*4.7. Protein Extraction and Western Blot*

Cassava leaves were harvested and ground by liquid nitrogen and phosphate buffer solution (pH 7.4). The extracted solution was centrifuged at 12,000 rpm and 4 °C for 10 min. The supernatant was boiled with SDS-PAGE sample loading buffer (P0015, Biyotime, Shanghai, China) for 5 min. After centrifugation for 10 min, the supernatant could be used in Western blot. The Western blot assay was performed according to a previous study [53]. Briefly, the protein samples were loaded into 12% polyacrylamide gel and separated by electrophoresis. The polyacrylamide gel was transferred to a PVDF membrane (475855-1R, Millpore, Massachusetts, America) by a Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell (1703940, Bio-rad, Hercules, America). Then, the PVDF membrane was blocked and incubated in 5% skim milk with anti-*GFP* antibody (AG281, Biyotime, Haimen, China).

## *4.8. GUS Staining and Activity Detection*

*GUS* staining and *GUS* activity assay were carried out according to a previous study with slight modifications [48]. The infiltrated cassava leaves were harvested and immersed into *GUS* staining solution (50 mM NaH2PO4, 50 mM Na2HPO4, 10 mM EDTA-Na2, 0.5 mM K4[Fe(CN)6], 0.5 mM K3[Fe(CN)6], 0.1% Triton-X100, and 2 mM X-Gluc; pH 7.0) with vacuum infiltration for 0.5 h in the dark, and then the plant leaves were incubated in the dark at 37 °C for at least 12 h. After staining, the leaves were immersed into 70% alcohol to remove chlorophyll.

The infiltrated leaves were harvested and ground by liquid nitrogen and phosphate buffer solution (pH 7.4). The extracted solution was centrifuged (12,000 rpm, 10 min, 4 °C) and the supernatant was used for further assay. The extraction was incubated in 1 mM 4-Methylumbelliferyl-b-D-glucuronide (4-MUG) at 37 °C, the reaction mixture was taken out per 5 min, and Na2CO<sup>3</sup> was added to stop the reaction. The samples were detected by a microplate system (Infinite M200 Pro, TECAN, Hombrechtikon, Switzerland) with 365 nm excitation and 455 nm emission. The *GUS* activity was calculated by the standard curve made by different concentrations of 4-methylumbelliferone (4-MU).
