Nanodrop photometer

C-terminally amidated peptides R-DIM-P-LF11-322 (PFWRIRIRRPRRIRIRWFP-NH₂, M = 2677.4 g/mol) and R-DIM-P-LF11-334 (PWRIRIRRPRRIRIRWP-NH2, M = 2382.4 g/mol) derived from Lactoferricin were purchased from PolyPeptide Group (San Diego, CA, USA). A purity of higher than 96% for all peptides had been determined by RP-HPLC. Peptide stock solutions were prepared in acetic acid (0.1%, v/v) to an approximate concentration of 3 mg/mL and treated by ultrasonication at 15 min for better solubility. Peptide concentration was determined by measurement of UV absorbance of tryptophan at a wavelength of 280 nm using a NanoDrop photometer (ND 1000, Peqlab, VWR International, Inc., Erlangen, Germany). All peptide stocks were stored at 4 °C until use.

RNA was isolated from basophils with a purity ≥99% using the “Direct-zol RNA MiniPrep Kit” from Zymo Research and reversely transcribed into cDNA using the “RevertAid H Minus First Strand cDNA synthesis Kit” (Thermo Scientific). A minimum of 5 × 10⁶ basophils was required to obtain sufficient amount of RNA. The RNA was either stored at −80°C or subjected immediately to the reverse transcription reaction. DNA concentration and quality was controlled with the Nanodrop photometer (Peqlab). Specific TLR cDNA was amplified by the following PCR protocol: pre-denaturation step at 95°C for 15 min followed by 40 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 60 s, followed by a final extension step at 72°C for 10 min before cooling to 4°C. Amplification was performed using the Maxima Hot Start DNA Polymerase (Thermo Scientific). Table 1 shows the sequences of the applied TLR-specific primers. A negative control reaction was performed without cDNA template and a positive control using specific primers for the housekeeping gene GAPDH. PCR products were analyzed on 1% agarose gels together with the “MassRuler DNA Ladder Mix” from Thermo Scientific (marker 1) and the “100 bp ladder” from Promega (marker 2).

Total RNA was extracted from the whole leaves of plants that were either B. cinerea-infected or mock-treated, by using the RNAiso Plus kit (Takara, Beijing, China) and following the manufacturer’s instructions. Quality and integrity of RNA were assessed by gel electrophoresis and A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀ ratios in a NanoDrop photometer (PeqLab, Germany). Pure and highly intact RNA samples were used for cDNA synthesis with Hifair^TM 1st Stranded cDNA Synthesis SuperMix for qPCR (gDNA digester plus) kit (Yeasen, Shanghai, China). The generated cDNA was then subjected to quantitative PCR (qPCR) with gene-specific primers (Supplementary Table S3), by using the TB Green^® Premix Ex Taq^TM II (Tli RNaseH Plus) kit (Takara). The qPCRs were implemented in a CFX Connect Real-Time System (Bio-Rad, United States), with three technical replicates in the same run with at least three biological replicates used. For normalization purposes, EXP (At4g26410) was used as an endogenous reference gene because it has high expression stability under varying plant stress conditions (Czechowski et al., 2005 (link); Liu S. et al., 2017 (link)). Primers used for reference genes and genes of interest are listed in Supplementary Table S3. The ΔΔCt method (Livak and Schmittgen, 2001 (link)) was used to calculate the relative expression levels of genes.