# The Infestation of Olive Fruits by _Bactrocera oleae_ ([PERSON])
## 2 Results and Discussion
### Effect of Bactrocera oleae Infestation on the Phenolic Profile of VOO
Thus, the reduction of olive \(\beta\)-glucosidase activity (the key phenolic forming enzyme) and/or the increase in olive PPO (the main oxidative enzyme) would cause a decrease in the final phenolic content of the oils. The involvement of PPO in the reduction of the phenolic content of the oils associated to _B. oleae_ infection was already suggested by [PERSON] al. [33] to explain the loss of phenolic content in oils obtained from infested olive fruits. Effect of Bactrocera oleae Infestation on Genes Controlling the Biosynthesis of Volatile and Phenolic Compounds  
A previous transcriptomic and proteomic study to investigate the molecular factors involved in infestation of cv. Moraiolo olives by _B. oleae_[40] found a substantial number of ESTs differentially expressed that were linked to wounding and to plant defense against biotic stress and abiotic stresses, as well as an important number of unidentified genes. More specifically, they found a remarkable enrichment of genes and proteins of the jasmonate signal transduction (LOX pathway) or phenylpropanol metabolism pathways, both of which have as final products volatile and phenolic compounds directly involved in the chemical defense against pathogens. Great gene expression differences were also found by [PERSON] et al. [24] between resistant and susceptible olive cultivars, suggesting the implication of a great number of metabolites and signaling pathways in response to _B. oleae_ infestation and pointing out the key role of some genes, such as those encoding \(\beta\)-glucosidase, in the defense system of the olive against this pest. Oleuropein activation by \(\beta\)-glucosidase is a well-known defense mechanism against herbivores. In fact, _B. oleae_ requires a complex transcriptomic response in collaboration with its symbiont _Candidatus Ervinia daciola_ in order to infect and feed on olive fruits with high phenolic content [41]. With this background, the level of expression of key genes within the biosynthesis pathways of volatile and phenolic compounds, such as LOX and \(\beta\)-glucosidase, was studied in cv. Picual. Both gene families had been previously studied in our research group [14; 42; 43]. Among the _LOX_ genes encoding proteins with 13-LOX activity, previous biochemical and gene expression data suggest the involvement of _Oep2 LOX2_ and to a lesser extent _Oep1 LOX2_ in the biosynthesis of VOO aroma. Similarly, previous biochemical and molecular characterization studies also point to the key role of _OeBGLU1A_ and _OeBGLU1B_ in the hydrolysis of phenolic glucosides of olive fruit that generate the main phenolic derivatives of VOO. Besides, based on the results obtained in the analysis of phenolic compounds in fruits and oils, we decided to study the possible involvement of _PPO_ genes in relation to the effect of _B. oleae_ infestation as well. Although different authors have reported the possible implication of PPO in the oxidation of phenolic compounds in olive, and its potential implication in the oxidative degradation of these compounds during the VOO production process, to date no PPO gene has been characterized in olive. Two _PPO_ genes, _OepPO1_ (GenBank accession number MW038828) and _OepPO2_ (GenBank accession number MW038829), putatively involved in the oxidative degradation of olive phenolic compounds, were identified in an olive transcriptome previously generated [44]. Both genes were synthesized, cloned, expressed in _E. coli_, and purified as described in the Material and Methods section. Functional characterization studies carried out with natural phenolic substrates from olive fruit and VOO showed that both recombinant proteins were active against orthodiphenyls, oleuropein, and hydroxytyrosol, but not to monophenols, such as tyrosol (data not shown). These data are in good agreement with the results previously obtained in in vitro oxidation experiments in which the incubations of VOO phenolic extracts with partially purified olive PPO from cv.