A Kunitz trypsin inhibitor from chickpea (Cicer arietinum L.) that exerts an antimicrobial effect on Fusarium oxysporum f.sp. ciceris

Abstract

Fusarium oxysporum f.sp. ciceris (Foc) is one of the most important fungal pathogens of chickpea and is regarded as a constant threat in tropical and subtropical countries. In order to correlate Fusarium wilt resistance/susceptibility in Cicer arietinum to the presence or absence of trypsin inhibitor (TI) in the crude extract, trypsin inhibitory assay (TIA) and in vitro activity of TI against Foc were studied. In the present study, a 20 kDa trypsin inhibitor was purified from Fusarium wilt resistant cultivar (viz. JG 2001-12) by ammonium sulfate precipitation, dialysis and chromatographies with Sephadex G-100 and Diethyl aminoethyl cellulose (DEAE-cellulose-52) ion-exchange column. Results of pathogenecity assay were found to be in correlation to the trypsin inhibitor assay where the Fusarium wilt resistant cultivar showed high trypsin inhibitory activity (99%) in the presence of trypsin enzyme using both natural and synthetic substrates. Preliminary studies using crude extracts of JG 2001-12 showed a decrease in radial growth of Foc. A 45%-82% reduction in conidium germination at 20 μg·mL-1 Cicer arietinum trypsin inhibitor (CaTI) concentration was observed, thereby, indicating the use of CaTI in suppression of pathogen and in its deployment through transgenic plants for the management of Fusarium wilt.

Share and Cite:

Nair, M. and Sandhu, S. (2013) A Kunitz trypsin inhibitor from chickpea (Cicer arietinum L.) that exerts an antimicrobial effect on Fusarium oxysporum f.sp. ciceris. Agricultural Sciences, 4, 585-594. doi: 10.4236/as.2013.411079.

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1] Varshney, R.K., Song, C., Saxena, R.K., Azam, S., Yu, S., Sharpe, A.G. Cannon, S., Baek, J., Rosen, B.D., Tar'an, B., Millan, T., Zhang, X., Ramsay, L.D., Iwata, A., Wang, Y., Nelson, W., Farmer, A.D., Gaur, P.M., Soderlund, C., Penmetsa, R.V., Xu, C., Bharti, A.K., He, W., Winter, P., Zhao, S., Hane, J.K.,Carrasquilla-Garcia, N., Condie, J.A., Upadhyaya, H.D., Luo, M.C., Thudi, M., Gowda, C.L., Singh, N.P., Lichtenzveig, J., Gali, K.K., Rubio, J., Nadarajan, N., Dolezel, J., Bansal, K.C., Xu, X., Edwards, D., Zhang, G., Kahl, G., Gil, J., Singh, K.B., Datta, S.K., Jackson, S.A., Wang, J. and Cook, D.R. (2013) Draft genome sequence of chickpea (Cicer arietinum) provides a resource for trait improvement. Nature Biotechnology, 31, 240-246. http://dx.doi.org/10.1038/nbt.2491
[2] Landa, B.B., Navas-Cortés, J.A., Jiménez-Gasco, M.M., Katan, J., Retig, B. and Jiménez-Díaz, R.M. (2006) Temperature response of chickpea cultivars to races of Fusarium oxysporum f. sp. ciceris, causal agent of Fusarium wilt. Plant Disease, 90, 365-374.
http://dx.doi.org/10.1094/PD-90-0365
[3] Haware, M.P., Nene, Y.L. and Natarajan, M. (1996) The survival of Fusarium oxysporum f. sp. ciceri in the soil in the absence of chickpea. Phytopathologia Mediterranea, 35, 9-12.
[4] Sharma, K.D. and Muehlbauer, F.J. (2007) Fusarium wilt of chickpea: Physiological specialization, genetics of resistance and resistance gene tagging. Euphytica, 157, 1-14. http://dx.doi.org/10.1007/s10681-007-9401-y
[5] Habib, H. and Fazili, K.M. (2007) Plant protease inhibitors: A defense strategy in plants. Biotechnology and Molecular Biology Review, 2, 68-85.
[6] Zavala, J.A., Patankar, A.G., Gase, K., Hui, D. and Baldwin, I.T. (2004) Manipulation of endogenous trypsin proteinase inhibitor production in Nicotiana attenuata demonstrates their function as antiherbivore defenses. Plant Physiology, 134, 1181-1190.
http://dx.doi.org/10.1104/pp.103.035634
[7] Srinivasan, A., Chougule, N.P., Giri, A.P., Gatehouse, J.A. and Gupta, V.S. (2005) Podborer (Helicoverpa armigera Hübn.) does not show speci?c adaptations in gut proteinases to dietary Cicer arietinum Kunitz proteinase inhibitor. Journal of Insect Physiology, 51, 1268-1276.
http://dx.doi.org/10.1016/j.jinsphys.2005.07.005
[8] Nair, M., Singh, S.S. and Babbar, A. (2013) Purification of trypsin inhibitor (TI) from seeds of Cicer arietinum (L.) and its insecticidal potentiality against Helicoverpa armigera (Hübner). Theoretical and Experimental Plant Physiology, Vol. 25, No. 2, pp. 141-152.
[9] Brock, F.M., Forsberg, C.W. and Buchanan-Smith, J.G. (1982) Proteolytic activity of rumen microorganisms and effects of proteinase inhibitors. Applied and Environmental Microbiology, 44, 561-569.
[10] Lowry, O., Rosbrough, N., Farr, A. and Randall, R. (1951) Protein measurement with the folin phenol reagent. Journal of Biological Chemistry, 193, 265-275.
[11] Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of head of bacteriophage T4. Nature, 227, 680-685. http://dx.doi.org/10.1038/227680a0
[12] Le, Q.T. and Katunuma, N. (2004) Determination of protease inhibitors by a reverse zymography method performed in a Tris-Tricine buffer system. Analytical Biochemistry, 324, 237-240.
http://dx.doi.org/10.1016/j.ab.2003.09.033
[13] Schwert, W.H. and Takenaka, Y. (1955) A spectrophotometric determination of trypsin and chymotrypsin. Biochimica Biophysica Acta, 16, 570-575.
http://dx.doi.org/10.1016/0006-3002(55)90280-8
[14] Gatehouse, A.M.R., Gatehouse, J.A. and Boulter, D. (1980) Isolation and characterization of trypsin inhibitors from cowpea (Vigna unguiculata). Phytochemistry, 19, 751-756. http://dx.doi.org/10.1016/0031-9422(80)85104-1
[15] Tremacoldi, C.R. and Pascholati, S.F. (2002) Detection of trypsin inhibitor in seeds of Eucalyptus urophylla and its influence on the in vitro growth of the fungi Pisolithus tinctorius and Rhizoctonia solani. Brazilian Journal of Microbiology, 33, 281-286.
http://dx.doi.org/10.1590/S1517-83822002000400001
[16] Spelbrink, R.E.J., Gerrits, P.J., Mooij, C. and Giuseppin, M.L.F. (2011) Quantitative Determination of Trypsin Inhibitory Activity in Complex Matrices. Open Food Science Journal, 5, 42-46.
http://dx.doi.org/10.2174/1874256401105010042
[17] Kakade, M.L., Simons, N. and Liener, I.E. (1969) An evaluation of natural vs. synthetic substrates for measureing the antitryptic activity of soybean samples. Cereal Chemistry, 46, 518-526.
[18] Woloshuk, C.P., Meulenhoff, J.S., Sela-Buurlage, M., van den Elzen, P.J.M. and Cornelissen, B.J.C. (1991) Pathogen-induced proteins with inhibitory activity toward Phytophthora infestans. Plant Cell, 3, 619-628.
http://dx.doi.org/10.1105/tpc.3.6.619
[19] Stintzi, A., Heitz, T., Prasad, V., Wiedemann-Merdinoglu, S., Kauffmann, S., Geoffroy, P., Legrand, M. and Fritig, B. (1993) Plant “Pathogenesis-related” proteins and their role in defense against pathogens. Biochimie, 75, 687-706. http://dx.doi.org/10.1016/0300-9084(93)90100-7
[20] Niderman, T., Genetet, I., Bruyère, T., Gees, R., Stintzi, A., Legrand, M., Fritig, B. and M?singer, E. (1995) Pathogenesis-related PR-1 proteins are antifungal. Plant Physiology, 108, 17-27.
http://dx.doi.org/10.1104/pp.108.1.17
[21] Chen, Z-Y., Brown, R.L., Russin, J.S., Lax, A.R. and Cleveland, T.E. (1999) A corn trypsin inhibitor with antifungal activity inhibits Aspergillus flavus a-amylase. Phytopathology, 89, 902-907.
http://dx.doi.org/10.1094/PHYTO.1999.89.10.902
[22] Dunaevskii, Y.E., Gladysheva, I.P., Pavlukova, E.B., Beliakova, G.A., Gladyschev, D.P., Papisova, A.I., Larionova, N.I. and Belozersky, M.A. (1997) The anionic protease inhibitor BBWI-1 from buckwheat seeds. Kinetic properties and possible biological role. Physiologia Plantarum, 100, 483-488.
http://dx.doi.org/10.1111/j.1399-3054.1997.tb01027.x
[23] Joshi, B., Sainani, M., Bastawade, K., Gupta, V.S. and Ranjekar, P.K. (1998) Cysteine protease inhibitor from pearl millet: A new class of antifungal protein. Biochemical and Biophysical Research Communications, 246, 382-387. http://dx.doi.org/10.1006/bbrc.1998.8625
[24] Chilosi, G., Caruso, C., Caporale, C., Leonardi, L., Bertini, L., Buzi, A., Nobile, M., Magro, P. and Buonocore, V. (2000) Antifungal activity of a Bowman-Birk-type trypsin inhibitor from wheat kernel. Journal of Phytopathology, 148, 477-481.
http://dx.doi.org/10.1046/j.1439-0434.2000.00527.x.
[25] Terras, F.R.G., Schoofs, H.M.E., Thevissen, K., Osborn, R.W., Vanderleyden, J., Cammue, B.P.A. and Broekaert, W.F. (1993) Synergistic enhancement of the antifungal activity of wheat and barley thionins by radish and oilseed rape 2S albumins and by barley trypsin inhibitors. Plant Physiology (Rockville), 103, 1311-1319.
[26] Lorito, M., Broadway, R.M., Hayes, C.K., Woo, S.L., Noviello, C., Williams, D.L. and Harman, G.E. (1994) Proteinase inhibitors from plants as a novel class of fungicides. Molecular Plant-Microbe Interactions, 7, 525-527. http://dx.doi.org/10.1094/MPMI-7-0525
[27] Huang, H., Qi, S.D., Qi, F., Wu, C.A., Yang, G.D. and Zheng, C.C. (2010) NtKTI1, a Kunitz trypsin inhibitor with antifungal activity from Nicotiana tabacum, plays an important role in tobacco's defense response. FEBS Journal, 277, 4076-4088.
http://dx.doi.org/10.1111/j.1742-4658.2010.07803.x
[28] Revina, T.A., Parfenov, I.A., Gvozdeva, E.L., Gerasimova, N.G. and Valueva, T.A. (2011) Chymotrypsin and trypsin inhibitor isolated from potato tubers. Prikladnaya Biokhimiya i Microbiologiya, 47, 265-271.

Copyright © 2024 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.