Puc57

The recombinant plasmid puc57 containing an ipaH specific fragment was synthesized by Tsingke Biotechnology Co., Ltd.; the plasmid DNA was diluted ten folds in nuclease-free water to obtain a series of concentrations ranging from 1.29×10⁷ copies/μL-1.29×10⁰ copies/μL. The plasmid solution was used as the reaction template for basic RPA and RPA-LFD and stored at -20°C before use. The RPA-LFD reaction was carried out under the previously described optimal conditions.

The Streptococcus pyogenes Cas9 gene and Agrobacterium tumefaciens VirD1, VirD2 and VirE2 genes were codon optimized for plants and synthesized by Tsingke (Beijing, China). The fused Cas9-VirD2 gene (CvD) was transferred to vector pUC57 (Tsingke, Beijing, China) flanked by the CaMV35S promoter and NOS terminator. The sgRNA fragment was flanked by the Arabidopsis thaliana U6-1/ Oryza sativa U6c promoter upstream, and the poly-T terminator downstream. The sgRNA and CvD cassettes were placed in tandem on vector psgRNA-CvD. The CvD gene in psgRNA-CvD was replaced with the Cas9 gene to generate vector psgRNA-Cas9. The VirE2 gene was transferred to vector pUC flanked by the CaMV35S promoter and E9 terminator to generate vector pVirE2. The VirD1 gene was introduced into binary vector pBI121 (Tsingke, Beijing, China) by replacing the β-glucuronidase (gusA) gene coding sequence to generate vector pVirD1. The 35S-VirD1-NOS expression cassette was amplified using primers containing EcoRI restriction sites, and the resulting product was transferred to vector pVirE2 (linearized with EcoRI) to generate tandem vector pVirD1E1 containing constructs 35S-VirD1-NOS and 35S-VirE2-E9. The vectors were constructed using the Trelief SoSoo Cloning Kit (Beijing, China) and all primers are listed in Supplementary Table S1.