Skip to main content
SpringerLink
Log in

Molecular characterization of the AtCXE8 gene, which promotes resistance to Botrytis cinerea infection

  • Original Article
  • Published:
Plant Biotechnology Reports Aims and scope Submit manuscript

Abstract

The relationships of sterase- and lipase-like proteins with three signaling molecules (salicylic acid, jasmonic acid, and ethylene) expressed during plant–pathogen interactions were studied. We isolated two carboxylesterase (AtCXE) genes, AtCXE8 and AtCXE9, from Arabidopsis thaliana. The AtCXE8 and AtCXE9 proteins possess carboxylesterase motifs (-GXSXG-) and catalytic triads (Ser, Asp, and His). We demonstrated that recombinant AtCXE8 and AtCXE9 proteins have both enzymatic activity and specific activity for p-nitrophenyl butyrate (C4) in vitro. Moreover, the enzymatic activity of recombinant AtCXE8 was twofold higher than that of AtCXE9. To gain a better understanding of the endogenous role of the AtCXE8 gene in Arabidopsis, we identified an enhancer trap T-DNA mutant (AtCXE8_KO) and used it to show that the AtCXE8 gene was induced in response to fungal infection. AtCXE8_KO plants were also more susceptible to infections than wild-type Col-0 plants. Moreover, overexpression of the AtCXE8 gene in transgenic Arabidopsis plants led to enhanced disease resistance against B. cinerea. Taken together, our data indicate that AtCXE8 plays a role in promoting resistance to fungal invasion.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7

Similar content being viewed by others

References

  • Alonso JM, Hirayama T, Roman G, Nourizadeh S, Ecker JR (1999) EIN2, a functional transducer of ethylene and stress responses in Arabidopsis. Science 25:2148–2152

    Google Scholar 

  • Alonso JM, Stepanova AN, Solano R, Wisman E, Ferrari S, Ausubel FM, Ecker JR (2003) Five components of the ethylene-response pathway identified in a screen for weak ethylene-insensitive mutants in Arabidopsis. Proc Natl Acad Sci USA 100:2992–2997

    Google Scholar 

  • Argueso CT, Ferreira FJ, Epple P, To JP, Hutchison CE, Schaller GE, Dangl JL, Kieber JJ (2012) Two-component elements mediate interactions between cytokinin and salicylic acid in plant immunity. PLoS Genet 8(1):e1002448

    Article  PubMed  CAS  Google Scholar 

  • Bechtold N, Ellis T, Pellctier G (1993) In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. C R Acad Sci 316:1194–1199

  • Bostock RM (2005) Signal crosstalk and induced resistance: straddling the line between cost and benefit. Annu Rev Phytopathol 43:545–580

    Article  PubMed  CAS  Google Scholar 

  • 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

    Article  PubMed  CAS  Google Scholar 

  • Cao H, Bowling SA, Gordon AS, Dong X (1994) Characterization of an Arabidopsis mutant that is nonresponsive to inducers of systemic acquired resistance. Plant Cell 6:1583–1592

    Google Scholar 

  • Cao H, Glazebrook J, Clarke JD, Volko S, Dong X (1997) The Arabidopsis NPR1 gene that controls systemic acquired resistance encodes a novel protein containing ankyrin repeats. Cell 88:57–63

    Google Scholar 

  • Chen H, Nelson RS, Sherwood JL (1994) Enhanced recovery of transformants of Agrobacterium tumefaciens after freeze–thaw transformation and drug selection. Biotechniques 16:664–668

    PubMed  CAS  Google Scholar 

  • Clarke JD, Volko SM, Ledford H, Ausubel FM, Dong X (2000) Roles of salicylic acid, jasmonic acid, and ethylene in cpr-induced resistance in Arabidopsis. Plant Cell 12:2175–2190

    Google Scholar 

  • Delaney TP, Friedrich L, Ryals JA (1995) Arabidopsis signal transduction mutant defective in chemically and biologically induced disease resistance. Proc Natl Acad Sci USA 92:6602–6606

    Google Scholar 

  • Dubreuil-Maurizi C, Vitecek J, Marty L, Branciard L, Frettinger P, Wendehenne D, Meyer AJ, Mauch F, Poinssot B (2011) Glutathione deficiency of the Arabidopsis mutant pad2-1 affects oxidative stress-related events, defense gene expression, and the hypersensitive response. Plant Physiol 157:2000–2012

    Google Scholar 

  • Ellis C, Turner JG (2001) The Arabidopsis mutant cev1 has constitutively active jasmonate and ethylene signal pathways and enhanced resistance to pathogens. Plant Cell 13:1025–1033

    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

    Google Scholar 

  • Feller G, Thiry M, Gerday C (1991) Nucleotide sequence of the lipase gene lip2 from the antarctic psychrotroph Moraxella TA144 and site-specific mutagenesis of the conserved serine and histidine residues. DNA Cell Biol 10:381–388

    Article  PubMed  CAS  Google Scholar 

  • Ferrari S, Plotnikova JM, De Lorenzo G, Ausubel FM (2003) Arabidopsis local resistance to Botrytis cinerea involves salicylic acid and camalexin and requires EDS4 and PAD2, but not SID2, EDS5 or PAD4. Plant J 35:193–205

    Google Scholar 

  • García-Andrade J, Ramírez V, Flors V, Vera P (2011) Arabidopsis ocp3 mutant reveals a mechanism linking ABA and JA to pathogen-induced callose deposition. Plant J 67:783–794

    Google Scholar 

  • Glazebrook J (2001) Genes controlling expression of defense responses in Arabidopsis—2001 status. Curr Opin Plant Biol 4:301–308

    Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

  • Glazebrook J, Zook M, Mert F, Kagan I, Rogers EE, Crute IR, Holub EB, Hammerschmidt R, Ausubel FM (1997) Phytoalexin-deficient mutants of Arabidopsis reveal that PAD4 encodes a regulatory factor and that four PAD genes contribute to downy mildew resistance. Genetics 146:381–392

    PubMed  CAS  Google Scholar 

  • Govrin EM, Levine A (2002) Infection of Arabidopsis with a necrotrophic pathogen, Botrytis cinerea, elicits various defense responses but does not induce systemic acquired resistance (SAR). Plant Mol Biol 48:267–276

    Google Scholar 

  • Hammond-Kosack KE, Jones JD (1996) Resistance gene-dependent plant defense responses. Plant Cell 8:1773–1791

    PubMed  CAS  Google Scholar 

  • Jakab G, Manrique A, Zimmerli L, Metraux JP, Mauch-Mani B (2003) Molecular characterization of a novel lipase-like pathogen-inducible gene family of Arabidopsis. Plant Physiol 132:2230–2239

    Article  PubMed  CAS  Google Scholar 

  • Jirage D, Tootle TL, Reuber TL, Frost LN, Feys BJ, Parker JE, Ausubel FM, Glazebrook J (1999) Arabidopsis thaliana PAD4 encodes a lipase-like gene that is important for salicylic acid signaling. Proc Natl Acad Sci USA 96:13583–13588

    Article  PubMed  CAS  Google Scholar 

  • Jones-Rhoades MW, Bartel DP (2004) Computational identification of plant microRNAs and their targets, including a stress-induced miRNA. Mol Cell 14:787–799

    Article  PubMed  CAS  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 Inter 14:80–85

    Article  CAS  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

    Google Scholar 

  • Kok RG, Christoffels VM, Vosman B, Hellingwerf KJ (1993) Growth-phase-dependent expression of the lipolytic system of Acinetobacter calcoaceticus BD413: cloning of a gene encoding one of the esterases. J Gen Microbiol 139:2329–2342

    Article  PubMed  CAS  Google Scholar 

  • Kunkel BN, Brooks DM (2002) Cross talk between signaling pathways in pathogen defense. Curr Opin Plant Biol 5:325–331

    Article  PubMed  CAS  Google Scholar 

  • Lamb C, Dixon RA (1997) The oxidative burst in plant disease resistance. Annu Rev Plant Physiol Plant Mol Biol 48:251–275

    Article  PubMed  CAS  Google Scholar 

  • Lamesch P, Berardini TZ, Li D, Swarbreck D, Wilks C, Sasidharan R, Muller R, Dreher K, Alexander DL, Garcia-Hernandez M, Karthikeyan AS, Lee CH, Nelson WD, Ploetz L, Singh S, Wensel A, Huala E (2012) The Arabidopsis Information Resource (TAIR): improved gene annotation and new tools. Nucleic Acids Res 40:D1202–D1210

  • Langin D, Laurell H, Holst LS, Belfrage P, Holm C (1993) Gene organization and primary structure of human hormone-sensitive lipase: possible significance of a sequence homology with a lipase of Moraxella TA144, an antarctic bacterium. Proc Natl Acad Sci USA 90:4897–4901

    Google Scholar 

  • Lawton K, Weymann K, Friedrich L, Vernooij B, Uknes S, Ryals J (1995) Systemic acquired resistance in Arabidopsis requires salicylic acid but not ethylene. Mol Plant Microbe Interact 8:863–870

    Google Scholar 

  • Luna E, Bruce TJ, Roberts MR, Flors V, Ton J (2012) Next-generation systemic acquired resistance. Plant Physiol 158:844–853

    Article  PubMed  CAS  Google Scholar 

  • Mukherjee M, Larrimore KE, Ahmed NJ, Bedick TS, Barghouthi NT, Traw MB, Barth C (2010) Ascorbic acid deficiency in arabidopsis induces constitutive priming that is dependent on hydrogen peroxide, salicylic acid, and the NPR1 gene. Mol Plant Microbe Interact 23:340–351

    Article  PubMed  CAS  Google Scholar 

  • Nawrath C, Métraux JP (1999) Salicylic acid induction-deficient mutants of Arabidopsis express PR-2 and PR-5 and accumulate high levels of camalexin after pathogen inoculation. Plant Cell 11:1393–1404

    Google Scholar 

  • Ng G, Seabolt S, Zhang C, Salimian S, Watkins TA, Lu H (2011) Genetic dissection of salicylic acid-mediated defense signaling networks in Arabidopsis. Genetics 189:851–859

    Google Scholar 

  • Ogawa M, Hanada A, Yamauchi Y, Kuwahara A, Kamiya Y, Yamaguchi S (2003) Gibberellin biosynthesis and response during Arabidopsis seed germination. Plant Cell 15:1591–1604

    Google Scholar 

  • Pan H, Liu S, Tang D (2011) HPR1, a component of the THO/TREX complex, plays an important role in disease resistance and senescence in Arabidopsis. Plant J 69:831–843

    Google Scholar 

  • Pande S, Galloway G, Gaur PM, Siddique KHM, Tripathi HS, Taylor P, MacLeod MWJ, Basandrai AK, Bakr A, Joshi S, Krishna Kishore G, Isenegger DA, Narayana Rao J, Sharma M (2006) Botrytis grey mould of chickpea: a review of biology, epidemiology and disease management. Aust J Agric Res 57:1137–1150

    Article  Google Scholar 

  • Penninckx IAMA, Eggermont K, Terras FRG, Thomma BPHJ, De Samblanx GW, Buchala A, Métraux 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

    Google Scholar 

  • Penninckx IAMA, Thomma BPHJ, Buchala A, Métraux JP, Broekaert WF (1998) Concomitant activation of jasmonate and ethylene response pathways is required for induction of a plant defensin gene in Arabidopsis. Plant Cell 10:2103–2114

    Google Scholar 

  • Pieterse CM, van Wees SC, Hoffland E, van Pelt JA, van Loon LC (1996) Systemic resistance in Arabidopsis induced by biocontrol bacteria is independent of salicylic acid accumulation and pathogenesis-related gene expression. Plant Cell 8:1225–1237

    Google Scholar 

  • Pieterse CMJ et al (2009) Networking by small-molecule hormones in plant immunity. Nat Chem Biol 5:308–316

    Article  PubMed  CAS  Google Scholar 

  • Rossi FR, Marina AGM, Romero FM, Gonzalez ME, Collado IG, Pieckenstain FL (2011) The sesquiterpene botrydial produced by Botrytis cinerea induces the hypersensitive response on plant tissues and its action is modulated by salicylic acid and jasmonic acid signaling. Mol Plant Microbe Interact 24:888–896

    Article  PubMed  CAS  Google Scholar 

  • Shen X, Liu H, Yuan B, Li X, Xu C, Wang S (2011) OsEDR1 negatively regulates rice bacterial resistance via activation of ethylene biosynthesis. Plant Cell Environ 34:179–191

    Article  PubMed  CAS  Google Scholar 

  • Spoel SH, Johnson JS, Dong X (2007) Regulation of tradeoffs between plant defenses against pathogens with different lifestyles. Proc Natl Acad Sci USA 47:18842–18847

    Google Scholar 

  • Sticher L, Mauch-Mani B, Métraux JP (1997) Systemic acquired resistance. Annu Rev Phytopathol 35:235–270

    Article  PubMed  CAS  Google Scholar 

  • Summermatter K, Sticher L, Metraux JP (1995) Systemic responses in Arabidopsis thaliana infected and challenged with Pseudomonas syringae pv syringae. Plant Physiol 108:1379–1385

    Google Scholar 

  • Thatcher LF, Anderson JP, Singh K (2005) Plant defence responses: what have we learnt from Arabidopsis? Funct Plant Biol 32:1–19

    Google Scholar 

  • Thomma BP, Eggermont K, Penninckx I, Mauch-Mani B, Vogelsang R, Cammue BPA, Broekaert WF (1998) Separate jasmonate-dependent and salicylate-dependent defense-response pathways in Arabidopsis are essential for resistance to distinct microbial pathogens. Proc Natl Acad Sci USA 95:15107–15111

    Google Scholar 

  • Thomma BPHJ, Eggermont K, Tierens KFMJ, Broekaert WF (1999a) Requirement of functional ethylene-insensitive 2 gene for efficient resistance of Arabidopsis to infection by Botrytis cinerea. Plant Physiol 121:1093–1101

    Google Scholar 

  • Thomma BPHJ, Nelissen I, Eggermont K, Broekaert WF (1999b) Deficiency in phytoalexin production causes enhanced susceptibility of Arabidopsis thaliana to the fungus Alternaria brassicicola. Plant J 19:163–171

    Google Scholar 

  • Tsuda K, Glazebrook J, Katagiri F (2008a) The interplay between MAMP and SA signaling. Plant Signal Behav 3:359–361

    Article  PubMed  Google Scholar 

  • Tsuda K, Sato M, Glazebrook J, Cohen JD, Katagiri F (2008b) Interplay between MAMP-triggered and SA-mediated defense responses. Plant J 53:763–775

    Article  PubMed  CAS  Google Scholar 

  • Ulmasov T, Hagen G, Guilfoyle TJ (1999) Dimerization and DNA binding of auxin response factors. Plant J 19:309–319

    Article  PubMed  CAS  Google Scholar 

  • van Wees SC, Luijendijk M, Smoorenburg I, van Loon LC, Pieterse CM (1999) Rhizobacteria-mediated induced systemic resistance (ISR) in Arabidopsis is not associated with a direct effect on expression of known defense-related genes but stimulates the expression of the jasmonate-inducible gene Atvsp upon challenge. Plant Mol Biol 41:537–549

    Article  PubMed  Google Scholar 

  • von Dahl CC, Winz RA, Halitschke R, Kühnemann F, Gase K, Baldwin IT (2007) Tuning the herbivore-induced ethylene burst: the role of transcript accumulation and ethylene perception in Nicotiana attenuata. Plant J 51:293–307

    Article  Google Scholar 

  • Wawrzynska A, Rodibaugh NL, Innes RW (2010) Synergistic activation of defense responses in Arabidopsis by simultaneous loss of the GSL5 callose synthase and the EDR1 protein kinase. Mol Plant Microbe Interact 23:578–584

    Article  PubMed  CAS  Google Scholar 

  • Xie DX, Feys BF, James S, Nieto-Rostro M, Turner JG (1998) COI1: an Arabidopsis gene required for jasmonate-regulated defense and fertility. Science 280:1091–1094

    Google Scholar 

  • Xie F, Xiao P, Chen D, Xu L, Zhang B (2012) MiRDeepFinder: a miRNA analysis tool for deep sequencing of plant small RNAs. Plant Mol Biol 80:75–84

    Article  CAS  Google Scholar 

  • Zarei A, Körbes AP, Younessi P, Montiel G, Champion A, Memelink J (2011) Two GCC boxes and AP2/ERF-domain transcription factor ORA59 in jasmonate/ethylene-mediated activation of the PDF1.2 promoter in Arabidopsis. Plant Mol Biol 75:321–331

    Google Scholar 

  • Zimmerli L, Métraux JP, Mauch-Mani B (2001) β-Aminobutyric acid-induced protection of Arabidopsis against the necrotrophic fungus Botrytis cinerea. Plant Physiol 126:517–523

    Google Scholar 

Download references

Acknowledgments

We thank the Arabidopsis Biological Research Center, The Arabidopsis Information Resource, and The University of Wisconsin Biotechnology Center for providing Arabidopsis thaliana Col-0, Col-6 ecotypes, ein2-1 mutant, and enhancer trap T-DNA mutants, respectively. We also thank Willem F. Broekaert (Katholieke Universiteit Leuven) for providing A. brassicicola strain IMI169558. This work was supported by the faculty research fund of Sejong University in 2011, the Bio-Industry Technology Development Program of iPET (111057-5), and the National Research Foundation of Korea (NRF) of the Korean government (MEST) (no. 2011-0001105) for S. Lee, and a Korea University Grant for W. Jeon.

Author information

Authors and Affiliations

  1. Department of Bioresource Engineering, Sejong University, Seoul, 143-747, Republic of Korea

    Sanghyeob Lee

  2. Plant Engineering Research Institute, Sejong University, Seoul, 143-747, Republic of Korea

    Sanghyeob Lee & Seongbin Hwang

  3. Department of Molecular Biology, Sejong University, Seoul, 143-747, Republic of Korea

    Seongbin Hwang

  4. College of Life Sciences and Biotechnology, Korea University, Seoul, 136-713, Republic of Korea

    Yong Weon Seo

  5. Institute of Life Science and Natural Resources, Korea University, Seoul, 136-713, Republic of Korea

    Woong Bae Jeon

  6. Bio-control Center, Jeonnam Bioindustry Foundation, Gokseong-gungun, Jenranam-do, 516-942, Republic of Korea

    Boung-Jun Oh

Authors
  1. Sanghyeob Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Seongbin Hwang
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yong Weon Seo
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Woong Bae Jeon
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Boung-Jun Oh
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Woong Bae Jeon or Boung-Jun Oh.

Electronic supplementary material

Below is the link to the electronic supplementary material.

11816_2012_253_MOESM1_ESM.pptx

Supplementary Figure 1. Expression profile of AtCXE8 gene. Microarray expression data was downloaded from Arabidopsis eFP Browser (http://bbc.botany.utoronto.ca/efp/cgi-bin/efpWeb.cgi). The mRNA transcripts levels of AtCXE8 by inoculation of P. infestans. X-axis represents timeseries after P. infestans inoculation. Y-axis represents that mRNA transcripts levels with inoculation of P. infestans compare to those of mock treatment (A). The mRNA transcripts levels of AtCXE8 of one and three hours after application of ACC, Zeatin, IAA, ABA, MJ and GA. X-axis represents all the applied chemicals in this experiment. Y-axis represents that mRNA transcripts levels with application of chemicals compare to those of mock treatment (B). The mRNA transcripts levels of AtCXE8 of one and twelve hours after application of cold, osmotic, salt and heat treatment. X-axis represents all the applied abiotic stressors in this experiment. Y-axis represents that mRNA transcripts levels with application of abiotic stressors compare to those of mock treatment (C). (PPTX 66 kb)

Supplementary Table 1. Comparison of sequence analysis between promoter sequences of AtCXE8 and AtCXE9 (XLSX 22 kb)

Supplementary Table 2. Analysis of miRNA binding site in promoter sequence of AtCXE8 (XLSX 9 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lee, S., Hwang, S., Seo, Y.W. et al. Molecular characterization of the AtCXE8 gene, which promotes resistance to Botrytis cinerea infection. Plant Biotechnol Rep 7, 109–119 (2013). https://doi.org/10.1007/s11816-012-0253-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11816-012-0253-0

Keywords

Search

Navigation