## 3 Results
### Crude Enzyme Extract of Empoasca (Matsumurasca) onukii Produced Geraniol from Greamyl Diphosphate  
Since the activity of tea leaves GES was not increased after _E. (M.) onukii_ attack, it was presumed that the geraniol accumulation may induced by GES which was from _E. (M.) onukii_. To investigate whether elicitors such as enzymes involved in geraniol formation were present in _E. (M.) onukii_, we extracted _E. (M.) onukii_ using buffer, and added GPP as the substrate. In contrast to controls (GPP or crude extract only), the reaction solution containing GPP and the insect extract specifically produced geraniol, but not other volatile monoterenes such as linalool. The GES activity was higher in the body part than in the head part of _E. (M.) onukii_ (Figure 2, Table S3). These results confirmed that _E. (M.) onukii_ contains GES, which generates geraniol from GPP. Figure 1: Effect of _Empoasca (Matsumurasca) (E. (M.) onukii_ attack on emission of geraniol and geraniol synthase activity in tea leaves. (**a**) Effect of _E. (M.) onukii_ attack on emission of geraniol in tea leaves in lab control experiments. (**b**) Effect of _E. (M.) onukii_ attack on geraniol synthase activity. The control (CK) was intact tea leaves without treatment. The levels of enzyme activity in the control were set as 1. Student \(t\) test was used to determine the differences between control and treatment. Data are expressed as mean \(\pm\) S.D. (n = 3) \({}^{\star}\), \(p\)\(\leq\) 0.05. Terpene synthase from Empoasca (Matsumurasca) onukii Was Able to Convert Greatyl Diphosphate to Geraniol  
Although typical terpene synthase was not found in insect genomic and transcriptomic sequences [22], the isoprenyl diphosphate synthases (IDSs) such as terpene synthases have been reported in _Phyllotrata strirolata_ and _Ips pini_[22; 25]. A full-length transcriptome of whole _E. (M.) onukii_ insect was sequenced with three generation PacBio sequencing technology (unpublished data). Three isoprenyl diphosphate synthases including two farnesyl diphosphate (FPPS, _EoFPPS1_ and _EoFPPS2_) homologue genes and one geranylgeranyl diphosphate synthase (GGPPS, _EoTPS_) homologue genes were found in this full-length transcriptome. These three genes were cloned from _E. (M.) onukii_ successfully. The full length of EoFPPS1 (GenBank Accession Number MH383157) was 1179 bps, encoding 392 amino acids. The full length of EoFPPS2 (GenBank Accession Number MH383158) was 1287 bps, encoding 428 amino acids. The full length of EoTPS (GenBank Accession Number MH383159) was 912 bps, encoding 303 amino acids. The calculated molecular masses of EoFPPS1, EoFPPS2, and EoTPS were 45, 49, and 35 kDa, respectively. Neighbor-joining tree was generated with other isoprenyl diphosphate synthases in insect. The two farnesyl diphosphate synthase, EoFPPS1 and EoFPPS2, belong to the FPP synthase family. They clustered together with the other characterized FPPS from beetles in phylogenetic trees and were closest to the _Phaedon cochariae_ FPPS (Figure 3). The geranylgeranyl diphosphate synthase, EoTPS, belongs to the GGPP synthase type III family (Figure 3) and it was closest with the _Choristoneura funiferana_ GGPPS in the phylogenetic analysis. These genes were heterologously expressed in _Escherichia coli_ (Figure 4a). Among these three genes, EoFPPS1 was predicted to be mitochondrion localized. To better determine the enzymatic activity of EoFPPS1, the ORF lacking pupative mitochondrial targeting peptide (the first 25 amino acids, EoFPPS1S) was also expressed in _E. coli_ (Figure 4a). The activity assay results showed that only EoTPS converted GPP to geraniol (Figure 4b, Figure S1). It was presumed that prokaryotic expression systems may not be suitable for functional protein expression. Therefore, EoFPPS1 and EoFPPS2 were also expressed in insect Sf9 cells. The results showed that there is no difference of geraniol formation between EoFPPS1 and empty vector, or between EoFPPS2 and empty vector.