Functional & Integrative Genomics

, Volume 19, Issue 3, pp 453–465 | Cite as

Transcriptomics analysis of propiconazole-treated Cochliobolus sativus reveals new putative azole targets in the plant pathogen

  • Deepika Somani
  • Ragini Adhav
  • Ramya Prashant
  • Narendra Y. KadooEmail author
Original Article


Cochliobolus sativus (anamorph: Bipolaris sorokiniana) is a filamentous fungus from the class Dothideomycetes. It is a pathogen of cereals including wheat and barley, and causes foliar spot blotch, root rot, black point on grains, head blight, leaf blight, and seedling blight diseases. Annual yields of these economically important cereals are severely reduced due to this pathogen attack. Evolution of fungicide resistant pathogen strains, availability of a limited number of potent antifungal compounds, and their efficacy are the acute issues in field management of phytopathogenic fungi. Propiconazole is a widely used azole fungicide to control the disease in fields. The known targets of azoles are the demethylase enzymes involved in ergosterol biosynthesis. Nonetheless, azoles have multiple modes of action, some of which have not been explored yet. Identifying the off-target effects of fungicides by dissecting gene expression profiles in response to them can provide insights into their modes of action and possible mechanisms of fungicide resistance. Moreover it can also reveal additional targets for development of new fungicides. Hence, we analyzed the global gene expression profile of C. sativus on exposure to sub-lethal doses of propiconazole in a time series. The gene expression patterns were confirmed using quantitative reverse transcriptase PCR (qRT-PCR). This study revealed overexpression of target genes from the sterol biosynthesis pathway supporting the reported mode of resistance against azoles. In addition, some new potential targets have also been identified, which could be explored to develop new fungicides and plant protection strategies.


Bipolaris sorokiniana Cochliobolus sativus Fungicide resistance Propiconazole RNA-seq Transcriptomics analysis 



ATP binding cassette


Counts per million


Differentially expressed genes


Effective concentration to give half maximal response


Fragments per kilobase million


Hours post treatment


Kyoto Encyclopedia of Genes and Genomes


Log2 fold change


National Center for Biotechnology Information


Potato dextrose agar


Quantitative reverse transcriptase polymerase chain reaction



DS acknowledges the Junior and Senior Research fellowships from the University Grants Commission (UGC), India. The authors thank Dr. Rajeev Kumar, Dept. of Agricultural Biotechnology & Molecular Biology, Dr. Rajendra Prasad Central Agricultural University, Pusa (India) for providing the D2 isolate of C. sativus and Dr. I.K. Kalappanwar, Dept. of Genetics and Plant Breeding, University of Agricultural Sciences, Dharwad (India) for help in microscopic identification of the pathogen. The Centre for Cellular and Molecular Platforms (C-CAMP), Bangalore (India) is acknowledged for RNA sequencing.


NK received financial support in the form of Council of Scientific and Industrial Research (CSIR), India (BSC 0117) and RP received financial support from Department of Biotechnology (DBT), India (GAP 304126).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Supplementary material

10142_2019_660_Fig8_ESM.png (2.4 mb)
Fig S1

Radial growth inhibition assay using PDA plates. a) Top and bottom view of culture at 2 ppm – 32 ppm concentration of propiconazole. b) Comparison of culture at 16 ppm and 32 ppm concentration of propiconazole with respect to control (picture taken on 6th day after inoculation) (PNG 2414 kb)

10142_2019_660_MOESM1_ESM.tif (3.1 mb)
High Resolution Image (TIF 3163 kb)
10142_2019_660_Fig9_ESM.png (326 kb)
Fig S2

Functional classification of differentially expressed transcripts (CC: Cellular Component, MF: Molecular Function, BP: Biological Process) (PNG 326 kb)

10142_2019_660_MOESM2_ESM.tif (410 kb)
High Resolution Image (TIF 409 kb)
10142_2019_660_Fig10_ESM.png (976 kb)
Fig S3

KEGG annotation of enzymes from DEGs to respective pathways at: (a) 3 hpt; (b) 6 hpt and (c) 12 hpt (PNG 976 kb)

10142_2019_660_MOESM3_ESM.tif (1.3 mb)
High Resolution Image (TIF 1367 kb)
10142_2019_660_Fig11_ESM.png (5.6 mb)
Fig S4

Functional protein association network of DEGs at 3 hpt, 6 hpt and 12 hpt from propiconazole treated culture (PNG 5774 kb)

10142_2019_660_MOESM4_ESM.tif (7.5 mb)
High Resolution Image (TIF 7689 kb)
10142_2019_660_Fig12_ESM.png (538 kb)
Fig S5

Fold change pattern of genes of steroid biosynthesis pathway. The numbers on Y-axis indicate the log2 fold change values (PNG 538 kb)

10142_2019_660_MOESM5_ESM.tif (637 kb)
High Resolution Image (TIF 637 kb)
10142_2019_660_MOESM6_ESM.xlsx (475 kb)
ESM 1 (XLSX 474 kb)


  1. Agarwal AK, Rogers PD, Baerson SR, Jacob MR, Barker KS, Cleary JD, Walker LA, Nagle DG, Clark AM (2003) Genome-wide expression profiling of the response to polyene, pyrimidine, azole, and echinocandin antifungal agents in Saccharomyces cerevisiae. J Biol Chem 278:34998–35015CrossRefPubMedGoogle Scholar
  2. Anders S, Pyl PT, Huber W (2015) HTSeq—a Python framework to work with high-throughput sequencing data. Bioinformatics 31:166–169CrossRefPubMedGoogle Scholar
  3. Anke T, Oberwinkler F, Steglich W, Schramm G (1977) The strobilurins-new antifungal antibiotics from the basidiomycete Strobilurus tenacellus. J Antibiot 30:806–810CrossRefPubMedGoogle Scholar
  4. Arendrup MC, Patterson TF (2017) Multidrug-resistant Candida: epidemiology, molecular mechanisms, and treatment. J Infect Dis 216:S445–S451CrossRefPubMedGoogle Scholar
  5. Ashman PJ, Mackenzie A, Bramley PM (1990) Characterization of ent-kaurene oxidase activity from Gibberella fujikuroi. Biochimica et Biophysica Acta (BBA) 1036:151–157CrossRefGoogle Scholar
  6. Babczinski P, Haselbeck A, Tanner W (1980) Yeast mannosyl transferases requiring dolichyl phosphate and dolichyl phosphate mannose as substrate: partial purification and characterization of the solubilized enzyme. Eur J Biochem 105:509–515CrossRefPubMedGoogle Scholar
  7. Balba H (2007) Review of strobilurin fungicide chemicals. J Environ Sci Health B 42:441–451CrossRefPubMedGoogle Scholar
  8. Bammert GF, Fostel JM (2000) Genome-wide expression patterns inSaccharomyces cerevisiae: comparison of drug treatments and genetic alterations affecting biosynthesis of ergosterol. Antimicrob Agents Chemother 44:1255–1265CrossRefPubMedPubMedCentralGoogle Scholar
  9. Becher R, Wirsel SGR (2012) Fungal cytochrome P450 sterol 14 alpha-demethylase (CYP51) and azole resistance in plant and human pathogens. Appl Microbiol Biotechnol 95:825–840. CrossRefPubMedGoogle Scholar
  10. Bennett V, Healy J (2008) Organizing the fluid membrane bilayer: diseases linked to spectrin and ankyrin. Trends Mol Med 14:28–36CrossRefPubMedGoogle Scholar
  11. Bolton MD, Ebert MK, Faino L, Rivera-Varas V, de Jonge R, Van de Peer Y, Thomma BP, Secor GA (2016) RNA-sequencing of Cercospora beticola DMI-sensitive and-resistant isolates after treatment with tetraconazole identifies common and contrasting pathway induction. Fungal Genet Biol 92:1–13CrossRefPubMedGoogle Scholar
  12. Cannon RD, Lamping E, Holmes AR, Niimi K, Tanabe K, Niimi M, Monk BC (2007) Candida albicans drug resistance–another way to cope with stress. Microbiology 153:3211–3217CrossRefPubMedGoogle Scholar
  13. Conesa A, Götz S, García-Gómez JM, Terol J, Talón M, Robles M (2005) Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21:3674–3676CrossRefGoogle Scholar
  14. de Waard MA, Andrade AC, Hayashi K, Hj S, Stergiopoulos I, Zwiers LH (2006) Impact of fungal drug transporters on fungicide sensitivity, multidrug resistance and virulence. Pest Manag Sci 62:195–207CrossRefPubMedGoogle Scholar
  15. Dubin H, Ginkel Mv (1991) The status of wheat diseases and disease research in warmer areas. Wheat for the nontraditional warm areas: a proceedings of the International Conference July 29–August 3 1990 Foz do Iguaçu, Brazil. CIMMYT, pp 125–145Google Scholar
  16. Duveiller E, Gilchrist L (1994) Production constraints due to Bipolaris sorokiniana in wheat: current situation and future prospects. Wheat in Warm Area, Rice-Wheat Farming Systems Dinajpur (Bangladesh) 13–15 Feb 1993Google Scholar
  17. Fischer B, Rummel G, Aldridge P, Jenal U (2002) The FtsH protease is involved in development, stress response and heat shock control in Caulobacter crescentus. Mol Microbiol 44:461–478CrossRefPubMedGoogle Scholar
  18. Fretland AJ, Omiecinski CJ (2000) Epoxide hydrolases: biochemistry and molecular biology. Chem Biol Interact 129:41–59CrossRefPubMedGoogle Scholar
  19. Gardiner DM, Stephens AE, Munn AL, Manners JM (2013) An ABC pleiotropic drug resistance transporter of Fusarium graminearum with a role in crown and root diseases of wheat. FEMS Microbiol Lett 348:36–45CrossRefPubMedGoogle Scholar
  20. Ghannoum MA (2000) Potential role of phospholipases in virulence and fungal pathogenesis. Clin Microbiol Rev 13:122–143CrossRefPubMedPubMedCentralGoogle Scholar
  21. Ghannoum MA, Rice LB (1999) Antifungal agents: mode of action, mechanisms of resistance, and correlation of these mechanisms with bacterial resistance. Clin Microbiol Rev 12:501–517CrossRefPubMedPubMedCentralGoogle Scholar
  22. Gu R, Fonseca S, Puskás LG, Hackler L Jr, Zvara Á, Dudits D, Pais MS (2004) Transcript identification and profiling during salt stress and recovery of Populus euphratica. Tree Physiol 24:265–276CrossRefPubMedGoogle Scholar
  23. Hargrove TY, Friggeri L, Wawrzak Z, Qi A, Hoekstra WJ, Schotzinger RJ, York JD, Guengerich FP, Lepesheva GI (2017) Structural analyses of Candida albicans sterol 14 alpha-demethylase complexed with azole drugs address the molecular basis of azole-mediated inhibition of fungal sterol biosynthesis. Journal of Biological Chemistry:jbc. M117. 778308Google Scholar
  24. Helliwell CA, Poole A, James Peacock W, Dennis ES (1999) Arabidopsis <em>ent</em>-kaurene oxidase catalyzes three steps of gibberellin biosynthesis. Plant Physiol 119:507–510. CrossRefPubMedPubMedCentralGoogle Scholar
  25. Joshi A, Chand R (2002) Variation and inheritance of leaf angle, and its association with spot blotch (Bipolaris sorokiniana) severity in wheat (Triticum aestivum). Euphytica 124:283–291CrossRefGoogle Scholar
  26. Kihara J, Sato A, Okajima S, Kumagai T (2001) Molecular cloning, sequence analysis and expression of a novel gene induced by near-UV light in Bipolaris oryzae. Mol Gen Genomics 266:64–71CrossRefGoogle Scholar
  27. Kim D, Pertea G, Trapnell C, Pimentel H, Kelley R, Salzberg SL (2013) TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol 14:R36CrossRefPubMedPubMedCentralGoogle Scholar
  28. Kim D, Salzberg SL (2011) TopHat-fusion: an algorithm for discovery of novel fusion transcripts. Genome Biol 12:R72CrossRefPubMedPubMedCentralGoogle Scholar
  29. Kumar J, Schäfer P, Hückelhoven R, Langen G, Baltruschat H, Stein E, Nagarajan S, Kogel KH (2002) Bipolaris sorokiniana, a cereal pathogen of global concern: cytological and molecular approaches towards better control. Mol Plant Pathol 3:185–195CrossRefPubMedGoogle Scholar
  30. Li X, Wang J, Manley JL (2005) Loss of splicing factor ASF/SF2 induces G2 cell cycle arrest and apoptosis, but inhibits internucleosomal DNA fragmentation. Genes Dev 19:2705–2714CrossRefPubMedPubMedCentralGoogle Scholar
  31. Liu T, Xu S, Liu L, Zhou F, Hou J, Chen J (2011) Cloning and characteristics of Brn1 gene in Curvularia lunata causing leaf spot in maize. Eur J Plant Pathol 131:211–219CrossRefGoogle Scholar
  32. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 25:402–408CrossRefGoogle Scholar
  33. Mehta Y, McNab A (1998) Constraints on the integrated management of spot blotch of wheat. Helminthosporium Blight of Wheat: Spot Blotch and Tan Spot. pp. 18-27Google Scholar
  34. Moore JT, Gaylor J (1969) Isolation and purification of an S-adenosylmethionine: Δ24-sterol methyltransferase from yeast. J Biol Chem 244:6334–6340PubMedGoogle Scholar
  35. Mullins JG, Parker JE, Cools HJ, Togawa RC, Lucas JA, Fraaije BA, Kelly DE, Kelly SL (2011) Molecular modelling of the emergence of azole resistance in Mycosphaerella graminicola. PLoS One 6:e20973CrossRefPubMedPubMedCentralGoogle Scholar
  36. Newman JW, Morisseau C, Hammock BD (2005) Epoxide hydrolases: their roles and interactions with lipid metabolism. Prog Lipid Res 44:1–51CrossRefPubMedGoogle Scholar
  37. Nuruzzaman M, Zhang R, Cao HZ, Luo ZY (2014) Plant pleiotropic drug resistance transporters: transport mechanism, gene expression, and function. J Integr Plant Biol 56:729–740CrossRefPubMedGoogle Scholar
  38. Oliveros JC (2007) VENNY. An interactive tool for comparing lists with Venn DiagramsGoogle Scholar
  39. Patel RK, Jain M (2012) NGS QC toolkit: a toolkit for quality control of next generation sequencing data. PLoS One 7:e30619CrossRefPubMedPubMedCentralGoogle Scholar
  40. Prasad R, Goffeau A (2012) Yeast ATP-binding cassette transporters conferring multidrug resistance. Annu Rev Microbiol 66:39–63CrossRefPubMedGoogle Scholar
  41. Ramakers C, Ruijter JM, Deprez RHL, Moorman AFM (2003) Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data. Neurosci Lett 339:62–66CrossRefGoogle Scholar
  42. Renes J, De Vries EGE, Nienhuis EF, Jansen PLM, Muller M (1999) ATP- and glutathione-dependent transport of chemotherapeutic drugs by the multidrug resistance protein MRP1. Br J Pharmacol 126:681–688CrossRefPubMedPubMedCentralGoogle Scholar
  43. Revie NM, Iyer KR, Robbins N, Cowen LE (2018) Antifungal drug resistance: evolution, mechanisms and impact. Curr Opin Microbiol 45:70–76CrossRefPubMedGoogle Scholar
  44. Rogers PD, Barker KS (2003) Genome-wide expression profile analysis reveals coordinately regulated genes associated with stepwise acquisition of azole resistance in Candida albicans clinical isolates. Antimicrob Agents Chemother 47:1220–1227CrossRefPubMedPubMedCentralGoogle Scholar
  45. Sanglard D, Odds FC (2002) Resistance of Candida species to antifungal agents: molecular mechanisms and clinical consequences. Lancet Infect Dis 2:73–85CrossRefPubMedGoogle Scholar
  46. Santos BS, da Silva LCN, Silva TD, Rodrigues JFS, Grisotto MAG, Correia MT, Napoleão TH, Silva MV, Paiva PMG (2016) Application of omics technologies for evaluation of antibacterial mechanisms of action of plant-derived products. Front Microbiol 7:1466PubMedPubMedCentralGoogle Scholar
  47. Secor GA, Rivera VV (2012) Fungicide resistance assays for fungal plant pathogens. Plant Fungal Pathogens: Methods and Protocols:385–392Google Scholar
  48. Song T-T, Zhao J, Ying S-H, Feng M-G (2013) Differential contributions of five ABC transporters to mutidrug resistance, antioxidion and virulence of Beauveria bassiana, an entomopathogenic fungus. PLoS One 8:e62179CrossRefPubMedPubMedCentralGoogle Scholar
  49. Soustre I, Letourneux Y, Karst F (1996) Characterization of the Saccharomyces cerevisiae RTA1 gene involved in 7-aminocholesterol resistance. Curr Genet 30:121–125CrossRefPubMedGoogle Scholar
  50. Strange RC, Spiteri MA, Ramachandran S, Fryer AA (2001) Glutathione-S-transferase family of enzymes. Mutat Res 482:21–26CrossRefPubMedGoogle Scholar
  51. Szklarczyk D, Franceschini A, Wyder S, Forslund K, Heller D, Huerta-Cepas J, Simonovic M, Roth A, Santos A, Tsafou KP (2014) STRING v10: protein–protein interaction networks, integrated over the tree of life. Nucleic Acids Research:gku1003Google Scholar
  52. Tarazona S, García F, Ferrer A, Dopazo J, Conesa A (2012) NOIseq: a RNA-seq differential expression method robust for sequencing depth biases. EMBnet Journal 17:18–19CrossRefGoogle Scholar
  53. Trapnell C, Pachter L, Salzberg SL (2009) TopHat: discovering splice junctions with RNA-Seq. Bioinformatics 25:1105–1111CrossRefPubMedPubMedCentralGoogle Scholar
  54. Trapnell C, Roberts A, Goff L, Pertea G, Kim D, Kelley DR, Pimentel H, Salzberg SL, Rinn JL, Pachter L (2012) Differential gene and transcript expression analysis of RNA-seq experiments with TopHat and Cufflinks. Nat Protoc 7:562–578CrossRefPubMedPubMedCentralGoogle Scholar
  55. Wang Q, He J, Lynn B, Rymond BC (2005) Interactions of the yeast SF3b splicing factor. Mol Cell Biol 25:10745–10754CrossRefPubMedPubMedCentralGoogle Scholar
  56. Wegulo SN (2012) Factors influencing deoxynivalenol accumulation in small grain cereals. Toxins 4:1157–1180CrossRefPubMedPubMedCentralGoogle Scholar
  57. Zhu N, Liu J, Yang J, Lin Y, Yang Y, Ji L, Li M, Yuan H (2016) Comparative analysis of the secretomes of Schizophyllum commune and other wood-decay basidiomycetes during solid-state fermentation reveals its unique lignocellulose-degrading enzyme system. Biotechnol Biofuels 9:42CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Biochemical Sciences DivisionCSIR-National Chemical LaboratoryPuneIndia
  2. 2.Academy of Scientific and Innovative Research (AcSIR)New DelhiIndia
  3. 3.Faculty of Health ScienceUniversity of MacauMacauChina

Personalised recommendations