3. Functional differentiation of TS family members as the driver for the high genomic potential for terpenoid biosynthesis inTrichoderma
We identified 16 TS-encoding genes in the genome of Tgam (Table1 ). For gene expression studies, we focused on 9 genes encoding TSs belonging to Class I – TS1, TS3, TS4, TS5, TS6, TS7, TS9, TS11 and TRI5 – which represented a high 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. In order to assess the environmental signals regulating the expression of TS genes selected in Tgam , the fungus was grown i) under different simulated stress conditions and in presence/absence of C source, ii) in a FHB scenario, and iii) in interaction with wheat roots.
We firstly investigated changes on TSs expression in 4-day-old liquid cultures of the fungus grown in MM, MM 0.9 % sucrose, MM 0.5 mM H202, MM with low N concentration, and MM 200 mM NaCl, using MM as reference control (Fig. 3a ). The availability of C source had contrasting effects on TS gene expression. Addition of 0.9% sucrose to the culture medium 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, addition of 0.5 mM H2O2 to the culture medium induced opposite changes on gene expression, indicating that regulation of TS genes occurs in different manners in response to oxidative stress. While expression of ts1 and ts11 did not change, ts3 andts6 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, addition of 200 mM NaCl to the medium down-regulated the expression of TS genes (0.02 to 0.33-fold), with the exception ofts1 , which expression was not affected. We did not detect transcripts of tri5 , ts5 or ts7 in any of the conditions tested.
Since Tgam is able to suppress Fgra on wheat spikes, we investigated whether a differential expression of TS genes ofTgam takes place in this scenario. To this end, we assessed relative changes in TS expression when the fungus was interacting withFgra on wheat spikes, where a reduction of FHB symptoms was observed (57.9 ± 4.7 % DS) compared to Fgra alone plants (88.3 ± 1.2 % DS), using spikes inoculated with Tgam alone as control (Fig. 3b ). Analysis revealed that ts1 , ts6 ,ts9 and ts11 were active when Tgam was on wheat spikes, regardless the presence/absence of the pathogen. Expression ofts1 , ts6 and ts9 did not change significantly between the two conditions, indicating that these genes are not particularly involved in the triple interaction in the conditions tested. Instead, ts11 was slightly up-regulated (1.45-fold) whenTgam 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 onts11 expression. These results indicate that Tgam did not induce prominent changes in terpene biosynthesis when interacted withFgra on wheat spikes, under the conditions tested.
In order to determine whether a differential regulation on terpene biosynthesis occurs when Tgam colonizes wheat roots, Tgamwas incubated for 3 days on PDA in presence of 1-day-old wheat seedlings, using Tgam grown alone on PDA under the same conditions as control. Root colonization affected TSs expression inTgam , which differentially regulated most of its TS genes, with the exception of ts7 , whose transcripts were not detected in any condition, and ts1 , which expression did not change (Fig.3c ). This clearly indicates a reprogramming in terpene biosynthesis in Tgam when colonizing wheat roots. In particular, a modulation in sesquiterpene biosynthesis occurred in Tgamduring root colonization. Whereas ts5 was slightly up-regulated (1.58-fold), the contact with the roots strongly repressed the expression of ts4 (0.03-fold) and in a lesser extent, that ofts3 (0.59-fold). Nevertheless, the most remarkably difference was found in the expression of tri5 , which was 137-fold up-regulated when the fungus was on the roots, suggesting that signals from the roots are responsible of triggering the expression of this gene inTgam . In addition, the interaction with the roots up-regulatedts6 (1.52-fold) and ts11 (2.58-fold). Since ts6 was predicted to encode an SQS, this suggests that triterpene biosynthesis was slightly enhanced in Tgam when colonizing the wheat roots. Interestingly, ts9 was found highly down-regulated (0.28-fold), thus suggesting a repression of indole diterpene biosynthesis in the fungus during the interaction with wheat roots.