Sequence analysis of OsAP79
The gene Os10g39300 has one exon and is predicted to encode a hydrolytic AP. The length of the coding sequence (CDS) is 1,185 bp, encoding 394 amino acids (AAs). Its deduced protein is predicted to have a signal peptide at AA 20 and 21, and analysis with PROSITE found that it has two active sites at AA 69 and 267.
To classify Os10g39300 among the AP genes, 96 rice AP protein sequences were selected to generate a phylogenetic tree using the neighbor-joining method of MEGA 4.0, as described by Chen et al. (2009). As a result, they were classified into six groups, among which groups A and D were the largest, while the other four groups contained only two to seven genes (Figure 1b). The sequence alignment analysis found that the protein encoded by Os10g39300 derived from rice lines RBPH16 and OsAP79 had the highest homologous similarity (bootstrap value up to 92). Therefore, Os10g39300 was annotated as OsAP79 . It belongs to category A, which usually has only one or no intron. In addition, OsAP10 , OsAP30 , OsAP63 , OsAP71 ,OsAP78 , OsAP79 , and OsAP81 were placed into the same class and had higher expression levels in the roots than in other tissues or organs (Chen et al. 2009).
To detect the genomic sequence of OsAP79 in different BPH resistant and susceptible rice varieties/lines, it was amplified and sequenced from four indica resistant varieties, RBPH16, PTB33, RBPH327, and ARC5984; one indica susceptible variety, 9311; and three japonica susceptible varieties, NIP, ZH11, and BaiMao. No differences were detected in the CDS of the four resistant strains and 9311 (Figure S1). However, nine single nucleotide polymorphisms (SNPs) of CDS and four AAs differed between indica and japonicarice varieties/lines (Figure S2). Furthermore, a 204-bp insertion was only detected in the promoter of all sequenced indica lines (RBPH16, PTB33, RBPH327, ARC5984, and 9311) where an auxin-responsive element (AuxRE) was present (Figure S3).
OsAP79 transgenic line development
To verify that OsAP79 is involved in rice resistance to BPH, an OE vector and a knockout pYLCRISPR/Cas9 vector of OsAP79 were generated and transformed into the rice variety NIP. Consequently, 24 OE transgenic seedlings of the T0 generation were generated and quantified by qRT-PCR using ubiquitin as the endogenous reference gene. All the checked seedlings were positive, and most of them (16/24) had one copy of the target gene. Four and two seedlings had four and three copies, respectively (Table S2). The other two seedlings had seven copies each.
In addition, 21 knockout transgenic seedlings of the T0generation were developed using the CRISPR/Cas9 technique, and 19 (90.5%) seedlings were successfully edited at the target sites verified by sequencing (Table S3). Compared with the wild type (WT), three types of mutations, homozygous, monoallelic and biallelic heterozygous, were detected in the T0 generation. Specifically, the first target site had 1 homozygous, 18 allelic heterozygous, and 2 WT plants, and the second had 6 homozygous and 15 WT plants. Thus, the mutation types, which included the deletion, insertion, or substitution of at least one nucleotide, were achieved. Among these, the 1 bp deletion type ranked first with 61.9% at the first target, and 28.6% at the second target (Figure S4a).