cDNA PK-profiling

Total RNA was extracted from MN841801–1 leaves using Tripure Reagent (Roche, Germany) according to the manufacturer’s instructions, and treated with Turbo RNase-free DNase (Ambion, Thermo Fisher Scientific, Whaltman, MA, USA) to remove all contaminating DNA. Poly-A⁺ RNA was then extracted from the total RNA using The Dynabeads Oligo (dT)₂₅ Kit (Invitrogen, Thermo Fisher Scientific, Whaltman, MA, USA). cDNA was synthesized from 40 ng Poly-A⁺ RNA employing the SMARTer PCR cDNA Synthesis Kit (Clontech, Takara Bio, Shiga, Japan) according to the manufacturer’s instructions.

PK profiling from cDNA of mock-inoculated plants was performed as reported in van der Linden et al. [16] with the modifications described by [21]. The restriction enzyme RsaI was used to digest 100 ng of cDNA. Adapters were then ligated to the restriction sites. The digestion of the cDNA and ligation of the adapters was performed in a single reaction at 37 °C. PK-specific fragments were PCR-amplified using primers directed towards specific sequences encoding amino acid motifs in conserved PK domains. The primers used were PK1Fa, PK1Fb, PK3Fa, PK3Fb, PK4R1a and PK4R1b, previously described by [20]. These were used in combination with adapter primers also described in [20]. The annealing temperature was set at 55 °C. The amplicons were then reamplified using the same adapter primers but labeled with the fluorochrome IRDye700 to visualize individual fragments in 6% denaturating polyacrylamide gels using the LI-COR 4300 DNA Analysis System (LI-COR Biosciences, Lincoln, NE, USA).

cDNA from an SSH library, obtained by [28], was also subjected to PK profiling.