Flg22

ROS production was monitored using a luminol-based assay (53 ). Leaf discs were made using a circular borer (diameter of 5 mm), and the collected leaf discs were incubated overnight in distilled water. For measurement of ROS production, leaf discs were placed in a 96-well plate containing 50 μl of distilled water and 50 μl of assay solution containing 400 μM luminol (FUJIFILM Wako Pure Chemical Corporation; 127-02581), 20 μg/ml peroxidase (Sigma–Aldrich; P6782), and either 400 μg/ml chitin (Sigma–Aldrich; C9752) or 2 μM flg22 (Invitrogen) were added to the wells. Luminescence was measured using a Luminoskan Ascent 2.1 (Thermo Fisher Scientific).

For genomic DNA and total RNA extraction, plant and fungal materials were powdered under liquid nitrogen using a mortar and pestle. Genomic DNA was isolated following the method of Doyle and Doyle (Doyle, 1987 ). For gene expression studies, seedlings were grown on solid half strength MS medium for 2 weeks and then transferred in six-well-plates containing liquid half-strength MS medium. After a recovery phase of 3 days, flg22 (Davids Biotechnology, Germany) was pipetted to each well containing MS medium with seedlings in a final concentration of 100 nM flg22 and harvested after 0, 2, 6, and 12 h. For all experiments, total RNA was extracted from plant or fungal materials using TRIzol (Invitrogen), and aliquots were used for cDNA synthesis with the qScript cDNA synthesis kit (Quanta Biosciences). Forty nanograms of genomic DNA or cDNA were used as template for quantitative real-time PCR (qPCR) analysis, using the SYBR Green JumpStart Taq ReadyMix (Sigma–Aldrich) and the 7500 FAST Real-Time PCR System under standard conditions (Applied Biosystems). For the detection of fungal and plant DNA, the primers ITS_F/ITS_R and UBQ4_F/UBQ4_R were used (Supplementary Table S1). The relative fungal genomic DNA or relative gene expression were calculated using the 2^-ΔCT method (Livak and Schmittgen, 2001 (link)).

N. benthamiana leaves were agroinfiltrated with desired constructs for transient protein expression. 36 hours after infiltration, either water control or 100nM flg22 (PhytoTechnology Laboratories, Shawnee Mission, USA) were injected to induce NbAcer31 and NbWRKY22 expression. Total RNA from agroinfiltrated leaf discs treated with flg22 for 1 hour was isolated with TRIzol reagent (Invitrogen, Carlsbad, USA). One microgram of total RNA was treated with DNase I (Invitrogen, Carlsbad, USA), followed by reverse transcription using a Super Script II reverse transcriptase (Invitrogen, Carlsbad, USA). qRT-PCR analysis was performed on an ABI Prism 7100 sequence detection system using Power SYBR Green reagents (Life Technologies, Carlsbad, USA). The N. benthamiana EF1 gene was used as an internal control for normalization [43 (link)]. Relative expression ratios were determined based on the comparative CT method (ΔΔCT) using the StepOne Software. Primers used in qRT-PCR are listed in S1 Table.