Fig 1.
A) A schematic 2D model of ROP10, depicting the G domain, hypervariable domain cysteine residues, polybasic region (PBR), and C-terminal GC-CG box. B) A 3D model of ROP10, emphasizing the cysteines in the G and hypervariable domains. C) A close-up 3D model of the G-domain cysteine residues.
Fig 2.
Evidence for S-acylation of ROP10 by palmitic and stearic acids.
A) Initial standards of ethyl palmitate (retention time: 31 min) and ethyl stearate (retention time: 33 min) derivatives of palmitate and stearate formed by hydrogenation. B) ROP10 is S-acylated by both palmitic and stearic acids. C) Mock-Arabidopsis Col-0 non-transgenic plants protein extract.
Fig 3.
The subcellular localization of ROP10 in stably expressing Arabidopsis plants is contingent upon the integrity of its hypervariable domain.
A-T) Maximum projection confocal images of His6-GFP-ROP10 and mutants. Green (GFP), magenta (PI), and overlay of GFP and PI signals before (C, G, K, O, and S) and after plasmolysis (D, H, L, P, and T). Note the absence of intracellular GFP fluorescence and the detached cell wall-labeled plasma membrane following plasmolysis in ROP10 (A-D) and rop10C160S (Q-T). Observe the intracellular fluorescence in rop10Δ183-197 (E, G, and H), rop10Δ200-204 (I, K, and L), and rop10C199S C205S (M, O, and P) hypervariable domain mutants. Bars are 10 µm.
Fig 4.
The constitutively active ROP10 and its G domain cysteine mutants are localized in the plasma membrane in stably expressing Arabidopsis plants.
A-L) Maximum projection confocal images of His6-GFP-rop10CA and G-domain cysteine mutants. Green (GFP), magenta (PI), and overlay of GFP and PI signals before (C, G and K) and after plasmolysis (D, H and L). Note the absence of intracellular GFP fluorescence in the detached cell wall-labeled plasma membrane following plasmolysis. Observe the changes in cell structure in all the images. Bars are 10 µm.
Fig 5.
The hypervariable domain, G-domain cysteine C160, and activation status influence the interaction between ROP10 and the plasma membrane.
Protein immunoblots, labeled with anti-GFP antibodies, depict the total protein before fractionation (Tot) soluble (Cyt) TSM and TIM membrane fractions of non-transgenic Arabidopsis (mock) and transgenic Arabidopsis plants expressing His6-GFP-ROP10 and rop10 mutants. The bars adjacent to each blot represent the densitometry of the bands in each fraction, calculated as a percentage of the total. See Fig S1 in S2 File Supporting Information – raw images for the full immunoblots.
Fig 6.
FRAP beam size analysis of the interaction dynamics of ROP10 and rop10 mutants with the membrane.
For each ROP10 variant, the specific transgenic lines employed are identical to those designated in the legend to Fig 7. A) Average τ values obtained in FRAP measurements (for sample representative FRAP curves, see Fig S6 in S1 File Supporting Information). Bars are means ± SD of multiple independent measurements, each conducted on a different cell (the number of measurements is depicted within each bar). Asterisks indicate significant differences (**, p ≤ 0.01, ****, p ≤ 0.001; one way ANOVA and Tukey’s post-hoc test) between the τ values of the indicated pairs of ROP10 variants, comparing separately the τ values obtained with the 40x objective (B, black lines) and the 63x objective (A, blue lines; **,p ≤ 0.01, ****,p ≤ 0.001 derived by one way ANOVA and Tukey’s post-hoc test). Apart of the differences between ROP10 WT or rop10C160 and the mutants, there were significant differences between the τ(63x) values of ROP10CA and the double mutants rop10CAC23S and rop10CAC160S (C, red lines). The mobile fraction (Rf) values did not show significant differences between all ROP10 variants (see Fig S8 in S1 File Supporting Information). B) FRAP beam-size bootstrap analysis. The studies employed 40x or 63x objectives, focusing the laser beam to larger (40x) and smaller (63x) Gaussian spots, with a beam size ratio of ω2(40x)/ω2(63x) = 2.28 ± 0.15 (n = 59 independent measurements) [33]. A τ(40x)/τ(63x) ratio similar to 2.28 is obtained for FRAP by lateral diffusion, while a τ ratio of 1 indicates recovery by exchange. The SD values for the τ ratios and beam-size ratios were calculated from the τ values shown in panel A, using bootstrap analysis (1,000 bootstrap resampling values). While the τ ratio of ROP10 was not significantly different from the 2.28 beam-size ratio, indicating recovery by lateral diffusion, the τ ratios of rop10CA and rop10C160S significantly deviated towards 1, suggesting a significant contribution by exchange to the FRAP. On the other hand, mutation of C160 or C23 on the background of rop10CA (rop10CAC160S and rop10CAC23S) returned the τ ratio to values not significantly different from the 2.28 beam-size ratio, indicating that the additional mutations returned the exchange rates to much slower values than the lateral diffusion. Asterisks indicate significant differences between the τ(40x)/τ(63x) ratio of a given ROP10 variant and the 2.28 beam-size ratio (**, p ≤ 0.02; ***, p ≤ 0.002; Student’s two-tailed t-test, comparing each τ ratio to the beam-size ratio).
Fig 7.
The intact hypervariable domain and G domain cysteine C160 are crucial for ROP10’s influence on cellular structure.
A) lobe number and B) circularity. Error bars – SD. Letters above bars in all correspond to statistically significant difference (one-way ANOVA and Tukey’s HSD test, p ≤ 0.05). Representative images are show in Figs 3 and 4. Fifty cells were measured for each cell line. For each ROP10 variant, two cell lines were generated and tested. The results shown here are for one line expressing the variant, as both lines gave identical results (Fig S10 in S1 File).