In the SSH library approximately 40% of the unigenes identified in the SSH library could be functionally annotated. Although a comparison between different works is difficult because of the increasing number of sequences in the databases, this proportion is lower than those reported in similar works about model plant species [16, 18], yet it is similar to other reports on species whose genomes have not yet been sequenced [25, 31]. Interestingly, the functional characterisation of the library indicated a higher representation of ESTs involved in the plant response to stress, including those related to biotic stress, such as wounding and pathogen attack, or abiotic stress, such as high or low temperature, drought and NaCl. Moreover, we identified transcripts involved in the production, signal transduction or response to hormones and molecules (e.g., jasmonic acid and ROS) that are related to plant resistance to herbivorous pests. A similar number of sequences of the library matched uncharacterised olive transcripts, suggesting that the olive response to B. oleae also involves novel or undescribed genes. The functional annotation of the library showed that a critical barrier for working with the olive is the dependence on models of biochemical pathways, gene ontology repertoires and genomic information that are primarily based on model species . These results also demonstrated that the SSH approach was an appropriate strategy to study the olive response. Furthermore, although many cellular processes and pathways inherent to pest resistance are evolutionarily conserved in plants , the library analysis suggested that the plasticity of signal transduction networks and the variety of defence compounds may be particularly pronounced for olive. This assumption is conceivable, considering not only the difference between this tree and model plants but also the strong co-evolutionary relationship of the specialist B. oleae with olive fruits. We anticipate that the uncharacterized unigenes may represent a reservoir of candidate defence genes and that their study may allow further insights into the mechanism of olive defence.
The proteomic analysis identified 19 proteins as differentially expressed after insect attack. Two of these entries were also identified in our transcriptomic dataset. As a percentage, the matching rate is similar to other studies based on the proteomic and transcriptomic analyses of non-model tree species [33–35]. Furthermore, considering that the low correlation between sequences deriving from mRNA or protein analytical approaches is usually explained by post-transcriptional, translational and/or post-translational regulation processes [36, 37], our data are suggestive of the importance and the possible magnitude of post-transcriptional events for gene regulation during pest defence . Finally, this comparison also shows that transcriptomic and proteomic data are complementary in plants, as in other organisms [39–43].
As plants present a variety of strategies against insects , it is expected that several molecular processes are involved in the defence mechanisms against the olive fruit fly. Functional annotation indicated that B. oleae larval feeding significantly diminished the abundance of various proteins related to photosynthesis and altered the amount of those related to carbohydrate metabolism, which included a reduced expression of serine hydroxymethyltransferase, which is important for photorespiration in mitochondria , and of enzymes related to carbohydrate catabolic processes. The downregulation of genes coding for photosynthetic proteins or the decline in the photosynthetic rate in attacked plants has been observed for different herbivores that feed on leaves . It is interesting that we observed a similar effect on sink organs, thus supporting the proposition that primary metabolites could also function as signals in pest defence pathways . A stress-related promotion of drupe maturation should also be taken into consideration . Transcriptomic analysis also demonstrated that unigenes responding to stress represent the largest functional class of upregulated olive genes during B. oleae attack. Under this condition, proteomics revealed increased accumulation of beta-glucosidase (as transcriptomics), major latex proteins, which have been already reported as defensive proteins against insects [48, 49], phosphogluconolactonase and 6-phosphogluconate dehydrogenase. A possible defensive role of the latter enzymes has been suggested . Taken together, the data indicated a metabolic shift towards defence during larval feeding . Olive direct defence employs a variety of inducible factors, which include genes that are known to be important in the determination of the plant resistance to herbivorous pests, such as those coding for proteinase inhibitors or hydrolytic enzymes (e.g., chitinases and glucosidases); however, PR genes are also activated. Approximately one-third of the functionally annotated unigenes are homologues to genes first described to be involved in plant–pathogen interactions. Overlap in the signalling pathways regulating pathogen-plant and insect-plant interactions has been reported in several instances . For the olive, such overlap is reasonable considering that B. oleae has long been known to be associated with different bacterial species [52, 53].
The production of compounds involved in direct defence should be mainly dependent on a network that includes reactive oxygen species and phytohormone signalling. The results from the transcriptomic and proteomic analyses were consistent in showing a remarkable enrichment of genes and proteins involved in the regulation of the redox status (such as the metallothionein-like proteins, GST, catalase, thioredoxins, and aldo-keto reductase), thus indicating that ROS production should be a relevant component of induced olive defence. The reduction of some proteins involved ROS metabolism is indicative of the plant cell effort to maintain homeostasis under stress condition, preventing direct damage from the possible production of highly reactive cytotoxic compounds . In the future, it will be interesting to ascertain if ROS production in drupes is also a consequence of the oxidative damage of membrane integrity due to lipid peroxidation. The functional classification indicated the presence of clones that are expected to be members of gene families involved in jasmonate signal transduction (e.g., lipoxygenase and the lipid transfer) or phenylpropanoid metabolism (i.e., the trans-cinnamate 4-hydroxylase and the Caffeoyl-o-methyltransferase) pathways, both of which produce compounds that, in plants, range from physical and chemical defence against biotic stressors to signal molecules involved in local and systemic signalling [55–57]. Overall, as was also reported for the interaction between the fungus S. oleaginea and olive , our data show an overlap of different pathways for the fruit fly response and denote that the olive responses to pathogens and herbivores should share a number of components at the signalling level .
Considering the unfeasibility of studying the olive-fruit fly interaction in controlled conditions, we recapitulated the expression analysis of twenty genes in drupes harvested in a different year. The real-time assay confirmed the differential expression of many but not all clones, as in other studies [32, 60], implying that the extent of drupe responses to larval feeding may depend on the amount of damage inflicted, on environmental conditions at the time in which insects feed on plants, and on plant resource availability and allocation . It is therefore relevant that, in replicate years and in two different cultivars, the most prominent gene activation was detected for trypsin inhibitors. Nonetheless, even though we could distinguish the transcripts of the two trypsin proteinase inhibitors in our qRT-PCR experiments, we cannot fully exclude the possibility that we monitored the activity of more than one transcript because serine inhibitors in plants belong to a large multigene family. In several plant species, proteinase inhibitors of the serine, cysteine and aspartic families are highly activated by larval feeding. Serine proteinases are the most relevant enzymes detected in the gut of Lepidoptera, Coleoptera, Hemiptera, Homoptera and Diptera . However, bioassays using serine protease inhibitors against Diptera are more limited than in other insect orders. It has been shown that the gut proteolytic systems of larvae of the Mediterranean fruit fly (C. capitata) rely mostly on basic proteinases, with trypsin-like serine proteinases being the most important . For these reasons, we argue that trypsin inhibitors should be a major element of drupe defence reaction. An in vivo assay against the olive fruit fly using purified inhibitors would clarify whether these molecules could be established as a novel insect control strategy, based on bio-compounds, on targeting the production of insect digestive enzymes by RNAi , on the selection of highly expressing olive genotypes, and on their combination.
Among the SSH-enriched cDNA clones, two sequences, coding for a chitinase and a PRp27-like protein, were selected for further functional characterisation. We isolated their full-length cDNA and studied their expression in response to biological (adult puncture and larval feeding of B. oleae) and physical (mechanical wounding) stress.
The Oe-Chi I protein has the two signature motifs of family 19 of the chitinases. Chitinases have long been considered a significant component of plant defence because of their direct action against chitin-containing pestiferous and pathogenic organisms. Furthermore, some of these enzymes are also involved in developmental processes or are associated with abiotic stress. The presence of a putative signal peptide and the gene expression analysis strongly support a role in drupe defence for Oe-Chi I. Specifically, taking into account the magnitude of gene activation, it is tempting to speculate that Oe-Chi I may not be exclusively involved in biological processes activated by “generic” mechanical damage but that this gene plays a specific role in the onset of the reaction against larval feeding. Although chitinases in plants are primarily associated with fungal resistance, there are various reports documenting their activation in plants following pest attack . Interestingly, a chitinase was found to be specifically activated by the application of Colorado beetle regurgitant, and the gene had the highest expression level after a continuous infestation . Further work, primarily directed towards the characterisation of the Oe-Chi I protein product, will be performed to understand its possible effect against phytophagous pests [66, 67].
The Oe-PRp27 gene was named after its high similarity to the Nicotiana tabacum NtPRp27, which codes for a secreted protein belonging to the Pathogenesis-Related 17 (PR17) family . Homologues of NtPRp27 have been found in a wide variety of flowering plants and because of their transcriptional activation in response to pathogen infection and various elicitors, these genes are classified as encoding PR proteins. However, the roles of the members of the PR17 family have not been fully elucidated, especially because these genes are activated by various forms of stress (drought, wounding, ABA, ethylene and MeJA) , and their constitutive expression in transgenic plants does not necessarily lead to an increased resistance to pathogens [29, 69]. It has been proposed that PR17 members may act in defence responses with relation to either cell wall metabolism or signal transduction , which would be consistent with the rapid activation of Oe-PR27 after wounding. Furthermore, Oe-PR27 is induced by B. oleae feeding and punctures, implying an involvement in insect defence. This result is not completely unexpected, as NtPR27 is also induced by JA and ethylene , and recently, the accumulation of an NtPR-like protein was reported during bacterial infection of grape . Although the mode of action and specificity of the PR17 family remain to be determined , our data support a possible broad function of members of this group in the early stages of defence, rather than as antibiotic components directly acting against invading pathogens.