Evaluation of BPH resistance in transgenic lines
To explore the role of OsAP79in BPH resistance, we evaluated BPH resistance in OE and knockout lines. A total of 15 OE T0 lines were surveyed for BPH resistance, and 12 were found to have higher plant survival rates than the WT (Figure S5a). Nine lines with the highest survival rates were used to evaluate their expression, and two lines, OE79-9 and OE79-12, were selected for further study (Figure S5b). This enhanced resistance to BPH with increased expression of the selected OE lines was further confirmed in the T1 and T2 families. The T1 lines of OE79-9 and OE79-12 showed enhanced resistance to BPH, with survival rates of 82.2% and 74.7%, respectively; however, it was only 32.9% for NIP (Figure S5c). The derived T2 lines also had significantly higher survival rates than the NIP lines (Figure 2a and 2c). Simultaneously, 20 knockout lines of T0 were assayed using the BPH resistance test (Figure S5d). The plant survival rates of all tested lines were lower than that of NIP (60%). Therefore, two independent homozygous lines (CR79-11 and CR79-18), containing frameshift mutations with 2 bp deletions or 1 bp insertions in the coding region, were selected for further phenotypic analysis (Figure S4b). The selected lines were detected to be without the CRISPR-Cas9 vector in the T2 generation. After inoculation with BPH at the seedling stage, the two lines exhibited enhanced susceptibility to BPH with survival rates of 10.4% and 9.5%, respectively, compared with that of NIP, which was 55.1% (Figure 2a and 2c). The same phenomenon was detected when the adult plants of the OE and knockout lines were surveyed for BPH resistance. After they were treated with BPH for 21 days, the plant survival rates were 84.5% and 86.0% for OE79-9 and OE79-12, respectively; however, they were 16.2% and 14.7% for CR79-11 and CR79-18, respectively (Figure 2b and 2d).
To further evaluate BPH resistance in OE and knockout lines, the BPH weight gain and honeydew excretion weight were surveyed. Consequently, the average BPH weight gain on OE79-9, OE79-12, CR79-11, and CR79-18 plants was -0.26 mg, 0.08 mg, 1.25 mg, and 1.48 mg, respectively, which was significantly different from that of NIP (0.63 mg) after 48 h of infestation (Figure 2e). The same result was obtained for the quantity of BPH honeydew excretion. After 48 h of treatment, there were 0.58 mg, 3.96 mg, 15.26 mg, 23.59 mg, and 7.8 mg on the lines of OE79-9, OE79-12, CR79-11, CR79-18, and NIP, respectively (Figure 2f). Taken together, these results demonstrate thatOsAP79 functions in an NIP genetic background and confers significantly positive resistance to BPH.