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Constitutive expression of a fungus-inducible carboxylesterase improves disease resistance in transgenic pepper plants

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Abstract

Main conclusion

Resistance against anthracnose fungi was enhanced in transgenic pepper plants that accumulated high levels of a carboxylesterase, PepEST in anthracnose-susceptible fruits, with a concurrent induction of antioxidant enzymes and SA-dependent PR proteins.

A pepper esterase gene (PepEST) is highly expressed during the incompatible interaction between ripe fruits of pepper (Capsicum annuum L.) and a hemibiotrophic anthracnose fungus (Colletotrichum gloeosporioides). In this study, we found that exogenous application of recombinant PepEST protein on the surface of the unripe pepper fruits led to a potentiated state for disease resistance in the fruits, including generation of hydrogen peroxide and expression of pathogenesis-related (PR) genes that encode mostly small proteins with antimicrobial activity. To elucidate the role of PepEST in plant defense, we further developed transgenic pepper plants overexpressing PepEST under the control of CaMV 35S promoter. Molecular analysis confirmed the establishment of three independent transgenic lines carrying single copy of transgenes. The level of PepEST protein was estimated to be approximately 0.002 % of total soluble protein in transgenic fruits. In response to the anthracnose fungus, the transgenic fruits displayed higher expression of PR genes, PR3, PR5, PR10, and PepThi, than non-transgenic control fruits did. Moreover, immunolocalization results showed concurrent localization of ascorbate peroxidase (APX) and PR3 proteins, along with the PepEST protein, in the infected region of transgenic fruits. Disease rate analysis revealed significantly low occurrence of anthracnose disease in the transgenic fruits, approximately 30 % of that in non-transgenic fruits. Furthermore, the transgenic plants also exhibited resistance against C. acutatum and C. coccodes. Collectively, our results suggest that overexpression of PepEST in pepper confers enhanced resistance against the anthracnose fungi by activating the defense signaling pathways.

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Abbreviations

APX:

Ascorbate peroxidase

CaMV:

Cauliflower Mosaic Virus

CXE:

Carboxylesterase

gDNA:

Genomic DNA

HR:

Hypersensitive response

JA:

Jasmonic acid

LB:

Left border

MeSA:

Methyl SA

PepEST:

Pepper esterase

PR:

Pathogenesis related

RB:

Right border

SA:

Salicylic acid

References

  • Alkan N, Fluhr R, Prusky D (2012) Ammonium secretion during Colletotrichum coccodes infection modulates salicylic and jasmonic acid pathways of ripe and unripe tomato fruit. Mol Plant Microbe Interact 25:85–96

    Article  CAS  PubMed  Google Scholar 

  • Apostol I, Heinstein PF, Low PS (1989) Rapid stimulation of an oxidative burst during elicitation of cultured plant cells: role in defense and signal transduction. Plant Physiol 90:109–116

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Aranda J, Cerqueira NM, Fernandes PA, Roca M, Tunon I, Ramos MJ (2014) The catalytic mechanism of carboxylesterases: a computational study. Biochemistry 53:5820–5829

    Article  CAS  PubMed  Google Scholar 

  • Bezier A, Lambert B, Baillieul F (2002) Cloning of a grapevine Botrytis-responsive gene that has homology to the tobacco hypersensitivity-related hsr203 J. J Exp Bot 53:2279–2280

    Article  CAS  PubMed  Google Scholar 

  • Bowling SA, Guo A, Cao H, Gordon AS, Klessig DF, Dong X (1994) A mutation in Arabidopsis that leads to constitutive expression of systemic acquired resistance. Plant Cell 6:1845–1857

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Chilton MD, Que Q (2003) Targeted integration of T-DNA into the tobacco genome at double-stranded breaks: new insights on the mechanism of T-DNA integration. Plant Physiol 133:956–965

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Cho KC, Han YJ, Kim SJ, Lee SS, Hwang OJ, Song PS, Kim YS, Kim JI (2011) Resistance to Rhizoctonia solani AG-2-2 (IIIB) in creeping bentgrass plants transformed with pepper esterase gene PepEST. Plant Pathol 60:631–639

    Article  CAS  Google Scholar 

  • Cui F, Li MX, Chang HJ, Mao Y, Zhang HY, Lu LX, Yan SG, Lang ML, Liu L, Qiao CL (2015) Carboxylesterase-mediated insecticide resistance: quantitative increase induces broader metabolic resistance than qualitative change. Pest Biochem Physiol 121:88–96

    Article  CAS  Google Scholar 

  • Cummins I, Landrum M, Steel PG, Edwards R (2007) Structure activity studies with xenobiotic substrates using carboxylesterases isolated from Arabidopsis thaliana. Phytochemistry 68:811–818

    Article  CAS  PubMed  Google Scholar 

  • De Buck S, Podevin N, Nolf J, Jacobs A, Depicker A (2009) The T-DNA integration pattern in Arabidopsis transformants is highly determined by the transformed target cell. Plant J 60:134–145

    Article  PubMed  Google Scholar 

  • Falk A, Feys BJ, Frost LN, Jones JD, Daniels MJ, Parker JE (1999) EDS1, an essential component of R gene-mediated disease resistance in Arabidopsis has homology to eukaryotic lipases. Proc Natl Acad Sci USA 96:3292–3297

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Feys BJ, Moisan LJ, Newman MA, Parker JE (2001) Direct interaction between the Arabidopsis disease resistance signaling proteins, EDS1 and PAD4. EMBO J 20:5400–5411

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Forouhar F, Yang Y, Kumar D, Chen Y, Fridman E, Park SW, Chiang Y, Acton TB, Montelione GT, Pichersky E, Klessig DF, Tong L (2005) Structural and biochemical studies identify tobacco SABP2 as a methyl salicylate esterase and implicate it in plant innate immunity. Proc Natl Acad Sci USA 102:1773–1778

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Fu ZQ, Dong X (2013) Systemic acquired resistance: turning local infection into global defense. Annu Rev Plant Biol 64:839–863

    Article  CAS  PubMed  Google Scholar 

  • Gershater MC, Edwards R (2007) Regulating biological activity in plants with carboxylesterases. Plant Sci 173:579–588

    Article  CAS  Google Scholar 

  • Gershater MC, Cummins I, Edwards R (2007) Role of a carboxylesterase in herbicide bioactivation in Arabidopsis thaliana. J Biol Chem 282:21460–21466

    Article  CAS  PubMed  Google Scholar 

  • Glazebrook J (2005) Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens. Annu Rev Phytopathol 43:205–227

    Article  CAS  PubMed  Google Scholar 

  • Hong JK, Jung HW, Kim YJ, Hwang BK (2000) Pepper gene encoding a basic class II chitinase is inducible by pathogen and ethephon. Plant Sci 159:39–49

    Article  CAS  PubMed  Google Scholar 

  • Ichinose Y, Hisayasu Y, Sanematsu S, Ishiga Y, Seki H, Toyoda K, Shiraishi T, Yamada T (2001) Molecular cloning and functional analysis of pea cDNA E86 encoding homologous protein to hypersensitivity-related hsr203J. Plant Sci 160:997–1006

    Article  CAS  PubMed  Google Scholar 

  • Kim YS, Lee HH, Ko MK, Song CE, Bae CY, Lee YH, Oh BJ (2001) Inhibition of fungal appressorium formation by pepper (Capsicum annuum) esterase. Mol Plant Microbe Interact 14:80–85

    Article  CAS  PubMed  Google Scholar 

  • Kim YS, Park JY, Kim KS, Ko MK, Cheong SJ, Oh BJ (2002) A thaumatin-like gene in nonclimacteric pepper fruits used as molecular marker in probing disease resistance, ripening, and sugar accumulation. Plant Mol Biol 49:125–135

    Article  CAS  PubMed  Google Scholar 

  • Ko MK, Jeon WB, Kim KS, Lee HH, Seo HH, Kim YS, Oh BJ (2005) A Colletotrichum gloeosporioides-induced esterase gene of nonclimacteric pepper (Capsicum annuum) fruit during ripening plays a role in resistance against fungal infection. Plant Mol Biol 58:529–541

    Article  CAS  PubMed  Google Scholar 

  • Ko MK, Soh H, Kim KM, Kim YS, Im K (2007) Stable production of transgenic pepper plants mediated by Agrobacterium tumefaciens. Hort Sci 42:1425–1430

    CAS  Google Scholar 

  • Kordic S, Cummins I, Edwards R (2002) Cloning and characterization of an S-formylglutathione hydrolase from Arabidopsis thaliana. Arch Biochem Biophys 399:232–238

    Article  CAS  PubMed  Google Scholar 

  • Kumar S, Fladung M (2002) Transgene integration in aspen: structures of integration sites and mechanism of T-DNA integration. Plant J 31:543–551

    Article  CAS  PubMed  Google Scholar 

  • Laizure SC, Herring V, Hu Z, Witbrodt K, Parker RB (2013) The role of human carboxylesterases in drug metabolism: have we overlooked their importance? Pharmacotherapy 33:210–222

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Marshall SD, Putterill JJ, Plummer KM, Newcomb RD (2003) The carboxylesterase gene family from Arabidopsis thaliana. J Mol Evol 57:487–500

    Article  CAS  PubMed  Google Scholar 

  • Oakeshott JG, Claudianos C, Russell RJ, Robin GC (1999) Carboxyl/cholinesterases: a case study of the evolution of a successful multigene family. Bio Essays 21:1031–1042

    CAS  Google Scholar 

  • Oh BJ, Ko MK, Kostenyuk I, Shin B, Kim KS (1999) Coexpression of a defensin gene and a thionin-like gene via different signal transduction pathways in pepper and Colletotrichum gloeosporioides interactions. Plant Mol Biol 41:313–319

    Article  CAS  PubMed  Google Scholar 

  • Oh IS, Park AR, Bae MS, Kwon SJ, Kim YS, Lee JE, Kang NY, Lee S, Cheong H, Park OK (2005) Secretome analysis reveals an Arabidopsis lipase involved in defense against Alternaria brassicicola. Plant Cell 17:2832–2847

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Park CJ, Kim KJ, Shin R, Park JM, Shin YC, Paek KH (2004) Pathogenesis-related protein 10 isolated from hot pepper functions as a ribonuclease in an antiviral pathway. Plant J 37:186–198

    Article  CAS  PubMed  Google Scholar 

  • Park SW, Kaimoyo E, Kumar D, Mosher S, Klessig DF (2007) Methyl salicylate is a critical mobile signal for plant systemic acquired resistance. Science 318:113–116

    Article  CAS  PubMed  Google Scholar 

  • Paulert R, Ebbinghaus D, Urlass C, Moerschbacher BM (2010) Priming of the oxidative burst in rice and wheat cell cultures by ulvan, a polysaccharide from green macroalgae, and enhanced resistance against powdery mildew in wheat and barley plants. Plant Pathol 59:634–642

    Article  CAS  Google Scholar 

  • Penninckx IA, Eggermont K, Terras FR, Thomma BP, De Samblanx GW, Buchala A, Metraux JP, Manners JM, Broekaert WF (1996) Pathogen-induced systemic activation of a plant defensin gene in Arabidopsis follows a salicylic acid-independent pathway. Plant Cell 8:2309–2323

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pontier D, Godiard L, Marco Y, Roby D (1994) hsr203J, a tobacco gene whose activation is rapid, highly localized and specific for incompatible plant/pathogen interactions. Plant J 5:507–521

    Article  CAS  PubMed  Google Scholar 

  • Pontier D, Tronchet M, Rogowsky P, Lam E, Roby D (1998) Activation of hsr203, a plant gene expressed during incompatible plant-pathogen interactions, is correlated with programmed cell death. Mol Plant Microbe Interact 11:544–554

    Article  CAS  PubMed  Google Scholar 

  • Prusky D, Kobiler I, Ardi R, Beno-Moalem D, Yakoby N, Keen NT (2000) Resistance mechanisms of subtropical fruits to Colletotrichum gloeosporioides. In: Prusky D, Freeman S, Dickman M (eds) Colletotrichum: Host specificity, pathology, and host-pathogen interaction. American Phytopathological Society Press, St Paul, pp 232–244

    Google Scholar 

  • Seo HH, Park S, Park S, Oh BJ, Back K, Han O, Kim JI, Kim YS (2014) Overexpression of a defensin enhances resistance to a fruit-specific anthracnose fungus in pepper. PLoS One 9:e97936

    Article  PubMed  PubMed Central  Google Scholar 

  • Shimizu K, Takahashi M, Goshima N, Kawakami S, Irifune K, Morikawa H (2001) Presence of an SAR-like sequence in junction regions between an introduced transgene and genomic DNA of cultured tobacco cells: its effect on transformation frequency. Plant J 26:375–384

    Article  CAS  PubMed  Google Scholar 

  • Thordal-Christensen H, Zhang Z, Wei Y, Collinge DB (1997) Subcellular localization of H2O2 in plants. H2O2 accumulation in papillae and hypersensitive response during the barley-powdery mildew interaction. Plant J 11:1187–1194

    Article  CAS  Google Scholar 

  • Torres MA, Jones JDG, Dangl JL (2006) Reactive oxygen species signaling in response to pathogens. Plant Physiol 141:373–378

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • van Kan JA (2006) Licensed to kill: the lifestyle of a necrotrophic plant pathogen. Trend Plant Sci 11:247–253

    Article  Google Scholar 

  • Wei R, Oeser T, Zimmermann W (2014) Synthetic polyester-hydrolyzing enzymes from thermophilic actinomycetes. Adv Appl Microbiol 89:267–305

    Article  PubMed  Google Scholar 

  • Zeng FS, Zhan YG, Zhao HC, Xin Y, Qi FH, Yang CP (2010) Molecular characterization of T-DNA integration sites in transgenic birch. Trees Struct Funct 24:753–762

    Article  CAS  Google Scholar 

  • Zhao Y, Park RD, Muzzarelli RA (2010) Chitin deacetylases: properties and applications. Mar Drugs 8:24–46

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhu QH, Ramm K, Eamens AL, Dennis ES, Upadhyaya NM (2006) Transgene structures suggest that multiple mechanisms are involved in T-DNA integration in plants. Plant Sci 171:308–322

    Article  CAS  PubMed  Google Scholar 

  • Zhu C, Wu J, He C (2010) Induction of chromosomal inversion by integration of T-DNA in the rice genome. J Genet Genomic 37:189–196

    Article  CAS  Google Scholar 

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Acknowledgments

We thank the Kumho Life Science Laboratory in Chonnam National University for providing plant growth facilities. This work was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (Grant no. NRF-2014R1A1A2057739 to J.-I. Kim; Grant no. NRF-2014R1A1A2007413 to Y.S. Kim) and Next-Generation BioGreen 21 Program, Rural Development Administration, Republic of Korea (Grant no. PJ01104001 to J.-I. Kim).

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Authors and Affiliations

  1. Department of Biotechnology and Kumho Life Science Laboratory, Chonnam National University, Gwangju, 500-757, Republic of Korea

    Moonkyung Ko, Jung Hyun Cho, Hyo-Hyoun Seo, Ha-Young Kang, Thai Son Nguyen, Hyun Cheol Soh, Young Soon Kim & Jeong-Il Kim

  2. Department of Biology, Chosun University, Gwangju, 501-759, Republic of Korea

    Hyun-Hwa Lee

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  1. Moonkyung Ko
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Ko, M., Cho, J.H., Seo, HH. et al. Constitutive expression of a fungus-inducible carboxylesterase improves disease resistance in transgenic pepper plants. Planta 244, 379–392 (2016). https://doi.org/10.1007/s00425-016-2514-6

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