434.

31. HondaS, SelkerEU (2008) Direct interaction between DNA methyltransferase DIM-2 and HP1 is required for DNA methylation in Neurospora crassa. Mol Cell Biol 28: 6044–6055.

32. RountreeMR, SelkerEU (2010) DNA methylation and the formation of heterochromatin in Neurospora crassa. Heredity 105: 38–44.

33. HiragamiK, FestensteinR (2005) Heterochromatin Protein 1: a pervasive controlling influence. Cell Mol Life Sci 62: 2711–2726.

34. TschierschB, HofmannA, KraussV, DornR, KorgeG, et al. (1994) The protein encoded by the Drosophila position-effect variegation suppressor gene Su(var)3-9 combines domains of antagonistic regulators of homeotic gene complexes. EMBO J 13: 3822–3831.

35. ZhangX, TamaruH, KhanSI, HortonJR, KeefeLJ, et al. (2002) Structure of the Neurospora SET domain protein DIM-5, a histone H3 lysine methyltransferase. Cell 111: 117–127.

36. ReaS, EisenhaberF, O'CarrollD, StrahlBD, SunZW, et al. (2000) Regulation of chromatin structure by site-specific histone H3 methyltransferases. Nature 406: 593–599.

37. IvanovaAV, BonaduceMJ, IvanovSV, KlarAJ (1998) The chromo and SET domains of the Clr4 protein are essential for silencing in fission yeast. Nat Genet 19: 192–195.

38. YinW, KellerNP (2011) Transcriptional regulatory elements in fungal secondary metabolism. J Microbiol 49: 329–339.

39. SmithKM, SancarG, DekhangR, SullivanCM, LiS, et al. (2010) Transcription factors in light and circadian clock signaling networks revealed by genome wide mapping of direct targets for Neurospora White Collar complex. Eukaryotic Cell 9: 1549–1556.

40. SmithKM, PhatalePA, SullivanCM, PomraningKR, FreitagM (2011) Heterochromatin is required for normal distribution of Neurospora CenH3. Mol Cell Biol 31: 2528–2542.

41. SeibothB, Karimi-AghchehR, PhatalePA, LinkeR, SauerDG, et al. (2012) The putative protein methyltransferase LAE1 controls 1 cellulase gene expression in Trichoderma reesei. Mol Microbiol 84: 1150–1164.

42. Karimi-AghchehR, BokJW, PhatalePA, SmithKM, BakerSE, et al. (2013) Functional analyses of Trichoderma reesei LAE1 reveal conserved and contrasting roles of this regulator. G3 (Bethesda) 3: 369–378.

43. WiemannP, SieberCM, von BargenKW, StudtL, NiehausEM, et al. (2013) Deciphering the cryptic genome: genome-wide analyses of the rice pathogen Fusarium fujikuroi reveal complex regulation of secondary metabolism and novel metabolites. PLoS Pathog 9: e1003475.

44. ConnollyLR, SmithKM, FreitagM (2013) The Fusarium graminearum Histone H3 K27 methyltransferase KMT6 regulates development and expression of secondary metabolite gene clusters. PLoS Genet 9: e1003916.

45. MikkelsenTS, KuM, JaffeDB, IssacB, LiebermanE, et al. (2007) Genome-wide maps of chromatin state in pluripotent and lineage-committed cells. Nature 448: 553–560.

46. BourrasS, MeyerM, GrandaubertJ, LapaluN, FudalI, et al. (2012) Incidence of genome structure, DNA asymmetry, and cell physiology on T-DNA integration in chromosomes of the phytopathogenic fungus Leptosphaeria maculans. G3 (Bethesda) 2: 891–904.

47. DaverdinG, RouxelT, GoutL, AubertotJN, FudalI, et al. (2012) Genome structure and reproductive behaviour influence the evolutionary potential of a fungal phytopathogen. PLoS Pathog 8: e1003020.

48. ChengX, CollinsRE, ZhangX (2005) Structural and sequence motifs of protein (histone) methylation enzymes. Annu Rev Biophys Biomol Struct 34: 267–294.

49. PalmerJM, PerrinRM, DagenaisTR, KellerNP (2008) H3K9 methylation regulates growth and development in Aspergillus fumigatus. Eukaryot Cell 7: 2052–2060.

50. FischleW, WangY, JacobsSA, KimY, AllisCD, et al. (2003) Molecular basis for the discrimination of repressive methyl-lysine marks in histone H3 by Polycomb and HP1 chromodomains. Genes Dev 17: 1870–1881.

51. TurckF, RoudierF, FarronaS, Martin-MagnietteML, GuillaumeE, et al. (2007) Arabidopsis TFL2/LHP1 specifically associates with genes marked by trimethylation of Histone H3 Lysine 27. PLoS Genet 3: 855–866.

52. ZhangX, GermannS, BlusBJ, KhorasanizadehS, GaudinV, et al. (2007) The Arabidopsis LHP1 protein colocalizes with histone H3 Lys27 trimethylation. Nat Struct Mol Biol 14: 869–871.

53. ExnerV, AichingerE, ShuH, WildhaberT, AlfaranoP, et al. (2009) The chromodomain of LIKE HETEROCHROMATIN PROTEIN 1 is essential for H3K27me3 binding and function during Arabidopsis development. PLoS One 4: e5335.

54. NielsenPR, NietlispachD, MottHR, CallaghanJ, BannisterA, et al. (200) Structure of the HP1 chromodomain bound to histone H3 methylated at lysine 9. Nature 416: 103–107.

55. DinantC, LuijsterburgMS (2009) The emerging role of HP1 in the DNA damage response. Mol Cell Biol 29: 6335–6340.

56. FischerT, CuiB, DhakshnamoorthyJ, ZhouM, RubinC, et al. (2009) Diverse roles of HP1 proteins in heterochromatin assembly and functions in fission yeast. Proc Natl Acad Sci U S A 106: 8998–9003.

57. KangJ, ChaudharyJ, DongH, KimS, BrautigamCA, et al. (2011) Mitotic centromeric targeting of HP1 and its binding to Sgo1 are dispensable for sister-chromatid cohesion in human cells. Mol Biol Cell 22: 1181–1190.

58. Reyes-DominguezY, BoediS, SulyokM, WiesenbergerG, StoppacherN, et al. (2012) Heterochromatin influences the secondary metabolite profile in the plant pathogen Fusarium graminearum. Fungal Genet Biol 49: 39–47.

59. Reyes-DominguezY, BokJW, BergerH, ShwabEK, BasheerA, et al. (2010) Heterochromatic marks are associated with the repression of secondary metabolism clusters in Aspergillus nidulans. Mol Microbiol 76: 1376–1386.

60. EissenbergJC, HartnettT (1993) A heat shock-activated cDNA rescues the recessive lethality of mutations in the heterochromatin-associated protein HP1 of Drosophila melanogaster. Mol Gen Genet 240: 333–338.

61. LuBY, EmtagePC, DuyfBJ, HillikerAJ, EissenbergJC (2000) Heterochromatin protein 1 is required for the normal expression of two heterochromatin genes in Drosophila. Genetics 155: 699–708.

62. HaldarS, SainiA, NandaJS, SainiS, SinghJ, et al. (2011) Role of Swi6/HP1 self-association-mediated recruitment of Clr4/Suv39 in establishment and maintenance of heterochromatin in fission yeast. J Biol Chem 286: 9308–9320.

63. LewisZA, AdhvaryuKK, HondaS, ShiverAL, KnipM, et al. (2010) DNA methylation and normal chromosome behavior in Neurospora depend on five components of a histone methyltransferase complex, DCDC. PLoS Genet 6: e1001196.

64. WoellmerA, Arteaga-SalasJM, HammerschmidtW (2012) BZLF1 governs CpG-methylated chromatin of Epstein-Barr Virus reversing epigenetic repression. PLoS Pathog 8: e1002902.

65. FlueckC, BartfaiR, NiederwieserI, WitmerK, AlakoBTF, et al. (2010) A major role for the Plasmodium falciparum ApiAP2 protein PfSIP2 in chromosome end biology. PLoS Pathog 6: e1000784.

66. KellerNP, HohnTM (1997) Metabolic pathway gene clusters in filamentous fungi. Fungal Genet Biol 21: 17–29.

67. FernandesM, KellerNP, AdamsTH (1998) Sequence-specific binding by Aspergillus nidulans AflR, a C6 zinc cluster protein regulating mycotoxin biosynthesis. Mol Microbiol 28: 1355–1365.

68. MichielseCB, van WijkR, ReijnenL, MandersEM, BoasS, et al. (2009) The nuclear protein Sge1 of Fusarium oxysporum is required for parasitic growth. PLoS Pathog 5: e1000637.

69. ZahiriA, HeimelK, WahlR, RathM, KämperJ (2010) The Ustilago maydis forkhead transcription factor Fox1 is involved in the regulation of genes required for the attenuation of plant defenses during pathogenic development. Mol Plant Microbe Interact 23: 1118–1129.

70. WaddingtonCH (1942) The epigenotype. Endeavour 1: 18–20.

71. KnipeDM, CliffeA (2008) Chromatin control of herpes simplex virus lytic and latent infection. Nat Rev Microbiol 6: 211–221.

72. LanzerM, FischerK, Le BlancqSM (1995) Parasitism and chromosome dynamics in protozoan parasites: is there a connection? Mol Biochem Parasit 70: 1–8.

73. Freitas-JuniorLH, Hernandez-RivasR, RalphSA, Montiel-CondadoD, Ruvalcaba-SalazarOK, et al. (2005) Telomeric heterochromatin propagation and histone acetylation control mutually exclusive expression of antigenic variation genes in malaria parasites. Cell 121: 25–36.

74. MerrickCJ, DuraisinghMT (2006) Heterochromatin-mediated control of virulence gene expression. Mol Microbiol 62: 612–620.

75. SmithDJ, BurnhamMK, BullJH, HodgsonJE, WardJM, et al. (1990) Beta-lactam antibiotic biosynthetic genes have been conserved in clusters in prokaryotes and eukaryotes. EMBO J 9: 741–747.

76. BokJW, KellerNP (2004) LaeA, a regulator of secondary metabolism in Aspergillus spp. Eukaryot Cell 3: 527–535.

77. PalmerJM, KellerNP (2010) Secondary metabolism in fungi: does chromosomal location matter? Curr Opin Microbiol 13: 431–436.

78. Broggini GAL (2007) Identification of apple scab avirulence gene AvrVg candidates. PhD Thesis. University of Zurich 112 pp.

79. de WitPJGM, van der BurgtA, OkmenB, StergiopoulosI, Abd-ElsalamKA, et al. (2012) The genomes of the fungal plant pathogens Cladosporium fulvum and Dothistroma septosporum reveal adaptation to different hosts and lifestyles but also signatures of common ancestry. PLoS Genet 8: e1003088.

80. ColemanJJ, RounsleySD, Rodriguez-CarresM, KuoA, WasmannCC, et al. (2009) The genome of Nectria haematococca: contribution of supernumerary chromosomes to gene expansion. PLoS Genet 5: e1000618.

81. PlettJM, MartinF (2012) Poplar root exudates contain compounds that induce the expression of MiSSP7 in Laccaria bicolor. Plant Signal Behav 7: 12–15.

82. BrakhageAA (2012) Regulation of fungal secondary metabolism. Nat Rev Microbiol 11: 21–32.

83. ThommaBP, BoltonMD, ClergeotPH, De WitPJGM (2006) Nitrogen controls in planta expression of Cladosporium fulvum Avr9 but no other effector genes. Mol Plant Pathol 7: 125–130.

84. BalesdentMH, AttardA, Ansan-MelayahD, DelourmeR, RenardM, et al. (2001) Genetic control and host range of avirulence toward Brassica napus cultivars Quinta and Jet Neuf in Leptosphaeria maculans. Phytopathology 91: 70–76.

85. MullinsED, ChenX, RomaineP, RainaR, GeiserDM, et al. (2001) Agrobacterium-Mediated Transformation of Fusarium oxysporum: an efficient tool for insertional mutagenesis and gene transfer. Phytopathology 91: 173–180.

86. GardinerDM, HowlettBJ (2004) Negative selection using thymidine kinase increases the efficiency of recovery of transformants with targeted genes in the filamentous fungus Leptosphaeria maculans. Curr Genet 45: 249–255.

87. Ansan-MelayahD, BalesdentMH, BueeM, RouxelT (1995) Genetic characterization of AvrLm1, the first avirulence gene of Leptosphaeria maculans. Phytopathology 85: 1525–1529.

88. BlaiseF, RémyE, MeyerM, ZhouL, NarcyJP, et al. (2007) A critical assessment of Agrobacterium tumefaciens-mediated transformation as a tool for pathogenicity gene discovery in the phytopathogenic fungus Leptosphaeria maculans. Fungal Genet Biol 44: 123–138.

89. BalesdentMH, LouvardK, PinochetX, RouxelT (2006) A large scale survey of races of Leptosphaeria maculans occurring on oilseed rape in France. Eur J Plant Pathol 114: 53–65.

90. GallC, BalesdentMH, RobinP, RouxelT (1994) Tetrad analysis of acid phosphatase, soluble protein patterns, and mating type in Leptosphaeria maculans. Phytopathology 84: 1299–1305.

91. AttardA, GoutL, RossS, ParlangeF, CattolicoL, et al. (2005) Truncated and RIP-degenerated copies of the LTR retrotransposon Pholy are clustered in a pericentromeric region of the Leptosphaeria maculans genome. Fungal Genet Biol 42: 30–41.

92. Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, U.S.A.

93. LiuYG, MitsukawaN, OosumiT, WhittierRF (1995) Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR. Plant J 8: 457–463.

94. MullerPY, JanovjakH, MiserezAR, DobbieZ (2002) Processing of gene expression data generated by quantitative real-time RT-PCR. BioTechniques 32: 1372–1379.

95. BolstadBM, IrizarryRA, AstrandM, SpeedTP (2003) A comparison of normalization methods for high density oligonucleotide array data based on variance and bias. Bioinformatics 19: 185–193.

96. IrizarryRA, HobbsB, CollinF, Beazer-BarclayYD, AntonellisKJ, et al. (2003) Exploration, normalization, and summaries of high density oligonucleotide array probe level data. Biostatistics 4: 249–264.

97. SimonA, BiotE (2010) ANAIS: analysis of NimbleGen arrays interface. Bioinformatics 26: 2468–2469.

98. FitzgeraldA, Van KanJA, PlummerKM (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: 963–971.

99. de GrootMJ, BundockP, HooykaasPJ, BeijersbergenAG (1998) Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nat Biotechnol 16: 839–842.

100. AltschulSF, GishW, MillerW, MyersEW, LipmanDJ (1990) Basic local alignment search tool. J Mol Biol 215: 403–410.

101. SturnA, QuackenbushJ, TrajanoskiZ (2002) Genesis: cluster analysis of microarray data. Bioinformatics 18: 207–208.

102. PapadopoulosJS, AgarwalaR (2007) COBALT: constraint-based alignment tool for multiple protein sequences. Bioinformatics 23: 1073–1079.

Štítky
Genetika Reprodukčná medicína

Článok vyšiel v časopise

PLOS Genetics


2014 Číslo 3
Najčítanejšie tento týždeň
Najčítanejšie v tomto čísle
Kurzy

Zvýšte si kvalifikáciu online z pohodlia domova

Získaná hemofilie - Povědomí o nemoci a její diagnostika
nový kurz

Eozinofilní granulomatóza s polyangiitidou
Autori: doc. MUDr. Martina Doubková, Ph.D.

Všetky kurzy
Prihlásenie
Zabudnuté heslo

Zadajte e-mailovú adresu, s ktorou ste vytvárali účet. Budú Vám na ňu zasielané informácie k nastaveniu nového hesla.

Prihlásenie

Nemáte účet?  Registrujte sa

#ADS_BOTTOM_SCRIPTS#