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