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Current Genetics

, Volume 64, Issue 2, pp 443–457 | Cite as

Functional analysis of diacylglycerol O-acyl transferase 2 gene to decipher its role in virulence of Botrytis cinerea

  • Esha Sharma
  • Pamil Tayal
  • Garima Anand
  • Piyush Mathur
  • Rupam KapoorEmail author
Original Article

Abstract

Gray mold disease inflicted by Botrytis cinerea is a serious menace responsible for significant economic loss worldwide. Due to its polyphagous nature, the pathogen has enthused inquisitiveness in researchers to unravel its complexity. Agrobacterium tumefaciens-mediated transformation was used to generate insertional mutants of Botrytis cinerea. A mutant (BCM-55) with disruption in a gene (BcDGAT2) that encodes for diacylglycerol O-acyl transferase 2 (DGAT2), showed enervated virulence on various hosts’ tissues. Enzyme DGAT2 is crucial in the final step of synthesis of triacylglycerol (TAG) that plays an important role in homeostasis of membrane and cellular processes. However, the role of DGAT2 has never been reported in a phytopathogenic fungus. In this study, BCM-55 was characterized to ascertain the role of DGAT2 in virulence of B. cinerea. The insertional mutant was defective in spore production and lacked sclerotia formation as a consequence of lower accumulation of TAG. A significant delay in spore germination in BCM-55 was accompanied with a low penetration potential. Hyphae of the mutant formed swollen endings with considerable impairment in penetration. Deletion of BcDGAT2 also led to increased sensitivity towards cell wall and membrane-disturbing agents. Furthermore, BCM-55 was deficient in the production of oxalic acid and showed lower activity of a cell wall-degrading enzyme, polygalacturonase. The role of BcDGAT2 in virulence was further confirmed by targeted deletion and complementation of the gene. The results insinuate a crucial role of BcDGAT2 in penetration and consequently virulence of B. cinerea. The study provides novel insights into plant–pathogen interactions that can be exploited to develop suitable disease management strategies.

Keywords

Agrobacterium tumefaciens-mediated transformation Botrytis cinerea Diacylglycerol O-acyl transferase Virulence Triacylglycerols 

Notes

Acknowledgements

We thank Dr. Praveen Verma (NIPGR, New Delhi) for providing binary vector pBIF-EGFP and Prof. S. C. Bhatla (Department of Botany, University of Delhi) for his guidance in conducting experiments on lipid analysis. The authors gratefully acknowledge Rasmus John Normand Frandsen for his directions in carrying out targeted deletion of gene. Rupam Kapoor thankfully acknowledges the grant-in-aid provided by Science and Engineering Research Board (SERB), Government of India. Esha Sharma and Garima Anand are thankful to Department of Science and Technology and University Grants Commission for providing fellowship.

Supplementary material

294_2017_752_MOESM1_ESM.pdf (645 kb)
Supplementary material 1 (PDF 644 kb)

References

  1. Aguayo C, Riquelme J, Valenzuela PDT, Hahn M, Moreno ES (2011) Bchex virulence gene of Botrytis cinerea: characterization and functional analysis. J Gen Plant Pathol 77:230–238. doi: 10.1007/s10327-011-0311-4 CrossRefGoogle Scholar
  2. Amselem J, Cuomo CA, van Kan JA, Viaud M, Benito EP, Couloux A, Coutinho PM, de Vries RP, Dyer PS, Fillinger S, Fournier E, Gout L, Hahn M, Kohn L, Lapalu N, Plummer KM, Pradier JM, Quevillon E, Sharon A, Simon A, ten Have A, Tudzynski B, Tudzynski P, Wincker P, Andrew M, Anthouard V, Beever RE, Beffa R, Benoit I, Bouzid O, Brault B, Chen Z, Choquer M, Collemare J, Cotton P, Danchin EG, Da Silva C, Gautier A, Giraud C, Giraud T, Gonzalez C, Grossetete S, Guldener U, Henrissat B, Howlett BJ, Kodira C, Kretschmer M, Lappartient A, Leroch M, Levis C, Mauceli E, Neuveglise C, Oeser B, Pearson M, Poulain J, Poussereau N, Quesneville H, Rascle C, Schumacher J, Segurens B, Sexton A, Silva E, Sirven C, Soanes DM, Talbot NJ, Templeton M, Yandava C, Yarden O, Zeng Q, Rollins JA, Lebrun MH, Dickman M (2011) Genomic analysis of the necrotrophic fungal pathogens Sclerotinia sclerotiorum and Botrytis cinerea. PLoS Genet 7:e1002230. doi: 10.1371/journal.pgen.1002230 CrossRefPubMedPubMedCentralGoogle Scholar
  3. Bhadauria V, Banniza S, Vandenberg A, Selvaraj G, Wei Y (2012) Peroxisomal alanine: glyoxylate aminotransferase AGT1 is indispensable for appressorium function of the rice blast pathogen, Magnaporthe oryzae. PLoS One 7:e36266. doi: 10.1371/journal.pone.0036266 CrossRefPubMedPubMedCentralGoogle Scholar
  4. Boyle NR, Page MD, Liu B, Blaby IK, Casero D, Kropat J, Cokus SJ, Hong-Hermesdorf A, Shaw J, Karpowicz SJ, Gallaher SD, Johnson S, Benning C, Pellegrini M, Grossman A, Merchant SS (2012) Three acyltransferases and nitrogen-responsive regulator are implicated in nitrogen starvation induced triacylglycerol accumulation in Chlamydomonas. J Biol Chem 287:15811–15825. doi: 10.1074/jbc.M111.334052 CrossRefPubMedPubMedCentralGoogle Scholar
  5. Brandhoff B, Simon A, Dornieden A, Schumacher J (2017) Regulation of conidiation in Botrytis cinerea involves the light-responsive transcriptional regulators BcLTF3 and BcREG1. Curr Genet. doi: 10.1007/s00294-017-0692-9 PubMedGoogle Scholar
  6. Calvo AM, Hinze LL, Gardner HW, Keller NP (1999) Sporogenic effect of polyunsaturated fatty acids on development of Aspergillus spp. Appl Environ Microbiol 65:3668–3673 (PubMed: 10427064) PubMedPubMedCentralGoogle Scholar
  7. Calvo AM, Gardner HW, Keller NP (2001) Genetic connection between fatty acid metabolism and sporulation in Aspergillus nidulans. J Biol Chem 276:25766–25774. doi: 10.1074/jbc.M100732200 CrossRefPubMedGoogle Scholar
  8. Choquer M, Fournier E, Kunz C, Levis C, Pradier JM, Simon A, Viaud M (2007) Botrytis cinerea virulence factors: new insights into a necrotrophic and polyphageous pathogen. FEMS Microbiol Lett 277:1–10. doi: 10.1111/j.1574-6968.2007.00930.x CrossRefPubMedGoogle Scholar
  9. Combier JP, Melayah D, Raffier C, Gay G, Marmeisse R (2003) Agrobacterium tumefaciens-mediated transformation as a tool for insertional mutagenesis in the symbiotic ectomycorrhizal fungus Hebeloma cylindrosporum. FEMS Microbiol Lett 220:141–148. doi: 10.1016/S0378-1097(03)00089-2 CrossRefPubMedGoogle Scholar
  10. Cui Z, Gao N, Wang Q, Ren Y, Wang K, Zhu T (2015) BcMctA, a putative monocarboxylate transporter, is required for pathogenicity in Botrytis cinerea. Curr Genet 61:545–553. doi: 10.1007/s00294-015-0474-1 CrossRefPubMedGoogle Scholar
  11. Dahlqvist A, Ståhl U, Lenman M, Banas A, Lee M, Sandager L, Ronne H, Stymne S (2000) Phospholipid: diacylglycerol acyltransferase: an enzyme that catalyzes the acyl-CoA-independent formation of triacylglycerol in yeast and plants. PNAS 97:6487–6492. doi: 10.1073/pnas.120067297 CrossRefPubMedPubMedCentralGoogle Scholar
  12. Daniel J, Deb C, Dubey VS, Sirakova TD, Abomoelak B, Morbidoni HR, Kolattukudy PE (2004) Induction of a novel class of diacylglycerol acyltransferases and triacylglycerol accumulation in Mycobacterium tuberculosis as it goes into a dormancy-like state in culture. J Bacteriol 186:5017–5030. doi: 10.1128/JB.186.15.5017-5030.2004 CrossRefPubMedPubMedCentralGoogle Scholar
  13. Dey P, Mall N, Chattopadhyay A, Chakraborty M, Maiti MK (2014) Enhancement of lipid productivity in oleaginous Colletotrichum fungus through genetic transformation using the yeast CtDGAT2b gene under model-optimized growth condition. PLoS One 9(4):e94472. doi: 10.1371/journal.pone.0094472 CrossRefPubMedPubMedCentralGoogle Scholar
  14. Duan ZB, Chen YX, Huang W, Shang YF, Chen PL, Wang CS (2013) Linkage of autophagy to fungal development, lipid storage and virulence in Metarhizium robertsii. Autophagy 9:538–549. doi: 10.4161/auto.23575 CrossRefPubMedPubMedCentralGoogle Scholar
  15. Durman SB, Menendez AB, Godeas AM (2005) Variation in oxalic acid production and mycelial compatibility within field populations of Sclerotinia sclerotiorum. Soil Biol Biochem 37:2180–2184. doi: 10.1016/j.soilbio.2005.03.017 CrossRefGoogle Scholar
  16. Dӧehlemann G, Berndt P, Hahn M (2006) Different signalling pathways involving a G-alpha protein, cAMP and a MAP kinase control germination of Botrytis cinerea conidia. Mol Microbiol 59:821–835. doi: 10.1111/j.1365-2958.2005.04991.x CrossRefGoogle Scholar
  17. Fillinger S, Elad Y (2016) Botrytis—the fungus, the pathogen and its management in agricultural systems. Springer. doi: 10.1007/978-3-319-23371-0 Google Scholar
  18. Gao Q, Shang Y, Huang W, Wang C (2013) Glycerol-3-phosphate acyltransferase contributes to triacylglycerol biosynthesis, lipid droplet formation, and host invasion in Metarhizium robertsii. Appl Environ Microbiol 79:7646–7653. doi: 10.1128/AEM.02905-13 CrossRefPubMedPubMedCentralGoogle Scholar
  19. Giesbert S, Schumacher J, Kupas V, Espino J, Segmüller N, Haeuser-Hahn I, Schreier PI, Tudzynski P (2012) Identification of pathogenesis-associated genes by T DNA–mediated insertional mutagenesis in Botrytis cinerea: a Type 2A phosphoprotein phosphatase and an SPT3 transcription factor have significant impact on virulence. MPMI 25:481–495. doi: 10.1094/MPMI-07-11-0199 CrossRefPubMedGoogle Scholar
  20. Guenther JC, Hallen-Adams HE, Bücking H, Shachar-Hill Y, Trail F (2009) Triacylglyceride metabolism by Fusarium graminearum during colonization and sexual development on wheat. MPMI 22:1492–1503. doi: 10.1094/MPMI-22-12-1492 CrossRefPubMedGoogle Scholar
  21. Hahn M (2014) The rising threat of fungicide resistance in plant pathogenic fungi: Botrytis as a case study. J Chem Biol 7:133–141. doi: 10.1007/s12154-014-0113-1 CrossRefPubMedPubMedCentralGoogle Scholar
  22. Hara A, Radin NS (1978) Lipid extraction of tissues with a low-toxicity solvent. Anal Biochem 90:420–426. doi: 10.1016/0003-2697(78)90046-5 CrossRefPubMedGoogle Scholar
  23. Huang SL, Kohmoto K (1991) A simple method for isolating single fungal spores. Bulletin of the Faculty of Agriculture-Tottori University, TottoriGoogle Scholar
  24. Jin Y, McFie PJ, Banman SL, Brandt C, Stone SJ (2014) Diacylglycerol acyltransferase-2 (DGAT2) and monoacylglycerol acyltransferase-2 (MGAT2) interact to promote triacylglycerol synthesis. J Biol Chem 289:28237–28248. doi: 10.1074/jbc.M114.571190 CrossRefPubMedPubMedCentralGoogle Scholar
  25. Khan MR, Anwer MA, Shahid S (2011) Management of gray mold of chickpea, Botrytis cinerea with bacterial and fungal biopesticides using different modes of inoculation and application. Biol Control 57:13–23. doi: 10.1016/j.biocontrol.2011.01.004 CrossRefGoogle Scholar
  26. Kumari S, Tayal P, Sharma E, Kapoor R (2014) Analyses of genetic and pathogenic variability among Botrytis cinerea isolates. Microbiol Res 169:862–872. doi: 10.1016/j.micres.2014.02.012 CrossRefPubMedGoogle Scholar
  27. Lakin-Thomas PL, Gooch VD, Ramsdale M (2001) Rhythms of differentiation and diacylglycerol in Neurospora. Philos Trans R Soc Lond B Biol Sci 356:1711–1715. doi: 10.1098/rstb.2001.0966 CrossRefPubMedPubMedCentralGoogle Scholar
  28. Lardizabal KD, Mai JT, Wagner NW, Wyrick A, Voelker T, Hawkins DJ (2001) DGAT2 is a new diacylglycerol acyltransferase gene family Purification, cloning, and expression in insect cells of two polypeptides from Mortierella ramanniana with diacylglycerol acyltransferase activity. J Biol Chem 276:38862–38869. doi: 10.1074/jbc.M106168200 CrossRefPubMedGoogle Scholar
  29. Leroux P (2004) Chemical control of Botrytis and its resistance to chemical fungicides. In: Elad Y, Williamson B, Tudzynski P, Delen N (eds) Botrytis: biology, pathology and control. Kluwer Academic Publisher, Dordrecht, pp 195–222. doi: 10.1007/978-1-4020-2626-3_12 Google Scholar
  30. Liu Q, Siloto RMP, Snyder CL, Weselake RJ (2011) Functional and topological analysis of yeast acyl-CoA:diacylglycerol acyltransferase2, an endoplasmic reticulum enzyme essential for triacylglycerol biosynthesis. J Biol Chem 286:13115–13126. doi: 10.1074/jbc.M110.204412 CrossRefPubMedPubMedCentralGoogle Scholar
  31. Mavraganis I, Meesapyodsuk D, Vrinten P, Smith M, Qiu X (2010) Type II diacylglycerol acyltransferase from Claviceps purpurea with ricinoleic acid, a hydroxyl fatty acid of industrial importance, as preferred substrate. Appl Environ Microbiol 76:1135–1142. doi: 10.1128/AEM.02297-09 CrossRefPubMedGoogle Scholar
  32. Nakajima M, Akutsu K (2014) Virulence factors of Botrytis cinerea. J Genet Plant Pathol 80:15–23. doi: 10.1007/s10327-013-0492-0 CrossRefGoogle Scholar
  33. Nizam S, Singh K, Verma PK (2010) Expression of the fluorescent proteins DsRed and EGFP to visualize early events of colonization of the chickpea blight fungus Ascochyta rabiei. Curr Genet 56:391–399. doi: 10.1007/s00294-010-0305-3 CrossRefPubMedGoogle Scholar
  34. Nizam S, Verma S, Singh K, Aggarwal R, Srivastava KD, Verma PK (2012) High reliability transformation of the wheat pathogen Bipolaris sorokiniana using Agrobacterium tumefaciens. J Microbiol Methods 88:386–392. doi: 10.1016/j.mimet.2012.01.004 CrossRefPubMedGoogle Scholar
  35. Oakes J, Brackenridge D, Colletti R, Daley M, Hawkins DJ, Xiong H, Mai J, Screen SE, Val D, Lardizabal K, Gruys K, Deikman J (2011) Expression of fungal diacylglycerol acyltransferase2 genes to increase kernel oil in maize. Plant Physiol 155:1146–1157. doi: 10.1104/pp.110.167676 CrossRefPubMedPubMedCentralGoogle Scholar
  36. Ortiz-Urquiza A, Keyhani NO (2015) Stress response signaling and virulence: insights from entomopathogenic fungi. Curr Genet 61:239–249. doi: 10.1007/s00294-014-0439-9 CrossRefPubMedGoogle Scholar
  37. Pontecorvo GV, Roper JA, Hemmons LM, MacDonald KD, Bufton AWJ (1953) The genetics of Aspergillus nidulans. Adv Genet 5:141–238. doi: 10.1016/S0065-2660(08)60408-3 PubMedGoogle Scholar
  38. Rolland S, Jobic C, Fevre M, Bruel C (2003) Agrobacterium mediated transformation of Botrytis cinerea, simple purification of monokaryotic transformants and rapid conidia-based identification of the transfer-DNA host genomic DNA flanking sequences. Curr Genet 44:164–171. doi: 10.1007/s00294-003-0438-8 CrossRefPubMedGoogle Scholar
  39. Romanazzi G, Smilanick JL, Feliziani E, Droby S (2016) Integrated management of postharvest gray mold on fruit crops. Postharvest Biol Technol 113:69–76. doi: 10.1016/j.postharvbio.2015.11.003 CrossRefGoogle Scholar
  40. Sadat MA, Jeon J, Mir AA, Choi J, Choi J, Lee YH (2014) Regulation of cellular diacylglycerol through lipid phosphate phosphatases is required for pathogenesis of the rice blast fungus Magnaporthe oryzae. PloS One 9(6):e100726. doi: 10.1371/journal.pone.0100726 CrossRefPubMedPubMedCentralGoogle Scholar
  41. Sambrook J, Russell D (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor. doi: 10.1016/0092-8674(90)90210-6 Google Scholar
  42. Sandager L, Gustavsson MH, Ståhl U, Dahlqvist A, Wiberg E, Banas A, Lenman M, Ronne H, Stymne S (2002) Storage lipid synthesis is non-essential in yeast. J Biol Chem 277:6478–6482. doi: 10.1074/jbc.M109109200 CrossRefPubMedGoogle Scholar
  43. Schumacher J (2016) Signal transduction cascades regulating differentiation and virulence in Botrytis cinerea. In: Fillinger S, Elad Y (eds) Botrytis—the fungus, the pathogen and its management in agricultural systems. Springer, Berlin, pp 247–267. doi: 10.1007/978-3-319-23371-0_13 CrossRefGoogle Scholar
  44. Schumacher J, Tudzynski P (2012) Morphogenesis and infection in Botrytis cinerea. In: Morphogenesis and pathogenicity in fungi. Springer, Berlin, pp 225–241. doi: 10.1007/978-3-642-22916-9_11
  45. Schumacher J, Simon A, Cohrs KC, Viaud M, Tudzynski P (2014) The transcription factor BcLTF1 regulates virulence and light responses in the necrotrophic plant pathogen Botrytis cinerea. PLoS Genet 10:e1004040. doi: 10.1371/journal.pgen.1004040 CrossRefPubMedPubMedCentralGoogle Scholar
  46. Shea JM, Del Poeta M (2006) Lipid signaling in pathogenic fungi. Curr Opin Microbiol 9:352–358. doi: 10.1016/j.mib.2006.06.003 CrossRefPubMedGoogle Scholar
  47. Shockey JM, Gidda SK, Chapital DC, Kuan JC, Dhanoa PK, Bland JM, Rothstein SJ, Mullen RT, Dyer JM (2006) Tung tree DGAT1 and DGAT2 have non-redundant functions in triacylglycerol biosynthesis and are localized to different subdomains of the endoplasmic reticulum. Plant Cell 18:2294–2313. doi: 10.1105/tpc.106.043695 CrossRefPubMedPubMedCentralGoogle Scholar
  48. Singh A, Del Poeta M (2011) Lipid signalling in pathogenic fungi. Cell Microbiol 13:177–185. doi: 10.1111/j.1462-5822.2010.01550.x CrossRefPubMedGoogle Scholar
  49. Sørensen LQ, Lysøe E, Larsen JE, Khorsand-Jamal P, Nielsen KF, Frandsen RJN (2014) Genetic transformation of Fusarium avenaceum by Agrobacterium tumefaciens mediated transformation and the development of a USER-Brick vector construction system. BMC Mol Biol 15:15. doi: 10.1186/1471-2199-15-15 CrossRefPubMedPubMedCentralGoogle Scholar
  50. Steinhauer J, Gijón MA, Riekhof WR, Voelker DR, Murphy RC, Treisman JE (2009) Drosophila lysophospholipid acyltransferases are specifically required for germ cell development. Mol Biol Cell 20:5224–5235. doi: 10.1091/mbc.E09-05-0382 CrossRefPubMedPubMedCentralGoogle Scholar
  51. Stone SJ, Myers HM, Watkins SM, Brown BE, Feingold KR, Elias PM, Farese RV (2004) Lipopenia and skin barrier abnormalities in DGAT2-deficient mice. J Biol Chem 279:11767–11776. doi: 10.1074/jbc.M311000200 CrossRefPubMedGoogle Scholar
  52. Temme N, Oeser B, Massaroli M, Heller J, Simon A, Gonzalez Collado I, Viaud M, Tudzynski P (2012) BcAtf1, a global regulator, controls various differentiation processes and phytotoxin production in Botrytis cinerea. Mol Plant Pathol 13:704–718. doi: 10.1111/J.1364-3703.2011.00778.X CrossRefPubMedGoogle Scholar
  53. Ten Have A, Tenberge KB, Benen JAE, Tudzynski P, Visser J (2002) The contribution of cell wall degrading enzymes to pathogenesis of fungal plant pathogens. In: Esser KBJ (ed) The mycota agricultural applications XI. Springer, Heidelberg, pp 341–358. doi: 10.1007/978-3-662-03059-2_17 CrossRefGoogle Scholar
  54. Thines E, Weber RWS, Talbot NJ (2000) MAP kinase and protein kinase A–dependent mobilization of triacylglycerol and glycogen during appressorium turgor generation by Magnaporthe grisea. Plant Cell 12:1703–1718. doi: 10.1105/tpc.12.9.1703 PubMedPubMedCentralGoogle Scholar
  55. Turchetto-Zolet AC, Christoff AP, Kulcheski FR, Loss-Morais G, Margis R, Margis-Pinheiro M (2016) Diversity and evolution of plant diacylglycerol acyltransferase (DGATs) unveiled by phylogenetic, gene structure and expression analyses. Genet Mol Biol 39:524–538. doi: 10.1590/1678-4685-GMB-2016-0024 CrossRefPubMedPubMedCentralGoogle Scholar
  56. Wang ZY, Thornton CR, Kershaw MJ, Li DB, Talbot NJ (2003) The glyoxylate cycle is required for temporal regulation of virulence by the plant pathogenic fungus Magnaporthe grisea. Mol Microbiol 47:1601–1612. doi: 10.1046/j.1365-2958.2003.03412.x CrossRefPubMedGoogle Scholar
  57. Wang X, Devaiah SP, Zhang W, Welti R (2006) Signaling functions of phosphatidic acid. Prog Lipid Res 45:250–278. doi: 10.1016/j.plipres.2006.01.005 CrossRefPubMedGoogle Scholar
  58. Weete JD (2012) Lipid biochemistry of fungi and other organisms. Springer. doi: 10.1007/978-1-4757-0064-0 Google Scholar
  59. Weiberg A, Wang M, Lin FM, Zhao H, Zhang Z, Kaloshian I, Huang H, Jin H (2013) Fungal small RNAs suppress plant immunity by hijacking host RNA interference pathways. Science 342:118–123. doi: 10.1126/science.1239705 CrossRefPubMedPubMedCentralGoogle Scholar
  60. White D, Chen W (2006) Genetic transformation of Ascochyta rabiei using Agrobacterium mediated transformation. Curr Genet 49:272–280. doi: 10.1007/s00294-005-0048-8 CrossRefPubMedGoogle Scholar
  61. Williamson B, Tudzynski B, Tudzynski P, van Kan JAL (2007) Botrytis cinerea: the cause of grey mould disease. Mol Plant Pathol 8:561–580. doi: 10.1111/J.1364-3703.2007.00417.X CrossRefPubMedGoogle Scholar
  62. Yadav KK, Rajasekharan R (2016) The transcription factor GCN4 regulates PHM8 and alters triacylglycerol metabolism in Saccharomyces cerevisiae. Curr Genet 62:841–851. doi: 10.1007/s00294-016-0590-6 CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  • Esha Sharma
    • 1
  • Pamil Tayal
    • 1
  • Garima Anand
    • 1
  • Piyush Mathur
    • 1
  • Rupam Kapoor
    • 1
    Email author
  1. 1.Department of BotanyUniversity of DelhiDelhiIndia

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