Protein-protein interaction assays

Interactions between RPG and CERBERUS were assayed using the yeast two- hybrid system as previously described [22]. The yeast strain AH109 was transformed with the constructs in destination vectors using lithium acetate transformation (Yeast Protocols Handbook PT3024-1, Clontech). The transformants were grown on synthetic defined medium (0.67% yeast nitrogen base, 2% Bacto-agar and amino acid mix) without the appropriate auxotrophic markers after gradient dilution. These assays were repeated three times.

For split-luciferase complementation imaging assays in N. benthamiana leaves,

LUCc-RPG was co-expressed with CERBERUS-LUCn, ARM-LUCn, in N. benthamiana leaves via agroinfiltration with p19, which inhibits gene silencing. The transformed plants were grown in a growth chamber. Two days after infiltration, images were captured by CCD (TANON 5200, China) after 1 mM luciferin (Promega) was sprayed onto the leaves. All images were acquired using the same exposure settings. Each interaction group was validated with three replicates, and two or three independent experiments were performed.

For co-immunoprecipitation (Co-IP) assays, GFP-RPG and CERBERUS-mCherry were co-expressed in N. benthamiana leaves. The samples were harvested 60 h after agroinfiltration, and approximately 0.6 g of plant tissue was extracted with 2 ml lysis buffer (50 mM Tris-MES at pH 8.0, 0.5 M sucrose, 1 mM MgCl₂, 10 mM EDTA, 5 mM DTT, 0.2% NP-40, 1 mM phenylmethanesulfonyl fluoride (PMSF] and proteinase inhibitor cocktail tablet (Roche]) for 15 min then centrifuged at 12,000 rpm for 10 min. The supernatants were collected for Co-IP. Samples were incubated for 1.5 h with 30 μL Anti-RFP Affinity beads 4FF (Cat. SA072C, SMART) at 4°C on a rotating wheel, then centrifuged at 2000 rpm for 2 min at 4°C. The beads were washed and analyzed by immunoblotting using anti-mCherry (Cat.T0090, Affinity Biosciences, Cincinnati, OH, USA) and anti-GFP antibody (Cat. M20004L, Abmart). Approximately 10 μL of lysis buffer containing total protein was loaded as the input control.

For BiFC assays, the construct pAtUBI10:nVenus-CERBERUS/pLjUBI1:cVenus-RPG was expressed in N. benthamiana leaves by agroinfiltration with p19. Transformed plants were grown in a growth chamber, and images were captured two to three days later by laser scanning confocal microscopy (Leica TCS SP8). The BiFC construct was also expressed in L. japonicus Gifu or M. truncatula sunn-1 by hairy root transformation. The transgenic hairy roots were scored based on the NLS-DsRed marker and inoculated with M. loti MAFF303099/RFP or Sm1021/mCherry (OD₆₀₀: 0.001). Images were analyzed at 5–7 dpi. The filter sets for excitation and emission were 514 nm and 524 to 545 nm, respectively, 561 nm for Venus, and 600 to 630 nm for DsRed. All BiFC experiments were repeated twice, and five leaves or roots were analyzed each time.