## 3 Results
### Arabidopsis Overexpressing TaPS Produced \(\beta\)-Patchoulene to Repel S. Exigua Larvae  
To explore the biological function _in planta_, _TaPS_ was overexpressed in Arabidopsis under control of the 35S promoter. The RT-PCR analysis indicated that _TaPS_ was successfully expressed in Arabidopsis (Figure 3A). Further GC-MS analysis also showed \(\beta\)-patchoulene production (\(\sim\)130 ng/g FW) in two transgenic Arabidopsis lines (Figure 3B,C). These results proved that TaPS catalyzed \(\beta\)-patchoulene formation as a sesquiterpene synthase _in planta_, consistent with its biochemical activity in microbial metabolic engineering system (Figure 1).  
Figure 2: Inducible gene expression of _TaPS_ and \(\beta\)-patchoulene accumulation in wheat. (**A**) qRT-PCR analysis of _TaPS_ in wheat treated with various elicitations including UV irradiation, 50 \(\upmu\)M methyl jasmonate (MeJA), 5 mg/L alamethicin, pathogen infection with _F. gramitearum_ spore inoculation or herbivory with beet armyworm larvae feeding. Untreated wheat leaves were used as the control. Relative gene expression level of TaPS in roots and stems were also shown. Gene expression of _TaPS_ was normalized with wheat _Actin_, the endogenous control gene. Error bars indicate SE (\(\mu=3\)). Different lowercase letters indicate significant difference (Tukey HSD test, \(p<0.05\)). (**B**) GC-MS chromatograms of wheat leaf extracts with alamethicin treatment (ALA). CK is the control wheat leaves without any treatments. The induced \(\beta\)-patchoulene and three other sesquiterpenens (peak 1–3) were labeled. Mass spectra and putative identification of peak 1–3 were shown in Figure S2. IS indicates the internal standard.  
Sesquiterpenes have been reported to exhibit various functions particularly anti-herbivory activity in plants, being repellent and/or antifeedant or attracting predators or parasitoids [15; 16; 17; 18; 19]. \(\beta\)-patchoulene is the component of essential oil from _P. cablin_ and has not been identified to be involved in plant defense. We used transgenic Arabidopsis plants overexpressing _TaPS_ to investigate the anti-herbivory activity of \(\beta\)-patchoulene. In the dual-choice feeding preference assay, _S. exigua_ larvae preferred WT over transgenic leaves (Figure 4A). After 24 h, leaf consumption by larvae on WT leaves was significantly higher than _TaPS_ overexpression lines (Figure 4B), indicating repellent activity of \(\beta\)-patchoulene produced by _TaPS_ overexpression in Arabidopsis. The potential toxicity of \(\beta\)-patchoulene on larvae was also explored and no significant growth inhibition was observed when larvae fed transgenic Arabidopsis (Figure S3), suggesting that \(\beta\)-patchoulene might not exert a direct toxicity to _S. exigua_ larvae.  
Figure 3: Overexpression of _TaPS_ in Arabidopsis. (**A**) RT-PCR analysis of _TaPS_ gene expression in transgenic Arabidopsis. _Actin_ was used as the control gene; (**B**) GC-MS analysis of \(\beta\)-patchoulene production in two transgenic Arabidopsis lines (TaPS-OE#2 and #5). IS indicates the internal standard; (**C**) head space volatile analysis by GC-MS for _TaPS_ transgenic Arabidopsis plants.