Isolation and Expression Analysis of a Defensin Gene from Strawberry (Fragaria×ananassa cv. Paros)

Authors
1 Department of Agronomy and Plant Breeding, Faculty of Agriculture, University of Kurdistan, Sanandaj, Islamic Republic of Iran.
2 Department of Agricultural Biotechnology, Faculty of Agriculture,University of Kurdistan, Sanandaj, Iran
3 Department of Animal Science, Faculty of Agriculture, University of Kurdistan, Sanandaj, Islamic Republic of Iran.
Abstract
Plant defensins are the cysteine-rich peptides that are encoded by small multi-gene families in the plant kingdom. In this study, we designed primers based on conserved regions of defensin genes to clone and identify defensin genes in strawberry (Fragaria×ananassa cv. Paros) by reverse transcription PCR technique. Sequence analysis showed that the deduced amino acid had significant similarity to other plant defensins from NCBI database and designated FaDef1. The predicted strawberry defensin protein encodes a 54 aa protein of 6.18 kDa, pI 9.22 and eight conserved cysteine residues with desired space conservation with other amino acids. Semi quantitative expressions of FaDef1 were analyzed in root, stem, leaf, flower, and fruit in three strawberry cultivars, namely, Queenelisa, Camarosa, and Paros. The results showed that the FaDef1 expression patterns were similar in different tissues of the three cultivars. The higher amount of relative expression of FaDef1 was in fruit and there was no observable expression in the root. The expression of FaDef1 increased after wounding and salicylic acid treatment. The expression level was higher in developed fruits compared to that of immature fruits. In fruits infected with the Gray mold agent (Botrytis cinerea), the expression of FaDef1 showed significant increase by development of disease symptom. Taken together, these results suggest that FaDef1 is both responsive to biotic stress signal compounds and strawberry B. cinerea and may be used as a candidate gene for engineering plants against gray mold.

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1. Amil-Ruiz, F., Blanco-Portales, R., Muñoz-Blanco, J. and Caballero, J. L. 2011. The Strawberry Plant Defense Mechanism: A Molecular Review. Plant Cell Physiol., 52: 1873-1903.
2. Antoniw, J. and White, R. 1980. The Effects of Aspirin and Polyacrylic Acid on Soluble Leaf Proteins and Resistance to Virus Infection in Five Cultivars of Tobacco. J.Phytopathol., 98: 331-341.
3. Bahramnejad, B., Erickson, L., Chuthamat, A. and Goodwin, P. 2009. Differential Expression of Eight Defensin Genes of Nicotiana benthamiana Following Biotic Stress, Wounding, Ethylene, and Benzothiadiazole Treatments. Plant Cell Rep., 28: 703-717.
4. Casado-Díaz, A., Encinas-Villarejo, S., Santos, B. d. l., Schilirò, E., Yubero-Serrano, E. M., Amil-Ruíz, F., Pocovi, M. I., Pliego-Alfaro, F., Dorado, G. and Rey, M. 2006. Analysis of Strawberry Genes Differentially Expressed in Response to Colletotrichum Infection. Physiologia Plantarum, 128: 633-650.
5. Chiang, C. and Hadwiger, L. 1991. The Fusarium solani-Induced Expression of a Pea Gene Family Encoding High Cysteine Content Proteins. Mol. Plant Microbe Interact., 4: 324–331.
6. Debnath, S. C. and Teixeira da Silva, J. 2007. Strawberry Culture in Vitro: Applications in Genetic Transformation and Biotechnology. Fruit Vegetable Cereal Sci. Biotechnol., 1: 1-12.
7. Do, H. M., Lee, S. C., Jung, H. W., Sohn, K. H. and Hwang, B. K. 2004. Differential Expression and in Situ Localization of a Pepper Defensin (CADEF1) Gene in Response to Pathogen Infection, Abiotic Elicitors and Environmental Stresses in Capsicum annuum. Plant Sci., 166: 1297-1305.
8. Fedorova, M., van de Mortel, J., Matsumoto, P. A., Cho, J., Town, C. D., VandenBosch, K. A., Gantt, J. S. and Vance, C. P. 2002. Genome-Wide Identification of Nodule-Specific Transcripts in the Model Legume Medicago truncatula. Plant Physiol., 130: 519-537.
9. González, G., Fuentes, L., Moya-León, M. A., Sandoval, C. and Herrera, R. 2013. Characterization of Two PR Genes from Fragaria chiloensis in Response to Botrytis Cinerea Infection: A Comparison with Fragaria x Ananassa. Physiol. Mol. Plant Pathol., 82: 73-80.
10. Gu, Q., Kawata, E. E., Morse, M.-J., Wu, H.-M. and Cheung, A. Y. 1992. A Flower-Specific cDNA Encoding a Novel Thionin in Tobacco. Mol. General Genet. (MGG), 234: 89-96.
11. Hanks, J. N., Snyder, A. K., Graham, M. A., Shah, R. K., Blaylock, L. A., Harrison, M. J. and Shah, D. M. 2005. Defensin Gene Family in Medicago truncatula: Structure, Expression and Induction by Signal Molecules. Plant Mol. Biol., 58: 385-399.
12. Henrik, U., James, G. and Thomson, Y. 2009. Plant Defensins. Plant Signal Behav., 4: 1010-1012.
13. Hirakawa, H., Shirasawa, K., Kosugi, S., Tashiro, K., Nakayama, S., Yamada, M., Kohara, M., Watanabe, A., Kishida, Y. and Fujishiro, T. 2014. Dissection of the Octoploid Strawberry Genome by Deep Sequencing of the Genomes of Fragaria Species. DNA Res., 21: 169-181.
14. Koike, M., Okamoto, T., Tsuda, S. and Imai, R. 2002. A Novel Plant Defensin-Like Gene of Winter Wheat Is Specifically Induced during Cold Acclimation. Biochm. Biophys. Res. Commun., 298: 46-53.
15. Komori, T., Yamada, S. and Imaseki, H. 1997. A cDNA Clone Forc-Thionin from Nicotiana paniculata (Accession no AB005250; PGR97–132). Plant Physiol., 115: 314.
16. Lai, F. -M., DeLong, C., Mei, K., Wignes, T. and Fobert, P. R. 2002. Analysis of the DRR230 Family of Pea Defensins: Gene Expression Pattern and Evidence of Broad Host-Range Antifungal Activity. Plant Sci., 163: 855-864.
17. Lawton, K., Weymann, K., Friedrich, L., Vernooij, B., Uknes, S. and Ryals, J. 1995. Systemic Acquired Resistance in Arabidopsis Requires Salicylic Acid but Not Ethylene. MPMI- Mol. Plant Microbe Interact., 8: 863-870.
18. Lay, F. and Anderson, M. 2005. Defensins-Components of the Innate Immune System in Plants. Curr. Protein Peptide Sci., 6: 85-101.
19. Lay, F. T., Brugliera, F. and Anderson, M. A. 2003. Isolation and Properties of Floral Defensins from Ornamental Tobacco and Petunia. Plant Physiol., 131: 1283-1293.
20. Lee, S. C., Kim, Y. J. and Hwang, B. K. 2001. A Pathogen-Induced Chitin-Binding Protein Gene from Pepper: Its Isolation and Differential Expression in Pepper Tissues Treated with Pathogens, Ethephon, Methyl Jasmonate or Wounding. Plant Cell Physiol., 42: 1321-1330.
21. Maitra, N. and Cushman, J. C. 1994. Isolation and Characterization of a Drought-Induced Soybean cDNA Encoding a D95 Family Late-Embryogenesis-Abundant Protein. Plant Physiol., 106: 805.
22. Manners, J., Penninckx, I., Vermaere, K., Kazan, K., Brown, R., Morgan, A., Maclean, D., Curtis, M., Cammue, B. and Broekaert, W. 1998. The Promoter of the Plant Defensin Gene PDF1.2 from Arabidopsisis Systemically Activated by Fungal Pathogens and Responds to Methyl Jasmonate but Not to Salicylic Acid. Plant Mol. Biol., 38: 1071–1080.
23. Mazzara, M. and James, D. J. 2000. The Influence of Photoperiodic Growth Condition on Isolation of RNA from Strawberry (Fragaria×ananassa Duch.) Tissue. Mol. Biotechnol., 15: 237-241.
24. Mello, E. O., Ribeiro, S. F., Carvalho, A. O., Santos, I. S., Da Cunha, M., Santa-Catarina, C. and Gomes, V. M. 2011. Antifungal Activity of PvD1 Defensin Involves Plasma Membrane Permeabilization, Inhibition of Medium Acidification, and Induction of ROS in Fungi Cells. Curr. Microbiol., 62: 1209-1217.
25. Meyer, B., Houlne, G., Pozueta-Romero, J., Schantz, M. -L. and Schantz, R. 1996. Fruit-Specific Expression of a Defensin-Type Gene Family in Bell Pepper (Upregulation during Ripening and upon Wounding). Plant Physiol., 112: 615-622.
26. Mirouze, M., Sels, J., Richard, O., Czernic, P., Loubet, S., Jacquier, A., François, I. E., Cammue, B., Lebrun, M. and Berthomieu, P. 2006. A Putative Novel Role for Plant Defensins: A Defensin from the Zinc Hyper-Accumulating Plant, Arabidopsis halleri, Confers Zinc Tolerance. Plant J., 47: 329-342.
27. Parashina, E., Serdobinskii, L., Kalle, E., Lavrova, N., Avetisov, V., Lunin, V. and Naroditskii, B. 2000. Genetic Engineering of Oilseed Rape and Tomato Plants Expressing a Radish Defensin Gene. Russian J. Plant Physiol., 47: 417-423.
28. Park, H. C., Kang, Y. H., Chun, H. J., Koo, J. C., Cheong, Y. H., Kim, C. Y., Kim, M. C., Chung, W. S., Kim, J. C. and Yoo, J. H. 2002. Characterization of a Stamen-Specific cDNA Encoding a Novel Plant Defensin in Chinese Cabbage. Plant Mol. Biol., 50: 57-68.
29. Penninckx, I. A., Thomma, B. P., Buchala, A., Métraux, J.-P. and Broekaert, W. F. 1998. Concomitant Activation of Jasmonate and Ethylene Response Pathways Is Required for Induction of a Plant Defensin Gene in Arabidopsis. Plant Cell Online, 10: 2103-2113.
30. Pervieux, I., Bourassa, M., Laurans, F., Hamelin, R. and Séguin, A. 2004. A Spruce Defensin Showing Strong Antifungal Activity and Increased Transcript Accumulation after Wounding and Jasmonate Treatments. Physiol. Mol. Plant Pathol., 64: 331-341.
31. Reymond, P., Weber, H., Damond, M. and Farmer, E. E. 2000. Differential Gene Expression in Response to Mechanical Wounding and Insect Feeding in Arabidopsis. Plant Cell Online, 12: 707-719.
32. Schenk, P. M., Kazan, K., Wilson, I., Anderson, J. P., Richmond, T., Somerville, S. C. and Manners, J. M. 2000. Coordinated Plant Defense Responses in Arabidopsis Revealed by Microarray Analysis. Proceed. Nat. Acad. Sci., 97: 11655-11660.
33. Silverstein, K. A., Graham, M. A., Paape, T. D. and VandenBosch, K. A. 2005. Genome Organization of More than 300 Defensin-Like Genes in Arabidopsis. Plant Physiol., 138: 600-610.
34. Terras, F. R., Eggermont, K., Kovaleva, V., Raikhel, N. V., Osborn, R. W., Kester, A., Rees, S. B., Torrekens, S., Van Leuven, F. and Vanderleyden, J. 1995. Small Cysteine-Rich Antifungal Proteins from Radish: Their Role in Host Defense. Plant Cell Online, 7: 573-588.
35. Tesfaye, M., Silverstein, K. A., Nallu, S., Wang, L., Botanga, C. J., Gomez, S. K., Costa, L. M., Harrison, M. J., Samac, D. A. and Glazebrook, J. 2013. Spatio-Temporal Expression Patterns of Arabidopsis thaliana and Medicago truncatula Defensin-Like Genes. PloS One, 8: e58992.
36. Thaler, J. S., Owen, B. and Higgins, V. J. 2004. The Role of the Jasmonate Response in Plant Susceptibility to Diverse Pathogens with a Range of Lifestyles. Plant Physiol., 135: 530-538.
37. Thomma, B. P. and Broekaert, W. F. 1998. Tissue-Specific Expression of Plant Defensin Genes PDF2. 1 and PDF2. 2 in Arabidopsis thaliana. Plant Physiol. Biochem., 36: 533-537.
38. Thomma, B. P., Cammue, B. P. and Thevissen, K. 2002. Plant Defensins. Planta, 216: 193-202.
39. Thomma, B. P., Eggermont, K., Tierens, K. F.-J. and Broekaert, W. F. 1999. Requirement of Functional Ethylene-Insensitive 2 Gene for Efficient Resistance of Arabidopsis to Infection by Botrytis cinerea. Plant Physiol., 121: 1093-1101.
40. Uknes, S., Mauch-Mani, B., Moyer, M., Potter, S., Williams, S., Dincher, S., Chandler, D., Slusarenko, A., Ward, E. and Ryals, J. 1992. Acquired Resistance in Arabidopsis. Plant Cell Online, 4: 645-656.
41. Uknes, S., Winter, A. M., Delaney, T., Vernooij, B., Morse, A., Friedrich, L., Nye, G., Potter, S., Ward, E. and Ryals, J. 1993. Biological Induction of Systemic Acquired Resistance in Arabidopsis. MPMI- Mol. Plant Microbe Interact., 6: 692-698.
42. Ward, E. R., Uknes, S. J., Williams, S. C., Dincher, S. S., Wiederhold, D. L., Alexander, D. C., Ahl-Goy, P., Metraux, J. -P. and Ryals, J. A. 1991. Coordinate Gene Activity in Response to Agents that Induce Systemic Acquired Resistance. Plant Cell Online, 3: 1085-1094.
43. White, R. 1979. Acetylsalicylic Acid (Aspirin) Induces Resistance to Tobacco Mosaic Virus in Tobacco. Virol., 99: 410-412.
44. Yamada, S., Komori, T. and Imaseki, H. 1997. cDNA Cloning of c-Thionin from Nicotiana Excelsior (Accession no AB005266; PGR97–131). Plant Physiol., 115: 314.