SUPPLEMENTARY DATA
Figure S1. The coding sequence (CDS) alignment of OsAP79derived from rice varieties/lines RBPH16, PTB33, RBPH327, ARC5984, 9311 and NIP.
Figure S2. The protein sequence alignment of OsAP79derived from rice varieties/lines RBPH16, PTB33, RBPH327, ARC5984, 9311 and NIP.
Figure S3. The promoter sequence alignment of OsAP79derived from rice varieties/lines RBPH16, PTB33, RBPH327, ARC5984, 9311, NIP, ZH11 and BaiMao.
Figure S4. (a) The number of OsAP79 knockout plants generated by CRISPR/Cas9 system. (b) The mutation information of two independent OsAP79 knockout lines. Arrows label the target sites. Red box indicated the 1bp deletion or 1bp insertion.
Figure S5. (a) Seedling survival rate of overexpressed (OE) T0 lines of OsAP79 . (b) Relative expression ofOsAP79 in OE T0 lines. (c) Seedling survival rate of OE T1 lines of OsAP79 . (d) Seedling survival rate of knockout T0 lines of OsAP79 . Asterisks in (a-d) represent significant differences based on one-way ANOVA and Tukey test (*, P < 0.05; **, P < 0.01).
Figure S6. Rice protoplasts were transfected with OsAP79-GFP. The transfected cells were examined under a confocal microscope. The images were then processed using ZEISS ZEN confocal microscope software. Overlapping fluorescence signals show that OsAP79 localizes to the endoplasmic reticulum (ER). Bright-field images are shown on the right. Scale bars, 10μm.
Figure S7. Principal component analysis (PCA) scores plots rice leaf sheath metabolites associated with BPH infestation. Metabolites of overexpression (OE) (a) or knockout (c) and Nipponbare (NIP) infested with BPH for 0 h (control), 48 h and 72 h. Metabolites of OE (b) or knockout (d) and NIP infested with BPH.
Figure S8. PLS-DA score plots and loading plots of rice leaf sheath and metabolites. Overexpression (OE) line of OsAP79 (a), knockout line of OsAP79 (c), and Nipponbare (NIP) (e). PLS-DA score plots obtained from metabolic profiles of leaf sheaths of OE line with BPH treatments for 0 h (control), 48 h and 72 h, symbols filled circles OE line with BPH treatments for 0 h, filled squares OE line with BPH treatments for 48 h, filled triangle OE line with BPH treatments for 72 h. Metabolites of PLS-DA loading plot OE line of OsAP79 (b), knockout line of OsAP79 (d) and NIP (f).
Figure S9. (a) Nipponbare (NIP), overexpression (OE) and knockout (CR) lines were applied to Xoc infestation. Scale bars, 5cm. (b) NIP, OE and CR lines were applied to Xoo infestation. Scale bars, 1cm. Lesion length detection after Xoc (c) andXoo (d) infestation. Asterisks in (c,d) represent significant differences based on one-way ANOVA and Tukey test (*, P< 0.05; **, P < 0.01).
Figure S10. (a) Seedling survival rate after cold tolerance treatment.. (b) Nipponbare (NIP), overexpression (OE) and knockout lines were treated at 5 °C for 5 days and recovered at room temperature (25 °C) for 7 days. Scale bars, 5cm.
Supplemental Table S1. Relative expression of aspartic protease (AP) genes detected in transcriptome after BPH treatment.
Supplemental Table S2 . Estimating the copy number of transgenes in overexpression (OE) plants by quantitative real-time PCR.
Supplemental Table S3 . DNA sequence alignment for wild type (WT) and mutant of knockout plants. Base deletion and insertion are represented with ‘-’ and ‘+’, respectively.
Supplemental Table S4 . Primers used in this study
Supplemental Table S5 . Relative peak area of identified metabolites in rice leaf shealth
Supplemental Table S6 . Metabolites relative peak area (mean ± SEM) measured by GC–MS from rice leaf sheath samples and the changes of metabolites