## 3 Results  
### Characterization of TSs Provides an Overview of the Terpenoid Biosynthetic Potential in Trichoderma  
We used an in-silico approach in order to assign putative functions to 387 TS-encoded proteins currently found in the genomes of _Trichoderma_ spp., which were found distributed in 15 functional groups. TSs sharing conserved domains and metal-binding motifs clustered in the same phylogenetic group, each one highlighted in a different color (Figure 1). TSs accession numbers, TS-content per each species showing specific portions of the TS inventory, and phylogenetic tree including protein accession numbers are available in Table S4 and Figure S1. \begin{table}
\begin{tabular}{c c c} \hline \hline _JGI Id._ & **Name** & **Putative function** \\ \hline Trigam1[5596 & TC4 & bifunctional HAD-like \\ \hline Trigam1[4742 & **TR15** & **trichodiene synthase** \\ \hline Trigam1[162 & **TS5** & **pentalenene synthase** \\ \hline Trigam1[9843 & **TS7** & **sesquiTS** \\ \hline Trigam1[1824 & **TS4** & **sesquiTS** \\ \hline Trigam1[4947 & **TS3** & **sesquiTS** \\ \hline Trigam1[5367 & **TS1** & **uncharacterized 4** \\ \hline Trigam1[340 & **TS6** & **squiene synthase** \\ \hline Trigam1[3208 & TC3 & GGTase I \\ \hline Trigam1[5139 & TC5 & GGTase II \\ \hline Trigam1[3927 & TC1 & FPP transferase \\ \hline Trigam1[4065 & TS8 & GGPP synthase \\ \hline Trigam1[2917 & TS10 & FPP synthase \\ \hline Trigam1[9898 & **TS9** & **indole diTS** \\ \hline Trigam1[8345 & TC2 & oxidosqualene cyclase \\ \hline Trigam1[6072 & **TS11** & **uncharacterized 5** \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Terpene synthase genes found in the genome of _T. gamsii_ T6085. _JGI_ accession numbers of each protein are shown in the first column. Protein names are shown in the second column. Putative protein functions derived from computational analysis are shown in the third column. Terpene synthases used in gene expression analyzed are shown in bold. Analysis revealed specific TSs sharing N-terminal HAD-like (Pfam 13419; PTHR43611:SF3) and C-terminal TS domains (IPR008930) (light blue color in Figure 1), which seems to be exclusive of species belonging to the clade Viride (Table S4). Although they did not cluster with known TSs, the presence of both Class I DDxxE and Class II DxDTT motifs indicates they are bifunctional enzymes. We found a vast group of sesquiterpene synthases (sesquiTSs) belonging to the TRI5 superfamily (Pfam 06330) (dark green color in Figure 1), which was particularly represented in species of Viride clade (Table S4). It contains 7 TRI5 (PIRSF001388), 15 longiborneol synthases, and two groups of proteins that did not cluster with known TSs (uncharacterized group 1 and 2, respectively). The phylogenetic distribution of TRI5 (Table S4) indicates that it is not a monophyletic trait in _Trichoderma_, opening questions about its evolutionary origin. Species of clade Viride were the only lacking longiborneol  
Figure 1: Genomic inventory for terpenoid biosynthesis in _Trichoderma_ spp. Terpene synthase (TS) proteins sharing conserved domains are highlighted in different colors: HAD-like (light blue), TRI5 (dark green), terpene synthase C (light green), squalene synthase-phytoeen synthase (orange), preynyl transferase (red), squalene/hopene cyclase (light and dark brown), kaurene synthase and/or ent-copalyl diphosphate synthase (grey), and polyprenyl synthase and/or TS C (dark blue). Putative functions of terpene cyclases (TCs) and prenyl transferase (PTs) were assigned based on phylogenetic analysis performed with terpene synthase proteins with known function from filamentous fungi (Table S2) [53, 54, 55, 56, 57, 58, 59]. Proteins which did not clustered with any known protein were designed as uncharacterized TSs. Aspartate-rich motifs of Class I, Class II and Bifunctional enzymes were identified in the amino-acidic sequences of each group of proteins. Bootstrap values \(>\) 50 are shown in the correspondent branches of the tree.