In general, ABA suppresses plant resistance mechanisms by antagonizing SA- and JA/ET-dependent immune responses , thereby promoting susceptibility . In addition, negative regulation involving systemic acquired resistance activation and ABA synthesis has been documented . Genome-wide transcriptional profiling studies have been valuable in the study of hormonal signaling during plant–pathogen interactions because they enable researchers to monitor the activation or suppression of multiple pathways simultaneously. We used hybridization-based microarray data obtained from tomato fruit infected with B. cinerea to characterize the patterns of expression of genes involved in hormone biosynthesis and signaling to infer the potential role of stress hormones in fruit–pathogen interactions. The expression pro- files of important genes were validated and extended by qRT-PCR using independent biological material at different stages of infection. We integrated the gene expression results with susceptibility phenotypes of fruit compromised in hormone synthesis and perception, in order to provide a model describing how ET, SA, JA and ABA influence the susceptibility of tomato fruit to B. cinerea.Genes that have been previously described as involved in the synthesis, modification, signaling, drainage collection pot and response of ET, SA, JA, and ABA were selected based on their functional annotation from the Arabidopsis Hormone Database 2.0 .
The amino acid sequences of the 414 selected genes were retrieved from the Arabidopsis TAIR10 collection and used as queries in a BLASTP search against all of the predicted proteins in the tomato genome sequence . A total of 326 sequences with identity greater than 60% and with alignment coverage more than 70% of the query length were considered putative tomato homologs of the Arabidopsis hormonerelated proteins. In addition, the sequences of 19 known tomato protein gene sequences related to ET synthesis and signaling pathways were added to the dataset. Corresponding unigene sequences and Affymetrix array chip probes were then obtained, respectively, from GenBank and Affymetrix to extract the normalized hybridization values from the microarray analysis of Botrytis cinerea-infected tomato fruit at the MG and RR stages and at 1 day post-inoculation . The resulting dataset was used to identify significant fold changes in ET, SA, JA, and ABA-related genes that are in common or uniquely regulated by infection of MG and RR fruit by B. cinerea and by ripening of healthy fruit.B. cinerea was provided by Dr. J. A. L. van Kan . Conidia, collected from sporulating cultures grown on 1% potato dextrose agar , were counted and diluted to 500 conidia μL−1 for inoculations. Fruit were disinfected and inoculated as in Cantu et al. . Briefly, on the day of harvest fruit were surface sterilized by submersion in a solution of 10% bleach followed by three deionized water rinses.
At the time of inoculation fruit were wounded at seven sites to a depth of 2 mm and a diameter of 1 mm. Six out of the seven sites were inoculated with 10μL of a water suspension containing 5000 conidia of B. cinerea and the seventh site was mock-inoculated with 10μL of sterile water . Healthy fruit were not wounded or inoculated. All fruit samples were incubated at 20◦C in high humidity. Susceptibility was determined daily for 3 dpi as disease incidence . The evaluation of susceptibility was repeated with three separate harvests of fruit using 10–15 fruits per experiment. The significance of the susceptibility data was analyzed by ANOVA followed by Tukey’s post-hoc test using R . For percentage values, statistical analysis was carried out after angular transformation.To confirm the gene expression changes identified in the reanalysis of the microarray hybridization data, additional MG and RR fruit were inoculated as above with B. cinerea or kept uninoculated . Fruit pericarp and epidermal tissues were collected after 1 and 3 days post-inoculation and high-quality RNA was isolated. Five biological replicates were produced per sample and each replicate consisted of independent pools of 3–5 fruits. Two grams of tissue per sample were ground in liquid nitrogen and 10 mL of the RNA extraction buffer were added. The samples were immediately incubated for 5 min at 65◦C. Two extractions with one equal volume of chloroform:isoamyl alchohol followed by centrifugation at 4000 rpm for 45 min at 4◦C were performed. The supernatant was recovered and 1/10 volume of 1M KOAc was added followed by centrifugation at 4000 rpm for 20 min at 4◦C.
The supernatant was collected and 1/4 volume of 10 M LiCl was added. Samples were incubated overnight at −20◦C and then centrifuged at 4000 rpm for 45 min at 4◦C. The supernatant was discarded and the RNA pellet was further purified using the RNeasy Plant Mini Kit . DNAse treatment was done in column during the purification step. The RNA was resuspended in 35μL of nuclease-free water. The RNA concentration and purity were measured using NanoDrop 2000c Spectrophotometer . Although the complete sequence of the tomato genome is available , an integration of genome annotations with functional information is required to assign biological importance to gene sequences and generate a framework for the study of developmental processes and signaling networks. The study of stress hormonal pathways in tomato fruit has focused mainly on the characterization of ET-related genes involved in the initiation of ripening . The roles of the stress hormones, SA, JA, and ABA, for the outcomes of fruit infections have not been extensively investigated. We previously used microarray hybridization technology to characterize the expression changes of ripening-related genes in relation to the increased susceptibility to B. cinerea of ripe fruit. Using RNA from tomato fruit at two ripening stages, MG and RR at 1 dpi with B. cinerea, we profiled the expression of several cell wall modifying genes and few hormone-related genes . The shortage of functional annotations for genes represented on the microarray has limited the identification of genes involved in hormonal pathways related to stress and pathogen responses. Here we report the identification of a set of 345 hormonerelated tomato genes, which includes 19 known ET-related genes and 326 tomato genes that show significant homology to Arabidopsis genes involved in ET, SA, JA, and ABA pathways; the re-annotation of the hormone-related genes on the Affymetrix Tomato Chip, and the transcriptional changes of these hormonal-related genes in response to B. cinerea using published microarray results . Hormone-related Arabidopsis gene sequences were retrieved from the AHD 2.0 and BLASTP searches were used to identify their homologous copies in the tomato genome . We selected the AHD 2.0 because it is currently the most comprehensive and up-to-date database of hormone-related genes; it includes 1318 gene accessions for eight different plant hormones, which had been extracted from 906 scientific papers published before August 2010. From this database, we identified 128 genes related to ET, 72 genes related to SA, 55 genes related to JA, and 159 genes related to ABA pathways . Among the homologous tomato genes identified, 141 genes were found to be expressed in tomato fruit based on the microarray data. Of these 141 genes, we focused on those with significant changes in expression that were in common during infection of tomato fruit by B. cinerea regardless of the ripening stage, round plastic pot that were responses to B. cinerea but are specific to the ripening stage and phenotype of the fruit , and that were common in response to infection and as a consequence of ripening. As result, we identified 65 stress hormone-related genes that showed differential expression in response to B. cinerea . Relative expression changes of 20 hormone-related genes were measured by qRT-PCR using independent preparations of RNA from B. cinerea-infected and equivalent healthy tomato fruit at MG and RR stages, in order to validate the results from the microarray analysis .
Additionally, gene expression was measured at 3dpi to determine whether the up- or down-regulation of the expression of these genes is maintained or modified as infection progresses . For the 20 genes analyzed, 88% of all expression comparisons, i.e., infection of MG fruit , infection of RR fruit , and ripening were observed in both the microarray and in the qRT-PCR data. However, by qRT-PCR only 59% of the gene expression changes were significant , mostly because of inter-sample variability ; in fact, the qRT-PCR coef- ficient of variation was almost three times higher than the microarray CV . Even with the high CV of the qRT-PCR experiments, there was a strong correlation between the microarray and the qRT-PCR data . In the following sections, the expression profiles of genes involved in ET, JA, SA, and ABA biosynthesis and signaling are presented and discussed in light of the susceptibility to B. cinerea of fruit that are either hormone-insensitive or hormone-deficient.B. cinerea . Three patterns of transcriptional reprogramming were identified in the microarray analysis: increased expression of S-adenosyl-L-methionine synthetase genes, LeSAMS1, and LeSAMS3, which decline during ripening of healthy fruit ; up-regulation of two members of the 1-aminocyclopropane-1- carboxylic acid synthase gene family; and downregulation of an ACC oxidase gene in B. cinerea infected MG fruit. Increases in LeSAMS1 and LeSAMS3 expression have been detected in tomato vegetative tissues under high salinity conditions and following ABA treatment, suggesting a link between SAM and stress tolerance . Besides being a substrate for ET synthesis, SAM is also utilized for the production of polyamines and is the primary methyl-donor for modi- fication of essential macromolecules . Both ET and PAs, and possibly the relative concentrations of each, mediate biotic and abiotic stress responses in fruit and vegetative tissues . PAs have been shown to reduce the rate of fruit ripening while ET accelerates it . Therefore, enhanced SAM production and changes in the relative synthesis or abundance of ET/PA may be associated with resistance to pathogen infection, particularly in MG fruit for which the upregulation of LeSAMS3 after B. cinerea inoculation was validated by qRT-PCR; expression increased further at a later time during the infection process . Tomato ACS and ACO isoforms are differentially expressed depending on the developmental process; some are specifically associated with ripening while others act preferentially in vegetative tissues and immature fruit . These expression patterns relate to different systems of ET production, described later. From the microarray analysis, premature increased expression of two ACS genes involved in the tomato ripening process, LeACS1a and LeACS2, occurs in B. cinerea-infected MG fruit, which might suggest that pathogen infections activate the synthesis of ET, thereby accelerating the onset of the ripening process and subsequently inducing susceptibility as proposed by Cantu et al. . On the other hand, down-regulation of the ET bio-synthetic gene LeACO5 only in MG fruit as consequence of infection can be interpreted as a counteracting effort by the plant to control the pathogen-induced increase in ET production. Infection of fruit affects the expression of 40% of the ET signaling components that are transcribed in fruit . Expression of the ET receptors LeETR4, LeETR5 and NR decrease after pathogen inoculation at both fruit ripening stages , and the down-regulation was validated in RR fruit at 1 and 3 days after B. cinerea infection for both LeETR5 and NR genes . ET receptors are negative regulators of the signaling pathway , and both their de-phosphorylation and degradation are induced upon ET binding, thereby activating responses to the hormone . However, during fruit ripening, increases in the transcript levels of these receptors do not correlate with protein accumulation or receptor activity . Therefore, the impact on ET perception caused by the down-regulation of the expression of the ET receptors observed during infection of fruit should be evaluated further by examining receptor protein levels and phosphorylation state. For example, the reduction in ET sensitivity caused by mutation in the NR receptor was shown to enhance resistance of tomato leaves to several pathogens and to reduce susceptibility of tomato fruit to B. cinerea infection . The expression of the primary ET response factors LeEIL3 and LeEIL4 is suppressed as a consequence of exposure of tomato fruit to B. cinerea and up-regulated during fruit ripening . The down-regulation after fruit infection was validated for LeEIL4 , while for LeEIL3 only the suppression in infected MG fruit was statistically significant . The LeEIL1-4 genes encode redundant transcription factors that bind to secondary response elements in order to activate downstream ET responses .