Skip to main content
. 2020 May 20;32(8):2639–2659. doi: 10.1105/tpc.19.00752

Figure 6.

Figure 6.

KAI2 and D14 Interact with SMXL Proteins through the D1M Domains.

(A) to (E) Y2H assays for KAI2 and D14 interactions with SMAX1 and SMXL7. KAI2 and D14 were fused to GAL4-BD. SMAX1, SMXL7, and their domains were fused to GAL4-AD. Yeast cells were co-transformed with KAI2/D14 and SMAX1 and SMXL7 (A), SMAX1 only (B), SMAX1 and its domains (C), SMXL7 and its domains (D), and both SMAX1 and SMXL7 domains (E). Serial 10-fold dilutions of yeast cultures were spotted onto selective growth medium that was supplemented with 10 μM GR24, 10 μM KARs, or 0.02% acetone control. Images show growth after 3 d at 30°C. -A, -Ade; -H, -His; -L, -Leu; -T, -Trp.

(F) and (G) Split-LUC complementation assay for interactions between D1M domains of SMAX1 and SMXL7 with KAI2 (F) and D14 (G). N. benthamiana leaves were transiently cotransformed with A. tumefaciens strains carrying cLUC, nLUC, or indicated fusions as well as a strain carrying an mCherry transgene as a transformation control. Luminescence was measured 15 min after treatment with 10 μM KAR2, 10 μM rac-GR24, or 0.02% (v/v) acetone control and normalized against mCherry fluorescence. n = 10 to 12 leaf discs. *P < 0.01, Mann–Whitney U test, comparisons to control treatment.

(H) Diagram of the pRATIO3212-SMXL7 ratiometric reporter system. The 35S promoter drives expression of a multicistronic transcript encoding an SMXL7-mScarlet-I fusion that is separately translated from the Venus fluorescent protein due to the *F2A peptide. Relative fluorescence from the SMXL7-mScarlet-I reporter and the Venus reference after transient expression of the SMXL7 ratiometric system in N. benthamiana is shown. Leaf discs were treated with 0.02% (v/v) acetone or 10 μM KAR1, KAR2, and rac-GR24 for 12 h before measurement. n = 5 to 6 leaf discs.

(I) Relative fluorescence after transient expression of pRATIO1212-SMXL7 and -SMXL7*S1167-593 in N. benthamiana and overnight treatment with 10 μM KAR1, 10 μM rac-GR24, or 0.01% (v/v) acetone control. SMXL7*S1167-593 is a chimera in which amino acids 183 to 618 of SMXL7 have been exchanged for amino acids 167 to 593 from SMAX1. n = 8 to 10 leaf discs. *P < 0.01, **P < 0.001, Mann–Whitney U test, comparisons to control treatment.