A transcriptomic approach highlights induction of secondary metabolism in citrus fruit in response to Penicillium digitatum infection

González-Candelas et al. BMC Plant Biology 2010, 10:194 http://www.biomedcentral.com/1471-2229/10/194 RESEARCH ARTICLE Open Access A transcriptomic approach highlights induction of secondary metabolism in citrus fruit in response to Penicillium digitatum infection Luis González-Candelas1*, Santiago Alamar1, Paloma Sánchez-Torres1,2, Lorenzo Zacarías1, Jose F Marcos1 Abstract Background: Postharvest losses of citrus fruit due to green mold decay, caused by the fungus Penicillium digitaum, have a considerable economic impact. However, little is known about the molecular processes underlying the response of citrus fruit to P. digitatum. Results: Here we describe the construction of a subtracted cDNA library enriched in citrus genes preferentially expressed in response to pathogen infection followed by cDNA macroarray hybridization to investigate gene expression during the early stages of colonization of the fruit’s peel by P. digitatum. Sequence annotation of clones from the subtracted cDNA library revealed that induction of secondary and amino acid metabolisms constitutes the major response of citrus fruits to P. digitatum infection. Macroarray hybridization analysis was conducted with RNA from either control, wounded, ethylene treated or P. digitatum infected fruit. Results indicate an extensive overlap in the response triggered by the three treatments, but also demonstrated specific patterns of gene expression in response to each stimulus. Collectively our data indicate a significant presence of isoprenoid, alkaloid and phenylpropanoid biosynthetic genes in the transcriptomic response of citrus fruits to P. digitatum infection. About half of the genes that are up-regulated in response to pathogen infection are also induced by ethylene, but many examples of ethylene-independent gene regulation were also found. Two notable examples of this regulation pattern are the genes showing homology to a caffeine synthase and a berberine bridge enzyme, two proteins involved in alkaloid biosynthesis, which are among the most induced genes upon P. digitatum infection but are not responsive to ethylene. Conclusions: This study provided the first global picture of the gene expression changes in citrus fruit in response to P. digitatum infection, emphasizing differences and commonalities with those triggered by wounding or exogenous ethylene treatment. Interpretation of the differentially expressed genes revealed that metabolism is redirected to the synthesis of isoprenes, alkaloids and phenylpropanoids. Background Citrus is one of the most economically important fruit crops in the world. Harvested fruits are usually stored before they reach the market for fresh consumption. During this postharvest period fruits are subjected to both biotic and abiotic stress conditions. Penicillium digitatum, the causal agent of green mold, is the major pathogen of citrus fruit worldwide during postharvest * Correspondence: 1 Departamento de Ciencia de los Alimentos, Instituto de Agroquímica y Tecnología de Alimentos (IATA-CSIC), Apartado de Correos 73, Burjassot, E46100-Valencia, Spain Full list of author information is available at the end of the article storage. Control of this fungus is mostly based on the use of chemical fungicides, such as imazalil or thiabendazol. However, problems associated with the use of these compounds are leading to the development of new and safer control alternatives, which mostly rely on biological control microorganisms, physical or chemical treatments either as standalone or combined treatments. Another approach focuses on improving the natural defense capability of the fruit. However, despite the economic relevance of losses due to P. digitatum infection, there have been few studies directed to unravel citrus fruit responses to pathogen invasion or to elicitors that increase resistance against pathogen infection. © 2010 González-Candelas et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. González-Candelas et al. BMC Plant Biology 2010, 10:194 http://www.biomedcentral.com/1471-2229/10/194 It is well known that the flavedo (outer colored part of the rind) is more resistant to P. digitatum than the albedo (inner white part) [1,2]. This fact has been classically associated with the presence of both preformed and induced antifungal compounds in the flavedo [3]. Moreover, the concentration of some phytoalexins, such as scoparone, increases in the flavedo in response to P. digitatum attack, although a much higher induction is achieved by treatments that increase resistance in the fruit [4,5]. Other responses triggered by this fungus include the induction of PR proteins, such as b-1,3-glucanase and chitinase, and phenylalanine ammonia lyase (PAL), which catalyzes the first step in the phenylpropanoid pathway [1,6-8]. Ethylene is a major modulator of many processes in plants, including regulation of defense responses to pathogen attack [9]. Increase in ethylene production by pathogen infection is a well characterized process. In citrus fruit, infection with P. digitatum enhances ethylene emission, which is provided by both the fruit and the fungus [10,11]. Many of the aforementioned responses of citrus fruit to P. digitatum infection are at least partially dependent on this hormone [12]. The relevance of ethylene in the defense response has also been shown by the increased susceptibility to the pathogen when ethylene perception was blocked by the ethylene antagonist 1-methyl cyclopropene[12,13]. Being P. digitatum a successful pathogen of citrus fruit, it must be able to overcome the fruit’s defense barriers. Thus, besides triggering different defense responses in the host, it is also able to suppress different lines of defense. The first evidence of this suppression of defenses was the observation that ethylene-mediated induction of PAL was greatly reduced in the presence of the fungus [14]. We have previously shown that this suppression seems to involve posttranscriptional regulation because Pal gene induction was not accompanied by induction of enzyme activity [1]. Production of reactive oxygen species (ROS) has also been shown to be suppressed in citrus fruit inoculated with P. digitatum, whereas inoculation with P. expansum, a closely related species but non-pathogenic on citrus fruit, triggers the production of ROS at attempted penetration sites [15]. On the other hand, the activity of different enzymes involved in the metabolism of ROS decreases in P. digitatum-infected fruit, albeit flavedo and albedo exhibit different patterns of enzyme inactivation [1]. Construction of cDNA libraries and generation of ESTs have become useful tools to identify plant genes responsive to pathogens [16-19]. The Citrus Fun (...truncated)