2.13 Subcellular localization analysis of CsTPS1 and CsTPS1-AS proteins
Subcellular localization assays of CsTPS1 and CsTPS1-AS proteins were performed following the procedure described in a previous study (Wang et al., 2019a). Briefly, binary vectors (pCHNP-eYFP/mCherry) were constructed with several elements on the pCAMBIA1300 backbone (CAMBIA, Canberra, Australia). The amplified fragments were introduced into pCHNP-eYFP with the NcoI site using in-fusion technology. The empty vector pCHNP-mCherry was used as a negative control. Agrobacterium tumefaciens strain GV3101 carrying the construct for the transient expression of individual mCherry and CsTPS1 EYFP and CsTPS1-AS EYFP fusion proteins was mixed and infiltrated into the leaves of tobacco. Images were taken using a laser confocal fluorescent microscope (Lecia DMi8, Germany). The EYFP, mCherry fluorescence, and chloroplast autofluorescence were analyzed at excitation wavelengths of 488 nm, 561 nm, and 561 nm and emission wavelengths of 500–530 nm, 580–620 nm, and 680–720 nm, respectively.