Messa a punto di un metodo per l'individuazione di β-1,3- glucanasi e chitinasi attraverso Native PAGE su estratti di piante di Prunus avium L. inoculate con Phytophthora spp.
DOI:
https://doi.org/10.12899/asr-878Keywords:
resistance, glucanase, chitinase, Prunus avium, Phytophthora spp.Abstract
Tecnique to detect on ß-1,3- glucanase and chitinasi by native PAGE, in Prunus avium (L.)clones inoculated with Phytophthora
spp. ß-1,3-glucanase and chitinase activities have been detected by native PAGE (poliacrylamide gel electrophoresis) on leaf extracts of wild cherry (P. avium) inoculated with different species of Phytophthora spp.. These pathogens are aggressive in wild cherry and they can cause wiltings and death of the plant. These two PR (pathogen-related) proteins are normally produced by plants as first resistance strategy towards pathogens. The PR activities are involved in ifal cell wall degradation and growth. Usually, the techniques to detect them are based on colorimetric assay performed with the total protein extracts or in the poliacrylamide gels, through expensive procedures. In this work a direct colorimetric detection on a Carboximetil (cm)-Curdlan RBB (Remazol Brilliant Blu) and Glycol Chitosan substrate, is used to reveal ß-1,3-glucanase and chitinasi respectively. This protocol resulted to be very sensible in detecting the two proteins, very simply, reliable and cheap.
References
Andersen M D., Jensen A., Robertus J.D., Leah R., Skriver K., 1997 – Heterologous expression and characterization of wild-type and mutant forms of a 26 kDa endochitinase from barley (Hordeum vulgare L.). Biochemical journal 322: 815-822.
Barzanti P., Biancalani F., De Rogatis A., Ghelardini L., Guerri S., Santini A., 2004 – Indagini preliminari per la messa a punto di test precoci di resistenza a Phytophthora sp. in alcuni cloni italiani di ciliegio da legno (Prunus avium L.). Forest@ 1 (2): 135-140.
Bradford M.M., 1976 – A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein- dye binding. Analytical Biochemistry 72: 248-254.
Cote F., Letarte J., Grenier J., Trudel J., Asselin A., 1989 – Detection of ß-1,3-glucanase activity after native polyacrylamide gel electrophoresis: application to tobacco pathogenesis-related proteins. Electrophoresis 10 (7): 527-529.
Deborah S.D., Palaniswami A., Velazhahan R., 2001 – Differential induction of chitinase and ß-1,3-glucanase in rice in response to inoculation with pathogen (Rhizoctonia solani) and non-pathogen (Pestalotia palmarum). Acta Phytopathologica et Entomologica Hungarica 36.
Ducci F., Canciani L., Biondi S., 1987 – Prospettive di miglioramento, propagazione e coltivazione del ciliegio da legno (Prunus avium L.). Monti e Boschi, XXXVIII 2: 14-19.
Ducci F., Tocci A., Veracini A., 1988 – Sintesi del registro del materiale di base di Prunus avium L. in Italia centro settentrionale, Basilicata e Calabria. Annali dell’Istituto Sperimentale per la Selvicoltura. XIX, Arezzo: 265-303.
Ducci F., Santi F., 1996 – Cloni naturali di ciliegio selvatico (Prunus avium L.): loro significato in foresta e per l’arboricoltura da legno. Sherwood 14: 11-16.
Ducci F., Santi F., 1997 – The distribution of clones in managed and unmanaged populations of wild cherry (Prunus avium L.). Canadian Journal of Forest Research 27: 1998-2004.
Egea C., Dickinson M.J., Candela M., Candela M.E., 1999 – ß-1,3-Glucanase isoenzymes and genes resistant and susceptible pepper (Capsicum annum) culitvars infected with Phytophthora capsici. Physiologia plantarum 107: 312-318.
Erwin D.C., Ribeiro O.K., 1996 – Phytophthora diseases worldwide. APS Press, St. Paul, Minnesota, 562 p.
Gohel V., Vyas P., Chhatpar H.S., 2005 – Activity staining method of chitinase on chitin agar plate through polyacrylamide gel electrophoresis. African Journal of Biotechnology 4 (1): 87-90.
Gohel V., Singh A., Vimal M., Ashwini P., Chhatpar H.S., 2006 – Bioprospecting and antifungal potential of chitinolytic microorganisms. African Journal of Biotechnology 5 (2): 54-72.
Graham M.Y., Weidner K., Wheeler K., Pelow M.J., Graham T.L., 2003 – Induced expression of pathogenesis-related protein genes in soybean by wounding and the Phytophthora sojae cell wall glucan elicitor. Physiological and molecular plant pathology 63: 141-149.
Habereder H., Schroeder G., Ebel J., 1989 – Rapid induction of phenilalanine ammonia-lyase and chalcone synthase mRNAs during fungus infection of soybean (Glicine max L.) roots or elicitor treatment of soybean cell cultures at the onset of phytoalexin synthesis. Planta 177: 58-65.
Hammatt N., Grant N.J., 1997 – Micropropagation of mature British wild cherry. Plant, Cell, Tissue and Organ Culture 47: 103-110.
Kalix S., Buchenauer H., 1995 – Direct detection of ß-1,3 glucanase in plant extracts by polyacrilamide gel electrophoresis. Electrophoresis 16: 1016-1018.
Kauffmann S., Legrand M., Geoffroy P., Fritig B., 1987 – Biological function of, pathogenesis related proteins: four PR proteins of tobacco have ß-1,3-glucanase activity. The EMBO journal 6 (11): 3209-3212.
Kim Z.S., 1979 – Inheritance of leucine aminopeptidase and acid phosphatase isozymes in beech (Fagus sylvatica L.). Silvae Genetica 28: 2-3.
Kim Y.J., Hwang B.K., 1994 – Differential accumulation of ß-1,3-glucanase and chitinase isoforms in pepper stems infected by compatible and incompatible isolates of Phytophthora capsici. Phisiological and molecular plant pathology 45: 195-209.
Kim Y.J., Hwang B.K., 1997 – Isolation of a basic 34 kiloDalton ß-1-,3-glucanase with inhibitory activity against Phytophthora capsici from pepper stem. Phisiological and molecular plant pathology 50: 103-115.
Legrand M., Kauffmann S., Geoffroy P., Fritig B., 1987 – Biological function of pathogenesis-related proteins are chitinases. Proceedings of the National Academy of Science USA 84: 6750-6754.
Niederman T., Genette I., Bruyere T., Gees R., Stintzi A., Leg rand M., Fritig B., Mösinge r E., 1995 – Pathogenesis-related PR-1 proetins are antifungal. Plant physiology 108: 17-27.
Oelofse D., Duberry I.A., 1996 – Induction of defese responses in cultured tobacco cells by elicitors from Phytophthora nicotianae. International Journal of Biochemistry and Cell Biology 28: 295-301.
Pan S.Q., Ye X. S., Kùc J., 1989 – Direct detection of ß-1,3-glucanase isozymes on polyacrylamide electrophoresis and isoeletrofocusing gels. Analytical biochemistry 182: 136-140.
Pan S.Q., Ye X. S., Kùc J., 1991a – A technique for detection of chitinase, ß-1,3-glucanse, and protein patterns after a single separation using polyacrylamide gel electrophoresis or isoelectrofocusing. Phytopathology 81: 970-974.
Pan S.Q., Ye X. S., Kùc J., 1991b – Association of ß-1,3-glucanase activity and isoform pattern with systematic resistance to blue mould in tobacco induced by stem injection with peronospora tabacina or leaf inoculation with tobacco mosaic virus. Phisiological and molecular plant pathology 39: 25-39.
Punja Z.K., Zhang Y.E., 1993 – Plant chitinases and their role in resistance to fungal disease. Journal of nematology 25: 526-540.
Rasmussen U., Bojse n K., Coll inge D.B., 1992 – Cloning and characterizzation of a pathogen-induced chitinase in Brassica napus. Plant Molecular Biology 20: 277-287.
Selitrennikoff C.P., 2001 – Antifungal proteins. Applied and environmental microbiology 67 (7): 2883-2894.
Sible G.V., Marimuthu T., Rabidran R., Velazhaan R., 2004 – Induction of resistance in rice against Xanthomonas oryzae pv. oryzae by foliar application of cow dung water extract. Acta Phytopathologica et Entomologica Hungarica 39.
Van Loon L.C., Van Strien E.A., 1999 – The families of pathogenesisrelated proteins, their activties, and comparative analysis of PR-1 type proteins. Physiological and molecular plant pathology 55: 85-97.
Van West P., Appiah A.A., Gow N.A.R., 2003 – Advances in research on oomycete root pathogens. Physiological and molecular plant pathology 62: 99-113.
Wolosh uk C., Meule nhoff J.S., Sel a-Buurlage M., Van de Elzen P. J. M., Cornel isse n B.J.C., 1991 – Pathogen–induces proteins with inibitory activity toward Phytophthora infestans. The plant cell 3: 619-628.
Wu C.T., Bradford K., 2003 – Class I Chitinase and ß-1,3-glucanase are differentially regulated by wounding, methyl jasmonate, ethylene, and gibberellin in tomato seeds and leaves. Plant Physiology 133: 263-273.
Downloads
How to Cite
Issue
Section
License
Authors who publish with this journal agree to the following terms: Authors retain copyright and grant the journal right of first publication with the work simultaneously licensed under a Creative Commons Attribution License that allows others to share the work with an acknowledgement of the work's authorship and initial publication in this journal. Authors are able to enter into separate, additional contractual arrangements for the non-exclusive distribution of the journal's published version of the work (e.g., post it to an institutional repository or publish it in a book), with an acknowledgement of its initial publication in this journal. Authors are permitted and encouraged to post their work online (e.g., in institutional repositories or on their website) prior to and during the submission process, as it can lead to productive exchanges, as well as earlier and greater citation of published work (See The Effect of Open Access).
