Extraction of Vacuolar and Cell Wall Proteins

The extraction of vacuolar and cell wall-bound proteins from cucumber and Nicotiana benthamiana leaves essentially followed the protocol described in Link et al. [58 (link)]. Bound proteins were eluted from the resuspended cell wall fraction with 500 mM NaCl (Sangon, Shanghai, China) for 1 h at 4 °C, using an overhead shaker, followed by centrifugation at 10,000× g at 4 °C. Soluble and salt-eluted proteins (cell wall-bound fraction) were washed and concentrated by centrifugal filter (Millipore, Darmstadt, Germany) with 50 mM NaOAc (Sangon, Shanghai, China) buffer pH 5. Protein concentrations were determined by Bradford assay (Sangon, Shanghai, China). For total soluble carbohydrates extraction from cucumber seedlings, method as described by Wei et al. [59 (link)].