The elicitor-responsive gene for a GRAS family protein, CIGR2, suppresses cell death in rice inoculated with rice blast fungus via activation of a heat shock transcription factor, OsHsf23

Figures & data

Fig. 1. Transactivation of 5xGAL4-UAS::LUC reporter gene by GAL4-BD/CIGR2 in suspension-cultured rice cells.

Notes: (A) Structure of effector and reporter plasmids. (B) Transient LUC reporter assay in cultured cells of rice cv Nipponbare. The cDNA for GAL4-BD or CIGR2 fused with GAL4-BD was co-introduced with the reporter plasmid into rice cells. At 48 h, GUS activity per mg protein was measured, and presented as a relative value to the activity by GAL4-BD. An asterisk and error bars indicate significant difference in a t-test at p < 0.05 and SD, respectively.

Fig. 2. Activation of OsHsf23 by CIGR2.

Notes: (A) Leaves of sibling plants (T1) of a pTA7001/CIGR2 line (#7) were sprayed with DEX or ethanol (EtOH), total RNA was isolated according to the time course indicated at the top of the photograph and expression of OsHsf23 was monitored by RT-PCR. As a control, total RNA isolated from a vector control-line (#9) treated with DEX was processed in the same way. (B) Schematic presentation of effecter and reporter plasmid. (C) Transient GUS reporter assay in cultured cells of rice cv Nipponbare. Two kinds of effecter plasmid (EL2 or CIGR2) were co-introduced with the reporter plasmid into cultured cells of rice by particle bombardment. Horizontal axis indicates GUS activity at 48 h after injection. An asterisk and error bars indicate significant difference against the control effecter, EL2, in a t-test at p < 0.05 and SD, respectively.

Fig. 3. Ratio of granulated cells in the epidermis of leaf sheath of CIGR2-RNAi and OsHsf23-RNAi lines inoculated with an avirulent isolate of M. oryzae, P91-15B.

Notes: (A) Ratio of appressoria that induced granulation in the epidermal cells of CIGR2-RNAi at 48 hpi. Experiments were carried out two times using different lines of transgenic rice. Asterisks indicate the significant difference against non-transgenic rice (NT) in each experiment in Dunnett’s test at p < 0.05. #23 (underlined) indicates a null RNAi line. (B) Same as (A) except that OsHsf23-RNAi lines were tested. #20 (underlined) indicates a null RNAi line. Error bars indicate SD.

Fig. 4. Length of lesions formed on the leaves of CIGR2-RNAi lines.

Notes: The fourth leaves of intact seedlings of CIGR2-RNAi (A) and OsHsf23-RNAi (B) rice plants were spray-inoculated with an avirulent strain of M. oryzae (P91–15B) and lesions at 5 dpi were visualized by lacto-phenol/trypan blue staining. Ratios of lesion numbers whose longitudinal length were >1.0, 1.0–0.4 or <0.4 mm were indicated by black, gray, and white bars, respectively. #23 and #20 (underlined) indicate null RNAi lines of CIGR2 and OsHsf23, respectively.

Fig. 5. Leaf sheath assay of CIGR2-RNAi lines.

Notes: Leaf sheath of the lines were inoculated with an avirulent isolate of M. oryzae, P91-15B. Ratio of appressoria which penetrate ≧2, 1 or 0 cells of epidermis of leaf sheath was shown by black, gray, and white bars, respectively. Error bars indicate SD. No significant differences were found against results in non-transgenic rice (NT) in Dunnet’s test at p < 0.05. #23 (underlined) indicates a null RNAi line.