## 4 Discussion  
Overexpression of the IBT40841 _tri5_ gene resulted in an increase of the trichodermin production as a result of a higher level of _tri5_ transcript and also in an increase in the antibiotic activity against a large panel of yeast, and it negatively affected tomato plant growth and the lesions caused by _Botrytis cinerea_[13]. Two of the _tri5_ gene overexpression transformants used in the present work, Tb40 tri5 and Tb41 tri5, had an increased level of expression of the regulatory genes _tri6_ and _tri10_, and _tri4_, the oxygenase gene that controls conversion of trichodiene to trichodermol [11]. However, in the Tb41 tri5 transformant, the expression of _tri3_, _tri11_ and _tri12_ genes were also induced. In spite of these differences between the different transformantsants it is likely that the overproduction of trichodiene has a positive effect on the expression of the _tri4_ gene, the next gene in the biosynthetic pathway (Figure 5), but also in the expression of the _tri6_ and _tri10_ genes, that would regulate the expression of all the _tri_ genes. The production of tyrosol and hydroxytyrosol simultaneous to the increase of the trichodermin level in the _tri5_-overexpressing transformantsants can be explained as a response by cells to regulate the levels of farnesol:tyrosol, since both trichodiene and farnesol have FPP as precursor. When farnesol and tyrosol are present in direct competition, tyrosol does not alter the quorum-sensing activity of farnesol, even when the tyrosol is present at a 16-fold molar excess [31]. When tyrosol was added to the culture  
Figure 5: Schematic representation of the model of regulation of the trichothecenes harzianum A and trichodermin biosynthesis. The synthesis of diterpenes and triterpenes is indicated by yellow squares. The genes indicated in red are those studied in the present work. _tri6_ and _tri10_ are regulatoy genes. Farnesol and tyrosol are two regulatory and antagonistic molecules. The red arrows indicated positive regulation. The blue doted lines indicate possible inhibitory activities (Modified from [12; 27; 28; 29; 30]). media, the expression of the _tri5_, _tri4_, _tri6_ and _tri10_ genes was repressed, and this repressing effect persisted until at least the seventh day of culture (data not shown). However, this repressing effect of tyrosol on _tri_ gene expression was observed only when this compound was added to the culture medium but when tyrosol was produced by the _tri5_-overexpressing transformants, rather, there was a 2.4 fold increase in toxin production in the transformant Tb41 tri5 when compared to the wild-type strain [13]. Thus, without external addition of tyrosol a significant increase in the expression of the _tri_ genes was observed in the transformants (see Figure 1), probably because the effect of the endogenous tyrosol would be regulated by the intracellular farnesol, equilibrating both stimulating and repressing activities, respectively. The addition of external tyrosol would shift this equilibrium towards the stimulating effects of the tyrosol, resulting in a repression of the genes involved in the biosynthesis of trichodermin, as a secondary metabolite, and a stimulation of the primary metabolism biosynthetic pathways. In this sense, the increase in the level of expression of the _erg1_ gene in the _tri5_ overexpressing transformants can be understood in the same way, since _erg1_ encodes squalene epoxidase, the enzyme that uses squalene as substrate in the first step of the ergosterol biosynthesis. Squalene is the condensation product of two farnesyl pyrophosphates. Thus, the increase in the level of expression of _erg1_ gene will direct the FPP towards the ergosterol biosynthesis, which is needed for the fungal cell growth, one of the stimulating effects exerted by the tyrosol. This correlates well with the previous observation that farnesol biosynthesis inhibits the ergosterol pathway in _C. albicans_[32].