Taq plus master mix

About 2 ng of the pre-amplified cDNA of each cell was further amplified using 2 × Taq Plus Master Mix (Vazyme, Cat. P212), and then cloned into T vector (Transgen, Cat. CT111-01). Next, the ligated plasmid transferred into Trans5α chemically competent cell (Transgen, Cat. CD201-01) by heat shock. The M13 primer were used to identify positive clones inserted with cDNA fragments. Single clones of bacteria were collected for Sanger sequencing to identify the barcode sequence of each cell.

To obtain transgenic Arabidopsis thaliana lines, the exonic region of GmGSTU23 was amplified using gene-specific primers with XbaI and SacI sites (Table S2). GmGSTU23 was then linearly cloned into the pTF101.1 binary vector with a modified CaMV 35S promoter, which contained a phosphinothricin acetyltransferase (bar) resistance gene (phosphinothricin N-acetyltransferase, PAT) as selection marker for the transgenic line. The constructed vector was transformed into Agrobacterium strain GV3101 by electroporation (Gene Pulser Xcell™ Electroporation Systems, Hercules,CA, USA), and the target gene was transferred into the A. thaliana ecotype Columbia (Col-0) by flower dipping [89 (link)]. The T₀ generation of A. thaliana was identified by overexpression by herbicide (Liberty^®, Bayer, Leverkusen, Germany) spraying and PCR molecular identification. Molecular identification of the T₃ transgenic line was performed using the 2 × Taq Plus Master Mix (Vazyme, Nanjing, China) according to the manufacturer’s instructions. The specific primers used for PCR were designed based on the vector and the gene sequence (Table S2).

Bacterial genomic DNA was extracted using a TIANamp Bacteria DNA Kit (TIANGEN, Beijing, China). Small-scale plasmid DNA preparations were generated using a E.Z.N.A™ Plasmid Miniprep Kit (Omega Bio-Tek, Norcross, GA, USA). The DNA fragments were amplified in a C1000™ Thermal Cycler using the 2×Taq Plus Master Mix (Vazyme, Jiangsu Sheng, China) or I-5^TM 2 ×High Fidelity Master Mix (MCLAB, South San Francisco, CA, USA). Purification of DNA fragments from the PCR reaction and the restriction digests were performed using the DNA Fragment Purification Kit Ver.4.0 (TaKaRa, Shiga, Japan). In-fusion segments were recombined to linearized pK18mobsacB (EcoRI/BamHI) using a ClonExpress II One Step Cloning Kit (Vazyme, Jiangsu Sheng, China). Restriction enzymes were purchased from TaKaRa. Point mutations were performed according to manual of Fast mutagenesis system Kit (TransGen Biotech, Beijing, China).

The genomic DNA of each knockout cell line was extracted with a GeneJET Genomic DNA purification kit (Thermo Fisher Scientific). Primers were designed at 200 nt upstream and downstream of the cutting site on CDC20 exons. 200 ng of genomic DNA was used as a template for the amplification of the interested region by 2× Taq Plus Master Mix (Vazyme). Amplified genomic DNA was further cloned into a pTA2 vector according to the manufacturer’s instructions (TArget Clone, Toyobo). Six clones were picked and sequenced for each cell line.

The 8 TDTS genes were synthesized by GenScript Biotech (Shanghai, China). The other 15 TDTS genes were amplified from the gDNA of corresponding wild strains with primers shown in Tables S3 and S4. PCR reactions were performed with the Q5^® High-Fidelity DNA Polymerase (NEB biotech, Ipswich, MA, USA). pUARA4 was digested utilizing KpnI (NEB biotech). The ClonExpress^®II One Step Cloning Kit (Vazyme Biotech, Nanjing, China) was used to construct expression plasmids by inserting the PCR product of different TDTS genes into the KpnI restriction sites of pUARA4 through homologous recombination to produce heterologous expression plasmids, pUARA4-TDTS. The single clones were manually picked out and utilized as the template for PCR using 2 × Taq Plus Master Mix (Vazyme) with the primers listed in Table S4. Positive clones were sequenced by Tsingke Biotech to confirm the gene was inserted into the right restriction site with no mutations. Plasmid DNA was extracted using the Plasmid Mini Kit I (OMEGA, Norcross, GA, USA). The active TDTS sequences were deposited in the National Center for Biotechnology Information under accession number PP516626-35. The 13 inactive TDTS sequences deposited in the National Center for Biotechnology Information under accession number PP776614-26

The obtained D. citri-like isolates were confirmed based on their colony morphology, microscopic conidia features, and molecular characteristics [2 (link)]. Specifically, we used D. citri-specific primer pair Dc-F/Dc-R (5′-CCCTCGAGGCATCATTAC-3′/5′-ATGTTGCAGATGGTCAAATGG-3′) designed based on sequence variations in the Tub gene among 21 Diaporthe species from citrus [2 (link)]. Each PCR reaction was conducted in a final volume of 20 µL, containing 1 µL of template genomic DNA (~20 ng), 0.8 µL forward primer, 0.8 µL reverse primer, 10 µL of 2 × Taq Plus Master Mix (Vazyme Biotechnology Co. Hangzhou), and 7.4 µL sterile water. PCR cycling condition was performed as follows: 94 °C for 3 min, 31 cycles of 94 °C for 30 s, 60 °C for 30 s, 72 °C for 1 min, with a final extension of 3 min at 72 °C. The PCR products were analyzed by electrophoresis at 120 V for 30 min in a 1% (w/v) agarose gel and visualized under UV light after ethidium bromide staining. Genomic DNAs from two strains (ZJUD2 and ZJUD14), which were identified as D. citri [2 (link)], were used as positive controls. The genomic DNAs of Diaporthe citriasiana strain ZJUD31 and D. citrichinensis strain ZJUD40 [2 (link)] were used as negative controls. These four reference isolates are stored at the Institute of Biotechnology at Zhejiang University (Hangzhou, Zhejiang Province, China).

Total RNA was extracted using RNAiso Plus Reagent (Takara, 9109) according to the manufacturer’s protocol. cDNA was synthesized using the M-MLV reverse transcriptase (Vazyme, CHN). PCR reactions were conducted using the 2 × Taq Plus Master Mix (Vazyme, CHN) and set at an initial 95 °C for 3 min and then 22 (glyceraldehyde 3-phosphate dehydrogenase (GAPDH)) and 30 (TRIM39) cycles of 95 °C for 30 s, 56 °C for 30 s, 72 °C for 30 s, and a final extension at 72 °C for 5 min. The products were separated by agarose gel electrophoresis and visualized by ChemiDoc MP System (Bio-Rad, USA). GAPDH was used as an internal control. The primers used in the study are shown in Table S2.