Fig. 2.

Single T-DNA expresses RRS1-R-HF, RPS4-HA, and inducible wild-type AvrRps4 or AvrRps4 mutant variants. (A) Illustrative layout of the Super-ETI (SETI) construct. There are five individual expression units or Golden Gate Level 2 positional components listed, which are indicated as positions 1–5. Position 1, expression unit of the FastRed selection marker (Shimada et al., 2010). Positions 2 and 5, chimeric transactivator XVE (LexA-VP16-ER) and the corresponding LexA-inducible system to express AvrRps4 or its mutant variants under the control of β-estradiol (E2) treatment. Positions 3 and 4, full-length RRS1-R and RPS4 proteins with epitope tags His₆-Flag₃ and HA₆, respectively. All individual units used for construct assembly can be found in Supplementary Tables S2 and S3. (B) Protein accumulation of RRS1-R-HF (IB:Flag, black arrowhead) and RPS4-HA (IB:HA, white arrowhead) of SETI lines expressing AvrRps4 (SETI_WT) or mutant AvrRps4 KRVY-AAAA (SETI_KRVYmut). Seedlings were grown in liquid culture and induced with 50 μM E2 for 2 h at 7 days after germination (DAG). Ponceau staining of Rubisco large subunits was used as a loading control. (C) Seedling phenotype of the SETI Arabidopsis transgenic line at 14 DAG in GM medium containing mock (0.1% DMSO) or 50 μM E2. Col-0 was sown as control for the effect of E2 on seedling growth. Scale bar=0.5 cm. (D) Confocal images of SETI_WT, SETI_KRVYmut, SETI_eds1 root cells expressing AvrRps4–mNeon, and AvrRps4^KRVY-AAAA–mNeon induced by 50 μM E2 for 24 h. The mNeon channel shows nucleocytoplasmic localization of AvrRps4–mNeon and AvrRps4^KRVY-AAAA–mNeon. Bright field channel and a merged image of mNeon and the bright field channel are shown together. Scale bars=10 μm. (This figure is available in colour at JXB online.)