The full-length CDSs of potential SlFNSIs were amplified using polymerase chain reaction (PCR) from cDNA in conjunction with primers designed based on the sequences obtained from the tomato genome database (https://solgenomics.net/). The CDSs were cloned into a pDONR207, sequenced, and subsequently recombined into pDEST17 vector through Gateway cloning [45]. The potential SlFNSIs were expressed in E.coli strain BL21 grown at 37 °C in Luria-Bertani (LB) media containing 0.05 mg ml− 1 carbenicillin until the optical density at 600 nm reached 0.7–0.9. Recombinant proteins were expressed by induction with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) for 18 h at 16 °C. Cells from 40 ml of culture were harvested by centrifugation and resuspended in 3 ml of PBS buffer (pH 7.0) at 4 °C. Afterward, cell lysis was performed using an ultrasonic homogenizer and the lysates were recovered by centrifugation (10,000 g) for 20 min [55].

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