Antifungal activity of recombinant rice LTP2 on some phytopathogenic fungi

Document Type: Original Research Papers


1 National Institute of Genetic Engineering and Biotechnology, Tehran, Iran

2 National Institute of Genetic Engineering and Biotechnology


Plant lipid transfer proteins (LTPs) are members of the pathogenesis-related proteins (PR-14) and some of them exhibit activity against phytopathogenic fungi. To investigate whether rice LTP2 plays a role in antifungal activity, the coding region of an Iranian rice Ltp2 gene was cloned into expression vector pET24-d(+) and then expressed in Escherichia coli Rosetta strain (DE3). The potential antifungal activity of the purified LTP2 was tested on mycelial growth of some important phytopathogenic fungi. The results showed that the rice LTP2 inhibit the growth of Rhizoctonia solani, Botrytis cinerea,Sclerotinia sclerotiorum, Fusarium sporotrichioides and F. oxysporum. Also, the purified LTP2 protein was shown to strongly inhibit spore germination and consequential mycelia of Alternaria brassicola.


Main Subjects

1. Kader, J.C. (1996) Lipid-transfer proteins in plants. Annu. Rev. Plant Physiol Plant Mol. Biol., 47, 627–654.


2. Van Loon, L.C., Van Strien, E.A. (1999) The families of pathogenesis-related proteins, their activities and comparative analysis of PR-1 type proteins. Physiol. Mol. Plant Pathol., 55, 85-97.


3. José-Estanyol, M., Gomis-Rüth, F.X., Puigdomènech, P. (2004) The eight-cysteine motif, a versatile structure in plant proteins. PlantPhysiol. Biochem., 42(5),355-365.


4. Samuel, D., Liu, Y.J., Cheng, C.S., Lyu, P.C. (2002) Purification and characterization of a novel 7-kDa non-specific lipid transfer protein-2 from rice (Oryza sativa).  J. Biol. Chem., 277, 35267–35273.


5. Lee, J.Y., Min, K., Cha, H., Hwang, D.H.S.K.Y., Suh, S.W. (1998) Rice nonspecific lipid transfer protein: the 1.6A° crystal structure in the unliganded state reveals a small hydrophobic cavity.  J. Mol. Biol., 276, 437–48.


6. Lindorff-Larsen, K., Winther, J.R. (2001) Surprisingly high stability of barley lipid transfer protein, LTP1, towards denaturant, heat and proteases. FEBS Lett., 488, 145–148.


7. Kirubakaran, S.I., Mubarak Begum, S.M., Ulaganathan, K., Sakthivel, N. (2008) Characterization of a new antifungal lipid transfer protein from wheat. Plant Physiol. Bioch., 46, 918-927.


8. Liu, Y.J., Samuel, D., Lin, C.H., Lyu, P.C. (2002) Purification and characterization of a novel 7-kDa non-specific lipid transfer protein-2 from rice (Oryza sativa). Biochem. Biophys. Res. Commun., 294, 535–540.

9. Gorjanovic, S., Spillner, E., Beljanski, M., Gorjanovic, R., Pavlovic, M., Gojgic-Cvijovic, G. (2005) Malting barley grain non/specific lipid-transfer protein (ns-LTP): importance for grain protectio. J. Inst. Brew., 111(2), 99–104

10. Castro, M.S., Gerhardt, I.R., Orru, S., Pucci, P., Bloch, C.Jr. (2003) P urification and characterization of a small (7.3 kDa) putative lipid transfer protein from maize seeds. J. Chromatogr. B., 794, 109-114.

11. Segura, A., Moreno, M., Garcia-Olmedo, F. (1993) Purification and antipathogenic activity of lipid transfer proteins (LTPs) from the leaves of Arabidopsis and spinach. FEBS Lett., 332(3), 243-246.

12. Arondel, V., Vergnolle, C., Tchang, F., Kader, J.C. (1990) Biofunctional lipid-transfer: Fatty acid-binding proteins in plants. Mol. cell. Biochim., 98, 49-56.

13. Sterk, P., Booij, H., Scheleekens, G.A., Van Kammen, A., de Vries, S.C. (1991) Cell specific expression of the carrot EP2 lipid transfer protein gene. Plant Cell., 3, 907-921.

14. Coutos-Thevenot, P., Jouenne, T., Maes, O., Guerbette, F., Grosbois, M., Le Caer, J.P., Boulay, M., Deloire, A., Kader, J.C., Guerin, J. (1993) Four 9-kDa proteins excreted by somatic embryos of grapevine are isoforms of lipid transfer proteins. Eur. J Biochem., 217, 885-889.

15. Buhot, N., Douliez, J.P., Jacquemard, A., Marion, D., Tran, V., Maume, BF., et al. (2001) A lipid transfer protein binds to a receptor involved in the control of plant defence responses. FEBS Lett., 509, 27–30.

16. Torres-Schumann, S., Godoy, J.A., Pintor-Toro, J.A. (1992) A probable lipid transfer protein gene is induced by NaCl in stems of tomato plants. Plant Mol. Biol., 18,749-757.

17. Carvalho, A.O., Gomes, V.M. (2007) Role of plant lipid transfer proteins in plant cell physiology: A concise review. Peptides., 28, 1144-1153.

18.  Patkar, R. N., Chattoo, B. B. (2006) Transgenic indica rice expressing ns-LTP-like protein shows enhanced resistance to both fungal and bacterial pathogens. Mol. Breeding., 17, 159-171.

19. Roy-Barman, S., Sautter, C., Chattoo, B.B. (2006) Expression of the lipid transfer protein Ace-AMP1 in transgenic wheat enhances antifungal activity and defense responses. Transgenic Res., 15(4), 435-446.

20. Sarowar, S., Kim, Y.J., Kim, K.D., Hwang, B.K., Ok, S.H., Shin, J.S. (2009) Overexpression of lipid transfer protein (LTP) genes enhances resistance to plant pathogens and LTP functions in long-distance systemic signaling in tobacco. Plant Cell Rep., 28, 419-427

21. Marion, D., Douliez, J.P., Gautier, M.F., Elmorjani, K. (2004) Plant Food Allergens. In Mills, E.N.C., Shewry, P.R. (eds.), Plant lipid transfer proteins: relationships between allergenicity and structural, biological and technological properties. Blackwell Science, Oxford, UK,  pp. 57-69.

22. Bradford, M.M. (1976) A rapid and sensitive method for the quantiWcation of microgram quantities of protein utilizing the principle of protein–dye binding, Anal. Biochem., 72,  248-254.

23. Boutrot, F., Chantret, N., Gautier, M.F. (2008) Genome-wide analysis of the rice and arabidopsis non-specific lipid transfer protein (nsLtp) gene families and identification of wheat nsLtp genes by EST data mining. BMC Genomics., 9,86.

24. Adams, D.J. (2004) Fungal cell wall chitinase and glucanases. Microbiol., 50, 2029-2035.

25. Selitrennikoff, CP. (2001) Antifungal Proteins. Appl. Env. Microbiol., 67, 2883-2894.

26. Velazhahan, R., Jayaraj, J., Jeoung, J.M., Liang, G.H., Muthukrishnan, S. (2002) Purification and characterization of an antifungal thaumatin-like protein from sorghum (Sorghum bicolor (L) Moench) leaves. J. Plant Dis. Protect., 109, 452-461.

27. Edreva, A. (2005)Pathogenesis-related proteins: research progress in the last 15 years. Appl. Plant Physiology., 31, 105-124.

28. Van Loon, L.C. (2001) Book of abstracts. Spa, Belgium, pp. 9.

29. Gianotti, A., Rios, W.M., Soarescosta, A., Nogaroto, V., Carmona, A.K., Oliva, M.L.V., Andrade, S.S., Henrique-Silva, F. (2006) Recombinant expression, puriWcation, and functional analysis of two novel cystatins from sugarcane (Saccharumo Ycinarum). Protein Expres. Purif., 47, 483-489.

30. Gustafsson, C., Govindarajan, S., Minshull, J. (2004) Codon bias and heterologous protein expression. Trends Biotechnol., 22, 346-353.