Chitin-induced FRET analysis of LysM receptors

Leaves of Nicotiana tabacum cv. Xanthi were transformed by agroinfiltration (Norkunas et al., 2018 (link)) with the Agrobacterium tumefaciens strain GV3101 containing an overexpression cassette for VvLYK1-1, VvLYK1-2, VvLYK5-1, or VvLYK5-2 fused with either a C-terminal Cyan Fluorescent Protein (C_ter-CFP in the pH7CWG2 plasmid, Karimi et al., 2005 (link)) or a C-terminal Yellow Fluorescent Protein (C_ter-YFP in the pH7YWG2 plasmid, Karimi et al., 2005 (link)). Five days after (co-)infiltration, the abaxial side of N. tabacum leaves transiently expressing the different constructs was infiltrated with chitin or water (mock treatment). Observations were performed 30 min post-treatments (according to Cheval et al., 2020 (link)) using a Nikon A1-MP multiphoton microscope with an Apo IR x60 objective (NA: 1.27, water immersion, Nikon). Fluorescence lifetime imaging (FLIM) images were collected using a time-correlated single-photon counting (TCSPC) module (Picoquant). CFP excitation (820 nm with two-photon excitation) was provided by an IR laser (Chameleon, Coherent) delivering femtosecond pulses at a repetition rate of 80 MHz. Its resulting fluorescence emission was collected with a single photon avalanche diode (SPAD), using a band-pass emission filter FF01-494/20 (Semrock). TCSPC lifetime recording was performed over 200 temporal channels (final resolution: 0.64 ps). The FLIM analysis was performed on regions of interest (ROIs) drawn on the plasma membrane using the SymPhoTime (PicoQuant) software. Fluorescence lifetime values were calculated by fitting the tail of the CFP fluorescence decay with a bi-exponential model. Among the two generated lifetime constants (τ₁ and τ₂), only τ₁ was reported, as it was the most sensitive to the Förster resonance energy transfer (FRET) (Bègue et al., 2019 (link); Rosnoblet et al., 2021 (link)). The efficiency of the energy transfer was given by the following equation: $E(\%)=(1-\frac{F_{DA}}{F_D})\times 100$ , with FDA and FD representing the relative fluorescence lifetime of the donor in the presence or the absence of the acceptor, respectively. Three independent experiments were conducted.