3 Biotech

, 8:475 | Cite as

Lipopolysaccharide-induced priming enhances NO-mediated activation of defense responses in pearl millet challenged with Sclerospora graminicola

  • S. N. Lavanya
  • A. C. Udayashankar
  • S. Niranjan Raj
  • Chakrabhavi Dhananjaya Mohan
  • V. K. Gupta
  • C. Tarasatyavati
  • R. Srivastava
  • S. Chandra NayakaEmail author
Original Article


Lipopolysaccharide (LPS) elicitors isolated from Pseudomonas fluorescens UOM SAR 14 effectively induced systemic and durable resistance against pearl millet downy mildew disease caused by the oomycete Sclerospora graminicola. Rapid and increased callose deposition and H2O2 accumulation were evidenced in downy mildew susceptible seeds pre-treated with LPS (SLPS) in comparison with the control seedlings, which also correlated with expression of various other defense responses. Biochemical analysis of enzymes and quantitative real-time polymerase chain reaction data suggested that LPS protects pearl millet against downy mildew through the activation of plant defense mechanisms such as generation of nitric oxide (NO), increased expression, and activities of defense enzymes and proteins. Elevation of NO concentrations was shown to be essential for LPS-mediated defense manifestation in pearl millet and had an impact on the other downstream defense responses like enhanced activation of enzymes and pathogen-related (PR) proteins. Temporal expression analysis of defense enzymes and PR-proteins in SLPS seedlings challenged with the downy mildew pathogen revealed that the activity and expression of peroxidase, phenylalanine ammonia lyase, and the PR-proteins (PR-1 and PR-5) were significantly enhanced compared to untreated control. Higher gene expression and protein activities of hydroxyproline-rich glycoproteins (HRGPs) were observed in SLPS seedlings which were similar to that of the resistant check. Collectively, our results suggest that, in pearl millet-downy mildew interaction, LPS pre-treatment affects defense signaling through the central regulator NO which triggers the activities of PAL, POX, PR-1, PR-5, and HRGPs.


Pearl millet downy mildew Lipopolysaccharide Induced resistance Nitric oxide Defense enzymes PR-proteins gene expression 


Author contributions

LSN, UAC, NRS, and CNS conceived the project. CDM, GVK, TC, SR, and CNS designed the experiments. LSN, UAC, NRS, and CNS carried out the research and analysis of data. LSN, UAC, CDM, and CNS wrote the paper.

Compliance with ethical standards

Conflict of interest

On behalf of all authors, the corresponding author states that there is no conflict of interest.

Supplementary material

13205_2018_1501_MOESM1_ESM.doc (603 kb)
Supplementary material 1 (DOC 603 KB)
13205_2018_1501_MOESM2_ESM.pdf (282 kb)
Supplementary material 2 (PDF 281 KB)


  1. Beaudoin-Eagan LD, Thorpe TA (1985) Tyrosine and phenylalanine ammonia lyase activities during shoot initiation in tobacco callus cultures. Plant Physiol 78(3):438–441CrossRefGoogle Scholar
  2. Bowler C, Van Camp W, Van Montagu M, Inze D, Asada K (1994) Superoxide dismutase in plants. Crit Rev Plant Sci 13(3):199–218CrossRefGoogle Scholar
  3. Bradley DJ, Kjellbom P, Lamb CJ (1992) Elicitor-and wound-induced oxidative cross-linking of a proline-rich plant cell wall protein: a novel, rapid defense response. Cell 70(1):21–30CrossRefGoogle Scholar
  4. Brownleader MD, Ahmed N, Trevan M, Chaplin MF, Dey PM (1995) Purification and partial characterization of tomato extensin peroxidase. Plant Physiol 109(3):1115–1123CrossRefGoogle Scholar
  5. Coventry HS, Dubery IA (2001) Lipopolysaccharides from Burkholderia cepacia contribute to an enhanced defensive capacity and the induction of pathogenesis-related proteins in Nicotianae tabacum. Physiol Mol Plant Pathol 58(4):149–158CrossRefGoogle Scholar
  6. Davies HA, Daniels MJ, Dow JM (1997) Induction of extracellular matrix glycoproteins in Brassica petioles by wounding and in response to Xanthomonas campestris. Mol Plant Microbe Interact 10(7):812–820CrossRefGoogle Scholar
  7. de Pinto MC, Tommasi F, De Gara L (2002) Changes in the antioxidant systems as part of the signaling pathway responsible for the programmed cell death activated by nitric oxide and reactive oxygen species in tobacco Bright-Yellow 2 cells. Plant Physiol 130(2):698–708CrossRefGoogle Scholar
  8. Deepak S, Shailasree S, Kini RK, Hause B, Shetty SH, Mithöfer A (2007) Role of hydroxyproline-rich glycoproteins in resistance of pearl millet against downy mildew pathogen Sclerospora graminicola. Planta 226(2):323–333CrossRefGoogle Scholar
  9. Delledonne M (2005) NO news is good news for plants. Curr Opin Plant Biol 8(4):390–396CrossRefGoogle Scholar
  10. Desaki Y, Miya A, Venkatesh B, Tsuyumu S, Yamane H, Kaku H, Minami E, Shibuya N (2006) Bacterial lipopolysaccharides induce defense responses associated with programmed cell death in rice cells. Plant Cell Physiol 47(11):1530–1540CrossRefGoogle Scholar
  11. Dow M, Newman M-A, von Roepenack E (2000) The induction and modulation of plant defense responses by bacterial lipopolysaccharides. Ann Rev Phytopathol 38(1):241–261CrossRefGoogle Scholar
  12. Durner J, Wendehenne D, Klessig DF (1998) Defense gene induction in tobacco by nitric oxide, cyclic GMP, and cyclic ADP-ribose. Proc Natl Acad Sci 95(17):10328–10333CrossRefGoogle Scholar
  13. Erbs G, Newman M-A (2012) The role of lipopolysaccharide and peptidoglycan, two glycosylated bacterial microbe-associated molecular patterns (MAMPs), in plant innate immunity. Mol Plant Pathol 13(1):95–104CrossRefGoogle Scholar
  14. Felix G, Duran JD, Volko S, Boller T (1999) Plants have a sensitive perception system for the most conserved domain of bacterial flagellin. Plant J 18(3):265–276CrossRefGoogle Scholar
  15. Garcia-Brugger A, Lamotte O, Vandelle E, Bourque S, Lecourieux D, Poinssot B, Wendehenne D, Pugin A (2006) Early signaling events induced by elicitors of plant defenses. Mol Plant Microb Interact 19(7):711–724CrossRefGoogle Scholar
  16. Geetha N, Amruthesh K, Sharathchandra R, Shetty HS (2005) Resistance to downy mildew in pearl millet is associated with increased phenylalanine ammonia lyase activity. Funct Plant Biol 32(3):267–275CrossRefGoogle Scholar
  17. Gerber IB, Zeidler D, Durner J, Dubery IA (2004) Early perception responses of Nicotiana tabacum cells in response to lipopolysaccharides from Burkholderia cepacia. Planta 218(4):647–657CrossRefGoogle Scholar
  18. Guo J-H, Qi H-Y, Guo Y-H, Ge H-L, Gong L-Y, Zhang L-X, Sun P-H (2004) Biocontrol of tomato wilt by plant growth-promoting rhizobacteria. Biol Control 29(1):66–72CrossRefGoogle Scholar
  19. Hahlbrock K, Scheel D (1989) Physiology and molecular biology of phenylpropanoid metabolism. Ann Rev Plant Biol 40(1):347–369CrossRefGoogle Scholar
  20. Hammerschmidt R, Nicholson RL (1999) A survey of plant defense responses to pathogens. Induced plant defenses against pathogens and herbivores. APS Press, St. Minn, pp 55–72Google Scholar
  21. Jetiyanon K, Plianbangchang P (2013) Lipopolysaccharide of Enterobacter asburiae strain RS83: a bacterial determinant for induction of early defensive enzymes in Lactuca sativa against soft rot disease. Biol Control 67(3):301–307CrossRefGoogle Scholar
  22. Kang Z, Buchenauer H (2003) Immunocytochemical localization of cell wall-bound thionins and hydroxyproline-rich glycoproteins in Fusarium culmorum-infected wheat spikes. J Phytopathol 151(3):120–129CrossRefGoogle Scholar
  23. Keshavarz-Tohid V, Taheri P, Taghavi SM, Tarighi S (2016) The role of nitric oxide in basal and induced resistance in relation with hydrogen peroxide and antioxidant enzymes. J Plant Physiol 199:29–38CrossRefGoogle Scholar
  24. Lavanya SN, Niranjan-Raj S, Nayaka SC, Amruthesh KN (2017) Systemic protection against pearl millet downy mildew disease induced by cell wall glucan elicitors from Trichoderma hamatum UOM 13. J Plant Protect ResGoogle Scholar
  25. Li L, Steffens JC (2002) Overexpression of polyphenol oxidase in transgenic tomato plants results in enhanced bacterial disease resistance. Planta 215(2):239–247CrossRefGoogle Scholar
  26. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2 – ∆∆CT method. Methods 25(4):402–408CrossRefGoogle Scholar
  27. Lorrain S, Vailleau F, Balagué C, Roby D (2003) Lesion mimic mutants: keys for deciphering cell death and defense pathways in plants? Trends Plant Sci 8(6):263–271CrossRefGoogle Scholar
  28. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193(1):265–275PubMedGoogle Scholar
  29. Lyon G (2007) Agents that can elicit induced resistance. Induced resistance for plant defence: a sustainable approach to crop protection:9–29Google Scholar
  30. Manjunatha G, Raj SN, Shetty NP, Shetty HS (2008) Nitric oxide donor seed priming enhances defense responses and induces resistance against pearl millet downy mildew disease. Pesticide Biochem Physiol 91(1):1–11CrossRefGoogle Scholar
  31. Manjunatha G, Deepak S, Geetha PN, Niranjan-Raj S, Kini RK, Shetty HS (2009a) Hypersensitive reaction and P/HRGP accumulation is modulated by nitric oxide through hydrogen peroxide in pearl millet during Sclerospora graminicola infection. Physiol Mol Plant Pathol 74(2):191–198CrossRefGoogle Scholar
  32. Manjunatha G, Niranjan-Raj S, Prashanth GN, Deepak S, Amruthesh KN, Shetty HS (2009b) Nitric oxide is involved in chitosan-induced systemic resistance in pearl millet against downy mildew disease. Pest Manag Sci 65(7):737–743CrossRefGoogle Scholar
  33. Melotto M, Underwood W, Koczan J, Nomura K, He SY (2006) Plant stomata function in innate immunity against bacterial invasion. Cell 126(5):969–980CrossRefGoogle Scholar
  34. Mohamed K-H, Daniel T, Aurélien D, El-Maarouf-Bouteau H, Rafik E, Arbelet-Bonnin D, Biligui B, Florence V, Mustapha EM, François B (2015) Deciphering the dual effect of lipopolysaccharides from plant pathogenic Pectobacterium. Plant Signal Behav 10(3):e1000160CrossRefGoogle Scholar
  35. Nagarathna K, SHETTY SA, Shetty HS (1993) Phenylalanine ammonia lyase activity in pearl millet seedlings and its relation to downy mildew disease resistance. J Exp Bot 44(8):1291–1296CrossRefGoogle Scholar
  36. Nayaka SC, Shetty HS, Satyavathi CT, Yadav RS, Kishor PK, Nagaraju M, Anoop T, Kumar MM, Kuriakose B, Chakravartty N (2017) Draft genome sequence of Sclerospora graminicola, the pearl millet downy mildew pathogen. Biotechnol Rep 16:18–20CrossRefGoogle Scholar
  37. Newman M-A, Dow JM, Molinaro A, Parrilli M (2007) Invited review: priming, induction and modulation of plant defence responses by bacterial lipopolysaccharides. J Endotoxin Res 13(2):69–84CrossRefGoogle Scholar
  38. Niranjan-Raj S, Lavanya S, Amruthesh K, Niranjana S, Shetty HS (2011) Comparative evaluation of Pseudomonas fluorescens and their lipopolysaccharides as implicated in induction of resistance against pearl millet downy mildew. Arch Phytopathol Plant Protect 44(13):1285–1299CrossRefGoogle Scholar
  39. Noorbakhsh Z, Taheri P (2016) Nitric oxide: a signaling molecule which activates cell wall-associated defense of tomato against Rhizoctonia solani. Eur J Plant Pathol 144(3):551–568CrossRefGoogle Scholar
  40. Prakash HS, Nayaka CS, Kini KR (2014) Downy Mildew disease of pearl millet and its control. In: Future challenges in crop protection against fungal pathogens. Springer, pp 109–129Google Scholar
  41. Prockop DJ, Udenfriend S (1960) A specific method for the analysis of hydroxyproline in tissues and urine. Anal Biochem 1:228–239CrossRefGoogle Scholar
  42. Pushpalatha H, Mythrashree S, Shetty R, Geetha N, Sharathchandra R, Amruthesh K, Shetty HS (2007) Ability of vitamins to induce downy mildew disease resistance and growth promotion in pearl millet. Crop Protection 26(11):1674–1681CrossRefGoogle Scholar
  43. Raj SN, Lavanya S, Amruthesh K, Niranjana S, Reddy M, Shetty HS (2012) Histo-chemical changes induced by PGPR during induction of resistance in pearl millet against downy mildew disease. Biol Control 60(2):90–102CrossRefGoogle Scholar
  44. Ramamoorthy V, Viswanathan R, Raguchander T, Prakasam V, Samiyappan R (2001) Induction of systemic resistance by plant growth promoting rhizobacteria in crop plants against pests and diseases. Crop Protect 20(1):1–11CrossRefGoogle Scholar
  45. Sarosh BR, Sivaramakrishnan S, Shetty HS (2005) Elicitation of defense related enzymes and resistance by L-methionine in pearl millet against downy mildew disease caused by Sclerospora graminicola. Plant Physiol Biochem 43(8):808–815CrossRefGoogle Scholar
  46. Siddaiah CN, Satyanarayana NR, Mudili V, Gupta VK, Gurunathan S, Rangappa S, Huntrike SS, Srivastava RK (2017) Elicitation of resistance and associated defense responses in Trichoderma hamatum induced protection against pearl millet downy mildew pathogen. Sci Rep 7:43991CrossRefGoogle Scholar
  47. Siddaiah CN, Prasanth KVH, Satyanarayana NR, Mudili V, Gupta VK, Kalagatur NK, Satyavati T, Dai X-F, Chen J-Y, Mocan A (2018) Chitosan nanoparticles having higher degree of acetylation induce resistance against pearl millet downy mildew through nitric oxide generation. Sci Rep 8(1):2485CrossRefGoogle Scholar
  48. Silipo A, Molinaro A, Sturiale L, Dow JM, Erbs G, Lanzetta R, Newman M-A, Parrilli M (2005) The elicitation of plant innate immunity by lipooligosaccharide of Xanthomonas campestris. J Biol Chem 280(39):33660–33668CrossRefGoogle Scholar
  49. Singh S, Gopinath R (1985) A seedling inoculation technique for detecting downy mildew resistance in pearl millet. Plant Dis 69(7):582–584Google Scholar
  50. Smit F, Dubery IA (1997) Cell wall reinforcement in cotton hypocotyls in response to a Verticillium dahliae elicitor. Phytochemistry 44(5):811–815CrossRefGoogle Scholar
  51. Sujeeth N, Deepak S, Shailasree S, Kini RK, Shetty SH, Hille J (2010) Hydroxyproline-rich glycoproteins accumulate in pearl millet after seed treatment with elicitors of defense responses against Sclerospora graminicola. Physiol Mol Plant Pathol 74(3–4):230–237CrossRefGoogle Scholar
  52. Sun A, Li Z (2013) Regulatory role of nitric oxide in lipopolysaccharides-triggered plant innate immunity. Plant Sign Behav 8(1):1081–1096CrossRefGoogle Scholar
  53. Sun A, Nie S, Xing D (2012) Nitric oxide-mediated maintenance of redox homeostasis contributes to NPR1-dependent plant innate immunity triggered by lipopolysaccharides. Plant Physiol 160(2):1081–1096CrossRefGoogle Scholar
  54. Thakur RP, Rao VP, Sharma R (2011) Influence of dosage, storage time and temperature on efficacy of metalaxyl-treated seed for the control of pearl millet downy mildew. Eur J Plant Pathol 129(2):353–359CrossRefGoogle Scholar
  55. Uzma F, Mohan CD, Hashem A, Konappa NM, Rangappa S, Kamath PV, Singh BP, Mudili V, Gupta VK, Siddaiah CN, Chowdappa S, Alqarawi AA, Abd Allah EF (2018) Endophytic fungi-alternative sources of cytotoxic compounds: a review. Front Pharmacol 9:309. CrossRefPubMedPubMedCentralGoogle Scholar
  56. Van Loon L, Bakker P, Pieterse C (1998) Systemic resistance induced by rhizosphere bacteria. Ann Rev Phytopathol 36(1):453–483CrossRefGoogle Scholar
  57. Walters DR, Ratsep J, Havis ND (2013) Controlling crop diseases using induced resistance: challenges for the future. J Exp Bot 64(5):1263–1280CrossRefGoogle Scholar
  58. Wang J, Higgins VJ (2005) Nitric oxide modulates H2O2-mediated defenses in the Colletotrichum coccodes–tomato interaction. Physiol Mol Plant Pathol 67(3–5):131–137CrossRefGoogle Scholar
  59. Wendehenne D, Durner J, Klessig DF (2004) Nitric oxide: a new player in plant signalling and defence responses. Curr Opin Plant Biol 7(4):449–455CrossRefGoogle Scholar
  60. Williams R (1984) Downy mildews of tropical cereals.​ In: Advances in Plant pathology. Academic Press, London, pp 1–103Google Scholar
  61. Yadav H (2014) Project coordinators review: All India coordinated project on pearl millet-49th Annual Group MeetingGoogle Scholar
  62. York WS, Darvill AG, McNeil M, Stevenson TT, Albersheim P (1986) Isolation and characterization of plant cell walls and cell wall components. In: Methods in enzymology, vol 118. Elsevier, pp 3–40Google Scholar
  63. Zeidler D, Zähringer U, Gerber I, Dubery I, Hartung T, Bors W, Hutzler P, Durner J (2004) Innate immunity in Arabidopsis thaliana: lipopolysaccharides activate nitric oxide synthase (NOS) and induce defense genes. Proc Natl Acad Sci USA 101(44):15811–15816CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • S. N. Lavanya
    • 1
  • A. C. Udayashankar
    • 1
  • S. Niranjan Raj
    • 2
  • Chakrabhavi Dhananjaya Mohan
    • 3
  • V. K. Gupta
    • 4
  • C. Tarasatyavati
    • 5
  • R. Srivastava
    • 6
  • S. Chandra Nayaka
    • 1
    Email author
  1. 1.Department of Studies in BiotechnologyUniversity of MysoreMysoreIndia
  2. 2.Department of Studies in MicrobiologyKarnataka State Open UniversityMysoreIndia
  3. 3.Department of Studies in Molecular BiologyUniversity of MysoreMysoreIndia
  4. 4.ERA Chair of Green Chemistry, Department of Chemistry and Biotechnology, School of ScienceTallinn University of TechnologyTallinnEstonia
  5. 5.All India Coordinated Research Project on Pearl MilletIndian Council of Agricultural ResearchJodhpurIndia
  6. 6.International Crops Research Institute for the Semi-Arid Tropics (ICRISAT)PatancheruIndia

Personalised recommendations