Isolation and characterisation of a protein elicitor from Sclerospora graminicola and elicitor-mediated induction of defence responses in cultured cells of Pennisetum glaucum
R. G. Sharathchandra A , N. P. Geetha A , K. N. Amruthesh A , K. Ramachandra Kini A , B. R. Sarosh A , N. P. Shetty B and H. S. Shetty A CA Downy Mildew Research Laboratory, Department of Studies in Applied Botany and Biotechnology, University of Mysore, Manasagangotri Mysore-570 006, India.
B Section for Plant Pathology, The Royal Veterinary and Agricultural University, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark.
C Corresponding author. Email: hss@appbot.uni-mysore.ac.in
Functional Plant Biology 33(3) 267-278 https://doi.org/10.1071/FP05197
Submitted: 8 August 2005 Accepted: 10 October 2005 Published: 2 March 2006
Abstract
Sclerospora graminicola (Sacc.) Schroet., an oomycete pathogen of Pennisetum glaucum (L.) R.Br. infects the meristematic tissues of young seedlings. The motile zoospores from the sporangia encyst, germinate and penetrate the plant tissue. Resistance to the invading pathogen is governed by the specific recognition of conserved pathogen-associated proteins or elicitors. In the present study, a zoospore protein was isolated and purified to homogeneity by a combination of size exclusion and high-performance liquid chromatography (HPLC). The crude fractionated protein was able to elicit an array of defence responses in resistant and susceptible cells of pearl millet. Treatment of cultured cells of pearl millet with partially purified elicitor protein resulted in a rapid loss of cell viability in the resistant cells and the percentage of cell death was higher in the resistant than in the susceptible cells. Cultures of resistant cells showed a sharp increase in the extra cellular pH compared with susceptible cells when treated with the crude elicitor. Increased oxidative burst was also recorded in the cells treated with the crude elicitor. The purified elicitor showed unique properties. The purified protein was acidic with a pI of 5.6 as revealed by isoelectric focusing (IEF) and matrix-assisted laser desorption ionisation (MALDI) analysis showed that the elicitor had a molecular mass of 7040 daltons. The primary structure determined by N-terminal Edman degradation and searches with BLAST did not reveal similarities to any known plant pathogenic or oomycete elicitor. Higher activities of the important defence-related enzymes phenylalanine ammonia lyase (PAL) and peroxidase in the resistant cell cultures than in the susceptible cell cultures treated with the purified elicitor were clearly evident. Studies of gene expression by northern blotting with heterologus peroxidase, PAL and oxalate oxidase probes showed that the mRNA transcripts were strongly up-regulated in resistant cell cultures within 30 min of elicitor treatment. The purified elicitor also demonstrated a very strong concentration-dependent sterol binding. The purified elicitor protein belongs to a class of low molecular weight oomycete elicitors with sterol carrier properties. The identified low molecular weight protein elicitor displays unique properties that can be exploited for synthesis of novel molecules for eco-friendly crop protection.
Keywords: biological activity, cell suspension culture, protein elicitor, Sclerospora graminicola, sterol binding, transcripts.
Acknowledgments
This work was conducted under the Project on Systemic Acquired Resistance funded by the Danish International Development Agency under the Enhancement of Research Capacity Program (DANIDA ENRECA). We are grateful to Dr Eigil de Neergard, the Principal Responsible Leader of the DANIDA ENRECA Project for his co-operation during this study. We also thank Dr Hans J. Lyngs Jørgensen, Section for Plant Pathology, The Royal Veterinary and Agricultural University, Denmark for critical review of the manuscript. The facilities provided by Indian Council of Agricultural Research, Government of India through All India Coordinated Pearl Millet Improvement Project are also gratefully acknowledged. The authors also thank Department of Science and Technology, New Delhi, India for providing facilities for cell line maintenance.
Altschul SF,
Gish W,
Miller W,
Myers EW, Lipman DJ
(1990) Basic local alignment search tool. Journal of Molecular Biology 215, 403–410.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Able AJ,
Sutherland MW, Guest DI
(2003) Production of reactive oxygen species during non-specific elicitation, non-host resistance and field resistance expression in cultured tobacco cells. Functional Plant Biology 30, 91–99.
| Crossref | GoogleScholarGoogle Scholar |
Auclair C, Voisin E
(1987) Nitroblue tetrazolium reduction. In ‘CRC handbook of methods for oxygen radical research’. (Ed. RA Greenwald)
pp. 123–130. (CRC Press: Boca Raton)
Bailey JA,
Rowell PM, Arnold GM
(1980) The temporal relationship between host cell death, phytoalexin accumulation and fungal inhibition during hypersensitive reactions of Phaseolus vulgaris to Colletotrichum lindemuthianum. Physiological and Molecular Plant Pathology 17, 329–339.
Baillieul F,
Ruffay PD, Kauffman S
(2003) Molecular cloning and the biological activity of α-, β- and γ- megaspermin, the three elicitins secreted by Phytophthora megasperma. Plant Physiology 31, 55–66.
Becker J,
Nagel S, Tenhamken R
(2000) Cloning and expression and characterization of protein elicitors from the soyabean pathogenic fungus Phytophthora sojae. Journal of Phytopathology 148, 161–167.
| Crossref | GoogleScholarGoogle Scholar |
Blein JP,
Thevenot PC,
Marion D, Ponchet M
(2002) From elicitins to lipid transfer proteins: a new insight in cell signaling involved in plant defense mechanisms. Trends in Plant Science 7, 293–296.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Bos JIB,
Armstrong M,
Whisson SC,
Torto TA,
Ochwo M,
Birch PRJ, Kamoun S
(2003) Intraspecific comparative genomics to identify avirulence genes from Phytophthora. New Phytologist 159, 63–72.
| Crossref | GoogleScholarGoogle Scholar |
Bradford MM
(1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry 72, 248–254.
| Crossref |
PubMed |
Capasso R,
Cristino G,
Di Mario A,
Ferranti P, Parente A
(2001) Syringicin, a new α elicitin from an isolate of Phytophthora syringae pathogenic to citrus fruit. Phytochemistry 58, 257–262.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Cordelier S,
De Ruffray P,
Fritig B, Kauffmann S
(2003) Biological and molecular comparison between localized and systemic acquired resistance induced in tobacco by a Phytophthora megasperma glycoprotein elicitin. Plant Molecular Biology 51, 109–118.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Debarry M,
Marten I,
Ngezahayo A, Kolbe HA
(2005) Differential defense responses in sweet potato suspension culture. Plant Science 168, 1171–1179.
| Crossref | GoogleScholarGoogle Scholar |
Felbrich G,
Romanski A,
Varet A,
Blume B,
Brunner F,
Engelhardt S,
Felix G,
Kemmerling M,
Krzymowska M, Nurnberger T
(2002) NPP1, a Phytophthora-associated trigger of plant defense in parsley and Arabidopsis. The Plant Journal 32, 375–390.
| Crossref |
PubMed |
Geetha NP,
Amruthesh KN,
Sharathchandra RG, Shetty HS
(2005) Resistance to downy mildew in pearl millet is associated with increased phenylalanine ammonia lyase activity. Functional Plant Biology 32, 267–275.
| Crossref | GoogleScholarGoogle Scholar |
Hammerschmidt R,
Nuckles EM, Kuc J
(1982) Association of enhanced peroxidase activity with induced systemic resistance of cucumber to Colletotrichum lagenarium. Physiological and Molecular Plant Pathology 20, 73–83.
Jiang AE,
Rays HY,
Angus L,
Dawe AE,
Weide R,
Van Staveren M,
Nuss DL, Govers F
(2005) Elicitin genes in Phytophthora infestans are clustered and interspersed with various transposon-like elements. Molecular Genetics and Genomics 273, 20–32.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Jones DA, Takemoto D
(2004) Plant innate immunity — direct and indirect recognition of general and specific pathogen-associated molecules. Current Opinion in Immunology 16, 48–62.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Koehl J,
Obwald W,
Kohn H,
Elstner EF, Heiser I
(2003) Different responses of two tobacco cultivars and their cell suspension cultures to quercinin, a novel elicitin from Phytophthora quercina. Plant Physiology and Biochemistry 41, 261–269.
| Crossref | GoogleScholarGoogle Scholar |
Lascombe MB,
Poncet M,
Vernard P,
Milat ML,
Blein JP, Prange T
(2000) Crystallization and preliminary X-ray studies of oligandrin, a sterol-carrier elicitor from Pythium oligandrum. Acta Crystallographica D56, 1498–1500.
Lascombe MB,
Poncet M,
Vernard P,
Milat ML,
Blein JP, Prange T
(2002) The 1.45Å resolution structure of the cryptogein–cholesterol complex: a close-up view of a sterol carrier protein (SCP) active site. Acta Crystallographia D58, 1442–1447.
Lascombe MB,
Poncet M,
Cardin L,
Milat ML,
Blein JP, Prange T
(2004) Purification, crystallization and preliminary X-ray studies of sylvaticin, an elicitin-like protein from Pythium sylvaticum. Acta Crystallographia D60, 362–364.
Latijnhouwers M,
de Wit PJGM, Govers F
(2003) Oomycetes and fungi: similar weaponry to attack plants. Trends in Microbiology 11, 462–469.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Maniatis, T ,
Fritsch, EF ,
and
Sambrook, J (1982).
Medrano RME, Ham MLM
(2003) Analysis of elicitor-induced cell viability changes in Lycopersicum esculentum Mill. Suspension culture by different methods. In Vitro Cellular and Developmental Biology 39, 236–239.
| Crossref | GoogleScholarGoogle Scholar |
Neuhoff V,
Stamm R, Eibl H
(1985) Clear background and highly sensitive protein staining with coomassie blue dyes in polyacrylamide gels: a systematic analysis. Electrophoresis 6, 427–448.
| Crossref | GoogleScholarGoogle Scholar |
Nürnberger T, Brunner F
(2002) Innate immunity in plants and animals: emerging parallels between the recognition of general elicitors and pathogen-associated molecular patterns. Current Opinion in Plant Biology 5, 318–324.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Nürnberger T, Lipka V
(2005) Non-host resistance in plants: new insights into an old phenomenon. Molecular Plant Pathology 6, 335–345.
| Crossref | GoogleScholarGoogle Scholar |
Orsomando G,
Lorenzi M,
Ferrari E,
Chiara C,
Spinsi A, Ruggieri S
(2003) PcF protein from Phytophthora cactorum and its recombinant homologue elicit phenylalanine ammonia lyase. Cellular and Molecular Life Sciences 60, 470–476.
Ortmann I,
Sumowski G,
Bauknecht H, Moerschbacher BM
(2004) Establishment of a reliable protocol for the quantification of an oxidative burst in suspension-cultured wheat cells upon elicitation. Physiological and Molecular Plant Pathology 64, 227–232.
| Crossref | GoogleScholarGoogle Scholar |
Osman H,
Vauthrin S,
Mikes V,
Milat ML,
Panabieres F,
Marais A,
Brunie S,
Maume B,
Ponchet M, Blein JP
(2001) Mediation of elicitin activity in tobacco is assumed by elicitin–sterol complex. Molecular Biology of the Cell 12, 2825–2834.
| PubMed |
Pare PW,
Farag MA,
Krishnamachari V,
Zhang H,
Ryu C, Kloepper JW
(2005) Elicitors and priming agents initiate plant defense responses. Photosynthesis Research 85, 149–159.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Parker JE
(2003) Plant recognition of microbial patterns. Trends in Plant Science 8, 245–247.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Pemberton CL, Salmond GPC
(2004) The Nep1-like proteins — a growing family of microbial elicitors of plant necrosis. Molecular Plant Pathology 5, 353–359.
| Crossref | GoogleScholarGoogle Scholar |
Radman R,
Saez T,
Bucke C, Keshavarz T
(2003) Elicitation of plants and microbial cell systems. Biotechnology and Applied Biochemistry 37, 91–102.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Safeeulla, KM (1976).
Thakur RP,
Rao VP,
Wu BM,
Subbarao KV, Shetty HS , et al.
(2004) Host resistance stability to downy mildew in pearl millet and pathogenic variability in Sclerospora graminicola. Crop Protection 23, 901–908.
| Crossref | GoogleScholarGoogle Scholar |
Thatcher LF,
Anderson JP, Singh KB
(2005) Plant defence responses: what have we learnt from Arabidopsis? Functional Plant Biology 32, 1–19.
| Crossref | GoogleScholarGoogle Scholar |
Tiedemann AV
(1997) Evidence for a primary role of active oxygen species in induction of host cell death during infection of bean leaves with Botrytis cinerea. Physiological and Molecular Plant Pathology 50, 151–166.
| Crossref | GoogleScholarGoogle Scholar |
Tsukada K,
Ishizaka M,
Fusjisawa Y,
Iwasaki Y,
Yamaguchi T,
Minami E, Shibuya N
(2002) Rice receptor for chitin oligosaccharide elicitor does not couple to heterotrimeric G-protein: elicitor responses of suspension cultured rice cells from daikoku (dI) dwarf mutants lacking a functional G-protein α subunit. Physiologia Plantarum 116, 373–382.
| Crossref | GoogleScholarGoogle Scholar |
Tyler BM
(2002) Molecular basis of recognition between Phytophthora pathogens and their hosts. Annual Review of Phytopathology 40, 137–167.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Veit S,
Manfred W,
Nurnberger T,
Koch W, Seitz HU
(2001) A novel protein elicitor (PaNie) from Pythium aphanidermatum induces multiple defense responses in carrot, Arabidopsis and tobacco. Plant Physiology 127, 832–841.
| Crossref |
PubMed |
Wang YC,
Hu DW,
Zhang ZG,
Ma ZC, Li DB
(2003) Purification and immunocytolocalization of a novel Phytophthora boehmeriae protein inducing hypersensitive response and systemic acquired resistance in tobacco and Chinese cabbage. Physiological and Molecular Plant Pathology 63, 223–232.
| Crossref | GoogleScholarGoogle Scholar |
We ZM,
Laby RJ,
Zumoff CH,
Bauer DW,
He SY,
Collmer A, Beer SV
(1992) Harpin elicitor of the hypersensitive response produced by the plant pathogen Erwinia amylovora. Science 257, 85–88.
| PubMed |
Wilson RJ,
Baillies BK, Jones DA
(2005) ER retrieval of Avr9 compromises its elicitor activity consistent with perception of Avr9 at the plasma membrane. Molecular Plant Pathology 6, 193–197.
| Crossref | GoogleScholarGoogle Scholar |
Wolski EA,
Lima C,
Augusti R,
Daleo GR,
Andreu AB, de Lederkremer RM
(2005) An α glucan elicitor from the cell wall of biocontrol binucleate Rhizoctonia isolate. Carbohydrate Research 340, 619–627.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
Yamaguchi T,
Mashara Y,
Kodama O,
Okada M,
Matsumura M, Shibuya N
(2002) Two purified oligosaccharide elicitors, N-acetylchitohepaton and tetra glucosyl glucitol, derived from Magnoporthe grisea cell wall, synergistically activate phytoalexins in suspension cultured rice cells. Journal of Plant Physiology 159, 1147–1149.
| Crossref | GoogleScholarGoogle Scholar |
Yu LM
(1992) Elicitins from Phytophthora and the basic resistance in tobacco. Proceedings of the National Academy of Sciences USA 92, 4088–4094.
Zhao J,
Davis LC, Verpoorte R
(2005) Elicitor signal transduction leading to production of plant secondary metabolites. Biotechnology Advances 23, 283–333.
| Crossref | GoogleScholarGoogle Scholar | PubMed |
