Fast alkaline phosphatase

Genomic DNA was extracted using the Dynabeads^® DNA DIRECT Universal kit (Invitrogen) on a 24 h culture. For each PCR reaction, 1 μL of extracted genomic DNA was used with primers listed in Table 2. PCR for the species identification and MLST were performed using the TEMPase Hot Start 2X Master Mix Blue II (Ampliqon, Denmark) and the Maxima Hot Start Taq Polymerase (Fermentas, Waltham, MA, United States) was used for toxin-encoding gene amplification, both were performed in a 25 μL reaction following the manufacturer’s instruction. Reactions were done in a Veriti Thermal Cycler (Applied Biosystems, 96 Well Model 9902). PCR products were purified using 10 U Exonuclease I (Thermo Fisher Scientific, Waltham, MA, United States) and 0.5 U Fast Alkaline Phosphatase (Thermo Fisher Scientific, Waltham, MA, United States) and incubated at 37°C for 15 min followed by an inactivation step at 85°C for 15 min (Werle et al., 1994 (link)). Macrogen Europe (The Netherlands) sequenced the gene fragments using the same primer used for the PCR amplification.

Young leaf pieces were pooled in 2ml tubes containing two clean steel balls. Tubes were rapidly frozen in liquid nitrogen and grinded using a tissue-lyser (Retsch). Genomic DNA from varieties of the mini-panel was extracted using a modified CTAB and chloroform:isoamyl alcohol method (Doyle and Doyle, 1987 ). The DNA pellet was resuspended in ddH₂O and quantified using a NanoPhotometer Pearl (Implen). About 50–150ng of gDNA was used in each PCR reaction in a final volume of 13 µl. Primers were designed in order to sequence the coding regions (CDS) of Hd1, PRR37, Ghd7, Ghd8 and COL4. The components were as follows: 1.3 µl of 10X PCR buffer, 2 µl of 5M Betaine or 1 µl of 30% DMSO, 0.2 µl of 10mM dNTPs, 0.25 µl of each primer (1 μg/μl, diluted 1:10), 0.2 µl Taq polymerase, and ddH₂O to volume. For the amplification of fragments with high GC content, LA Taq and GC Buffers (Takara) were used following manufacturer’s instructions. Amplification reactions were run for 40 cycles. The PCR products were incubated with Exonuclease I (Thermo Scientific) and Fast Alkaline Phosphatase (Thermo Scientific) for 15min at 37°C. Enzymes were heat-deactivated for 15min at 85°C. Purified fragments were sequenced using specific primers.

To perform the AaAPK1 autophosphorylation assay, 3 µg of AaAPK1-HIS⁶ or the AtOST1-HIS⁶ protein was added into kinase assay buffer (20 mM Tris-HCl buffer, 100mMNaCl, 20 mM MgCl2, 2 mM DTT, with or without 10 mM of ATP) to a final volume of 25 µl. To analyse the phosphorylation of AabZIP1 by AaAPK1, 3 µg of the GST-AabZIP1 protein and 1 µg of the AaAPK1-HIS⁶ protein were simultaneously added to the kinase assay buffer to a final volume of 25 µl. Serving as the positive control, 3 µg GST-AtABF2 and 1 µg AtOST1-HIS⁶ were added to the same kinase assay buffer, to a final volume of 25 µl. For the alkaline phosphatase (AP) treatment, 1 µl of fast alkaline phosphatase (Thermo Fisher, Waltham, MA, USA) was added into the 25 µl kinase assay buffer. Samples were mixed gently and stored at 30 °C for 30 min. Samples were subsequently separated using 8% (w/v) SDS-PAGE, with 0.1 mM of MnCl₂ and 0.1 mM of Phos-tag (Wako, Tokyo, Japan), and the proteins were detected by Coomassie Blue staining.

Plasmid DNA was extracted from E. coli cells using the Wizard SV Plus Miniprep Kit (Promega) or the QIAprep Spin Miniprep Kit (Qiagen), following the manufacturer’s instructions. DNA was digested with FastDigest restriction enzymes (ThermoFisher) by incubating 0.5–1 µg DNA at 37°C for 1 hour, with 1× FastDigest buffer, 1 µL of the selected endonuclease, and 1 µL of Fast Alkaline Phosphatase (ThermoFisher), when necessary. DNA fragments were purified using the Wizard SV Plus Cleanup Kit (Promega), according to the manufacturer’s instructions. DNA ligations were performed by standard molecular biology techniques using T4 DNA Ligase (ThermoFisher). PCR reactions were performed using either Phusion DNA Polymerase for cloning purposes or DreamTaq Polymerase for colony screenings (both ThermoFisher), following the manufacturer’s instructions. For inverse PCR, one primer was 5′ phosphorylated using a T4 Polynucleotide Kinase (ThermoFisher) using recommended reaction conditions. After PCR amplification, 1 µL DpnI (NEB) was added to the PCR mix, which was incubated for 1 hour at 37°C followed by purification. Five microliters of the purified PCR product was used for ligation using T4 DNA Ligase followed by transformation.

To determine whether OsVQ13 was phosphorylated, we used Phos-tag^® SDS-PAGE (NARD Institute Ltd., Amagasaki, Japan). To achieve dephosphorylation, the beads were prepared in 50 µL of 1× alkaline phosphatase buffer (Thermo Fisher Scientific) and incubated with 2 units of fast alkaline phosphatase (Thermo Fisher Scientific) for 2 h. After incubation, the beads were then washed five times in 0.5 mL of extraction buffer, and the binding proteins were eluted with 2× SDS sample buffer.