Rneasy plant miniprep kit

Mutants of PAT14 were analyzed by genotyping PCR using the following primers: ZP1147/ZP1771 for PAT14, LB1/ZP1771 for pat14-1, ZP8/ZP1203 for pat14-2. For RT-PCR and quantitative real-time PCR analyses, total RNAs were extracted using the RNeasy Plant miniprep kit according to the manufacturer’s instructions (Qiagen). Reverse transcriptions were performed using Superscript^TM III Reverse Transcriptase with on-column DNase-I treatment (Invitrogen). The following primer pairs were used in RT-PCRs to characterize mutants or complemented mutants: F1/R1 for endogenous PAT14, ZP1599/ZP1600 for exogenous PAT14 or PAT14-C157S, ZP1506/ZP11 for ZmPAT14, and ZP1628/ZP2682 for TaPAT14. Arabidopsis ACTIN2 was used as the internal control for RT-PCRs49 (link). The qPCR analyses were performed as described36 (link). Primers in qRT-PCR analyses are as followed: ZP1581/ZP1582 for SAG13, ZP3139/ZP3140 for SAG12, ZP1498/ZP1499 for SAG21, ZP2190/ZP2191 for PAD4, ZP2319/ZP2320 for SID2, ZP2304/ZP2305 for PDF1.2, and ZP1583/ZP1584 for PR1. Primers are listed in Table S1.

Fungal mycelia were harvested, frozen in liquid nitrogen and ground with a table mill. RNA was isolated according to the instruction of the RNeasy® Plant Miniprep Kit (Qiagen). Approximately 0.8 μg of RNA was used for cDNA synthesis according to manufacturer's instructions of the QuantiTect® Reverse Transcription Kit (Qiagen). RT-PCR was performed with the MESA GREEN qPCR MasterMix Plus for SYBR Assay (Eurogentec) in a CFX Connect Real-Time System (BioRad). One microliter of 1:10 diluted cDNA was used per reaction. Two biological replicates with each three technical replicates were used. Primers are listed in Supplementary Table 5. Data were recorded with the CFX Manager software (BioRad). Expression of the housekeeping genes h2A, gpdA, and rps15 were used for normalization.

ECW-30R pepper plants were inoculated with derivatives of Xcv strain 85–10 (OD₆₀₀ of 0.05) expressing avrBs3 and mutant derivatives. RNA was extracted from ten leaf discs (diameter 0.28 cm), harvested 10 hpi, using the RNeasy Plant Miniprep kit (Qiagen, Hilden, Germany). RNA concentrations were determined with an ND-1000 spectrophotometer (NanoDrop Technologies, Rockland, DE, USA). cDNA was synthesized by reverse transcription using an oligo dT-primer and the Revert Aid First Strand Synthesis Kit (Fermentas). For RT-PCR of Bs3 the RT-PCR was performed as described using oligonucleotides Cand-7–01-fwd and Cand-7–01-rev for Bs3 and RS-EFrt-F1 and RS-EFrt-R1 for EF-1α (used for RT-PCR normalization) [7 (link)].

The total RNAs of the roots from the 4 days-after-germination (DAG) seedlings were isolated using a Qiagen RNeasy plant miniprep kit according to the manufacturer’s instructions. Oligo(dT)-primed cDNAs were synthesized using Superscript III reverse transcriptase with on-column DNase digestion (Invitrogen). Quantitative real-time PCRs were performed with the Bio-Rad CFX96 real-time system using a SYBR Green real-time PCR master mix (Toyobo) as described (Zhou et al., 2013 (link)). All the primers are listed in Supplementary Table S1.

Fungal tissues were harvested from cultures, which were incubated under conditions inducing the sexual cycle, after 3 days of incubation and ground with a table mill in powder. The RNA isolation was performed according to the instruction of the RNeasy Plant Miniprep Kit (Qiagen, Hilden, Germany). Approximately 0.8 µg of RNA was used for cDNA synthesis according to manufacturer’s instructions of the QuantiTect Reverse Transcription Kit (Qiagen); 1 µl of cDNA was used for semi-quantitative reverse-transcriptase polymerase chain reaction. The used primers are listed in the Supplementary file 6. Measurements were conducted in three independent biological replicates.

For RNA isolation strains were grown vegetatively or asexually. Mycelia was harvested through sterile filters (Miracloth) and immediately frozen in liquid nitrogen. Frozen mycelia were ground with a table mill (Retsch) directly before RNA extraction. RNA was extracted with the RNeasy Plant Miniprep Kit (Qiagen) according to manufacturer’s instructions. cDNA was transcribed from 0.8 μg RNA with the QuantiTect Reverse Transcription Kit (Qiagen).