Agroinfiltration-Based Transient Protein Expression in Nicotiana benthamiana Leaves for Subcellular Localization and Protein-Protein Interaction (BiFC) Assays

Use 4-6 week-old, pre-bolting N. benthamiana plants; infiltrate 3-4 fully expanded leaves per construct/combination across ≥3 independent plants (biological replicates). For each leaf, infiltrate defined zones with a marked syringe pattern. Include a co-expressed silencing suppressor (p19) in all infiltrations to boost expression. Image 2-4 days post-infiltration (dpi). For BiFC, run all combinatorial controls (nYFP-X + cYFP-Y, plus each with the complementary empty fragment, and a known positive interaction pair) in parallel on the same leaves/plants.

BSL-1. Agrobacterium is a plant pathogen — autoclave all cultures, tips, and infiltrated leaf waste; decontaminate surfaces with 70% ethanol. Acetosyringone (DMSO stock) and propidium iodide/DAPI (mutagenic/intercalating dyes) require gloves and careful disposal as chemical waste. Confocal lasers: never view the beam directly; follow laser-safety interlocks. Wear gloves, lab coat, eye protection. Contain transgenic plant material; bag and autoclave before disposal. MES/MgCl2 buffers are low-hazard.

Free fluorophore control (e.g., free GFP) marking cytoplasm + nucleus to verify expression and define background. Empty-fragment BiFC controls: nYFP-X + cYFP (empty) and nYFP (empty) + cYFP-Y to detect spurious self-assembly. A validated positive interaction pair (e.g., bZIP dimerization or a known interactor set) confirming the BiFC system works on these plants. Organelle marker co-infiltration (ER/Golgi/PM/nucleus mCherry markers) for localization assignment. p19-only and buffer-only infiltrations to define autofluorescence/wounding background. Image all controls with identical acquisition settings.

Strong, clear fluorescence appears in epidermal/mesophyll cells at 2-3 dpi. Localization patterns match expectation (e.g., nuclear, plasma-membrane outline, ER-network reticulate, cytoplasmic strands) and co-localize with the corresponding organelle marker (Pearson's R typically >0.7 for true co-localization). True interacting BiFC pairs reconstitute YFP in a high fraction of expressing cells with the expected subcellular distribution, while empty-fragment controls remain at background. Signal declines and necrosis increases after ~4-5 dpi.

• Nicotiana benthamiana plants, 4-6 weeks old, grown 25 °C, 16 h light
• Agrobacterium tumefaciens GV3101 (pMP90) or LBA4404 carrying binary constructs (fluorescent fusions / BiFC fragments)
• p19 silencing suppressor strain (e.g., from Tomato bushy stunt virus) in Agrobacterium
• Organelle markers (e.g., mCherry-tagged ER, Golgi, PM, nucleus markers) as co-localization references
• LB medium with antibiotics (rifampicin 50, gentamicin 25, plus vector selection)
• Infiltration buffer: 10 mM MES pH 5.6, 10 mM MgCl2, 150-200 µM acetosyringone
• Needleless 1 mL syringes
• Spectrophotometer for OD600
• Confocal microscope (e.g., 488 nm/514 nm/561 nm laser lines)
• DAPI or propidium iodide (optional counterstains)

To rapidly express tagged proteins (e.g., GFP/YFP/RFP fusions) in the epidermal and mesophyll cells of Nicotiana benthamiana via Agrobacterium-mediated transient transformation, enabling confocal imaging of subcellular localization, organelle marker co-localization, and bimolecular fluorescence complementation (BiFC) to assay candidate protein-protein interactions in planta within 2-4 days.

1. Streak Agrobacterium strains (constructs + p19) on LB agar with selection; grow 2 days at 28 °C.
2. Inoculate 5 mL LB cultures with antibiotics; shake overnight at 28 °C, 250 rpm.
3. Pellet cells at 4,000 × g, 10 min; resuspend in infiltration buffer.
4. Adjust each strain to OD600 ~0.5 (0.2-0.8 depending on construct); for BiFC, mix the two fragment strains 1:1, and add p19 to a final OD600 ~0.3-0.5 in every mix.
5. Incubate suspensions 1-3 h at room temperature in the dark to activate vir genes.
6. Using a needleless syringe, press against the abaxial leaf surface and gently depress to infiltrate the apoplast; mark infiltrated zones with a marker.
7. Return plants to the growth chamber (25 °C, 16 h light).
8. At 2-4 dpi, excise leaf discs from infiltrated zones and mount in water on a slide.
9. Image by confocal microscopy: GFP ex488/em500-530; YFP ex514/em525-560; RFP/mCherry ex561/em580-630; collect organelle-marker channels sequentially to avoid bleed-through.
10. For BiFC, image all combinations with identical laser/gain settings and compare reconstituted YFP signal against the empty-fragment and positive-pair controls.

Independent variables: construct/fusion identity, BiFC fragment combination, Agrobacterium OD600, time post-infiltration (dpi), presence of p19. Dependent variables: presence/intensity of fluorescence, subcellular localization pattern, co-localization (Pearson/Manders coefficients), fraction of cells showing reconstituted BiFC signal. Controlled variables: plant age/growth conditions, infiltration buffer composition, acetosyringone concentration, incubation time, confocal laser power/gain/pinhole, leaf position, mounting medium.

Co-infiltrating Agrobacterium strains carrying complementary nYFP- and cYFP-fused candidate proteins will reconstitute YFP fluorescence in N. benthamiana cells only when the two proteins physically interact, while non-interacting or empty-fragment controls will show no reconstituted signal above background.

Acquire confocal z-stacks with identical settings across conditions. Quantify fluorescence intensity and co-localization in Fiji (Coloc2 for Pearson's/Manders' coefficients on background-subtracted, thresholded images). For BiFC, score the proportion of expressing cells with reconstituted signal and measure mean YFP intensity in defined ROIs, normalized to a free-fluorophore expression control. Report representative images plus quantification across ≥3 leaves/plants. Use consistent look-up tables and include scale bars.

No or weak fluorescence: omit/insufficient p19 — always include the silencing suppressor; check Agrobacterium OD (try 0.4-0.6) and confirm construct by sequencing/Western. Patchy infiltration: leaves too old/turgid — use younger, well-watered plants and infiltrate in the morning. Strong autofluorescence/necrosis: imaging too late or OD too high — image at 2-3 dpi and lower OD600; chloroplast autofluorescence is far-red, so spectrally unmix. BiFC false positives: high-OD overexpression drives nonspecific assembly — use lowest OD giving signal and always run empty-fragment controls. Channel bleed-through in co-localization: use sequential scanning and single-fluorophore controls to set thresholds.

Compare BiFC signal intensity or percent-positive cells across combinations with one-way ANOVA and Tukey's HSD, or Kruskal-Wallis with Dunn's test if non-normal, using ≥3 biological replicates (plants) and multiple fields per replicate; α=0.05. For co-localization, compare Pearson's coefficients between true and randomized (Costes) controls with a paired test. Treat plant as the biological replicate unit (avoid pseudoreplication by averaging fields within a plant). Apply Benjamini-Hochberg correction across multiple pairwise BiFC comparisons.