Generation of constructs and transformation
To generate the OE construct of Os10g39300 , the full-length cDNA was amplified from the resistant line RBPH16, and cloned into pCAMBIA1301A with the CaMV35S promoter.
To knockout gene Os10g39300 , two target sites (31–49 bp and 910–927 bp) were selected in the exon region to generate the construct using the CRISPR-Cas9 technique (http://skl.scau.edu.cn/). The gRNA target oligonucleotides were synthesized by the Beijing Genomics Institute. Secondary structures of both sgRNAs were developed using CRISPR-P (version 2.0; http://crispr.hzau.edu.cn/CRISPR2/). The annealing products of target sense and target antisense oligonucleotides of sgRNA1 and sgRNA2 were ligated with pYLgRNA-OsU6a and pYLgRNA-OsU6b, respectively, using specific primers. Both PCR products were combined and purified using the TaKaRa MiniBEST Purification Kit Ver.4.0. The expression cassette was pooled, and the restriction ligation reaction was set up using the pYLCRISPR/Cas9 plasmid. The constructs were individually transformed into Agrobacterium strain EHA105.