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Identification and expression analysis of chitinase genes related to biotic stress resistance in Brassica

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Abstract

Brassica is a very important vegetable group because of its contribution to human nutrition and consequent economic benefits. However, biotic stress is a major concern for these crops and molecular biology techniques offer the most efficient of approaches to address this concern. Chitinase is an important biotic stress resistance-related gene. We identified three genes designated as Brassica chitinase like protein (BrCLP1), BrCLP2 and BrCLP3 from a full-length cDNA library of Brassica rapa cv. Osome. Sequence analysis of these genes confirmed that BrCLP1 was a class IV chitinase, and BrCLP2 and BrCLP3 were class VII chitinases. Also, these genes showed a high degree of homology with other biotic stress resistance-related plant chitinases. In expression analysis, organ-specific expression of all three genes was high except BrCLP1 in all the organs tested and BrCLP2 showed the highest expression compared to the other genes in flower buds. All these genes also showed expression during all developmental growth stages of Chinese cabbage. In addition, BrCLP1 was up-regulated with certain time of infection by Pectobacterium carotovorum subsp. carotovorum in Chinese cabbage plants during microarray expression analysis. On the other hand, expression of BrCLP2 and BrCLP3 were increased after 6 h post inoculation (hpi) but decreased from 12 hpi. All these data suggest that these three chitinase genes may be involved in plant resistance against biotic stresses.

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References

  1. Cardoza V, Stewart CN Jr (2004) Brassica biotechnology: progress in cellular and molecular biology. In Vitro Cell Dev Biol Plants 40:542–551

    Article  CAS  Google Scholar 

  2. Salunkhe DK, Kadam SS (1998) Handbook of vegetable science technology: production, composition, storage, and processing. Marcel Dekker Inc., New York

    Google Scholar 

  3. Agrios GN (1997) Plant pathology, 4th edn. Academic Press, San Diego

    Google Scholar 

  4. Park YS, Min HJ, Ryang SH, Oh KJ, Cha JS, Kim HY, Cho TJ (2003) Characterization of salicylic acid-induced genes in Chinese cabbage. Plant Cell Rep 21:1027–1034

    Article  PubMed  CAS  Google Scholar 

  5. Sela-Buurlage MB, Ponstein AS, Bres-Vloemans SA, Melchers LS, van den Elzen PJM, Comelissen BJC (1993) Only specific tobacco (Nicotiana tabacum) chitinases and-1, 3-glucanase exhibit antifungal activity. Plant Physiol 101:857–863

    PubMed  CAS  Google Scholar 

  6. Abeles FB, Bossart RP, Forrence LE, Habig WH (1971) Preparation and purification of glucanase and chitinase from bean leaves. Plant Physiol 47:129–134

    Article  PubMed  CAS  Google Scholar 

  7. Muthukrishnan S, Liang GH, Trick HN et al (2001) Pathogenesis-related proteins and their genes in cereals. J Plant Cell Tissue Organ Cult 64:93–114

    Article  CAS  Google Scholar 

  8. Wessels JGH, Seitsma JH (1981) Fungal cell walls: a survey. In: Tanner W, Loewus FA (eds) Encyclopedia of plant physiology, new series, vol 133B, plant carbohydrates II. Springer-Verlag, New York, pp 352–394

    Google Scholar 

  9. Roby D, Toppan A, Esquerre-Tugaye MT (1989) Systemic induction of chitinase activity and resistance in melon plants upon fungal infection or elicitor treatment. Physiol Mol Plant Pathol 33:409–417

    Article  Google Scholar 

  10. Wargo PM (1975) Lysis of the cell wall of Armillaria mellea by enzymes from forest trees. Physiol Plant Pathol 5:99–105

    Article  CAS  Google Scholar 

  11. Hadwiger LA, Beckman JM (1980) Chitosan as a component of pea-Fusarium solani interactions. Plant Physiol 66:205–211

    Article  PubMed  CAS  Google Scholar 

  12. Cota IE, Troncoso-Rojas R, Sotelo-Mundo R, Sánchez-Estrada A, Tiznado-Hernández ME (2007) Chitinase and b-1, 3-glucanase enzymatic activities in response to infection by Alternaria alternata evaluated in two stages of development in different tomato fruit varieties. Sci Hortic 2591:1–9

    Google Scholar 

  13. Wasano N, Konno K, Nakamura M, Hirayama C, Hattori M et al (2009) A unique latex protein, MLX56, defends mulberry trees from insects. Phytochemistry 70:880–888

    Article  PubMed  CAS  Google Scholar 

  14. Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucl Acids Res 8:4321–4325

    Article  PubMed  CAS  Google Scholar 

  15. Thompson JD, Higgins DG, Gibson TJ (1994) ClustalW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucl Acids Res 22:4673–4680

    Article  PubMed  CAS  Google Scholar 

  16. Park JI, Kumar TS, Ahmed NU, Nou IS (2010) Construction of full-length cDNA library and investigation of potential genes in Brassica rapa. J Basic Life Res Sci 10:6–11

    Google Scholar 

  17. Guruprasad K, Reddy BV, Pandit MW (1990) Correlation between stability of a protein and its dipeptide composition: a novel approach for predicting in vivo stability of a protein from its primary sequence. Protein Eng 4(2):155–161

    Article  PubMed  CAS  Google Scholar 

  18. Neuhaus JM (1999) Plant chitinases (PR-3, PR-4, PR-8, PR-11). In: Datta SK, Muthukrishnan S (eds) Pathogenesis-related protein in plants. CRC Press, Cambridge, pp 77–105

    Google Scholar 

  19. Neuhaus JM, Fritig B, Linthorst HJM, Meins F Jr, Mikkelsen JD, Ryals J (1996) A revised nomenclature for chitinase genes. Plant Mol Bio Rep 14(2):102–104

    Article  CAS  Google Scholar 

  20. Henrissat B, Davies G (1997) Structural and sequence-based classification of glycosyl hydrolases. Curr Opin Struct Biol 7:637–644

    Article  PubMed  CAS  Google Scholar 

  21. Cohen-Kupeic R, Chet I (1998) The molecular biology of chitin digestion. Curr Opin Biotechnol 9:270–277

    Article  Google Scholar 

  22. Rasmussen U, Bojsen K, Collinge DB (1992) Cloning and characterization of a pathogen-induced chitinase in Brassica napus. Plant Mol Biol 20(2):277–287

    Article  PubMed  CAS  Google Scholar 

  23. Robinson SP, Jacobs AK, Dry IB (1997) A class IV chitinase is highly expressed in grape berries during ripening. Plant Physiol 114(3):771–778

    Article  PubMed  CAS  Google Scholar 

  24. Troedsson UM, Terefework Z, Jarl-Sunesson CI (2006) Cloning of four chitinase genes and a lectin gene in Galega orientalis. Symbiosis 42(1):25–37

    CAS  Google Scholar 

  25. de A Gerhardt LB, Sachetto-Martins G, Contarini MG, Sandroni M, de P Ferreira R, de Lima VM, Cordeiro MC, de Oliveira DE, Margis-Pinheiro M (1997) Arabidopsis thaliana class IV chitinase is early induced during the interaction with Xanthomonas campestris. FEBS Lett 419(1):69–75

  26. Li J, Liu JY (2003) A novel cotton gene encoding a new class of chitinase. Acta Bot Sin 45(12):1489–1496

    CAS  Google Scholar 

  27. Kezuka Y, Kitazaki K, Itoh Y, Watanabe J, Takaha O, Watanabe T, Nishizawa Y, Nonaka T (2004) Crystallization and preliminary X-ray analysis of plant class I chitinase from rice. Protein Pept Lett 11(4):401–405

    Article  PubMed  CAS  Google Scholar 

  28. Fan J, Wang H, Feng D, Liu B, Liu H, Wang J (2007) Molecular characterization of plantain class I chitinase gene and its expression in response to infection by Gloeosporium musarum Cke and Massee and other abiotic stimuli. J Biochem 142(5):561–570

    Article  PubMed  CAS  Google Scholar 

  29. Leah R, Tommerup H, Svendsen I, Mundy J (1991) Biochemical and molecular characterization of three barley seed proteins with antifungal properties. J Biol Chem 266(3):1564–1573

    PubMed  CAS  Google Scholar 

  30. Zhang D, Hrmova M, Wan CH, Wu C, Balzen J, Cai W, Wang J, Densmore LD, Fincher GB, Zhang H, Haigler CH (2004) Members of a new group of chitinase-like genes are expressed preferentially in cotton cells with secondary walls. Plant Mol Biol 54(3):353–372

    Article  PubMed  CAS  Google Scholar 

  31. Li DM, Staehelin C, Wang WT, Peng SL (2010) Molecular cloning and characterization of a chitinase-homologous gene from Mikania micrantha infected by Cuscuta campestris. Plant Mol Biol Rep 28(1):90–101

    Article  CAS  Google Scholar 

  32. Hamel F, Bellemare G (1995) Characterization of a class I chitinase gene and of wound-inducible, root and flower-specific chitinase expression in Brassica napus. Biochim Biophys Acta 1263(3):212–220

    PubMed  Google Scholar 

  33. Krishnaveni S, Liang GH, Muthukrishnan S, Manickam A (1999) Purification and partial characterization of chitinases from sorghum seeds. Plant Sci 144(1):1–7

    Article  CAS  Google Scholar 

  34. Grison R, Grezes-Besset B, Schneider M, Lucante N, Olsen L, Leguay JJ, Toppan A (1996) Field tolerance to fungal pathogens of Brassica napus constitutively expressing a chimeric chitinase gene. Nat Biotechnol 4:643–646

    Article  Google Scholar 

  35. Oldach KH, Becker D, Lorz H (2001) Heterologous expression of the genes mediating enhanced fungal resistance in transgenic wheat. Mol Plant Microbe Interact 14:832–838

    Article  PubMed  CAS  Google Scholar 

  36. Lawrence SD, Novak NG (2006) Expression of poplar chitinase in tomato leads to inhibition of development in colorado potato beetle. Biotechnol Lett 28:593–599

    Article  PubMed  CAS  Google Scholar 

  37. Kitajima S, Kamei K, Taketani S, Yamaguchi M, Kawai F, Komatsu A, Inukai Y (2010) Two chitinase-like proteins abundantly accumulated in latex of mulberry show insecticidal activity. BMC Biochem 11:6

    Article  PubMed  Google Scholar 

  38. Libantova J, Kamarainen T, Moravcikova J, Matusikova I, Salaj J (2009) Detection of chitinolytic enzymes with different substrate specificity in tissues of intact sundew (Drosera rotundifolia L.): chitinases in sundew tissues. Mol Biol Rep 36(5):851–856

    Article  PubMed  CAS  Google Scholar 

  39. Neale AD, Wahleithner JA, Lund M, Bonnett HT, Kelly A, Meeks-Wagner DR, Peacock WJ, Dennis ES (1990) Chitinase, [beta]-1, 3-Glucanase osmotin, and extensin are expressed in tobacco explants during flower formation. Plant Cell 2:673–684

    Article  PubMed  CAS  Google Scholar 

  40. Samac DA, Hironaka CM, Yallaly PE, Shah DM (1990) Isolation and characterization of the genes encoding basic and acidic chitinase in Arabidopsis thaliana. Plant Physiol 93:907–914

    Article  PubMed  CAS  Google Scholar 

  41. del Campillo E, Lewis LN (1992) Occurrence of 9.5 cellulase and other hydrolases in flower reproductive organs undergoing major cell wall disruption. Plant Physiol 99:1015–1020

    Article  PubMed  Google Scholar 

  42. de Jong AJ, Heidstra R, Spanik HP, Hartog MV, Meijer EA, Hendriks T, Loschiavo F, Terzi M, Bisseling T, van Kammen A, de Vries SC (1993) Rhizobium lipooligosaccharides rescue a carrot somatic embryo mutant. Plant Cell 5:615–620

    Article  PubMed  Google Scholar 

  43. Van Loon LC, Van Strien EA (1999) The families of pathogenesis-related proteins, their activities, and comparative analysis of PR-1 type proteins. Physiol Mol Plant Pathol 55:85–97

    Article  Google Scholar 

  44. Van Loon LC (2001) The families of pathogenesis-related proteins. In: 6th international work-shop on PR-proteins, Spa, 20–24 May 2001, p 9

  45. Selitrennikoff CP (2001) Antifungal proteins. Appl Environ Microbiol 67:2883–2894

    Article  PubMed  CAS  Google Scholar 

  46. Datta K, Velazhahan R, Oliva N, Ona I, Mew T, Khush GS, Muthukrishnan S, Datta SK (1999) Over-expression of the cloned rice thaumatin-like protein (PR-5) gene in transgenic rice plants enhances environmental friendly resistance to Rhizoctonia solani causing sheath blight disease. Theor Appl Genet 98:1138–1145

    Article  CAS  Google Scholar 

  47. Fagoaga C, Rodrigo I, Conejero V, Hinarejos C, Tuset JJ, Arnau J, Pina JA, Navarro L, Peña L (2001) Increased tolerance to Phytophthora citrophthora in transgenic orange plants constitutively expressing a tomato pathogenesis related protein PR-5. Mol Breed 7:175–185

    Article  CAS  Google Scholar 

  48. Bachmann D, Rezzonico E, Retelska D, Chételat A, Schaerer S, Beffa R (1998) Improvement of potato resistance to Phytophthora infestans by overexpressing antifungal hydrolases. In: 5th international workshop on pathogenesis-related proteins. Signalling pathways and biological activities, Aussois, 29 March–2 April 1998

  49. Li WL, Faris JD, Muthukrishnan S, Liu DJ, Chen PD, Gill BS (2000) Isolation and characterization of novel cDNA clones of acidic chitinases and β-1, 3-glucanases from wheat spikes infected by Fusarium graminearum. Theor Appl Genet 102(2–3):353–362

    Google Scholar 

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Acknowledgments

This study was supported by the Cabbage Genomics assisted breeding supporting center research program funded by Ministry for Food, Agriculture, Forestry and Fisheries of the Korean Government. We wish to thank Genomics Division, Department of Agricultural Bio-resources, National Academy of Agricultural Science, Republic of Korea for providing microarray data.

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Authors and Affiliations

  1. Department of Horticulture, Sunchon National University, 413 Jungangno, Suncheon, Jeonnam, 540-742, Republic of Korea

    Nasar Uddin Ahmed, Jong-In Park, Thamilarasan Senthil Kumar, In-Ho Lee & Ill-Sup Nou

  2. Genomics Division, Department of Agricultural Bio-resources, National Academy of Agricultural Science, 150, Suin-ro, Gwonseon-gu, Suwon, Gyeonggi-do, 441-707, Republic of Korea

    Mi-Suk Seo & Beom-Seok Park

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  1. Nasar Uddin Ahmed
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Correspondence to Ill-Sup Nou.

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Nasar Uddin Ahmed and Jong-In Park contributed equally to this study.

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Ahmed, N.U., Park, JI., Seo, MS. et al. Identification and expression analysis of chitinase genes related to biotic stress resistance in Brassica . Mol Biol Rep 39, 3649–3657 (2012). https://doi.org/10.1007/s11033-011-1139-x

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  • DOI: https://doi.org/10.1007/s11033-011-1139-x

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