Understanding how genomes encode complex cellular and organismal behaviors has become the outstanding challenge of modern genetics. Unlike classical screening methods, analysis of genetic variation that occurs naturally in wild populations can enable rapid, genome-scale mapping of genotype to phenotype with a medium-throughput experimental design. Here we describe the results of the first genome-wide association study (GWAS) used to identify novel loci underlying trait variation in a microbial eukaryote, harnessing wild isolates of the filamentous fungus Neurospora crassa. We genotyped each of a population of wild Louisiana strains at 1 million genetic loci genome-wide, and we used these genotypes to map genetic determinants of microbial communication. In N. crassa, germinated asexual spores (germlings) sense the presence of other germlings, grow toward them in a coordinated fashion, and fuse. We evaluated germlings of each strain for their ability to chemically sense, chemotropically seek, and undergo cell fusion, and we subjected these trait measurements to GWAS. This analysis identified one gene, NCU04379 (cse-1, encoding a homolog of a neuronal calcium sensor), at which inheritance was strongly associated with the efficiency of germling communication. Deletion of cse-1 significantly impaired germling communication and fusion, and two genes encoding predicted interaction partners of CSE1 were also required for the communication trait. Additionally, mining our association results for signaling and secretion genes with a potential role in germling communication, we validated six more previously unknown molecular players, including a secreted protease and two other genes whose deletion conferred a novel phenotype of increased communication and multi-germling fusion. Our results establish protein secretion as a linchpin of germling communication in N. crassa and shed light on the regulation of communication molecules in this fungus. Our study demonstrates the power of population-genetic analyses for the rapid identification of genes contributing to complex traits in microbial species.
Vyšlo v časopise: Genome Wide Association Identifies Novel Loci Involved in Fungal Communication. PLoS Genet 9(8): e32767. doi:10.1371/journal.pgen.1003669 Kategorie: Research Article prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1003669
1. Read ND, Fleissner A, Roca MG, Glass NL (2010) Hyphal fusion. In: Cellular and Molecular Biology of Filamentous Fungi. Borkovich KA and Ebbole DJ, editors. Washington, D.C.: ASM Press. pp. 260–273.
2. SchmitJC, BrodyS (1976) Biochemical genetics of Neurospora crassa conidial germination. Bacteriol Revs 40 : 1–41.
3. RocaMG, ArltJ, JeffreeCE, ReadND (2005) Cell biology of conidial anastomosis tubes in Neurospora crassa. Eukaryot Cell 4 : 911–919.
4. RocaGM, ReadND, WhealsAE (2005) Conidial anastomosis tubes in filamentous fungi. FEMS Microbiol Letts 249 : 191–198.
5. SimoninA, Palma-GuerreroJ, FrickerM, GlassNL (2012) The physiological significance of network organization in fungi. Eukaryot Cell 11 : 1345–1352.
6. RoperM, SimoninA, HickeyPC, LeederA, GlassNL (2013) Nuclear dynamics in a fungal chimera. Proc Natl Acad Sci USA (in revision).
7. FleissnerA, SimoninAR, GlassNL (2008) Cell fusion in the filamentous fungus, Neurospora crassa. Methods Mol Biol 475 : 21–38.
8. WrightGD, ArltJ, PoonWC, ReadND (2007) Optical tweezer micromanipulation of filamentous fungi. Fungal Genet Biol 44 : 1–13.
9. ReadND, GoryachevAB, LichiusA (2012) The mechanistic basis of self-fusion between conidial anastomosis tubes during fungal colony initiation. Fungal Biol Rev 26 : 1–11.
10. PandeyA, RocaMG, ReadND, GlassNL (2004) Role of a mitogen-activated protein kinase pathway during conidial germination and hyphal fusion in Neurospora crassa. Eukaryot Cell 3 : 348–358.
11. MaerzS, ZivC, VogtN, HelmstaedtK, CohenN, et al. (2008) The nuclear Dbf2-related kinase COT1 and the mitogen-activated protein kinases MAK1 and MAK2 genetically interact to regulate filamentous growth, hyphal fusion and sexual development in Neurospora crassa. Genetics 179 : 1313–1325.
12. DettmannA, IllgenJ, MarzS, SchurgT, FleissnerA, et al. (2012) The NDR kinase scaffold HYM1/MO25 is essential for MAK2 map kinase signaling in Neurospora crassa. PLoS Genet 8: e1002950.
13. FleissnerA, SarkarS, JacobsonDJ, RocaMG, ReadND, et al. (2005) The so locus is required for vegetative cell fusion and postfertilization events in Neurospora crassa. Eukaryot Cell 4 : 920–930.
14. FleissnerA, LeederAC, RocaMG, ReadND, GlassNL (2009) Oscillatory recruitment of signaling proteins to cell tips promotes coordinated behavior during cell fusion. Proc Natl Acad Sci U S A 106 : 19387–19392.
15. GoryachevAB, LichiusA, WrightGD, ReadND (2012) Excitable behavior can explain the “ping-pong” mode of communication between cells using the same chemoattractant. BioEssays 34 : 259–266.
16. ReadND, LichiusA, ShojiJY, GoryachevAB (2009) Self-signalling and self-fusion in filamentous fungi. Curr Opin Microbiol 12 : 608–615.
17. PerkinsDD, TurnerBC, BarryEG (1976) Strains of Neurospora collected from nature. Evolution 30 : 281–313.
18. TurnerE, JacobsonDJ, TaylorJW (2010) Reinforced postmating reproductive isolation barriers in Neurospora, an Ascomycete microfungus. J Evol Biol 23 : 1642–1656.
19. DettmanJR, JacobsonDJ, TurnerE, PringleA, TaylorJW (2003) Reproductive isolation and phylogenetic divergence in Neurospora: comparing methods of species recognition in a model eukaryote. Evolution 57 : 2721–2741.
20. DettmanJR, JacobsonDJ, TaylorJW (2003) A multilocus genealogical approach to phylogenetic species recognition in the model eukaryote Neurospora. Evolution 57 : 2703–2720.
21. EllisonCE, HallC, KowbelD, WelchJ, BremRB, et al. (2011) Population genomics and local adaptation in wild isolates of a model microbial eukaryote. Proc Natl Acad Sci U S A 108 : 2831–2836.
22. MagwireMM, FabianDK, SchweyenH, CaoC, LongdonB, et al. (2012) Genome-wide association studies reveal a simple genetic basis of resistance to naturally coevolving viruses in Drosophila melanogaster. PLoS Genet 8: e1003057.
23. WeberAL, KhanGF, MagwireMM, TaborCL, MackayTF, et al. (2012) Genome-wide association analysis of oxidative stress resistance in Drosophila melanogaster. PLoS One 7: e34745.
24. MackayTF, RichardsS, StoneEA, BarbadillaA, AyrolesJF, et al. (2012) The Drosophila melanogaster genetic reference panel. Nature 482 : 173–178.
25. BrachiB, FaureN, HortonM, FlahauwE, VazquezA, et al. (2010) Linkage and association mapping of Arabidopsis thaliana flowering time in nature. PLoS Genet 6: e1000940.
26. ChanEK, RoweHC, CorwinJA, JosephB, KliebensteinDJ (2011) Combining genome-wide association mapping and transcriptional networks to identify novel genes controlling glucosinolates in Arabidopsis thaliana. PLoS Biol 9: e1001125.
27. AtwellS, HuangYS, VilhjalmssonBJ, WillemsG, HortonM, et al. (2010) Genome-wide association study of 107 phenotypes in Arabidopsis thaliana inbred lines. Nature 465 : 627–631.
28. FiliaultDL, MaloofJN (2012) A genome-wide association study identifies variants underlying the Arabidopsis thaliana shade avoidance response. PLoS Genet 8: e1002589.
29. MandelJR, NambeesanS, BowersJE, MarekLF, EbertD, et al. (2013) Association mapping and the genomic consequences of selection in sunflower. PLoS Genet 9: e1003378.
30. LitiG, LouisEJ (2012) Advances in quantitative trait analysis in yeast. PLoS Genet 8: e1002912.
31. Foulongne-OriolM (2012) Genetic linkage mapping in fungi: current state, applications, and future trends. Appl Microbiol Biotechnol 95 : 891–904.
32. HendricksKB, WangBQ, SchniedersEA, ThornerJ (1999) Yeast homologue of neuronal frequenin is a regulator of phosphatidylinositol-4-OH kinase. Nat Cell Biol 1 : 234–241.
33. DekaR, KumarR, TamuliR (2011) Neurospora crassa homologue of Neuronal Calcium Sensor-1 has a role in growth, calcium stress tolerance, and ultraviolet survival. Genetica 139 : 885–894.
34. BurgoyneRD (2007) Neuronal calcium sensor proteins: generating diversity in neuronal Ca2+ signalling. Nat Rev Neurosci 8 : 182–193.
35. TamuliR, KumarR, DekaR (2011) Cellular roles of neuronal calcium sensor-1 and calcium/calmodulin-dependent kinases in fungi. J Basic Microbiol 51 : 120–128.
36. GromadaJ, BarkC, SmidtK, EfanovAM, JansonJ, et al. (2005) Neuronal calcium sensor-1 potentiates glucose-dependent exocytosis in pancreatic beta cells through activation of phosphatidylinositol 4-kinase beta. Proc Natl Acad Sci U S A 102 : 10303–10308.
37. Kapp-BarneaY, MelnikovS, SheflerI, JerominA, Sagi-EisenbergR (2003) Neuronal calcium sensor-1 and phosphatidylinositol 4-kinase beta regulate IgE receptor-triggered exocytosis in cultured mast cells. J Immunol 171 : 5320–5327.
38. StrahlT, HamaH, DeWaldDB, ThornerJ (2005) Yeast phosphatidylinositol 4-kinase, Pik1, has essential roles at the Golgi and in the nucleus. J Cell Biol 171 : 967–979.
39. ConibearE, StevensTH (2000) Vps52p, Vps53p, and Vps54p form a novel multisubunit complex required for protein sorting at the yeast late Golgi. Mol Biol Cell 11 : 305–323.
40. BowmanBJ, DraskovicM, FreitagM, BowmanEJ (2009) Structure and distribution of organelles and cellular location of calcium transporters in Neurospora crassa. Eukaryot Cell 8 : 1845–1855.
41. BridgesD, MoorheadGB (2005) 14-3-3 proteins: a number of functions for a numbered protein. Sci STKE 2005: re10.
42. DemmelL, BeckM, KloseC, SchlaitzAL, GloorY, et al. (2008) Nucleocytoplasmic shuttling of the Golgi phosphatidylinositol 4-kinase Pik1 is regulated by 14-3-3 proteins and coordinates Golgi function with cell growth. Mol Biol Cell 19 : 1046–1061.
43. RadfordA (2004) Metabolic highways of Neurospora crassa revisited. Adv Genet 52 : 165–207.
44. BourneY, DannenbergJ, PollmannV, MarchotP, PongsO (2001) Immunocytochemical localization and crystal structure of human frequenin (neuronal calcium sensor 1). J Biol Chem 276 : 11949–11955.
45. De CastroE, NefS, FiumelliH, LenzSE, KawamuraS, et al. (1995) Regulation of rhodopsin phosphorylation by a family of neuronal calcium sensors. Biochem Biophys Res Commun 216 : 133–140.
46. DasonJS, Romero-PozueloJ, MarinL, IyengarBG, KloseMK, et al. (2009) Frequenin/NCS-1 and the Ca2+-channel alpha1-subunit co-regulate synaptic transmission and nerve-terminal growth. J Cell Sci 122 : 4109–4121.
47. MahsA, IschebeckT, HeiligY, StenzelI, HempelF, et al. (2012) The essential phosphoinositide kinase MSS-4 Is required for polar hyphal morphogenesis, localizing to sites of growth and cell fusion in Neurospora crassa. PloS One 7: e51454.
48. CappellSD, DohlmanHG (2011) Selective regulation of MAP kinase signaling by an endomembrane phosphatidylinositol 4-kinase. J Biol Chem 286 : 14852–14860.
49. TerBushDR, MauriceT, RothD, NovickP (1996) The Exocyst is a multiprotein complex required for exocytosis in Saccharomyces cerevisiae. EMBO J 15 : 6483–6494.
50. NewmanAP, ShimJ, Ferro-NovickS (1990) BET1, BOS1, and SEC22 are members of a group of interacting yeast genes required for transport from the endoplasmic reticulum to the Golgi complex. Mol Cell Biol 10 : 3405–3414.
51. KaurH, GanguliD, BachhawatAK (2012) Glutathione degradation by the alternative pathway (DUG pathway) in Saccharomyces cerevisiae is initiated by (Dug2p-Dug3p)2 complex, a novel glutamine amidotransferase (GATase) enzyme acting on glutathione. J Biol Chem 287 : 8920–8931.
52. PocsiI, PradeRA, PenninckxMJ (2004) Glutathione, altruistic metabolite in fungi. Adv Microb Physiol 49 : 1–76.
53. PrigentM, Boy-MarcotteE, ChesneauL, GibsonK, Dupre-CrochetS, et al. (2011) The RabGAP proteins Gyp5p and Gyl1p recruit the BAR domain protein Rvs167p for polarized exocytosis. Traffic 12 : 1084–1097.
54. LombardiR, RiezmanH (2001) Rvs161p and Rvs167p, the two yeast amphiphysin homologs, function together in vivo. J Biol Chem 276 : 6016–6022.
55. De AntoniA, SchmitzovaJ, TrepteHH, GallwitzD, AlbertS (2002) Significance of GTP hydrolysis in Ypt1p-regulated endoplasmic reticulum to Golgi transport revealed by the analysis of two novel Ypt1-GAPs. J Biol Chem 277 : 41023–41031.
56. RawlingsND, BarrettAJ (1994) Families of serine peptidases. Methods Enzymol 244 : 19–61.
57. BardwellL (2005) A walk-through of the yeast mating pheromone response pathway. Peptides 26 : 339–350.
58. KimH, BorkovichKA (2006) Pheromones are essential for male fertility and sufficient to direct chemotropic polarized growth of trichogynes during mating in Neurospora crassa. Eukaryot Cell 5 : 544–554.
59. TheisT, WeddeM, MeyerV, StahlU (2003) The antifungal protein from Aspergillus giganteus causes membrane permeabilization. Antimicrob Agents Chemother 47 : 588–593.
60. LeiterE, SzappanosH, OberparleiterC, KaisererL, CsernochL, et al. (2005) Antifungal protein PAF severely affects the integrity of the plasma membrane of Aspergillus nidulans and induces an apoptosis-like phenotype. Antimicrob Agents Chemother 49 : 2445–2453.
61. BorkovichKA, AlexLA, YardenO, FreitagM, TurnerGE, et al. (2004) Lessons from the genome sequence of Neurospora crassa: tracing the path from genomic blueprint to multicellular organism. Microbiol Mol Biol Rev 68 : 1–108.
62. JonesCA, Greer-PhillipsSE, BorkovichKA (2007) The response regulator RRG-1 functions upstream of a mitogen-activated protein kinase pathway impacting asexual development, female fertility, osmotic stress, and fungicide resistance in Neurospora crassa. Mol Biol Cell 18 : 2123–2136.
63. SaitoH, TatebayashiK (2004) Regulation of the osmoregulatory HOG MAPK cascade in yeast. J Biochem 136 : 267–272.
64. ConnellyCF, AkeyJM (2012) On the prospects of whole-genome association mapping in Saccharomyces cerevisiae. Genetics 191 : 1345–1353.
65. DunlapJC, BorkovichKA, HennMR, TurnerGE, SachsMS, et al. (2007) Enabling a community to dissect an organism: Overview of the Neurospora functional genomics project. Adv Genet 57 : 49–96.
66. ZhuC, GoreM, BucklerES, YuJ (2008) Status and prospects of association mapping in plants. Plant Genome 1 : 5–20.
67. TurnerE, JacobsonDJ, TaylorJW (2011) Genetic architecture of a reinforced, postmating, reproductive isolation barrier between Neurospora species indicates evolution via natural selection. PLoS Genet 7: e1002204.
68. McCluskeyK, WiestA, PlamannM (2010) The Fungal Genetics Stock Center: a repository for 50 years of fungal genetics research. J Biosci 35 : 119–126.
69. ColotHV, ParkG, TurnerGE, RingelbergC, CrewCM, et al. (2006) A high-throughput gene knockout procedure for Neurospora reveals functions for multiple transcription factors. Proc Natl Acad Sci USA 103 : 10352–10357.
70. McCluskeyK (2003) The fungal genetics stock center: From molds to molecules. Adv Appl Microbiol 52 : 245–262.
71. VogelHJ (1956) A convenient growth medium for Neurospora. Microbial Genetics Bulletin 13 : 42–46.
72. WestergaardM, MitchellHK (1947) Neurospora V. A synthetic medium favoring sexual reproduction. Amer J Bot 34 : 573–577.
73. MetzenbergRL (2004) Bird Medium: an alternative to Vogel Medium. Fungal Genet Newslett 51 : 19–20.
74. ChomczynskiP, SacchiN (1987) Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162 : 156–159.
75. BrowneKA (2002) Metal ion-catalyzed nucleic acid alkylation and fragmentation. J Am Chem Soc 124 : 7950–7962.
76. GalaganJE, CalvoSE, BorkovichKA, SelkerEU, ReadND, et al. (2003) The genome sequence of the filamentous fungus Neurospora crassa. Nature 422 : 859–868.
77. LiH, RuanJ, DurbinR (2008) Mapping short DNA sequencing reads and calling variants using mapping quality scores. Genome Res 18 : 1851–1858.
78. FreitagM, HickeyPC, RajuNB, SelkerEU, ReadND (2004) GFP as a tool to analyze the organization, dynamics and function of nuclei and microtubules in Neurospora crassa. Fungal Genet Biol 41 : 897–910.
79. FisherRA (1922) On the interpretation of χ2 from contingency tables, and the calculation of P. J Royal Stat Soc 85 : 87–94.
80. PriceMN, DehalPS, ArkinAP (2010) FastTree 2–approximately maximum-likelihood trees for large alignments. PloS One 5: e9490.
81. LetunicI, BorkP (2011) Interactive Tree Of Life v2: online annotation and display of phylogenetic trees made easy. Nucl Acids Res 39: W475–478.
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