Real-time PCR was conducted to quantify the mRNA, as described by Joshi et al. (2016a). Briefly, all of the primers used in this study were designed using the National Center for Biotechnology Information (NCBI) primer BLAST software.1 The generated primers were 100–120 bp in size and the melting temperatures of the primers were designed for 60°C, with a difference of less than 5°C for each primer pair (Supplementary Table 1). To exclude the possibility of non-specific binding, primer sequences were analyzed by BLAST analysis (using NCBI BLAST software) against the database for the genus Pectobacterium. The primer mixture for qRT-PCR contained 5 μL of Fast SYBR Green Master Mix (Applied Biosystems) and 0.8 μL (5 μM) of each forward and reverse primer. Then, a total of 3.4 μL (17 ng) of cDNA was added to each well, so that the total reaction mixture would be 10 μL for each well. Reactions were performed using a Step One Plus Real-Time PCR system (Applied Biosystems) with the following cycling parameters: holding stage, 95°C for 20 s; cycling stage, 40 cycles of 95°C for 3 s and 60°C for 30 s; and melting curve stage, 95°C for 15 s, 60°C for 1 min and 95°C for 15 s. The data were analyzed by the comparative CT (ΔΔCT) method, with expression normalized to the expression of the reference gene ffh, as described by Takle et al. (2007).

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