## 3 Results
### Characterization of TSs Provides an Overview of the Terpenoid Biosynthetic Potential in Trichoderma
These findings suggest the origin of a novel _tri5_-associated cluster in _T. gamsii_, which is likely involved in the biosynthesis of trichodiene derivates with unknown functions. According to this, _tri5_ could participate in two different sesquiterpene biosynthesis in _Trichoderma_. Functional Differentiation of TS Family Members as the Driver for the High Genomic Potential for Terpenoid Biosynthesis in Trichoderma  
We identified 16 TS-encoding genes in the genome of _Tgam_ (Table 1). For gene expression studies, we focused on nine genes encoding Class I TSs--_ts1_, _ts3_, _ts4_, _ts5_, _ts6_, _ts7_, _ts9_, _ts11_, and _tri5_--which represented a highly diverse functional group, well distributed across _Trichoderma_ spp., according to our analyses. Thus, we excluded genes encoding Class II proteins and those involved in the biosynthesis of terpene precursors and protein prenylation. We firstly investigated changes on TSs expression in 4-day-old liquid cultures of the fungus grown in modified MM (Figure 3a). The availability of C source had contrasting effects on TS gene expression. The addition of 0.9% sucrose did not change the transcript levels of _ts6_ and _ts11_. Nevertheless, _ts3_ was significantly down-regulated (0.1-fold), whereas an up-regulation was observed in _ts1_, _ts9_ and particularly, in _ts4_ (18.7-fold). In the same way, the addition of 0.5 mM H\({}_{2}\)O\({}_{2}\) induced the opposite changes on gene expression, indicating that regulation of TS genes occurs in different manners in response to oxidative stress. While the expression of _ts1_ and _ts11_ did not change, _ts3_ and _ts6_ were down-regulated (0.3-, 0.6-fold, respectively). In contrast, _ts9_, encoding a putative indole diTS, was up-regulated (2.7-fold), suggesting that biosynthesis of indole diterpenes might occur in response to oxidative stress in _Tgam_. We observed that N starvation tended to negatively regulate TS genes, although significant differences were only found in _ts3_ and _ts11_ (0.4-fold). Similarly, the addition of 200 mM NaCl down-regulated the expression of TS genes (0.02-0.33-fold), with the exception of _ts1_, whose expression was not affected. We did not detect transcripts of _tri5_, _ts5_ or _ts7_ in any of the conditions tested. Further, we investigated whether a differential expression of TS genes of _Tgam_ takes place in the presence of _Fgra_ on wheat spikes, where a reduction of FHB symptoms was observed (\(57.9\pm 4.7\%\) DS) compared to _Fgra_ alone plants (\(88.3\pm 1.2\%\) DS) (Figure 3b). Analysis revealed that _ts1_, _ts6_, _ts9_, and _ts11_ were active when _Tgam_ was on wheat spikes, regardless of the presence/absence of the pathogen. Expression of _ts1_, _ts6_ and _ts9_ did not change significantly between the two conditions, indicating that these genes are not particularly involved in this triple interaction. Instead, _ts11_ was slightly up-regulated (1.45-fold) when _Tgam_ was on spikes with _Fgra_, suggesting the presence of the pathogen could directly induce changes in its expression or could mediate physiological changes in spikes that promoted changes on _ts11_ expression. These results indicate that _Tgam_ did not induce changes in the pathogen. Figure 3: Gene expression studies of terpene synthase genes in _T. gamsii_ T6085 in different environment conditions. (**a**) Liquid cultures of _T. gamsii_ T6085 (_Tgam_) in different substrates. Total RNA was extracted from 4-day-old mycelium of _Tgam_ grown on minimal medium without sucrose (MM) (basal condition, \(2^{-\Delta\Delta\Gamma}=1\)), or MM with 0.9% sucrose, MM 0.5 mM H\({}_{2}\)O\({}_{2}\), MM with only 0.01% of nitrogen (N starvation), or 200 mM NaCl, respectively. Grey color bars represent relative expression values of TS genes on each condition. (**b**) Interaction of _Tgam_ with _F. gamsiream_ (_Fgra_) on wheat spikes. Total RNA was extracted from wheat spikes colonized by _Tgam_ alone (basal condition, \(2^{-\Delta\Delta\Gamma}=1\)) or by _Tgam_ + _Fgra_, 6 days after inoculation of the pathogen.