Abstract
The genetic-exchange process of some endophytic root fungi whose teleomorphs have not been discovered is unknown. To assess parasexuality in the root-colonizing fungus Glutinomyces brunneus, we investigated the transmission of genetic markers in this fungus. We generated strains resistant to benomyl and hygromycin B and performed co-cultivation; dual resistance was used as a selection marker for genetic exchange between hyphae. Co-cultivation of benomyl- and hygromycin B-resistant strains yielded significantly larger numbers of dual-resistant colonies than did co-cultivation of strains resistant to the same agent, which suggests genetic exchange between the two vegetative hyphae. This was not simply due to heterokaryosis because all dual-resistant strains were monokaryotic. Also, half of the analyzed dual-resistant strains were homozygous in β-tubulin gene which is associated with benomyl resistance; thus, their simple diploidization was rejected for these strains. During selective cultivation, temporal hyphal growth occurred from the point at which two hyphae touched, which stopped after 2 weeks; this was followed by growth of true dual-resistant hyphae. Transmission of resistance markers was more frequent during co-cultivation on 2% malt extract agar than on other media, which suggests a role of medium composition. Genetic exchange between resistant strains that did not share an ancestor was limited, which implies vegetative incompatibility in this fungus. Our findings suggest the partial presence of parasexual potential in G. brunneus.
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References
Arnau J, Oliver RP (1993) Inheritance and alteration of transforming DNA during an induced parasexual cycle in the imperfect fungus Cladosporium fulvum. Curr Genet 23:508–511. https://doi.org/10.1007/BF00312643
Bever JD, Platt TG, Morton ER (2012) Microbial population and community dynamics on plant roots and their feedbacks on plant communities. Annu Rev Microbiol 66:265–283. https://doi.org/10.1146/annurev-micro-092611-150107
Brauer KL, Robbers JE (1987) Induced parasexual processes in Claviceps sp strain SD58. Appl Environ Microbiol 53:70–73
Bundock P, Dulk-Ras A den, Beijersbergen A, Hooykaas PJ (1995) Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae. EMBO J 14: 3206–3214.
Crous PW, Wingfield MJ, Burgess TI et al (2017) Fungal planet description sheets: 625–715. Persoonia 39:270–467. https://doi.org/10.3767/persoonia.2017.39.11
Fischer-Harman V, Jackson KJ, Muñoz A, Shoji JY, Read ND (2012) Evidence for tryptophan being a signal molecule that inhibits conidial anastomosis tube fusion during colony initiation in Neurospora crassa. Fungal Genet Biol 49:896–902. https://doi.org/10.1016/j.fgb.2012.08.004
Garber R (1992) The parasexual cycle in Ustilago scabiosae (Ustilaginales). Int J Plant Sci:98–101. https://doi.org/10.1086/297010
Glémin S (2003) How are deleterious mutations purged? Drift versus nonrandom mating. Evolution:2678. https://doi.org/10.1554/03-406
Grünig C, Sieber T, Holdenrieder O (2001) Characterisation of dark septate endophytic fungi (DSE) using inter-simple-sequence-repeat-anchored polymerase chain reaction (ISSR-PCR) amplification. Mycol Res:24–32. https://doi.org/10.1017/S0953756200003658
Grünig CR, Duò A, Sieber TN (2006) Population genetic analysis of Phialocephala fortinii s.l. and Acephala applanata in two undisturbed forests in Switzerland and evidence for new cryptic species. Fungal Genet Biol 43:410–421. https://doi.org/10.1016/j.fgb.2006.01.007
Grünig CR, Duò A, Sieber TN, Holdenrieder O (2008) Assignment of species rank to six reproductively isolated cryptic species of the Phialocephala fortinii s.1.–Acephala applanata species complex. Mycologia 100:47–67. https://doi.org/10.3852/mycologia.100.1.47
Grünig CR, McDonald BA, Sieber TN, Rogers SO, Holdenrieder O (2004) Evidence for subdivision of the root-endophyte Phialocephala fortinii into cryptic species and recombination within species. Fungal Genet Biol 41:676–687. https://doi.org/10.1016/j.fgb.2004.03.004
Hatoh K, Izumitsu K, Morita A, Shimizu K, Ohta A et al (2013) Transformation of the mushroom species Hypsizigus marmoreus, Flammulina velutipes, and Grifola frondosa by an Agrobacterium-mediated method using a universal transformation plasmid. Mycoscience 54:8–12. https://doi.org/10.1016/j.myc.2012.08.002
Hood E, Gelvin S, Melchers L, Hoekema A (1993) New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Res:208–218. https://doi.org/10.1007/BF01977351
Ishikawa FH, Souza EA, Read ND, Roca MG (2010) Live-cell imaging of conidial fusion in the bean pathogen, Colletotrichum lindemuthianum. Fungal Biol 114:2–9. https://doi.org/10.1016/j.funbio.2009.11.006
Izumitsu K, Hatoh K, Sumita T, Kitade Y, Morita A et al (2012) Rapid and simple preparation of mushroom DNA directly from colonies and fruiting bodies for PCR. Mycoscience:396–401. https://doi.org/10.1007/s10267-012-0182-3
Jung MK, Wilder IB, Oakley BR (1992) Amino acid alternations in the benA (beta-tubulin) gene of Aspergillus nidulans that confer benomyl resistance. Cell Motil Cytoskeleton 22:170–174. https://doi.org/10.1002/cm.970220304
Käfer E (1961) The processes of spontaneous recombination in vegetative nuclei of Aspergillus nidulans. Genetics 46:1581–1609
Mandyam K, Jumpponen A (2005) Seeking the elusive function of the root-colonising dark septate endophytic fungi. Stud Mycol:173–189. https://doi.org/10.3114/sim.53.1.173.
Muller HJ (1964) The relation of recombination to mutational advance. Mutat Res Fundam Mol Mech Mutagen:2–9. https://doi.org/10.1016/0027-5107(64)90047-8
Nakamura N, Tanaka E, Tanaka C, Takeuchi-Kaneko Y (2018a) Localization of helotialean fungi on ectomycorrhizae of Castanopsis cuspidata visualized by in situ hybridization. Mycorrhiza 28:17–28. https://doi.org/10.1007/s00572-017-0803-y
Nakamura N, Hosoya T, Tanaka C, Takeuchi-Kaneko Y (2018b) Detection of a root-associated group of Hyaloscyphaceae (Helotiales) species that commonly colonizes Fagaceae roots and description of three new species in genus Glutinomyces. Mycoscience. https://doi.org/10.1016/j.myc.2018.02.010
Orbach MJ, Porro EB, Yanofsky C (1986) Cloning and characterization of the gene for beta-tubulin from a benomyl-resistant mutant of Neurospora crassa and its use as a dominant selectable marker. Mol Cell Biol 6:2452–2461. https://doi.org/10.1128/MCB.6.7.2452
Palma-Guerrero J, Huang I-CC, Jansson H-BB, Salinas J, Lopez-Llorca LV et al (2009) Chitosan permeabilizes the plasma membrane and kills cells of Neurospora crassa in an energy-dependent manner. Fungal Genet Biol 46:585–594. https://doi.org/10.1016/j.fgb.2009.02.010
Pontecorvo (1956) The parasexual cycle in fungi. Ann Rev Microbiol:393–400. https://doi.org/10.1146/annurev.mi.10.100156.002141
Roca MG, Weichert M, Siegmund U, Tudzynski P, Fleissner A (2012) Germling fusion via conidial anastomosis tubes in the grey mould Botrytis cinerea requires NADPH oxidase activity. Fungal Biol 116:379–387. https://doi.org/10.1016/j.funbio.2011.12.007
Schneider G, Chicken E, Becvarik R (2017) Package “NSM3.”
Sheir-Neiss G, Lai MH, Morris NR (1978) Identification of a gene for beta-tubulin in Aspergillus nidulans. Cell 15:639–647. https://doi.org/10.1016/0092-8674(78)90032-6
Toju H, Yamamoto S, Sato H, Tanabe AS (2013a) Sharing of diverse mycorrhizal and root-endophytic fungi among plant species in an oak-dominated cool-temperate forest. PLoS One 8:e78248. https://doi.org/10.1371/journal.pone.0078248
Toju H, Yamamoto S, Sato H, Tanabe AS, Gilbert GS et al (2013b) Community composition of root-associated fungi in a Quercus-dominated temperate forest: codominance of mycorrhizal and root-endophytic fungi. Ecol Evol 3:1281–1293. https://doi.org/10.1002/ece3.546
Tsukasaki W, Maruyama J-I, Kitamoto K (2014) Establishment of a new method to quantitatively evaluate hyphal fusion ability in Aspergillus oryzae. Biosci Biotechnol Biochem 78:1254–1262. https://doi.org/10.1080/09168451.2014.917262
Walton FJ, Idnurm A, Heitman J (2005) Novel gene functions required for melanization of the human pathogen Cryptococcus neoformans. Mol Microbiol 57:1381–1396. https://doi.org/10.1111/j.1365-2958.2005.04779.x
Yamada A, Katsuya K (1995) Mycorrhizal association of isolates from sporocarps and ectomycorrhizas with Pinus densiflora seedlings. Mycoscience:315–323. https://doi.org/10.1007/BF02268607
Yamamoto S, Sato H, Tanabe AS, Hidaka A, Kadowaki K et al (2014) Spatial segregation and aggregation of ectomycorrhizal and root-endophytic fungi in the seedlings of two Quercus species. PLoS One 9:e96363. https://doi.org/10.1371/journal.pone.0096363
Yan K, Dickman MB (1996) Isolation of a beta-tubulin gene from Fusarium moniliforme that confers cold-sensitive benomyl resistance. Appl Environ Microbiol 62:3053–3056
Yarden O, Katan T (1993) Mutations leading to substitutions at amino acids 198 and 200 of beta-tubulin that correlate with benomyl-resistance phenotypes of field strains of Botrytis cinerea. Phytopathology 83:1478–1483
Zaffarano PL, Duò A, Grünig CR (2010) Characterization of the mating type (MAT) locus in the Phialocephala fortinii s.l. –Acephala applanata species complex. Fungal Genet Biol 47:761–772. https://doi.org/10.1016/j.fgb.2010.06.001
Zaffarano PL, Queloz V, Duò A, Grünig CR (2011) Sex in the PAC: a hidden affair in dark septate endophytes? BMC Evol Biol 11:282. https://doi.org/10.1186/1471-2148-11-282
Zeigler RS, Scott RP, Leung H, Bordeos AA, Kumar J et al (1997) Evidence of parasexual exchange of DNA in the rice blast fungus challenges its exclusive clonality. Phytopathology:284–294. https://doi.org/10.1094/PHYTO.1997.87.3.284
Acknowledgments
We thank Dr. Alexander Idnurm for providing A. tumefaciens EHA105 harboring pPZPHYG2.
Funding
This work was financially supported by the Japan Society for the Promotion of Science KAKENHI (Grant Nos. 26257418 and 15H05249).
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Fig. S1
The tub2 gene DNA (a) and amino acid (b) sequences of the wild type strain (Fhi3), hygromycin B-resistant strain (Fhi3hyg), benomyl-resistant strain (Fhi3ben), and 8 dual-resistant strains (Dual resistant strains 1–8). The same sequence region of the independently obtained benomyl-resistant strains were also shown below (Fhi3ben2–Fhi3ben7). DNA bases or amino acids identical to that of the wild type strain was represented as dots. (PNG 1107 kb)
Fig. S2
The tub2 gene sequencing electropherogram of the benomyl-resistant strain (Fhi3ben), hygromycin B-resistant strain (Fhi3hyg), and 8 dual-resistant strains (a–h). The sites shown in boxes represent the T to C substitution site in Fhi3ben, which is related to benomyl-resistance. (PNG 972 kb)
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Nakamura, N., Tanaka, C. & Takeuchi-Kaneko, Y. Transmission of antibiotic-resistance markers by hyphal fusion suggests partial presence of parasexuality in the root endophytic fungus Glutinomyces brunneus. Mycol Progress 18, 453–462 (2019). https://doi.org/10.1007/s11557-018-1455-9
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DOI: https://doi.org/10.1007/s11557-018-1455-9