Original article
Developmental expression of stress response genes in Theobroma cacao leaves and their response to Nep1 treatment and a compatible infection by Phytophthora megakarya

https://doi.org/10.1016/j.plaphy.2005.04.006Get rights and content

Abstract

Developmental expression of stress response genes in Theobroma cacao leaves and their response to Nep1 and a compatible infection by Phytophthora megakarya were studied. Ten genes were selected to represent genes involved in defense (TcCaf-1, TcGlu1,3, TcChiB, TcCou-1, and TcPer-1), gene regulation (TcWRKY-1 and TcORFX-1), cell wall development (TcCou-1, TcPer-1, and TcGlu-1), or energy production (TcLhca-1 and TcrbcS). Leaf development was separated into unexpanded (UE), young red (YR), immature green (IG), and mature green (MG). Our data indicates that the constitutive defense mechanisms used by cacao leaves differ between different developmental stages. TcWRKY-1 and TcChiB were highly expressed in MG leaves, and TcPer-1, TcGlu-1, and TcCou-1 were highly expressed in YR leaves. TcGlu1,3 was highly expressed in UE and YR leaves, TcCaf-1 was highly expressed in UE leaves, and TcLhca-1 and TcrbcS were highly expressed in IG and MG leaves. NEP1 encodes the necrosis inducing protein Nep1 produced by Fusarium oxysporum and has orthologs in Phytophthora species. Nep1 caused cellular necrosis on MG leaves and young pods within 24 h of application. Necrosis was observed on YR leaves 10 days after treatment. Expression of TcWRKY-1, TcORFX-1, TcPer-1, and TcGlu-1 was enhanced and TcLhca-1 and TcrbcS were repressed in MG leaves after Nep1 treatment. Expression of TcWRKY-1 and TcORFX-1 was enhanced in YR leaves after Nep1 treatment. Infection of MG leaf disks by P. megakarya zoospores enhanced expression of TcGlu-1, TcWRKY-1, and TcPer-1 and repressed expression of TcChiB, TcLhca-1 and TcrbcS. Five of the six genes that were responsive to Nep1 were responsive to infection by P. megakarya. Susceptibility of T. cacao to P. megakarya includes altered plant gene expression and phytotoxic molecules like Nep1 may contribute to susceptibility.

Introduction

Several Phytophthora species, including P. megakarya Brasier and Griffin, P. palmivora (Butl.) Butler, P. citrophthora (R.H. Sm. and E. Sm.) Leonian, and P. capsici Leonian, attack the tropical tree Theobroma cacao L. (cacao) causing black pod disease. Symptoms include seedling blights, stem cankers, and pod rots [15], [56]. P. megakarya is the most aggressive of the four species on cacao and poses a major threat to cacao production in western Africa [15], [56]. A bioassay using leaf disks to screen for resistance to black pod in cacao reveled that increasing levels of necrosis were an indication of susceptibility to Phytophthora spp. [44], [47]. The reaction to Phytophthora spp. in the leaf disk assay is highly dependent upon the leaf's stage of development. Young cacao leaves are generally highly susceptible to attack by Phytophthora spp. [15]. Mature leaves were used in the leaf disk assay and they could be highly resistant to specific Phytophthora spp. depending on the cacao genotype [43], [46], [49]. Selection of resistance based on the response of leaf disks from mature cacao leaves to Phytophthora spp. zoospore inoculation has been correlated with pod resistance [44], [47], [51].

The extra-cellular protein Nep1 is produced by Fusarium oxysporum Schlechtend:Fr. f. sp. erythroxyli. Nep1 causes cell death in many different dicot plant species when applied as a foliar spray [38]. Orthologues of NEP1 (AF036580), the gene for Nep1 [42], have been identified in a broad range of microbes including several Phytophthora spp. (accession #-AF352031.1, AAK25828.1, AF320326.1), Pythium aphanidermatum (Edson) Fitzp (accession #-AF179598), and Bacillus halodurans (accession #-BAB04114.1). Although the importance of Nep1 in pathogenesis of F. oxysporum remains in question [12], Qutob et al. [46] demonstrated that Phytophthora sojae preferentially expresses PsojNIP during the necrotrophic phase of disease development on soybeans and therefore may function as a pathogenicity factor. In addition to cell death, the gene products of orthologues from the plant pathogens F. oxysporum, Nep1; Phytophthora spp., NPP1 and PsojNIP [26], [46]; and Pythium spp., PaNie and others [54], cause similar responses in host and nonhost dicot plant species. Plant cell cultures respond to Nep1 and NPP1 by altered ion channeling and induction of active oxygen [26], [34]. PaNie from P. aphanidermatum induces DNA laddering in carrot (Daucus carota L.), a primary measure for programmed cell death, in addition to production of the phytoalexin 4-hydroxybenzoic acid [54]. Foliar application of the combination of Nep1 with the plant pathogen Pleospora papaveracea enhances disease development on opium poppy (Papaver somniferum L.) [11].

Very little is known concerning the responses of cacao to biotic and abiotic stresses at the gene expression level. Recently Verica et al. [55] used subtraction library techniques to identify cacao expressed sequence tags responsive to inducers of resistance and to Nep1 treatment in mature green leaves although detailed expression data were not provided. In order to exploit genomic approaches to studying stress responses in cacao it is important to understand the influence of tissue developmental stage on gene expression. We have identified and cloned cDNA fragments showing altered expression in cacao leaves responding to pathogens and other stresses. Our primary objectives were to characterize the influence of leaf developmental stage on constitutive expression of stress response genes in T. cacao and to develop an understanding of the susceptible response of T. cacao to pathogens by characterizing the expression of nine cDNA clones in cacao leaves after treatment with Nep1, and after infection by P. megakarya.

Section snippets

Gene expression in during leaf development

Leaf development was separated into four stages (Fig. 1): Stage 1) unexpanded leaves (UE) less than 1 cm long with limited pigmentation, Stage 2) young red leaves (YR) 5–10 cm long and pliable, Stage 3) immature green leaves (IG) 10–20 cm long and pliable, and Stage 4) mature green leaves (MG) 10–20 cm and rigid. Large differences were detected in the constitutive expression levels of the ten genes being studied depending upon the developmental stage of the leaf (Fig. 2). TcWRKY-1 mRNA was most

Discussion

Nep1 caused necrosis in cacao leaves and pods in a time frame similar to that observed for Nep1 and related proteins in other plant species [9], [26], [34], [38], [46], [54]. The necrosis on cacao leaves was centered on stomata that serve as points of entry of Nep1 into the leaf [38]. This provides clear evidence that Nep1, when combined with Silwet-L77, penetrates through stomata and causes a very localized necrosis. It was unclear what the points of entry into the pods were. The amount of

Nep1 production

Nep1 was purified from culture filtrates of F. oxysporum f. sp. erythroxyli as previously described [9] and stored in buffer (20 mM MES, 300 mM KCl, pH 5.0) at –20 °C prior to use.

Plant production

Open pollinated seeds of T. cacao variety comun (Lower Amazon Amelonado type) were collected by Alan Pomella from established plantings at the Almirante Cacau, Inc. farm (Itabuna, Bahia, Brazil). Seeds were planted in 15.2 cm pots filled with a soilless mix (2:2:1, sand/perlite/promix); seedlings were grown in ambient

References (56)

  • G. Antúnez De Mayolo, Genetic engineering of Theobroma cacao and molecular studies on cacao defense responses, Ph.D....
  • A.E. Arnold et al.

    Fungal endophytes limit pathogen damage in a tropical tree

    Proc. Natl. Acad. Sci. USA

    (2003)
  • H. Ashihara et al.

    Biosynthesis of caffeine in leaves of coffee

    Plant Physiol.

    (1996)
  • S.M. Assmann

    Heterotrimeric and unconventional GTP binding proteins in plant cell signaling

    Plant Cell

    (2002)
  • B.A. Bailey

    Purification of a protein from culture filtrates of Fusarium oxysporum that induces ethylene and necrosis in leaves of Erythroxylum coca

    Phytopathology

    (1995)
  • B.A. Bailey et al.

    The influence of ethylene and tissue age on the sensitivity of Xanthi tobacco leaves to a Trichoderma viride xylanase

    Plant Cell Physiol.

    (1995)
  • B.A. Bailey et al.

    Nep1 Protein from Fusarium oxysporum enhances biological control of opium poppy by Pleospora papaveracea

    Phytopathology

    (2000)
  • B.A. Bailey et al.

    Expression of NEP1 by Fusarium oxysporum f. sp. erythroxyli after gene replacement and overexpression using polyethylene glycol-mediated transformation

    Phytopathology

    (2002)
  • J.G. Bishop et al.

    Rapid evolution in plant chitinases: molecular targets of selection in plant–pathogen coevolution

    Proc. Natl. Acad. Sci. USA

    (2000)
  • G.P. Bolwell et al.

    The apoplastic oxidative burst in response to biotic stress in plants: a three-component system

    J. Exp. Bot.

    (2002)
  • J.H. Bowers, B.A. Bailey, P.K. Hebbar, S. Sanogo, R.D. Lumsden, The impact of plant diseases on world chocolate...
  • J.H. Bowers et al.

    Relationship between inoculum level of Phytophthora capsici and mortality of pepper

    Phytopthology

    (1991)
  • S. Chang et al.

    A simple and efficient method for isolating RNA from pine trees

    Plant Mol. Biol. Rep.

    (1993)
  • J.H. Christensen et al.

    The syringaldazine-oxidizing peroxidase PXP 3–4 from poplar xylem: cDNA isolation, characterization and expression

    Plant Mol. Biol.

    (2001)
  • E. Delannoy et al.

    Activity of class III peroxidases in the defense of cotton to bacterial blight

    Mol. Plant Microbe Interact.

    (2003)
  • I.P. De Leon et al.

    Involvement of the Arabidopsis alpha-DOX1 fatty acid dioxygenase in protection against oxidative stress and cell death

    Plant J.

    (2002)
  • J. Dong et al.

    Expression profiles of the Arabidopsis WRKY gene superfamily during plant defense response

    Plant Mol. Biol.

    (2000)
  • J. Ehlting et al.

    Identification of 4-coumarate:coenzyme A ligase (4CL) substrate recognition domains

    Plant J.

    (2001)
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