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Narnaviruses: novel players in fungal–bacterial symbioses


Rhizopus microsporus is an early-diverging fungal species with importance in ecology, agriculture, food production, and public health. Pathogenic strains of R. microsporus harbor an intracellular bacterial symbiont, Mycetohabitans (formerly named Burkholderia). This vertically transmitted bacterial symbiont is responsible for the production of toxins crucial to the pathogenicity of Rhizopus and remarkably also for fungal reproduction. Here we show that R. microsporus can live not only in symbiosis with bacteria but also with two viral members of the genus Narnavirus. Our experiments revealed that both viruses replicated similarly in the growth conditions we tested. Viral copies were affected by the developmental stage of the fungus, the substrate, and the presence or absence of Mycetohabitans. Absolute quantification of narnaviruses in isolated asexual sporangiospores and sexual zygospores indicates their vertical transmission. By curing R. microsporus of its viral and bacterial symbionts and reinfecting bacteria to reestablish symbiosis, we demonstrate that these viruses affect fungal biology. Narnaviruses decrease asexual reproduction, but together with Mycetohabitans, are required for sexual reproductive success. This fungal–bacterial-viral system represents an outstanding model to investigate three-way microbial symbioses and their evolution.

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Fig. 1: Discovery of narnaviruses in Rhizopus microsporus.
Fig. 2: Quantification of RmNV-20S and RmNV-23S along development and reproduction of R. microsporus.
Fig. 3: Generation and characterization of narnaviruses-free (nv−), bacteria-free (b−), and bacteria-reinfected (b*) fungal strains.
Fig. 4: Effect of narnaviruses in the asexual and sexual reproduction of R. microsporus.


  1. 1.

    Bonfante P, Desirò A. Who lives in a fungus? The diversity, origins and functions of fungal endobacteria living in Mucoromycota. ISME J. 2017;11:1727–35.

    PubMed  PubMed Central  Google Scholar 

  2. 2.

    Pawlowska TE, Gaspar ML, Lastovetsky OA, Mondo SJ, Real-Ramirez I, Shakya E, et al. Biology of fungi and their bacterial endosymbionts. Annu Rev Phytopathol. 2018;56:289–309.

    CAS  PubMed  Google Scholar 

  3. 3.

    Partida-Martínez LP. The fungal holobiont: evidence from early diverging fungi. Environ Microbiol. 2017;19:2919–23.

    PubMed  Google Scholar 

  4. 4.

    Partida-Martinez LP, Groth I, Schmitt I, Richter W, Roth M, Hertweck C. Burkholderia rhizoxinica sp. nov. and Burkholderia endofungorum sp. nov., bacterial endosymbionts of the plant-pathogenic fungus Rhizopus microsporus. Int J Syst Evol Microbiol. 2007;57:2583–90.

    CAS  PubMed  Google Scholar 

  5. 5.

    Lackner G, Möbius N, Scherlach K, Partida-Martinez LP, Winkler R, Schmitt I, et al. Global distribution and evolution of a toxinogenic Burkholderia-Rhizopus symbiosis. Appl Environ Microbiol. 2009;75:2982–6.

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 6.

    Partida-Martinez LP. A model for bacterial-fungal interactions. Saarbrücken: LAP LAMBERT Academic Publishing; 2013.

    Google Scholar 

  7. 7.

    Estrada-de Los Santos P, Palmer M, Chávez-Ramírez B, Beukes C, Steenkamp ET, Briscoe L, et al. Whole genome analyses suggests that Burkholderia sensu lato contains two additional novel genera (Mycetohabitans gen. nov., and Trinickia gen. nov.): implications for the evolution of diazotrophy and nodulation in the burkholderiaceae. Genes. 2018;9:389.

    PubMed Central  Google Scholar 

  8. 8.

    Oren A, Garrity GM. List of new names and new combinations previously effectively, but not validly, published. Int J Syst Evol Microbiol. 2016;66:2463–6.

    PubMed  Google Scholar 

  9. 9.

    Partida-Martinez LP, Hertweck C. Pathogenic fungus harbours endosymbiotic bacteria for toxin production. Nature. 2005;437:884–8.

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10.

    Lackner G, Moebius N, Partida-Martinez L, Hertweck C. Complete genome sequence of Burkholderia rhizoxinica, an endosymbiont of Rhizopus microsporus. J Bacteriol. 2011;193:783–4.

    CAS  Google Scholar 

  11. 11.

    Lackner G, Moebius N, Partida-Martinez LP, Boland S, Hertweck C. Evolution of an endofungal lifestyle: deductions from the Burkholderia rhizoxinica genome. BMC Genom. 2011;12:210.

    CAS  Google Scholar 

  12. 12.

    Horn F, Üzüm Z, Möbius N, Guthke R, Linde J, Hertweck C. Draft genome sequences of symbiotic and nonsymbiotic Rhizopus microsporus strains CBS 344.29 and ATCC 62417. Genome Announc. 2015;3:e01370-14.

    PubMed  PubMed Central  Google Scholar 

  13. 13.

    Lastovetsky OA, Gaspar ML, Mondo SJ, LaButti KM, Sandor L, Grigoriev IV, et al. Lipid metabolic changes in an early divergent fungus govern the establishment of a mutualistic symbiosis with endobacteria. Proc Natl Acad Sci USA. 2016;113:15102–7.

    CAS  PubMed  Google Scholar 

  14. 14.

    Partida-Martinez LP, Hertweck C. A gene cluster encoding rhizoxin biosynthesis in “Burkholderia rhizoxina”, the bacterial endosymbiont of the fungus Rhizopus microsporus. ChemBioChem. 2007;8:41–5.

    CAS  PubMed  Google Scholar 

  15. 15.

    Partida-Martinez LP, Looß CF, de, Ishida K, Ishida M, Roth M, Buder K, et al. Rhizonin, the first mycotoxin isolated from the zygomycota, is not a fungal metabolite but is produced by bacterial endosymbionts. Appl Environ Microbiol. 2007;73:793–7.

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Niehs SP, Scherlach K, Hertweck C. Genomics-driven discovery of a linear lipopeptide promoting host colonization by endofungal bacteria. Org Biomol Chem. 2018;16:8345–52.

    PubMed  Google Scholar 

  17. 17.

    Niehs SP, Dose B, Scherlach K, Roth M, Hertweck C. Genomics-driven discovery of a symbiont-specific cyclopeptide from bacteria residing in the rice seedling blight fungus. Chembiochem Eur J Chem Biol. 2018;19:2167–72.

    CAS  Google Scholar 

  18. 18.

    Partida-Martinez LP, Monajembashi S, Greulich K-O, Hertweck C. Endosymbiont-dependent host reproduction maintains bacterial–fungal mutualism. Curr Biol. 2007;17:773–7.

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 19.

    Mondo SJ, Lastovetsky OA, Gaspar ML, Schwardt NH, Barber CC, Riley R, et al. Bacterial endosymbionts influence host sexuality and reveal reproductive genes of early divergent fungi. Nat Commun. 2017;8:1843.

    PubMed  PubMed Central  Google Scholar 

  20. 20.

    Moebius N, Üzüm Z, Dijksterhuis J, Lackner G, Hertweck C. Active invasion of bacteria into living fungal cells. eLife. 2014;3:e03007.

    PubMed  PubMed Central  Google Scholar 

  21. 21.

    Lackner G, Moebius N, Hertweck C. Endofungal bacterium controls its host by an hrp type III secretion system. ISME J. 2011;5:252–61.

    CAS  PubMed  Google Scholar 

  22. 22.

    Bermúdez-Barrientos, J. Roberto. Exploring the molecular mechanisms maintaining the Rhizopus microsporus—Burkholderia rhizoxinica symbiosis. Irapuato: CINVESTAV-Irapuato; 2016.

  23. 23.

    Rodriguez-Cousiño N, Esteban LM, Esteban R. Molecular cloning and characterization of W double-stranded RNA, a linear molecule present in Saccharomyces cerevisiae. Identification of its single-stranded RNA form as 20S RNA. J Biol Chem. 1991;266:12772–8.

    PubMed  Google Scholar 

  24. 24.

    Esteban LM, Rodriguez-Cousiño N, Esteban R. T double-stranded RNA (dsRNA) sequence reveals that T and W dsRNAs form a new RNA family in Saccharomyces cerevisiae. Identification of 23S RNA as the single-stranded form of T dsRNA. J Biol Chem. 1992;267:10874–81.

    CAS  PubMed  Google Scholar 

  25. 25.

    Wejksnora PJ, Haber JE. Ribonucleoprotein particle appearing during sporulation in yeast. J Bacteriol. 1978;134:246–60.

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 26.

    Hillman BI, Cai G. The family narnaviridae: simplest of RNA viruses. In: Ghabrial SA, editor. Advances in virus research, Vol 86: Mycoviruses. San Diego: Elsevier Academic Press Inc.; 2013. pp 149–76.

  27. 27.

    Rodríguez-Cousiño N, Solórzano A, Fujimura T, Esteban R. Yeast positive-stranded virus-like RNA replicons. 20 S and 23 S RNA terminal nucleotide sequences and 3’ end secondary structures resemble those of RNA coliphages. J Biol Chem. 1998;273:20363–71.

    PubMed  Google Scholar 

  28. 28.

    García-Cuéllar MP, Esteban R, Fujimura T. RNA-dependent RNA polymerase activity associated with the yeast viral p91/20S RNA ribonucleoprotein complex. RNA. 1997;3:27–36.

    PubMed  PubMed Central  Google Scholar 

  29. 29.

    Matsumoto Y, Fishel R, Wickner RB. Circular single-stranded RNA replicon in Saccharomyces cerevisiae. Proc Natl Acad Sci USA. 1990;87:7628–32.

    CAS  PubMed  Google Scholar 

  30. 30.

    Cai G, Myers K, Fry WE, Hillman BI. A member of the virus family narnaviridae from the plant pathogenic oomycete Phytophthora infestans. Arch Virol. 2012;157:165–9.

    CAS  PubMed  Google Scholar 

  31. 31.

    Osaki H, Sasaki A, Nomiyama K, Tomioka K. Multiple virus infection in a single strain of Fusarium poae shown by deep sequencing. Virus Genes. 2016;52:835–47.

    CAS  PubMed  Google Scholar 

  32. 32.

    Lye L-F, Akopyants NS, Dobson DE, Beverley SM. A narnavirus-like element from the trypanosomatid protozoan parasite Leptomonas seymouri. Microbiol Resour Announc. 2016;4:e00713–16.

    Google Scholar 

  33. 33.

    Grybchuk D, Akopyants NS, Kostygov AY, Konovalovas A, Lye L-F, Dobson DE, et al. Viral discovery and diversity in trypanosomatid protozoa with a focus on relatives of the human parasite Leishmania. Proc Natl Acad Sci USA. 2018;115:E506–15.

    CAS  PubMed  Google Scholar 

  34. 34.

    Shi M, Lin X-D, Tian J-H, Chen L-J, Chen X, Li C-X, et al. Redefining the invertebrate RNA virosphere. Nature. 2016;540:539–43.

    CAS  PubMed  Google Scholar 

  35. 35.

    Fauver JR, Akter S, Morales AIO, Black WC, Rodriguez AD, Stenglein MD, et al. A reverse-transcription/RNase H based protocol for depletion of mosquito ribosomal RNA facilitates viral intrahost evolution analysis, transcriptomics and pathogen discovery. Virology. 2019;528:181–97.

    CAS  PubMed  Google Scholar 

  36. 36.

    Richaud A, Frézal L, Tahan S, Jiang H, Blatter JA, Zhao G, et al. Vertical transmission in Caenorhabditis nematodes of RNA molecules encoding a viral RNA-dependent RNA polymerase. Proc Natl Acad Sci USA. 2019;116:24738–47.

    CAS  PubMed  Google Scholar 

  37. 37.

    Zoll J, Verweij PE, Melchers WJG. Discovery and characterization of novel Aspergillus fumigatus mycoviruses. PLoS ONE. 2018;13:e0200511.

    PubMed  PubMed Central  Google Scholar 

  38. 38.

    Niu Y, Yuan Y, Mao J, Yang Z, Cao Q, Zhang T, et al. Characterization of two novel mycoviruses from Penicillium digitatum and the related fungicide resistance analysis. Sci Rep. 2018;8:5513.

    PubMed  PubMed Central  Google Scholar 

  39. 39.

    Ghignone S, Salvioli A, Anca I, Lumini E, Ortu G, Petiti L, et al. The genome of the obligate endobacterium of an AM fungus reveals an interphylum network of nutritional interactions. ISME J. 2012;6:136–45.

    CAS  PubMed  Google Scholar 

  40. 40.

    Uehling J, Gryganskyi A, Hameed K, Tschaplinski T, Misztal PK, Wu S, et al. Comparative genomics of Mortierella elongata and its bacterial endosymbiont Mycoavidus cysteinexigens. Environ Microbiol. 2017;19:2964–83.

    CAS  PubMed  Google Scholar 

  41. 41.

    Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, et al. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol. 2012;61:539–42.

    PubMed  PubMed Central  Google Scholar 

  42. 42.

    Kumar S, Stecher G, Tamura K. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol. 2016;33:1870–4.

    CAS  Google Scholar 

  43. 43.

    Dimmic MW, Rest JS, Mindell DP, Goldstein RA. rtREV: an amino acid substitution matrix for inference of retrovirus and reverse transcriptase phylogeny. J Mol Evol. 2002;55:65–73.

    CAS  PubMed  Google Scholar 

  44. 44.

    Zhang L, Fu Y, Xie J, Jiang D, Li G, Yi X. A novel virus that infecting hypovirulent strain XG36-1 of plant fungal pathogen Sclerotinia sclerotiorum. Virol J. 2009;6:96.

    PubMed  PubMed Central  Google Scholar 

  45. 45.

    Niu Y, Zhang T, Zhu Y, Yuan Y, Wang S, Liu J, et al. Isolation and characterization of a novel mycovirus from Penicillium digitatum. Virology. 2016;494:15–22.

    CAS  PubMed  Google Scholar 

  46. 46.

    Routhier E, Bruenn JA. Functions of conserved motifs in the RNA-dependent RNA polymerase of a yeast double-stranded RNA virus. J Virol. 1998;72:4427–9.

    CAS  PubMed  PubMed Central  Google Scholar 

  47. 47.

    King AMQ, Adams MJ, Carstens EB, Lefkowitz EJ. Virus Taxonomy: Ninth Report of the International Committee on Taxonomy of Viruses. San Diego: Elsevier Academic Press; 2012.

  48. 48.

    Ghabrial SA, Castón JR, Jiang D, Nibert ML, Suzuki N. 50-plus years of fungal viruses. Virology. 2015;479–80:356–68.

    Google Scholar 

  49. 49.

    Hillman BI, Suzuki N. Viruses of the chestnut blight fungus, Cryphonectria parasitica. Adv Virus Res. 2004;63:423–72.

    CAS  PubMed  Google Scholar 

  50. 50.

    Spatafora JW, Chang Y, Benny GL, Lazarus K, Smith ME, Berbee ML, et al. A phylum-level phylogenetic classification of zygomycete fungi based on genome-scale data. Mycologia. 2016;108:1028–46.

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51.

    Dolja VV, Koonin EV. Metagenomics reshapes the concepts of RNA virus evolution by revealing extensive horizontal virus transfer. Virus Res. 2018;244:36–52.

    CAS  PubMed  Google Scholar 

  52. 52.

    Ikeda Y, Shimura H, Kitahara R, Masuta C, Ezawa T. A novel virus-like double-stranded RNA in an obligate biotroph arbuscular mycorrhizal fungus: a hidden player in mycorrhizal symbiosis. Mol Plant-Microbe Interact. 2012;25:1005–12.

    CAS  Google Scholar 

  53. 53.

    Neupane A, Feng C, Feng J, Kafle A, Buecking H, Lee Marzano S-Y. Metatranscriptomic analysis and in silico approach identified mycoviruses in the arbuscular mycorrhizal fungus Rhizophagus spp. Viruses-Basel. 2018;10:707.

    CAS  Google Scholar 

  54. 54.

    Bianciotto V, Lumini E, Bonfante P, Vandamme P. ‘Candidatus glomeribacter gigasporarum’ gen. nov., sp. nov., an endosymbiont of arbuscular mycorrhizal fungi. Int J Syst Evol Microbiol. 2003;53:121–4.

    CAS  Google Scholar 

  55. 55.

    Turina M, Ghignone S, Astolfi N, Silvestri A, Bonfante P, Lanfranco L. The virome of the arbuscular mycorrhizal fungus Gigaspora margarita reveals the first report of DNA fragments corresponding to replicating non-retroviral RNA viruses in fungi. Environ Microbiol. 2018;20:2012–25.

    CAS  PubMed  Google Scholar 

  56. 56.

    Li CX, Zhu JZ, Gao BD, Zhu HJ, Zhou Q, Zhong J. Characterization of a novel ourmia-like mycovirus infecting Magnaporthe oryzae and implications for viral diversity and evolution. Viruses. 2019;11:223.

    CAS  PubMed Central  Google Scholar 

  57. 57.

    Bidartondo MI, Read DJ, Trappe JM, Merckx V, Ligrone R, Duckett JG. The dawn of symbiosis between plants and fungi. Biol Lett. 2011;7:574–7.

    PubMed  PubMed Central  Google Scholar 

  58. 58.

    Desirò A, Faccio A, Kaech A, Bidartondo MI, Bonfante P. Endogone, one of the oldest plant-associated fungi, host unique Mollicutes-related endobacteria. New Phytol. 2015;205:1464–72.

    PubMed  Google Scholar 

  59. 59.

    Lutzoni F, Nowak MD, Alfaro ME, Reeb V, Miadlikowska J, Krug M, et al. Contemporaneous radiations of fungi and plants linked to symbiosis. Nat Commun. 2018;9:5451.

    CAS  PubMed  PubMed Central  Google Scholar 

  60. 60.

    Roossinck MJ. Evolutionary and ecological links between plant and fungal viruses. New Phytol. 2019;221:86–92.

    PubMed  Google Scholar 

  61. 61.

    Andika IB, Wei S, Cao C, Salaipeth L, Kondo H, Sun L. Phytopathogenic fungus hosts a plant virus: a naturally occurring cross-kingdom viral infection. Proc Natl Acad Sci USA. 2017;114:12267–72.

    CAS  PubMed  Google Scholar 

  62. 62.

    Dolatabadi S, Walther G, Ende van den AHGG, Hoog de GS. Diversity and delimitation of Rhizopus microsporus. Fungal Divers. 2013;64:145–63.

    Google Scholar 

  63. 63.

    Ghabrial SA. Origin, adaptation and evolutionary pathways of fungal viruses. Virus Genes. 1998;16:119–31.

    CAS  PubMed  PubMed Central  Google Scholar 

  64. 64.

    Hamid MR, Xie J, Wu S, Maria SK, Zheng D, Assane Hamidou A, et al. A novel deltaflexivirus that infects the plant fungal pathogen, Sclerotinia sclerotiorum, can be transmitted among host vegetative incompatible strains. Viruses. 2018;10:295.

    PubMed Central  Google Scholar 

  65. 65.

    Chagnon P-L. Ecological and evolutionary implications of hyphal anastomosis in arbuscular mycorrhizal fungi. FEMS Microbiol Ecol. 2014;88:437–44.

    CAS  PubMed  Google Scholar 

  66. 66.

    de Novais CB, Pepe A, Siqueira JO, Giovannetti M, Sbrana C, de Novais CB, et al. Compatibility and incompatibility in hyphal anastomosis of arbuscular mycorrhizal fungi. Sci Agric. 2017;74:411–6.

    Google Scholar 

  67. 67.

    Itabangi H, Sephton-Clark PCS, Zhou X, Insua I, Probert M, Correia J, et al. A bacterial endosymbiont enables fungal immune evasion during fatal mucormycete infection. 2019:584607.

  68. 68.

    Cai G, Fry WE, Hillman B. PiRV-2 stimulates sporulation in Phytophthora infestans. Virus Res. 2019;271:197674.

    CAS  PubMed  Google Scholar 

  69. 69.

    Nuss DL. Mycoviruses, RNA silencing, and viral RNA recombination. Adv Virus Res. 2011;80:25–48.

    CAS  PubMed  PubMed Central  Google Scholar 

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Authors acknowledge María Nélida Vázquez Sánchez for technical support; Luis Delaye for advice on the phylogenetic analyses, as well as Karin Grothe, Robert Winkler, and the three anonymous referees for their useful comments. LPPM is thankful to Consejo Nacional de Ciencia y Tecnología (CONACyT) in Mexico, which financed most of this research with grant FOINS-2015-01-006.

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LPPM, ANEV, JRBB, and CAG: designed and planned research; ANEV, JRBB, JFCR, GCL, FMC, AMV, DACP, LPPM: performed experiments and analyzed the data; SJM and TEP generated RNA-Seq libraries and provided R. microsporus wild-type strains; LPPM secured funding; LPPM and ANEV wrote the paper; all authors read, improved, and approved the final version of this document.

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Correspondence to Laila P. Partida-Martínez.

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Espino-Vázquez, A.N., Bermúdez-Barrientos, J.R., Cabrera-Rangel, J.F. et al. Narnaviruses: novel players in fungal–bacterial symbioses. ISME J 14, 1743–1754 (2020).

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