DOI QR코드

DOI QR Code

Overexpression of cysteine protease in transgenic Brassica rapa enhances resistance to bacterial soft rot and up-regulate the expression of various stress-regulated genes

  • Jung, Yu-Jin (Institute of Genetic Engineering, Hankyong National University) ;
  • Kang, Kwon-Kyoo (Department of Horticulture, Hankyong National University)
  • Received : 2010.09.03
  • Accepted : 2010.09.06
  • Published : 2010.09.30

Abstract

Cysteine proteases have been known as a critical factor in plant defense mechanisms in pineapple, papaya, or wild fig. Papain or ficin is one kind of cysteine proteases that shows toxic effects to herbivorous insects and pathogenic bacteria. However, resistance to bacterial soft rot of plants genetically engineered with cysteine protease has been little examined thus far. We cloned a cysteine protease cDNA from Ananas comosus and introduced the gene into Chinese cabbage (Brassica rapa) under the control of the cauliflower mosaic virus 35S promoter. The transgene was stably integrated and actively transcribed in transgenic plants. In comparisons with wild-type plants, the $T_2$ and $T_3$ transgenic plants exhibited a significant increase in endo-protease activity in leaves and enhanced resistance to bacterial soft rot. A cDNA microarray analysis revealed that several genes were more abundantly transcribed in the transgenic than in the wild type. These genes encode a glyoxal oxidase, PR-1 protein, PDF1, protein kinase, LTP protein, UBA protein and protease inhibitor. These results suggest an important role for cysteine protease as a signaling regulator in biotic stress signaling pathways, leading to the build-up of defense mechanism to pathogenic bacteria in plants.

Keywords

References

  1. Alonso JM, Granell A (1995) A putative vacuolar processing protease is regulated by ethylene and also during fruit ripening in Citrus fruit. Plant Physiol. 109:541-547 https://doi.org/10.1104/pp.109.2.541
  2. Andres M, Montesano M, Koiv V, Tapio-Palva E (2001) Transgenic plants producing the bacterial pheromone N-acylhomoserine lactone exhibit enhanced resistance to the bacterial phytopathogen Erwinia carotovora. Mol. Plant-Microbe Interact. 14:1035-1042 https://doi.org/10.1094/MPMI.2001.14.9.1035
  3. Barrett AJ, Rawlings ND, Davies EM, Macheleidt M, Salvesen GE, Turk V (1986) In: Barrett, A. J. and Salvesen, G. E. eds. Proteinase inhibitors. Amsterdam Science Publishers 12: 515-569
  4. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Anal. Biochem. 72:248-254 https://doi.org/10.1016/0003-2697(76)90527-3
  5. Buchanan-Wollaston V and Ainsworth C (1997) Leaf senescence in Brassica napus, cloning of senescence related genes by subtractive hybridization. Plant Mol. Biol. 33:821-843 https://doi.org/10.1023/A:1005774212410
  6. Chen HJ, Huang DJ, Hou WC, Lui JH and Lin YH (2006) Molecular cloning and characterization of a granulin-containing cysteine protease SPCP 3 from sweet potato (Ipomoea batatas) senescent leaves. J. Plant Physiol. 163:863-876 https://doi.org/10.1016/j.jplph.2005.08.008
  7. Cui J, Jander G, Racki LR, Kim PD, Pierce NE and Ausubel FM (2002) Signals involved in Arabidopsis resistance to Trichoplussiani caterpillars induced by virulent and avirulent strains of the phytopathogen Pseudomonas syringae. Plant Physiol. 129:551-564 https://doi.org/10.1104/pp.010815
  8. D’ Silva I, Poirier GG and Heath MC (1998) Activation of cysteine proteases in cowpea plants during the hypersensitive responsea form of programmed cell death. Exp. Cell Res. 245:389-399 https://doi.org/10.1006/excr.1998.4256
  9. Eason JR, Ryan DJ, Pinkney TT and O’Donoghue EM (2002) Programmed cell death during flower senescence: isolation and characterization of cysteine proteinase from Sendersonia aurantiaca. Functional Plant Biol. 29:1055-1064 https://doi.org/10.1071/PP01174
  10. Gepstein S, Sabehi G, Carp M J, Hajouj T, Nesher MFO, Yariv I, Dor C and Bassani M (2003) Large-scale identification of leaf senescence-associated genes. The Plant J. 36:629-642 https://doi.org/10.1046/j.1365-313X.2003.01908.x
  11. Granell A, Cercos M, Carbonell J (1988) Plant cysteine proteinases in germination and senescence. In: Barrett, A. J., Rawlings, D. D. and Woessner, J. F. eds. The handbook of proteolytic enzymes. San Diego: Academic Press, 578-583
  12. Guerrero C, Calle M, Reid MS, Valpuesta V (1998) Analysis of the expression of two thiolprotease genes from daylily (Hemero callis spp) during flower senescence. Plant Mol. Biol. 36:565-571 https://doi.org/10.1023/A:1005952005739
  13. Harrak H, Azelmat S, Baker EN, Tabaeizadeh Z (2001) Isolation and characterization of gene encoding a drought-induced cysteine protease in tomato (Lycopersicon esculentum). Genome 44:368-374 https://doi.org/10.1139/gen-44-3-368
  14. He YK (1990) Plant regeneration from Ri T-DNA transformed roots of cabbage. Chin J. Biotechnol. 6:131-137
  15. Hennin C, Hofte M, Diederichsen E (2001) Functional expression of Cf9 and Avr9 genes in Brassica napus induces enhanced resistance to Leptosphaeria maculans. Mol. Plant Microbe Interact. 14:1075-1085 https://doi.org/10.1094/MPMI.2001.14.9.1075
  16. Holwerda BC, Rogers JC (1992) Purification and characterization of aleurain. Plant Physiol. 99:848-855 https://doi.org/10.1104/pp.99.3.848
  17. Hughes TR, Mao M, Jones AR, Burchard J, Linsley PS (2001) Expression profiling using microarrays fabricated by an ink-jet oligonucleotide synthesizer. Nat. Biotechnol. 19:342-347
  18. Kalde M, Barth M, Somssich IE, Lippok B (2003) Members of the Arabidopsis WRKY group III transcription factors are part of different plant defence signaling pathways. Mol. Plant Microbe Interact. 16:295-305 https://doi.org/10.1094/MPMI.2003.16.4.295
  19. Kardailsky IV, Brevin NJ (1996) Expression of cysteine protease genes in pea nodules development and sencescene. Mol. Plant-Microbe Interact. 9:689-695 https://doi.org/10.1094/MPMI-9-0689
  20. Keller H, Pamboukdjian N, Ponchet M, Poupet A, Delon R, Verrier JL, Roby D, Ricci P (1999) Pathogen-induced elicitin production in transgenic tobacco generates a hypersensitivity response and nonspecific disease resistance. Plant Cell 11:223-235 https://doi.org/10.1105/tpc.11.2.223
  21. Kim LJ, Brian JJ, Antony B, Carolyn JS (2003) The fasciclin-like arabinoglactan proteins of Arabidopsis. A multigene family of putative cell adhesion molecules. Plant Physiol. 133:1911-1925 https://doi.org/10.1104/pp.103.031237
  22. Koizumi M, Yamaguchi-Shinozaki K, Tsuji H, Shinozaki K (1993) Structure and expression of two genes that encode distinct drought-inducible cysteine proteinase in Arabidopsis thaliana. Gene 29:175-182 https://doi.org/10.1016/0378-1119(84)90178-1
  23. Laemmli NK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685
  24. Ling J, Kojima T, Shiraiwa M, Takahara H (2003) Cloning of two cysteine proteinase genes: CysP1 and CysP2, from soybean cotyledons by cDNA representational difference analysis. Biochimica et Biophysica Acta 1627:129-139 https://doi.org/10.1016/S0167-4781(03)00082-4
  25. Linthorst HJM, Vanderdoes C, Brederode FT, Bol JF (1993) Circadian expression and induction by wounding of tobacco genes for cysteine proteinase. Plant Mol. Biol. 21:685-694 https://doi.org/10.1007/BF00014551
  26. Lohman KN, Gan S, John MC, Amasino RM (1994) Molecular analysis of natural leaf senescence in Arabidopsis thaliana. Physiologia Plantarum 92:322-328 https://doi.org/10.1111/j.1399-3054.1994.tb05343.x
  27. Markus A, Belastegui-Macadam X, Kaldenhoff R (2006) An attack of the plant parasite Cuscuta reflexa induces the expression of attAGP, an attachment protein of the host tomato. The Plant J. 48:548-556 https://doi.org/10.1111/j.1365-313X.2006.02897.x
  28. Murashige T, Skoog E (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15:473-497 https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
  29. Muta E, Okamoto Y, Ota S (1997) Cloning and sequencing of cysteine proteinase in Ananas comosus. Accession No. GI2351106 from NCBI database
  30. Naito Y, Fujie M, Usami S, Murooka Y, Yamada T (2000) The involvement of cysteine proteinase in the nodule development in Chinese milk vetch infected with Mesorhizobium huakuii subsp. Rengei. Plant Physiol. 124:1087-1096 https://doi.org/10.1104/pp.124.3.1087
  31. Okamoto T, Shimada T, Hara-Nishimura I, Nishimura M, Minamikawa T (2003) C-terminal KDEL sequence of a KDEL-tailed cysteine proteinase (sulfhydryl-endopeptidase) is involved in formation of KDEL vesicle and in efficient vacuolar transport of sulfhydryl-endopeptidase. Plant Physiol. 132:1892-1900 https://doi.org/10.1104/pp.103.021147
  32. Park HC, Kang YH, Chun HJ, Koo JC, Cheong YH, Kim CY, Kim MC, Chung WS, Kim JC, Yoo JH, Koo YD, Koo SC, Lim CO, Lee SY, Cho MJ (2002) Characterization of a stamen-specific cDNA encoding a novel plant defensin in Chinese cabbage. Plant Mol. Biol. 50:59-69
  33. Ren J, Petzoldt R, Dickson MH (2001) Screening and identification of resistance to bacterial soft rot in Brassica rapa. Euphytica 118:271-280 https://doi.org/10.1023/A:1017522501229
  34. Rizhsky L, Liang H, Mittler R (2002) The combined effect of drought and heat shock on gene expression in tobacco. Plant Physiol. 130:1143-1151 https://doi.org/10.1104/pp.006858
  35. Saijo Y, Hata S, Kyozuka J, Shimanoto K, Izui K (2000) Overexpression of a single $Ca^{+2}$ dependent protein kinase confers both cold and salt/ drought tolerance on rice plants. Plant J. 23:319-327 https://doi.org/10.1046/j.1365-313x.2000.00787.x
  36. Schaffer MA, Fischer RL (1988) Analysis of mRNAs that accumulates in response to low temperature identifies a thiolprotease gene in tomato. Plant Physiol. 87:431-436 https://doi.org/10.1104/pp.87.2.431
  37. Schmid M, Simpson D, Gietl C (1999) Programmed cell death in castor bean endosperm is associated with the accumulation and release of a cysteine endopeptidase from ricinosomes. Proceedings of the National Academy of Sciences, USA 96:14159-14164 https://doi.org/10.1073/pnas.96.24.14159
  38. Schmid M, Simpson D, Sarioglu H, Lottspeich F, Gietl C (2001) The ricinosomes of senescing plant tissue bud from the endoplasmic reticulum. Proceedings of the National Academy of Sciences, USA 98:5353-5435 https://doi.org/10.1073/pnas.061038298
  39. Shen S, Li Q, He SY, Barker KR., Li D, Hunt AG (2000) Conversion of compatible plant-pathogen interactions into incompatible interactions by expression of the Pseudomonas syringae pv. syringae 61 hrmA gene in transgenic tobacco plants. Plant J. 23:205-213 https://doi.org/10.1046/j.1365-313x.2000.00772.x
  40. Shimada T, Hiraiwa N, Nishimura M, Hara-Nishimura I (1994) Vacuolar processing enzyme of soybean that converts proproteins to the corresponding mature forms. Plant and Cell Physiol. 35:713-718 https://doi.org/10.1093/oxfordjournals.pcp.a078648
  41. Solomon M, Belenghi B, Delledonne M, Menachem E, Levine A. (1999) The involvement of cysteine proteases and protease inhibitor genes in the regulation of programmed cell death in plants. The Plant Cell 11:431-444 https://doi.org/10.2307/3870871
  42. Toyooka K, Okamoto T, Minamikawa T (2000) Mass transport of a proform of a KDEL-tailed cysteine proteinase (SH-EP) to protein storage vacuoles by endoplasmic reticulum-derived vesicle is involved in protein mobilization in germinating seeds. J. of Cell Biol. 148:453-563 https://doi.org/10.1083/jcb.148.3.453
  43. Ueda T, Seo S, Ohashi Y, Hashimoto J (2000) Circadian and senescence-enhanced expression of a tobacco cysteine protease gene. Plant Mol. Biol. 44:649-657 https://doi.org/10.1023/A:1026546004942
  44. Verberne MC, Verpoorte R, Bol JF, Mercado-Blanco J, Linthorst HJM (2000) Overproduction of salicylic acid in plants by bacterial transgenes enhances pathogen resistance. Nat. Biotechnol. 18:779-783 https://doi.org/10.1038/77347
  45. Wretblad S, Bohman S, Dixelius C (2003) Overexpression of a Brassica nigra cDNA gives enhanced resistance to Leptosphaeria maculans in B. napus. Mol. Plant Microbe Interact. 16:477-484 https://doi.org/10.1094/MPMI.2003.16.6.477
  46. Xu XF, Chye LM. (1999) Expression of cysteine proteinase during developmental events associated with programmed cell death in brinjal. The Plant J. 17:321-328 https://doi.org/10.1046/j.1365-313X.1999.00370.x
  47. Yoshida R, Hobo T, Ichimura K, Mizoguchi T, Takahashi F, Aronso J, Ecker JR, Shinozaki K (2002) ABA-activated SnRK2 protein kinase is required for dehydration stress signaling in Arabidopsis. Plant Cell Physiol. 43:1473-1483 https://doi.org/10.1093/pcp/pcf188
  48. Zhou J, Loh YT, Bressan RA, Martin GB (1995) The tomato gene pti1 encodes a serine/threonine kinase that is phosporylated by Pto and is involved in the hypersensitive response. Cell 15:925-935

Cited by

  1. Enhanced resistance to bacterial pathogen in transgenic tomato plants expressing cathelicidin antimicrobial peptide vol.18, pp.3, 2013, https://doi.org/10.1007/s12257-013-0392-3