*4.11. Real-Time RT-PCR*

To determine whether KOF112 induces plant defense response in grapevines, transcriptional alteration of genes encoding two PR proteins, class IV chitinase and β-1,3-glucanase, was evaluated in KOF112-treated grapevine leaves. Three leaf disks each having a diameter of 13 mm were cut out from Koshu leaves using a cork polisher and placed upside down on moistened filter paper in square Petri dishes (140 mm × 100 mm). Ten microliters of KOF112 solution (1 × 108 cfu/mL, containing 10% SCD) was dropped onto four locations on the abaxial surface of the leaf disk. Ten percent SCD medium or *Agrobacterium* sp. isolate CHB3 (1 × 108 cfu/mL, containing 10% SCD) was used as control. After incubation at 22 ◦C for 24 h and 48 h in an incubator (11.8 Wm<sup>−</sup>2/16 h/d), the disks were homogenized in a mortar containing liquid nitrogen using a pestle. Total RNA isolation from the pulverized samples was performed using NucleoSpin RNA Plant (Takara, Shiga, Japan), and purification was carried out using Fruit-mate for RNA Purification (Takara). First-strand cDNA was synthesized from the total RNA using PrimeScript RT Master Mix (Perfect Real Time) (Takara). Real-time RT-PCR was performed using an SYBR Premix Ex Taq II (Perfect Real Time) (Takara) with a Thermal Cycler Dice Real Time System (Takara). PCR conditions were as follows: incubation at 95 ◦C for 30 s, followed by 40 cycles at 95 ◦C for 5 s and at 60 ◦C for 45 s. The primers used for amplification were as follows: ubiquitin primers (5 -GTGGTATTATTGAGCCATCCTT-3 and 5 -AACCTCCAATCCAGTCATCTAC-3 , GenBank accession no. BN000705); class IV chitinase primers (5 -CAATCGGGTCCTTGTGATTC-3 and 5 -CAAGGCACTGAGAAACGCT-3 , Gen-Bank accession no. U97522), and β-1,3-glucanase primers (5 -GAATCTGTTCGATGCCATGC-3 and 5 -GCATTATCAACCGTAGTCCC-3 , GenBank accession no. DQ267748). Ubiquitin primers were used as the reference gene to normalize each gene expression because ubiquitin gene expression was stable in the grapevine leaves [52]. Using the standard curve method of Thermal Cycler Dice Real Time System Single Software ver. 3.00 (Takara), gene expression levels were determined as the number of amplification cycles needed to reach a fixed threshold and are expressed as relative values to ubiquitin.
