Reaction buffer

Tissue samples were digested in 25 mM NaOH/0.2 mM EDTA at 65°C overnight in a water bath, and then neutralized with 40 mM Tris HCl (pH 5.0). Isolated genomic DNA was amplified to detect the wild-type (WT, Itln1⁺) or the targeted Itln1^trap allele using the following primers: Itln1^trap (5’-GAGATGGCGCAACGCAATTAATG-3’, 5’-GAAAGCTAAAGCTAAACCCTGGGTGG-3’) and Itln1⁺ (5’- AAGTCCTCTGATAGAGCAGTGCTTGC-3’, 5’- CAAGACCTGAAAGGCAGAAACAACC-3’). PCR: Briefly, sample DNA and primers were mixed with Taq DNA Polymerase, reaction buffer, and dNTP(s) according to the manufacturer’s protocol (New England Biolabs, Ipswich, MA). PCR reactions were as follows: 94°C for 5 min, 40 cycles [94°C 30 sec, 55°C 30 sec, and 72°C, 40 sec], and 72°C for 5 min. The PCR products were assessed by agarose gel electrophoresis, where the Itln1⁺ allele produces a 340 BP product, and the Itln1^trap allele produces a 439 BP product.

Root genomic DNA was also used for host plant genetic analysis. Sixteen previously described microsatellite loci [28 (link), 29 (link)] allowed observations of polymorphisms in M. sinensis and M. floridulus (Table S3). DNA was amplified by PCR cycling with an initial denaturation of 5 min at 95 °C, followed by 35 cycles of 1 min at 95 °C (denaturation), 1 min at a primer-specific annealing temperature, and 1 min at 72 °C (extension), with a final extension at 72 °C for 10 min. The reaction mixture (10 µl) contained 10 × reaction buffer (New England Biolabs, Beverly, MA), 2 mM MgSO₄, 0.125 µM dNTPs, 0.25 µM of each primer 0.5 U of Taq DNA polymerase (New England Biolabs) and 40 ng template DNA. Forward primers were then fluorescently labeled so that they could be used for automated genotyping. The PCR products were treated with poly(A) at 65 °C for 30 min, then diluted in ddH₂O if too concentrated and sized using the LIZ500 internal sizing standard on an ABI 3130xl automated DNA sequencer with GENEMAPPER V4.0 software (Applied Biosystems, Foster City, CA).

Eight microsatellites markers (SSR) were chosen: g1-K04, e1-020, e4-D03, e1-021, g2-J08, e3-B02, g2-G12, g2-L17 [21 (link)]. DNA extract (5 μL of 10 ng/μL) from each plant was added to 20 μL of a master mix [2.5 μL 10× reaction buffer (NEB), 0.5 μL 25 mM dNTPs (NEB), 0.5 μL 10 mM Primer 1, 0.5 μL 10 mM Primer 2, 0.15 μL 5 U/μL Taq DNA polymerase (NEB), and 15.85 μL UHQ water]. Forward primers are 6-FAM-labeled at the 5 end PCRs were conducted with a 2720 Thermal Cycler (Applied Biosystems, Waltham, MA, USA), with the following program: one cycle of 5 min at 95 °C, 35 cycles of 30 s at 95 °C, 30 s at 52 °C, 1 min at 68 °C, and a final extension step of 10 min at 68 °C. PCR products were screened by capillary electrophoresis using the ABI 3730 XL (Applied Biosystems) of the GENTYANE Genotyping and Sequencing platform (Clermond-Ferrand, France). Electrophoregrams were analyzed with the Gene-Mapper software (Applied Biosytems). A phylogenic Tree was drawn using the DARwin software, with the Unweighted Neighbor joining method.

The synthesized crRNAs were diluted to 10 μM with nuclease-free water. Diluted crRNAs were pre-incubated with AsCas12a proteins (IDT, 1081069) or LbCas12a proteins (IDT, 10007922) to form the ribonucleoproteins (RNPs) in the 1 × NEB 2.0 buffer using a 1:1.2 ratio of Cas12a:crRNA. The final concentration of RNP was designed to 1 μM. After 15 min incubation at room temperature, the RNPs were mixed with 1 μL 10 × Reaction Buffer (NEB 2.0), 1 μg purified plasmid (Supplementary Data File 1), 10 U NdeI restriction enzyme, and added with nuclease-free water up to 10 μL. The reaction was incubated at 37 °C for 30 min. The digestion products were analyzed by running 1% agarose gel. For kinetics study, 10 nM dsDNA substrate was incubated with various amount of A/Z-AsCas12a RNPs (20 nM, 40 nM, 100 nM, and 500 nM). Cleavage reactions were sampled at distinct time points (10 s, 20 s, 60 s, 100 s, and 600 s), and substrate cleavage was assessed through capillary electrophoresis (Agilent).

Twenty-four INDELs larger than 28 bp were selected for the development of genome-wide INDEL markers: one in the middle of the short arm (p) and one in the middle of the long arm (q) for each chromosome. The forward and reverse primers targeting these INDEL-carrying sequences were designed using the generic primer interface of BatchPrimer3 (https://probes.pw.usda.gov/batchprimer3/). The criteria were as follows: product size 100–300 bp, primer size 18–27 bp, GC content 20–80%. The sequences of the reference genome [Oryza sativa (rice)] and all primer pairs were BLAST searched against NCBI sequence database (https://blast.ncbi.nlm.nih.gov/Blast.cgi) to verify the PCR product in the reference.
The 24 pairs of primers were synthesized by IDT (Integrated DNA Technologies). PCRs were conducted in 25 μL reactions containing 2.5 μL 10X reaction buffer (NEB), 0.125 μl Taq (NEB), 0.5 μL of 25 mM dNTP (Amersco), 0.5 μL of 10 μM primers, 10 nanograms of SR86 and reference rice DNA and 20.375 μL nuclease-free water (NEB). PCR reactions were carried out as follows: 95 °C, 30 s; 30 cycles of (95 °C, 30 s; 52 °C, 30 s; 68 °C, 30 s) and 5 min extension at 68 °C. PCR products were analyzed with a Bioanalyzer 2100 according to the manufacturer’s instructions (Agilent).

Each reaction mixture contained 2 μg of recombinant GST-tagged TTP (wild type or mutants) served as substrates, 3 μl of 10X reaction buffer (New England Biolabs), 30 μM of ATP, and kinases including ERK2 (New England Biolabs), p38 alpha (SignalChem), RSK1 (SignalChem), and MK2 (SignalChem) in a final volume of 30 μl. The kinases can be from cell extracts. 300 μg of LPS-treated RAW264.7 whole cell extracts were incubated with GSH-Sepharose bound 2 μg of GST-TTP in the buffer containing 20 mM HEPES, pH 7.7, 75 mM NaCl, 0.5 mM MgCl₂, 0.1 mM EDTA, 0.05% Triton X-100, 0.5 mM DTT, 20 mM β-glycerolphosphate, 0.1 mM Na₃VO₄, 1 μg/ml leupeptin, 1 μg/ml pepstatin A, 100 μg/ml PMSF. The mixture was rotated at 4 °C for 3 h and pelleted by centrifugation at 10,000×g for 20 s. After 4 × 1-ml washes in HEPES binding buffer (20 mM HEPES, pH 7.7, 50 mM NaCl, 2.5 mM MgCl₂, 0.1 mM EDTA, 0.05% Triton X-100), the beads were resuspended in 30 μl for kinase assay. The reaction mixtures were incubated at 30 °C for 30 min and stopped by adding one volume of protein sample buffer. Samples were subjected to SDS-PAGE for western blotting with anti-phospho-S316 and ponceau S staining.