P per plant protein extraction kit

To verify protein production during transient expression in N. benthamiana, total proteins were extracted using the P‐PER Plant Protein Extraction Kit (Thermo Scientific) and Protease Inhibitor Cocktail Kit (Thermo Scientific) from the agroinfiltrated N. benthamiana leaves 60 hr after inoculation, following the manufacturer's instructions. The proteins were separated using 15% sodium dodecyl sulphate polyacrylamide electrophoresis gels, and transient protein expression in N. benthamiana was assessed using anti‐FLAG M2 antibody (Sigma) and detected using the Pierce ECL western blotting substrate (Thermo Scientific).

The working solution (WS) from the P-PER plant protein extraction kit (Thermo Scientific, Massachusetts, USA) was prepared according to the manufacturer’s instructions with the addition of 10 mm DTT and cOmplete ULTRA protease inhibitor cocktail tablets from Roche (Basel, Switzerland). H. hemerocallidea plant material (corm, leaf and flower) was individually crushed in liquid nitrogen using a sterile mortar and pestle. The WS was then mixed with 80 mg of crushed plant tissue in the provided polypropylene mesh bags, in which the mixture was further homogenised mechanically. The homogenous mixture was centrifuged for 5 minutes at 5,000 x g at room temperature. The lower aqueous layer, containing the extracted soluble proteins, was transferred to a clean Eppendorf tube. The protein was quantified using the Qubit Fluorometer 2.0 from Life Technologies (California, USA) and then subjected to electrophoresis as described in the “Sodium-dodecyl sulphate polyacrylamide gel electrophoresis” section.

Soluble proteins from plant leaves were extracted using the P-PER plant protein extraction kit (Thermo Fisher Scientific) according to the manufacturer’s instructions, and their protein concentration was determined by Bradford assay. Fluorescence intensity of the extracted proteins was measured using a Tecan Infinite M1000Pro microplate reader (Tecan, Männedorf, Switzerland). The excitation/emission spectra were 488/507 nm for eGFP, 514/527 nm for eYFP, and 587/610 nm for mCherry. Each fluorescence protein amount was determined using standard curves generated from serial dilutions of His-tagged recombinant FPs (Supplementary Figure 2), which were purified as previously described (Lee et al., 2019 (link)). Quantification was performed after dilution of leaf soluble proteins so that fluorescence signals fell within the linear range of the standard curve. Data are mean of three biological replicates, each from an independent experiment, and two technical replicates were performed for each biological replicate. Primers used in the cloning of recombinant proteins are listed in Supplementary Table 1.

The extraction of total protein and small molecules was carried out following the manufacturer’s instruction of the P-PER^® Plant Protein Extraction Kit, Thermo Scientific. The matured seeds were collected from plant grown in green house. The collected samples were treated with liquid nitrogen to make powder and suspended with protein extraction buffer (25mM Tris-HCl pH 7.5; 500mM NaCl; 10% Glycerol; 1mM EDTA; 1mM PMSF; 0.5% Tween-20; 1mM β-ME). The suspended solutions were homogenized and sonicated on ice. The suspension was centrifuged to sediment the debris other than protein and aqueous layer (total protein) was collected and quantified with protein estimation method [29 (link)]

Fully expanded leaves of Chardonnay were detached from plants and laid in 10-cm Petri dishes with moistened filter papers. The leaves were sprayed with 2% (w/v) GFB or C. glutamicum extract and incubated in the light at 23°C for 24 or 48 h as indicated in Fig. 1B and Fig. 2B. The leaves were then frozen and ground into a fine powder in an MM 300 mixer (Qiagen). Total protein was extracted from the powder with a P-PER plant protein extraction kit (Thermo, Rockford, IL, USA) according to the manufacturer's manual. The protein extracts were then diluted with reaction buffer (0.1 M Na₂HPO₄/NaH₂PO₄, pH 6.0, with 10 mM dithiothreitol), and total protein was quantified with a BCA Protein Assay kit (Thermo).
Chitinase activity was measured using 40 mM 4-methylumbelliferyl β-d-N,N′,N″-triacetylchitotrioside (4-MU-(GlcNAc)₃; Sigma, St. Louis, MO, USA) as a substrate. Leaf protein extract (50 µL, 0.5 mg/mL) was mixed with 50 µL of substrate solution and incubated at 37°C for 90 min. The reaction was terminated by the addition of 100 µL of 1 M Gly-NaOH (pH 10.2). The hydrolyzed 4-MU was monitored by spectrofluorometer (SpectraMax M2; Molecular Devices, Sunnyvale, CA, USA) with excitation at 360 nm and emission at 450 nm. One unit (U) of chitinase activity was defined as the amount of enzyme that catalyzed the conversion of 1 µmol of 4-MU-(GlcNAc) per minute under the assay conditions.

The primary antibody against plastocyanin was obtained from agrisera (www.agrisera.com). Total protein was extracted from the flag leaf at heading stage and shoot of three week old plants using the reagents of P-PER^® Plant Protein Extraction Kit (Thermo Scientific) after homogenizing 30 mg tissue in liquid nitrogen in motor and pestle. Ten µg of protein was subjected to the Tricene-SDS-PAGE (18%) for separation and transferred to nitrocellulose membrane by electro-blotting. The blots were developed using the Pierce^® Fast Western Blot Kit SuperSignal^® west femto substrate (Thermo Scientific) following the instructions specified by the manufacturer.