3.2 Expression levels of eight candidate CsGES genes in pathogen-infected tea plants
Given that geraniol has been reported to function as an antifungal compound (Li et al., 2017; Kalagatur et al., 2018; Tang et al., 2018), changes in the abundance of geraniol in response to C. gloeosporioides and Neopestalotiopsis sp. infection were characterized using GC–MS. The geraniol content in the infected leaves significantly increased after 24 and 48 h of infection (Figure 1B), indicating that geraniol might play a role in activating defense-signaling pathways following fungal attack in tea plants. To determine which candidates are involved in the biosynthesis of geraniol, gene-specific primers (Table S1) of these eight genes were designed, and the expression of these genes in response to pathogen infection was analyzed 0, 12, 24, and 48 h after infection with C. gloeosporioides and Neopestalotiopsis sp. (Figure 1C,D). To verify the specificity of the primers, the abundances of the transcripts of the eight candidate genes were analyzed, and their products were verified by agarose gel electrophoresis (Figure 1E). One clear band was observed for seven genes (CsTPS2–CsTPS8 ), whereas three clear bands were observed for CsTPS1 (Figure 1E), which indicates the presence of an AS form of CsTPS1 in tea plants that is expressed in response to fungal attack.
To verify the presence of the AS forms of CsTPS1, the full-length sequences and the shorter AS forms of CsTPS1 were obtained from young leaves of C. sinensis var. sinensis cv.Shuchazao using gene-specific primer pairs (Table S1) (Wei et al., 2018). The whole-length CsTPS1 contains a 1758-bp open reading frame (Figure S1) that encoded 585 amino acids (Figure S2); there were 83 fewer amino acids in the AS form (referred to as CsTPS1-AS ) (Figure 2A and Figure S2). The AS form of CsTPS1 was confirmed based on an AS database for tea plants (TeaAS,http://www.teaas.cn/index.php) (Mi et al., 2021). The expression of CsTPS1 and its AS isoform (CsTPS1 -AS ) was quantified in response to pathogen infection. To further verify whether CsTPS1 -AS is expressed in tea plants in response to pathogen infection. The new specific quantitative primers for CsTPS1 -AS andCsTPS1 were redesigned (Table S1and Figure S2). The expression ofCsTPS1 -AS and CsTPS1 was quantified using RT-PCR, respectively. With the exception of CsTPS1 -AS , the expression of none of the eight candidates was induced in tea plants following pathogen infection (Figure 1C,D,F). The expression ofCsTPS1-AS was significantly induced in response to infection with both C. gloeosporioides and Neopestalotiopsis sp.,which is consistent with changes in the content of geraniol in infected leaves. Therefore, the roles of CsTPS1 and its AS forms in geraniol biosynthesis and the response to pathogen infection were studied.
To verify that CsTPS1-AS is involved in regulating geraniol biosynthesis and disease resistance in tea plants, the expression of CsTPS1 -AS in infected tea plants was determined at various points after infection in repeated experiment (Figure 2B). The expression of CsTPS1-AS was significantly increased under pathogen infection compared with the control, especially at 24 and 48 h, which is consistent with changes in the content of geraniol in leaves infected with the two pathogens (Figure 1B). Overall, these findings indicate thatCsTPS1 -AS might be involved in the biosynthesis of geraniol in response to pathogen infection in tea plants.