A dispensable paralog of succinate dehydrogenase subunit C mediates standing resistance towards a subclass of SDHI fungicides in Zymoseptoria tritici

English version

Autoři: Diana Steinhauer ^aff001; Marie Salat ^aff001; Regula Frey ^aff001; Andreas Mosbach ^aff001; Torsten Luksch ^aff001; Dirk Balmer ^aff001; Rasmus Hansen ^aff002; Stephanie Widdison ^aff002; Grace Logan ^aff002; Robert A. Dietrich ^aff003; Gert H. J. Kema ^aff004; Stephane Bieri ^aff001; Helge Sierotzki ^aff001; Stefano F. F. Torriani ^aff001; Gabriel Scalliet ^aff001
Působiště autorů: Syngenta Crop Protection AG, Stein, Switzerland ^aff001; Syngenta Jealott’s Hill Int. Research Centre, Bracknell Berkshire, United Kingdom ^aff002; Syngenta Biotechnology Inc., Research Triangle Park, North Carolina, United States of America ^aff003; Wageningen University and Research, The Netherlands ^aff004
Vyšlo v časopise: A dispensable paralog of succinate dehydrogenase subunit C mediates standing resistance towards a subclass of SDHI fungicides in Zymoseptoria tritici. PLoS Pathog 15(12): e32767. doi:10.1371/journal.ppat.1007780
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1007780

Souhrn

Succinate dehydrogenase inhibitor (SDHI) fungicides are widely used for the control of a broad range of fungal diseases. This has been the most rapidly expanding fungicide group in terms of new molecules discovered and introduced for agricultural use over the past fifteen years. A particular pattern of differential sensitivity (resistance) to the stretched heterocycle amide SDHIs (SHA-SDHIs), a subclass of chemically-related SDHIs, was observed in naïve Zymoseptoria tritici populations not previously exposed to these chemicals. Subclass-specific resistance was confirmed at the enzyme level but did not correlate with the genotypes of the succinate dehydrogenase (SDH) encoding genes. Mapping and characterization of the molecular mechanisms responsible for standing SHA-SDHI resistance in natural field isolates identified a gene paralog of SDHC, termed ZtSDHC3, which encodes for an alternative C subunit of succinate dehydrogenase, named alt-SDHC. Using reverse genetics, we showed that alt-SDHC associates with the three other SDH subunits, leading to a fully functional enzyme and that a unique Qp-site residue within the alt-SDHC protein confers SHA-SDHI resistance. Enzymatic assays, computational modelling and docking simulations for the two SQR enzymes (altC-SQR, WT_SQR) enabled us to describe enzyme-inhibitor interactions at an atomistic level and to propose rational explanations for differential potency and resistance across SHA-SDHIs. European Z. tritici populations displayed a presence (20–30%) / absence polymorphism of ZtSDHC3, as well as differences in ZtSDHC3 expression levels and splicing efficiency. These polymorphisms have a strong impact on SHA-SDHI resistance phenotypes. Characterization of the ZtSDHC3 promoter in European Z. tritici populations suggests that transposon insertions are associated with the strongest resistance phenotypes. These results establish that a dispensable paralogous gene determines SHA-SDHIs fungicide resistance in natural populations of Z. tritici. This study paves the way to an increased awareness of the role of fungicidal target paralogs in resistance to fungicides and demonstrates the paramount importance of population genomics in fungicide discovery.

Klíčová slova:

Genomics – Transposable elements – Fungal genetics – Genetic loci – Europe – Polymerase chain reaction – Mitochondria – Fungicides

Zdroje

1. Lamberth C, Jeanmart S, Luksch T, Plant A. Current challenges and trends in the discovery of agrochemicals. Science. 2013;341(6147):742–6. Epub 2013/08/21. doi: 10.1126/science.1237227 [pii]. 23950530

2. Horsefield R, Yankovskaya V, Sexton G, Whittingham W, Shiomi K, Omura S, et al. Structural and computational analysis of the quinone-binding site of complex II (succinate-ubiquinone oxidoreductase): a mechanism of electron transfer and proton conduction during ubiquinone reduction. Journal of Biological Chemistry. 2006;281(11):7309–16. doi: 10.1074/jbc.M508173200 16407191

3. Zhu XL, Xiong L, Li H, Song XY, Liu JJ, Yang GF. Computational and experimental insight into the molecular mechanism of carboxamide inhibitors of succinate-ubquinone oxidoreductase. ChemMedChem. 2014;9(7):1512–21. Epub 2014/03/29. doi: 10.1002/cmdc.201300456 24678033

4. Lancaster CR. Succinate:quinone oxidoreductases: an overview. Biochim Biophys Acta. 2002;1553(1–2):1–6. Epub 2002/01/23. doi: S0005272801002407 [pii]. doi: 10.1016/s0005-2728(01)00240-7 11803013

5. Scalliet G, Boehler M, J. B, Geen PS, Kilby PM, Fonné-Pfister R. SDHIs and the Fungal Succinate Dehydrogenase. In: Dehne HW, Deising HB, Gisi U, Kuck KH, Russell PE, Lyr H, editors. Modern Fungicides and Antifungal Compounds VI; DPG-Verlag, Braunschweig, Germany 2011. p. 171–8.

6. Schmeling BV, Kulka M. Systemic fungicidal activity of 1,4-oxathiin derivatives. Science. 1966;152(3722):659–60. Epub 1966/04/29. doi: 152/3722/659 [pii] doi: 10.1126/science.152.3722.659 17779512

7. Snel M, Schmeling BV, Edgington LV. Fungitoxicity and structure-activity relationships of some oxathiin and thiazole derivatives. Phytopathology. 1970;60:1164–9. doi: 10.1094/phyto-60-1164 5529696

8. Stammler G, Brix H-D, Glaettli A, Semar M, Schoefl U, editors. Biological properties of the carboxamide boscalid including recent studies on its mode of action. Proceedings, 16th International congress of Plant Protection; 2007; Glasgow.

9. FRAC. FRAC MoA Poster 2019-final FRAC web2019. Available from: http://www.frac.info/docs/default-source/publications/frac-mode-of-action-poster/frac-moa-poster-2019-final.pdf

10. Sierotzki H, Scalliet G. A review of current knowledge of resistance aspects for the next-generation succinate dehydrogenase inhibitor fungicides. Phytopathology. 2013;103(9):880–7. Epub 2013/04/19. doi: 10.1094/PHYTO-01-13-0009-RVW 23593940

11. Torriani SF, Melichar JP, Mills C, Pain N, Sierotzki H, Courbot M. Zymoseptoria tritici: A major threat to wheat production, integrated approaches to control. Fungal Genet Biol. 2015;79:8–12. Epub 2015/06/21. doi: 10.1016/j.fgb.2015.04.010 [pii]. 26092783

12. Skinner W, Bailey A, Renwick A, Keon J, Gurr S, Hargreaves J. A single amino-acid substitution in the iron-sulphur protein subunit of succinate dehydrogenase determines resistance to carboxin in Mycosphaerella graminicola. Curr Genet. 1998;34(5):393–8. Epub 1998/12/31. doi: 10.1007/s002940050412 9871122

13. Fraaije BA, Bayon C, Atkins S, Cools HJ, Lucas JA, Fraaije MW. Risk assessment studies on succinate dehydrogenase inhibitors, the new weapons in the battle to control Septoria leaf blotch in wheat. Mol Plant Pathol. 2012;13(3):263–75. Epub 2011/09/22. doi: 10.1111/j.1364-3703.2011.00746.x 21933337

14. Scalliet G, Bowler J, Luksch T, Kirchhofer-Allan L, Steinhauer D, Ward K, et al. Mutagenesis and functional studies with succinate dehydrogenase inhibitors in the wheat pathogen Mycosphaerella graminicola. PLoS One. 2012;7(4):e35429. Epub 2012/04/27. doi: 10.1371/journal.pone.0035429 [pii]. 22536383; PubMed Central PMCID: PMC3334918.

15. Ishii H, Miyamoto T, Ushio S, Kakishima M. Lack of cross-resistance to a novel succinate dehydrogenase inhibitor, fluopyram, in highly boscalid-resistant isolates of Corynespora cassiicola and Podosphaera xanthii. Pest Management Science. 2011;67(4):474–82. doi: 10.1002/ps.2092 21394880

16. Leroux P, Gredt M, Leroch M, Walker AS. Exploring mechanisms of resistance to respiratory inhibitors in field strains of Botrytis cinerea, the causal agent of gray mold. Applied & Environmental Microbiology. 2010;76(19):6615–30. doi: 10.1128. PubMed PMID: AEM.00931-10.

17. Rehfus A, Strobel D, Bryson R, Stammler G. Mutations in sdh genes in field isolates of Zymoseptoria tritici and impact on the sensitivity to various succinate dehydrogenase inhibitors. Plant Pathology. 2018;67(1):175–80. doi: 10.1111/ppa.12715

18. Dooley H, Shaw MW, Spink J, Kildea S. The effect of succinate dehydrogenase inhibitor/azole mixtures on selection of Zymoseptoria tritici isolates with reduced sensitivity. Pest Manag Sci. 2016;72(6):1150–9. Epub 2015/08/14. doi: 10.1002/ps.4093 26269125

19. FRAC. sdhi Fungicides working group 2019 [cited 2019]. Available from: http://www.frac.info/working-group/sdhi-fungicides

20. Klappach K, Zito R, Bryson R, Stammler G, Semar M, Mehl M, et al. Succinate Dehydrogenase Inhibitor (SDHI) Working Group 2019 [cited 2018]. Meeting on December 11/2, 2018, Protocol of the discussions and use recommendations of the SDHI Working Group of the Fungicide Resistance Action Committee (FRAC)]. Available from: http://www.frac.info/docs/default-source/sdhi-wg/sdhi-meeting-minutes/minutes-of-the-2018-sdhi-meeting-11-12th-of-december-2018-with-recommendations-for-2019.pdf

21. Yamashita M, Fraaije B. Non-target site SDHI resistance is present as standing genetic variation in field populations of Zymoseptoria tritici. Pest Manag Sci. 2018;74(3):672–81. Epub 2017/10/13. doi: 10.1002/ps.4761 29024365; PubMed Central PMCID: PMC5814837.

22. Torriani SF, Brunner PC, McDonald BA, Sierotzki H. QoI resistance emerged independently at least 4 times in European populations of Mycosphaerella graminicola. Pest Manag Sci. 2009;65(2):155–62. Epub 2008/10/04. doi: 10.1002/ps.1662 18833571

23. Kema GHJ, Mirzadi Gohari A, Aouini L, Gibriel HAY, Ware SB, van den Bosch F, et al. Stress and sexual reproduction affect the dynamics of the wheat pathogen effector AvrStb6 and strobilurin resistance. Nat Genet. 2018;50(3):375–80. Epub 2018/02/13. doi: 10.1038/s41588-018-0052-9 [pii]. 29434356

24. Fraaije BA, Cools HJ, Fountaine J, Lovell DJ, Motteram J, West JS, et al. Role of Ascospores in Further Spread of QoI-Resistant Cytochrome b Alleles (G143A) in Field Populations of Mycosphaerella graminicola. Phytopathology. 2005;95(8):933–41. Epub 2008/10/24. doi: 10.1094/PHYTO-95-0933 18944416

25. Cools HJ, Fraaije BA. Update on mechanisms of azole resistance in Mycosphaerella graminicola and implications for future control. Pest Manag Sci. 2013;69(2):150–5. Epub 2012/06/26. doi: 10.1002/ps.3348 22730104

26. Cools HJ, Mullins JG, Fraaije BA, Parker JE, Kelly DE, Lucas JA, et al. Impact of recently emerged sterol 14{alpha}-demethylase (CYP51) variants of Mycosphaerella graminicola on azole fungicide sensitivity. Appl Environ Microbiol. 2011;77(11):3830–7. Epub 2011/04/12. doi: 10.1128/AEM.00027-11 AEM.00027-11 [pii]. 21478305; PubMed Central PMCID: PMC3127603.

27. Cools HJ, Bayon C, Atkins S, Lucas JA, Fraaije BA. Overexpression of the sterol 14alpha-demethylase gene (MgCYP51) in Mycosphaerella graminicola isolates confers a novel azole fungicide sensitivity phenotype. Pest Manag Sci. 2012;68(7):1034–40. Epub 2012/03/14. doi: 10.1002/ps.3263 22411894

28. Omrane S, Audeon C, Ignace A, Duplaix C, Aouini L, Kema G, et al. Plasticity of the MFS1 Promoter Leads to Multidrug Resistance in the Wheat Pathogen Zymoseptoria tritici. mSphere. 2017;2(5). Epub 2017/11/01. doi: e00393-17 [pii] doi: 10.1128/mSphere.00393-17 mSphere00393-17 [pii]. 29085913; PubMed Central PMCID: PMC5656749.

29. Omrane S, Sghyer H, Audeon C, Lanen C, Duplaix C, Walker AS, et al. Fungicide efflux and the MgMFS1 transporter contribute to the multidrug resistance phenotype in Zymoseptoria tritici field isolates. Environ Microbiol. 2015;17(8):2805–23. Epub 2015/01/30. doi: 10.1111/1462-2920.12781 25627815

30. Roohparvar R, De Waard MA, Kema GH, Zwiers LH. MgMfs1, a major facilitator superfamily transporter from the fungal wheat pathogen Mycosphaerella graminicola, is a strong protectant against natural toxic compounds and fungicides. Fungal Genet Biol. 2007;44(5):378–88. Epub 2006/11/17. doi: S1087-1845(06)00176-9 [pii] doi: 10.1016/j.fgb.2006.09.007 17107817

31. Roohparvar R, Mehrabi R, Van Nistelrooy JG, Zwiers LH, De Waard MA. The drug transporter MgMfs1 can modulate sensitivity of field strains of the fungal wheat pathogen Mycosphaerella graminicola to the strobilurin fungicide trifloxystrobin. Pest Manag Sci. 2008;64(7):685–93. Epub 2008/03/28. doi: 10.1002/ps.1569 18366066

32. Gutierrez-Alonso O, Hawkins NJ, Cools HJ, Shaw MW, Fraaije BA. Dose-dependent selection drives lineage replacement during the experimental evolution of SDHI fungicide resistance in Zymoseptoria tritici. Evol Appl. 2017;10(10):1055–66. Epub 2017/11/21. doi: 10.1111/eva.12511 [pii]. 29151860; PubMed Central PMCID: PMC5680630.

33. Krishnan P, Meile L, Plissonneau C, Ma X, Hartmann FE, Croll D, et al. Transposable element insertions shape gene regulation and melanin production in a fungal pathogen of wheat. BMC Biol. 2018;16(1):78. Epub 2018/07/18. doi: 10.1186/s12915-018-0543-2 [pii]. 30012138; PubMed Central PMCID: PMC6047131.

34. Emanuelsson O, Nielsen H, Brunak S, von Heijne G. Predicting subcellular localization of proteins based on their N-terminal amino acid sequence. J Mol Biol. 2000;300(4):1005–16. Epub 2000/07/13. doi: 10.1006/jmbi.2000.3903 [pii]. 10891285

35. Grandaubert J, Bhattacharyya A, Stukenbrock EH. RNA-seq-Based Gene Annotation and Comparative Genomics of Four Fungal Grass Pathogens in the Genus Zymoseptoria Identify Novel Orphan Genes and Species-Specific Invasions of Transposable Elements. G3 (Bethesda). 2015;5(7):1323–33. Epub 2015/04/29. doi: 10.1534/g3.115.017731g3.115.017731 [pii]. 25917918; PubMed Central PMCID: PMC4502367.

36. Rudd JJ, Kanyuka K, Hassani-Pak K, Derbyshire M, Andongabo A, Devonshire J, et al. Transcriptome and metabolite profiling of the infection cycle of Zymoseptoria tritici on wheat reveals a biphasic interaction with plant immunity involving differential pathogen chromosomal contributions and a variation on the hemibiotrophic lifestyle definition. Plant Physiol. 2015;167(3):1158–85. Epub 2015/01/18. doi: 10.1104/pp.114.255927 [pii]. 25596183; PubMed Central PMCID: PMC4348787.

37. Fraaije BA, Bayon C, Atkins S, Cools HJ, Lucas JA, Fraaije MW. Risk assessment studies on succinate dehydrogenase inhibitors, the new weapons in the battle to control Septoria leaf blotch in wheat. Molecular Plant Pathology. 2011. Epub 20 SEP 2011. doi: 10.1111/j.1364-3703.2011.00746.x 21933337

38. Plissonneau C, Hartmann FE, Croll D. Pangenome analyses of the wheat pathogen Zymoseptoria tritici reveal the structural basis of a highly plastic eukaryotic genome. BMC Biol. 2018;16(1):5. Epub 2018/01/13. doi: 10.1186/s12915-017-0457-4 [pii]. 29325559; PubMed Central PMCID: PMC5765654.

39. Hickman AB, Dyda F. DNA Transposition at Work. Chemical Reviews. 2016;116(20):12758–84. doi: 10.1021/acs.chemrev.6b00003 27187082

40. Alvarez-Ponce D, Sabater-Muñoz B, Toft C, Ruiz-González MX, Fares MA. Essentiality Is a Strong Determinant of Protein Rates of Evolution during Mutation Accumulation Experiments in Escherichia coli. Genome Biology and Evolution. 2016;8(9):2914–27. doi: 10.1093/gbe/evw205 27566759

41. Palma-Guerrero J, Torriani SF, Zala M, Carter D, Courbot M, Rudd JJ, et al. Comparative transcriptomic analyses of Zymoseptoria tritici strains show complex lifestyle transitions and intraspecific variability in transcription profiles. Mol Plant Pathol. 2016;17(6):845–59. Epub 2015/11/27. doi: 10.1111/mpp.12333 26610174

42. Simões K, Hawlik A, Rehfus A, Gava F, Stammler G. First detection of a SDH variant with reduced SDHI sensitivity in Phakopsora pachyrhizi. Journal of Plant Diseases and Protection. 2018;125(1):21–6. doi: 10.1007/s41348-017-0117-5

43. Kupfer DM, Drabenstot SD, Buchanan KL, Lai H, Zhu H, Dyer DW, et al. Introns and splicing elements of five diverse fungi. Eukaryot Cell. 2004;3(5):1088–100. Epub 2004/10/08. doi: 10.1128/EC.3.5.1088-1100.2004 15470237; PubMed Central PMCID: PMC522613.

44. Fouché S, Badet T, Oggenfuss U, Plissonneau C, Francisco CS, Croll D. Stress-driven transposable element de-repression dynamics in a fungal pathogen. bioRxiv. 2019:633693. doi: 10.1101/633693

45. Mogi T, Kawakami T, Arai H, Igarashi Y, Matsushita K, Mori M, et al. Siccanin Rediscovered as a Species-Selective Succinate Dehydrogenase Inhibitor. The Journal of Biochemistry. 2009;146(3):383–7. doi: 10.1093/jb/mvp085 19505951

46. Nose K, Endo A. Mode of action of the antibiotic siccanin on intact cells and mitochondria of Trichophyton mentagrophytes. J Bacteriol. 1971;105(1):176–84. Epub 1971/01/01. 4250609; PubMed Central PMCID: PMC248339.

47. Miyadera H, Shiomi K, Ui H, Yamaguchi Y, Masuma R, Tomoda H, et al. Atpenins, potent and specific inhibitors of mitochondrial complex II (succinate-ubiquinone oxidoreductase). Proceedings of the National Academy of Sciences of the United States of America. 2003;100(2):473–7. doi: 10.1073/pnas.0237315100 12515859

48. Moghaddam MB, Gross T, Becker A, Vilcinskas A, Rahnamaeian M. The selective antifungal activity of Drosophila melanogaster metchnikowin reflects the species-dependent inhibition of succinate-coenzyme Q reductase. Sci Rep. 2017;7(1):8192. Epub 2017/08/16. doi: 10.1038/s41598-017-08407-x [pii]. 28811531; PubMed Central PMCID: PMC5557811.

49. D'Costa VM, King CE, Kalan L, Morar M, Sung WW, Schwarz C, et al. Antibiotic resistance is ancient. Nature. 2011;477(7365):457–61. Epub 2011/09/02. doi: 10.1038/nature10388 [pii]. 21881561

50. Iwata F, Shinjyo N, Amino H, Sakamoto K, Islam MK, Tsuji N, et al. Change of subunit composition of mitochondrial complex II (succinate-ubiquinone reductase/quinol-fumarate reductase) in Ascaris suum during the migration in the experimental host. Parasitol Int. 2008;57(1):54–61. Epub 2007/10/16. doi: S1383-5769(07)00103-1 [pii] doi: 10.1016/j.parint.2007.08.002 17933581

51. Roos MH, Tielens AG. Differential expression of two succinate dehydrogenase subunit-B genes and a transition in energy metabolism during the development of the parasitic nematode Haemonchus contortus. Mol Biochem Parasitol. 1994;66(2):273–81. Epub 1994/08/01. doi: 0166-6851(94)90154-6 [pii]. doi: 10.1016/0166-6851(94)90154-6 7808477

52. Elorza A, Roschzttardtz H, Gomez I, Mouras A, Holuigue L, Araya A, et al. A nuclear gene for the iron-sulfur subunit of mitochondrial complex II is specifically expressed during Arabidopsis seed development and germination. Plant Cell Physiol. 2006;47(1):14–21. Epub 2005/10/27. doi: pci218 [pii] doi: 10.1093/pcp/pci218 16249327

53. Szeto SS, Reinke SN, Oyedotun KS, Sykes BD, Lemire BD. Expression of Saccharomyces cerevisiae Sdh3p and Sdh4p paralogs results in catalytically active succinate dehydrogenase isoenzymes. J Biol Chem. 2012;287(27):22509–20. Epub 2012/05/11. doi: 10.1074/jbc.M112.344275 M112.344275 [pii]. 22573324; PubMed Central PMCID: PMC3391083.

54. Szeto SS, Reinke SN, Sykes BD, Lemire BD. Mutations in the Saccharomyces cerevisiae succinate dehydrogenase result in distinct metabolic phenotypes revealed through (1)H NMR-based metabolic footprinting. Journal of Proteome Research. 2010;9(12):6729–39. doi: 10.1021/pr100880y 20964315

55. Gebert N, Gebert M, Oeljeklaus S, von der Malsburg K, Stroud DA, Kulawiak B, et al. Dual function of Sdh3 in the respiratory chain and TIM22 protein translocase of the mitochondrial inner membrane. Mol Cell. 2011;44(5):811–8. Epub 2011/12/14. doi: 10.1016/j.molcel.2011.09.025 [pii]. 22152483

56. Brunner PC, Stefansson TS, Fountaine J, Richina V, McDonald BA. A Global Analysis of CYP51 Diversity and Azole Sensitivity in Rhynchosporium commune. Phytopathology. 2016;106(4):355–61. Epub 2015/12/02. doi: 10.1094/PHYTO-07-15-0158-R 26623995

57. Hawkins NJ, Cools HJ, Sierotzki H, Shaw MW, Knogge W, Kelly SL, et al. Paralog re-emergence: a novel, historically contingent mechanism in the evolution of antimicrobial resistance. Mol Biol Evol. 2014;31(7):1793–802. Epub 2014/04/16. doi: 10.1093/molbev/msu134 [pii]. 24732957; PubMed Central PMCID: PMC4069618.

58. Lendenmann MH, Croll D, McDonald BA. QTL mapping of fungicide sensitivity reveals novel genes and pleiotropy with melanization in the pathogen Zymoseptoria tritici. Fungal Genet Biol. 2015;80:53–67. Epub 2015/05/17. doi: 10.1016/j.fgb.2015.05.001 [pii]. 25979163

59. Bowler J, Scott E, Tailor R, Scalliet G, Ray J, Csukai M. New capabilities for Mycosphaerella graminicola research. Mol Plant Pathol. 2010;11(5):691–704. Epub 2010/08/11. doi: 10.1111/j.1364-3703.2010.00629.x [pii]. 20696006

60. Skinner W, Bailey A, Renwick A, Keon J, Gurr S, Hargreaves J. A single amino-acid substitution in the iron-sulphur protein subunit of succinate dehydrogenase determines resistance to carboxin in Mycosphaerella graminicola. Current Genetics. 1998;34(5):393–8. doi: 10.1007/s002940050412 9871122

61. Hellens R, Mullineaux P, Klee H. Technical Focus: a guide to Agrobacterium binary Ti vectors. Trends Plant Sci. 2000;5(10):446–51. Epub 2000/10/25. doi: S1360-1385(00)01740-4 [pii]. doi: 10.1016/s1360-1385(00)01740-4 11044722

62. Hood EE, Gelvin SB, Melchers LS, Hoekema A. NewAgrobacterium helper plasmids for gene transfer to plants. Transgenic Research. 1993;2(4):208–18. doi: 10.1007/bf01977351

63. Waalwijk C, Mendes O, Verstappen EC, de Waard MA, Kema GH. Isolation and characterization of the mating-type idiomorphs from the wheat septoria leaf blotch fungus Mycosphaerella graminicola. Fungal Genet Biol. 2002;35(3):277–86. Epub 2002/04/04. doi: 10.1006/fgbi.2001.1322 [pii]. 11929216

64. Kema GHJ, Verstappen ECP, Todorova M, Waalwijk C. Successful crosses and molecular tetrad and progeny analyses demonstrate heterothallism in Mycosphaerella graminicola. Current Genetics. 1996;30(3):251–8. doi: 10.1007/s002940050129 8753655

65. Wu TD, Nacu S. Fast and SNP-tolerant detection of complex variants and splicing in short reads. Bioinformatics. 2010;26(7):873–81. Epub 2010/02/12. doi: 10.1093/bioinformatics/btq057 20147302; PubMed Central PMCID: PMC2844994.

66. Miller NA, Kingsmore SF, Farmer A, Langley RJ, Mudge J, Crow JA, et al. Management of High-Throughput DNA Sequencing Projects: Alpheus. J Comput Sci Syst Biol. 2008;1:132. Epub 2008/12/26. doi: 10.4172/jcsb.1000013 20151039; PubMed Central PMCID: PMC2819532.

67. Vilella AJ, Severin J, Ureta-Vidal A, Heng L, Durbin R, Birney E. EnsemblCompara GeneTrees: Complete, duplication-aware phylogenetic trees in vertebrates. Genome Res. 2009;19(2):327–35. Epub 2008/11/26. doi: 10.1101/gr.073585.107 19029536; PubMed Central PMCID: PMC2652215.

68. Sievers F, Wilm A, Dineen D, Gibson TJ, Karplus K, Li W, et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol Syst Biol. 2011;7:539. Epub 2011/10/13. doi: 10.1038/msb.2011.75 21988835; PubMed Central PMCID: PMC3261699.

69. Guindon S, Dufayard JF, Lefort V, Anisimova M, Hordijk W, Gascuel O. New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst Biol. 2010;59(3):307–21. Epub 2010/06/09. doi: 10.1093/sysbio/syq010 20525638

70. Letunic I, Bork P. Interactive tree of life (iTOL) v3: an online tool for the display and annotation of phylogenetic and other trees. Nucleic Acids Res. 2016;44(W1):W242–5. Epub 2016/04/21. doi: 10.1093/nar/gkw290 27095192; PubMed Central PMCID: PMC4987883.

71. Zarnack K, Maurer S, Kaffarnik F, Ladendorf O, Brachmann A, Kamper J, et al. Tetracycline-regulated gene expression in the pathogen Ustilago maydis. Fungal Genet Biol. 2006;43(11):727–38. Epub 2006/07/18. doi: S1087-1845(06)00109-5 [pii] doi: 10.1016/j.fgb.2006.05.006 16843015

72. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–54. Epub 1976/05/07. doi: 0003-2697(76)90527-3 [pii]. doi: 10.1006/abio.1976.9999 942051

73. Shevchenko A, Tomas H, Havli J, Olsen JV, Mann M. In-gel digestion for mass spectrometric characterization of proteins and proteomes. Nature Protocols. 2007;1:2856. doi: 10.1038/nprot.2006.468 17406544

74. Gerber PR, Muller K. MAB, a generally applicable molecular force field for structure modelling in medicinal chemistry. J Comput Aided Mol Des. 1995;9(3):251–68. Epub 1995/06/01. doi: 10.1007/bf00124456 7561977

75. Cole JC, Korb O, McCabe P, Read MG, Taylor R. Knowledge-Based Conformer Generation Using the Cambridge Structural Database. Journal of Chemical Information and Modeling. 2018;58(3):615–29. doi: 10.1021/acs.jcim.7b00697 29425456

76. Zhao Y, Truhlar DG. A new local density functional for main-group thermochemistry, transition metal bonding, thermochemical kinetics, and noncovalent interactions. The Journal of Chemical Physics. 2006;125(19):194101. doi: 10.1063/1.2370993 17129083

77. Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheeseman JR, et al. Gaussian 09 Revision A.02: Gaussian Inc. Wallingford CT 2009; 2009.