Metarhizium robertsii ammonium permeases (MepC and Mep2) contribute to rhizoplane colonization and modulates the transfer of insect derived nitrogen to plants


Autoři: Soumya Moonjely aff001;  Xing Zhang aff002;  Weiguo Fang aff002;  Michael J. Bidochka aff001
Působiště autorů: Department of Biological Sciences, Brock University, St. Catharines, ON Canada aff001;  Institute of Microbiology, Zhejiang University, Hangzhou, China aff002
Vyšlo v časopise: PLoS ONE 14(10)
Kategorie: Research Article
doi: 10.1371/journal.pone.0223718

Souhrn

The endophytic insect pathogenic fungi (EIPF) Metarhizium promotes plant growth through symbiotic association and the transfer of insect-derived nitrogen. However, little is known about the genes involved in this association and the transfer of nitrogen. In this study, we assessed the involvement of six Metarhizium robertsii genes in endophytic, rhizoplane and rhizospheric colonization with barley roots. Two ammonium permeases (MepC and Mep2) and a urease, were selected since homologous genes in arbuscular mycorrhizal fungi were reported to play a pivotal role in nitrogen mobilization during plant root colonization. Three other genes were selected on the basis on RNA-Seq data that showed high expression levels on bean roots, and these encoded a hydrophobin (Hyd3), a subtilisin-like serine protease (Pr1A) and a hypothetical protein. The root colonization assays revealed that the deletion of urease, hydrophobin, subtilisin-like serine protease and hypothetical protein genes had no impact on endophytic, rhizoplane and rhizospheric colonization at 10 or 20 days. However, the deletion of MepC resulted in significantly increased rhizoplane colonization at 10 days whereas ΔMep2 showed increased rhizoplane colonization at 20 days. In addition, the nitrogen transporter mutants also showed significantly higher 15N incorporation of insect derived nitrogen in barley leaves in the presence of nutrients. Insect pathogenesis assay revealed that disruption of MepC, Mep2, urease did not reduce virulence toward insects. The enhanced rhizoplane colonization of ΔMep2 and ΔMepC and insect derived nitrogen transfer to plant hosts suggests the role of MepC and Mep2 in Metarhizium-plant symbiosis.

Klíčová slova:

Deletion mutation – Fungal genetics – Fungi – Insects – Larvae – Metarhizium – Ureases – Barley


Zdroje

1. Moonjely S, Barelli L, Bidochka MJ. Insect pathogenic fungi as endophytes. In: Lovett B, St. Leger RJ, editors. Insect pathogenic fungi as endophytes. Academic Press Inc.; 2016. p. 107–35.

2. Ortiz-Urquiza A, Keyhani NO. Action on the surface: Entomopathogenic fungi versus the insect cuticle. Insects. 2013;4(3):357–74. doi: 10.3390/insects4030357 26462424

3. Ortiz-Urquiza A, Keyhani NO. Molecular genetics of Beauveria bassiana infection of insects. In: Lovett B, St. Leger RJ, editors. Molecular genetics of Beauveria bassiana infection of insects. Academic Press Inc.; 2016. p. 165–249.

4. Behie SW, Bidochka MJ. Endophytic insect-parasitic fungi translocate nitrogen directly from insects to plants. Science (80). 2012;336:1576–7.

5. Behie SW, Bidochka MJ. Ubiquity of insect-derived nitrogen transfer to plants by endophytic insect-pathogenic fungi: an additional branch of the soil nitrogen cycle. Appl Environ Microbiol. 2014;80(5):1553–60. doi: 10.1128/AEM.03338-13 24334669

6. Behie SW, Moreira CC, Sementchoukova I, Barelli L, Zelisko PM, Bidochka MJ. Carbon translocation from a plant to an insect-pathogenic endophytic fungus. Nat Commun. 2017;8:14245. doi: 10.1038/ncomms14245 28098142

7. Fang W, St Leger RJ. Mrt, a gene unique to fungi, encodes an oligosaccharide transporter and facilitates rhizosphere competency in Metarhizium robertsii. Plant Physiol. 2010;154(3):1549–57. doi: 10.1104/pp.110.163014 20837701

8. Liao X, Fang W, Lin L, Lu H-L, St Leger RJ. Metarhizium robertsii produces an extracellular invertase (MrINV) that plays a pivotal role in rhizospheric interactions and root colonization. PLoS One. 2013;8(10):e78118. doi: 10.1371/journal.pone.0078118 24205119

9. Ellerbeck M, Schüßler A, Brucker D, Dafinger C, Loos F, Brachmann A. Characterization of three ammonium transporters of the glomeromycotan fungus Geosiphon pyriformis. Eukaryot Cell. 2013;12(11):1554–62. doi: 10.1128/EC.00139-13 24058172

10. Montanini B, Moretto N, Soragni E, Percudani R, Ottonello S. A high-affinity ammonium transporter from the mycorrhizal ascomycete Tuber borchii. Fungal Genet Biol. 2002;36(1):22–34. doi: 10.1016/S1087-1845(02)00001-4 12051892

11. López-Pedrosa A, González-Guerrero M, Valderas A, Azcón-Aguilar C, Ferrol N. GintAMT1 encodes a functional high-affinity ammonium transporter that is expressed in the extraradical mycelium of Glomus intraradices. Fungal Genet Biol. 2006;43(2):102–10. doi: 10.1016/j.fgb.2005.10.005 16386437

12. Pérez-Tienda J, Testillano PS, Balestrini R, Fiorilli V, Azcón-Aguilar C, Ferrol N. GintAMT2, a new member of the ammonium transporter family in the arbuscular mycorrhizal fungus Glomus intraradices. Fungal Genet Biol. 2011;48(11):1044–55. doi: 10.1016/j.fgb.2011.08.003 21907817

13. Govindarajulu M, Pfeffer PE, Jin H, Abubaker J, Douds DD, Allen JW, et al. Nitrogen transfer in the arbuscular mycorrhizal symbiosis. Nature. 2005;435(7043):819–23. doi: 10.1038/nature03610 15944705

14. Holsbeeks I, Lagatie O, Van Nuland A, Van De Velde S, Thevelein JM. The eukaryotic plasma membrane as a nutrient-sensing device. Trends Biochem Sci. 2004;29(10):556–64. doi: 10.1016/j.tibs.2004.08.010 15450611

15. Wang C, Hu G, St. Leger RJ. Differential gene expression by Metarhizium anisopliae growing in root exudate and host (Manduca sexta) cuticle or hemolymph reveals mechanisms of physiological adaptation. Fungal Genet Biol. 2005;42(8):704–18. doi: 10.1016/j.fgb.2005.04.006 15914043

16. Freimoser FM, Screen S, Bagga S, Hu G, St Leger RJ. Expressed sequence tag (EST) analysis of two subspecies of Metarhizium anisopliae reveals a plethora of secreted proteins with potential activity in insect hosts. Microbiology. 2003;149(1):239–47.

17. Hu X, Xiao G, Zheng P, Shang Y, Su Y, Zhang X, et al. Trajectory and genomic determinants of fungal-pathogen speciation and host adaptation. Proc Natl Acad Sci. 2014;111(47):16796–801. doi: 10.1073/pnas.1412662111 25368161

18. Gao Q, K J, Ying SH, Zhang Y, Xiao G, Shang Y, et al. Genome sequencing and comparative transcriptomics of the model entomopathogenic fungi Metarhizium anisopliae and M. acridum. PLoS Genet. 2011;7(1):e1001264. doi: 10.1371/journal.pgen.1001264 21253567

19. Wang C, St. Leger RJ. The MAD1 adhesin of Metarhizium anisopliae links adhesion with blastospore production and virulence to insects, and the MAD2 adhesin enables attachment to plants. Eukaryot Cell. 2007;6(5):808–16. doi: 10.1128/EC.00409-06 17337634

20. Xu C, Zhang X, Qian Y, Chen X, Liu R, Zeng G, et al. A high-throughput gene disruption methodology for the entomopathogenic fungus Metarhizium robertsii. PLoS One. 2014;9(9):e107657. doi: 10.1371/journal.pone.0107657 25222118

21. Zhang S, Xia YX, Kim B, Keyhani NO. Two hydrophobins are involved in fungal spore coat rodlet layer assembly and each play distinct roles in surface interactions, development and pathogenesis in the entomopathogenic fungus, Beauveria bassiana. Mol Microbiol. 2011;80(3):811–26. doi: 10.1111/j.1365-2958.2011.07613.x 21375591

22. Sevim A, Donzelli BGG, Wu D, Demirbag Z, Gibson DM, Turgeon BG. Hydrophobin genes of the entomopathogenic fungus, Metarhizium brunneum, are differentially expressed and corresponding mutants are decreased in virulence. Curr Genet. 2012;58(2):79–92. doi: 10.1007/s00294-012-0366-6 22388867

23. Moonjely S, Keyhani NO, Bidochka MJ. Hydrophobins contribute to root colonization and stress responses in the rhizosphere-competent insect pathogenic fungus Beauveria bassiana. Microbiology. 2018;164(4):517–28. doi: 10.1099/mic.0.000644 29517481

24. Greenfield M, Gómez-Jiménez MI, Ortiz V, Vega FE, Kramer M, Parsa S. Beauveria bassiana and Metarhizium anisopliae endophytically colonize cassava roots following soil drench inoculation. Biol Control. 2016;95:40–8. doi: 10.1016/j.biocontrol.2016.01.002 27103778

25. Fernandes ÉKK, Keyser CA, Rangel DEN, Foster RN, Roberts DW. CTC medium: A novel dodine-free selective medium for isolating entomopathogenic fungi, especially Metarhizium acridum, from soil. Biol Control. 2010;54(3):197–205.

26. Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res. 2004;32(5):1792–7. doi: 10.1093/nar/gkh340 15034147

27. Tamura K, Stecher G, Peterson D, Filipski A, Kumar S. MEGA6: Molecular evolutionary genetics analysis version 6.0. Mol Biol Evol. 2013;30(12):2725–9. doi: 10.1093/molbev/mst197 24132122

28. Ronquist F, Huelsenbeck JP. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics. 2003;19(12):1572–4. doi: 10.1093/bioinformatics/btg180 12912839

29. Teichert S, Rutherford JC, Wottawa M, Heitman J, Tudzynski B. Impact of ammonium permeases MepA, MepB, and MepC on nitrogen-regulated secondary metabolism in Fusarium fujikuroi. Eukaryot Cell. 2008;7(2):187–201. doi: 10.1128/EC.00351-07 18083831

30. Monahan BJ, Fraser JA, Hynes MJ, Davis MA. Isolation and characterization of two ammonium permease genes, meaA and mepA, from Aspergillus nidulans. Eukaryot Cell. 2002;1(1):85–94. doi: 10.1128/EC.1.1.85-94.2002 12455974

31. Shnaiderman C, Miyara I, Kobiler I, Sherman A, Prusky D. Differential activation of ammonium transporters during the accumulation of ammonia by Colletotrichum gloeosporioides and its effect on appressoria formation and pathogenicity. Mol Plant-Microbe Interact. 2013;26(3):335–55.

32. Javelle A, Morel M, Rodríguez-Pastrana BR, Botton B, André B, Marini AM, et al. Molecular characterization, function and regulation of ammonium transporters (Amt) and ammonium-metabolizing enzymes (GS, NADP-GDH) in the ectomycorrhizal fungus Hebeloma cylindrosporum. Mol Microbiol. 2003;47(2):411–30. doi: 10.1046/j.1365-2958.2003.03303.x 12519192

33. Monahan BJ, Askin MC, Hynes MJ, Davis MA. Differential expression of Aspergillus nidulans ammonium permease genes is regulated by GATA transcription factor AreA. Eukaryot Cell. 2006;5(2):226–37. doi: 10.1128/EC.5.2.226-237.2006 16467464

34. Screen S, Bailey A, Charnley K, Cooper R, Clarkson J. Isolation of a nitrogen response regulator gene (nrr1) from Metarhizium anisopliae. Gene. 1998;221(1):17–24. doi: 10.1016/s0378-1119(98)00430-2 9852945

35. Xu JR, Hamer JE. MAP kinase and cAMP signaling regulate infection structure formation and pathogenic growth in the rice blast fungus Magnaporthe grisea. Genes Dev. 1996;10(21):2696–706. doi: 10.1101/gad.10.21.2696 8946911

36. Chen X, Xu C, Qian Y, Liu R, Zhang Q, Zeng G, et al. MAPK cascade-mediated regulation of pathogenicity, conidiation and tolerance to abiotic stresses in the entomopathogenic fungus Metarhizium robertsii. Environ Microbiol. 2016;18(3):1048–62. doi: 10.1111/1462-2920.13198 26714892

37. Fang W, Pava-ripoll M, Wang S, St Leger R. Protein kinase A regulates production of virulence determinants by the entomopathogenic fungus, Metarhizium anisopliae. Fungal Genet Biol. 2009;46(3):277–85. doi: 10.1016/j.fgb.2008.12.001 19124083

38. Hawkins BJ, Robbins S. pH affects ammonium, nitrate and proton fluxes in the apical region of conifer and soybean roots. 2010;238–47.

39. Zhu J, Ying S-H, Feng M-G. The Pal pathway required for ambient pH adaptation regulates growth, conidiation, and osmotolerance of Beauveria bassiana in a pH-dependent manner. Appl Microbiol Biotechnol. 2016;100(10):4423–33. doi: 10.1007/s00253-016-7282-5 26754817

40. Liao X O’Brien TR, Fang W, St Leger RJ. The plant beneficial effects of Metarhizium species correlate with their association with roots. Appl Genet Mol Biotechnol. 2014;98(16):7089–96.

41. Bagga S, Hu G, Screen SE, St Leger RJ. Reconstructing the diversification of subtilisins in the pathogenic fungus Metarhizium anisopliae. Gene. 2004;324:159–69. doi: 10.1016/j.gene.2003.09.031 14693381

42. St Leger RJ, Joshi L, Bidochka MJ, Roberts DW. Construction of an improved mycoinsecticide overexpressing a toxic protease. Proc Natl Acad Sci USA. 1996;93(13):6349–54. doi: 10.1073/pnas.93.13.6349 8692818

43. Sreedhar L, Kobayashi DY, Bunting TE, Hillman BI, Belanger FC. Fungal proteinase expression in the interaction of the plant pathogen Magnaporthe poae with its host. Gene. 1999;235(1):121–9.

44. Reddy P V, Lam CK, Belanger FC. Mutualistic fungal endophytes express a proteinase that is homologous to proteases suspected to be important in fungal pathogenicity. Plant Physiol. 1996;111(4):1209–18. doi: 10.1104/pp.111.4.1209 8756501

45. Bryant MK, Schardl CL, Hesse U, Scott B. Evolution of a subtilisin-like protease gene family in the grass endophytic fungus Epichlo festucae. BMC Evol Biol. 2009;9(1):168.

46. Pava-Ripoll M, Angelini C, Fang W, Wang S, Posada FJ, St Leger R. The rhizosphere-competent entomopathogen Metarhizium anisopliae expresses a specific subset of genes in plant root exudate. Microbiology. 2011;157(1):47–55.

47. Kim S, Ahn IP, Rho HS, Lee YH. MHP1, a Magnaporthe grisea hydrophobin gene, is required for fungal development and plant colonization. Mol Microbiol. 2005;57(5):1224–37. doi: 10.1111/j.1365-2958.2005.04750.x 16101997

48. Viterbo A, Chet I. TasHyd1, a new hydrophobin gene from the biocontrol agent Trichoderma asperellum, is involved in plant root colonization. Mol Plant Pathol. 2006;7(4):249–58. doi: 10.1111/j.1364-3703.2006.00335.x 20507444

49. Izumitsu K, Kimura S, Kobayashi H, Morita A, Saitoh Y, Tanaka C. Class I hydrophobin BcHpb1 is important for adhesion but not for later infection of Botrytis cinerea. J Gen Plant Pathol. 2010;76(4):254–60.

50. Dubey MK, Jensen DF, Karlsson M. Hydrophobins are required for conidial hydrophobicity and plant root colonization in the fungal biocontrol agent Clonostachys rosea. BMC Microbiol. 2014;14(1):18.

51. Brunner-Mendoza C, del Rocío Reyes-Montes M, Moonjely S, Bidochka MJ, Toriello C. A review on the genus Metarhizium as an entomopathogenic microbial biocontrol agent with emphasis on its use and utility in Mexico. Biocontrol Sci Technol. 2018;1–20.

52. Ortiz-Urquiza A, Keyhani NO. Stress response signaling and virulence: insights from entomopathogenic fungi. Curr Genet. 2015;61(3):239–49. doi: 10.1007/s00294-014-0439-9 25113413

53. Geremia RA, Goldman GH, Jacobs D, Ardiles W, Vila SB, Van Montagu M, et al. Molecular characterization of the proteinase‐encoding gene, prb1, related to mycoparasitism by Trichoderma harzianum. Mol Microbiol. 1993;8(3):603–13. doi: 10.1111/j.1365-2958.1993.tb01604.x 8326868

54. Li J, Li Y, Yang J, Dong L, Tian B, Yu Z, et al. New insights into the evolution of subtilisin-like serine protease genes in Pezizomycotina. BMC Evol Biol. 2010;10(1):68.

55. Porto M, Leão C, Vieira Tiago P, Dini Andreote F, Luiz De Araújo W, Tinti De Oliveira N. Differential expression of the pr1A gene in Metarhizium anisopliae and Metarhizium acridum across different culture conditions and during pathogenesis. Genet Mol Biol. 2015;38(1):86–92. doi: 10.1590/S1415-475738138120140236 25983629

56. Harrison R, Papp B, Pal C, Oliver SG, Delneri D. Plasticity of genetic interactions in metabolic networks of yeast. Proc Natl Acad Sci U S A. 2007;104(7):2307–12. doi: 10.1073/pnas.0607153104 17284612


Článek vyšel v časopise

PLOS One


2019 Číslo 10