Figure 4.
ES20 Directly Interacts with CESA6.
(A) to (F) ES20 interacts with CESA6, but not CESA6P595S, in a DARTS assay. Representative protein immunoblots of DARTS assays for YFP-CESA6 with ES20 (A), YFP-CESA6 with ampicillin (C), and YFP-CESA6P595S with ES20 (E). Quantitative analysis of DARTS assays for YFP-CESA6 with ES20 (B), YFP-CESA6 with ampicillin (D), and YFP-CESA6P595S with ES20 (F). ES20 protects YFP-CESA6, but not YFP-CESA6P595S from degradation by proteases.
(G) to (I) Central cytoplasmic domain of CESA6 interacts with ES20 and UDP-Glc in an MST assay. Purified GFP-tagged CESA6 central cytoplasmic domain (GFP-CESA6c) with a His-SUMO tag (see [G], lane 2). Thermophoresis binding curve showing a direct interaction between GFP-CESA6c and ES20 (H). Thermophoresis binding curve showing a direct interaction between GFP-CESA6c and UDP-Glc (I). FNorm, normalized fluorescence .
(J) to (L) Central cytoplasmic domain of CESA6P595S interacts with ES20 and UDP-Glc in an MST assay. Purified GFP-CESA6P595Sc with a His-SUMO tag (see [J], lane 2). Thermophoresis binding curve showing a direct interaction between GFP-CESA6P595Sc and ES20 (K). Thermophoresis binding curve showing a direct interaction between GFP-CESA6P595Sc and UDP-Glc (L). FNorm, normalized fluorescence .
(M) and (N) UDP-Glc treatment partially complemented the root swelling caused by ES20. Representative images of seedlings treated with DMSO (0.1%), ES20 (0.8 μM), UDP-Glc (1 mM), and ES20 (0.8 μM) + UDP-Glc (1 mM; see [M]). Quantification of root width (see [N]) at the elongation zone of seedlings with different treatments as shown in (M). The letters (a to c) in (N) indicate statistically significant differences (P < 0.05) determined by one-way analysis of variance tests followed by Tukey’s multiple comparison tests in different samples. Values are means ± sd, with n = 6 in (B), (D), and (F); n = 3 in (H), (I), (K), and (L); and n = 16 in (N). In (B), *, P < 0.05 and **, P < 0.01, by two-tailed Student’s t test. Bar in (M) = 100 μm.