Fig 1.
Rescue of the trichome phenotype.
Representative pictures of rosette leaves of (A) Col-0, (B) spi-4, and (C) M20. Higher magnification REM pictures of (D) Col-0, (E) spi-4, and (F) M20. (G) Ten trichomes at the top half of ten leaves were analyzed for the number of twisted branches per trichome in Col-0, spi-4, and M20. The significance of the data was tested by a two-tailed T-test and is indicated by lowercase letters (p<0.001).
Fig 2.
Root hair phenotype of Col-0, spi-4, and the M20 line.
Light microscopy images of (A) Col-0, (B) spi-4, and (C) M20 roots grown on ½ MS agar plates. The scale bar displays 500 μm. (D) Barplot displaying root hair lengths of Col-0, spi-4, and M20. The significance of the data (n = 30 cells) was tested by a two-tailed T-test and is indicated by lowercase letters (p<0.001).
Fig 3.
The effect of salt stress on Col-0, spi-4, and the suppressor line M20.
Seeds of the respective lines were germinated on ½ MS medium. After visible germination, approx. 50 seeds were transferred to ½ MS plates (control) or ½ MS plates supplemented with 125 mM NaCl. Three independent technical replicates were analyzed, and green vs. bleached seedlings were counted after one week. (A) Representative pictures of treated plants; (B) Frequency of phenotypes. The significance of the data sets (n = 3 replicates) was tested by a Mann-Whitney-U test and is indicated by lowercase letters (p<0.1).
Fig 4.
Complementation of the suppressor line M20.
(A) REM picture of trichomes in the suppressor line M20 (spi-4 rabe1c). Expression of RABE1C from a 3.3 kb genomic fragment rescues the rabe1c mutation leading to the spi-4 phenotype (cf. Fig 1): (B) REM picture of a trichome showing the restored spi phenotype. The scale bar in (A) and (B) displays 100 μm. (C) Overview of trichomes on a young rosette leaf showing the restored spi phenotype. (D) Schematic representation of the RABE1C gene (At3g46060) and transcript in the M20 line. M20 RABE1C exhibits a G>A mutation at the border of intron two to exon three, which leads to an 18 bp deletion in the coding sequence after splicing. Sequencing was carried out by GATC/Eurofins, Ebersberg.
Fig 5.
Rescue of the twisted trichome phenotype of spi-4 with RABE1CS29N.
(A) Ten trichomes at the top half of ten leaves were analyzed for the number of twisted branches in spi-4 RABE1CS29N. Data for Col-0, spi-4, and M20 from Fig 1G were added for comparison. The significance of the data was tested by a two-tailed T-test and is indicated by lowercase letters (p<0.001). (B) Rosette leaf of spi-4 RABE1CS29N.
Fig 6.
Co-localization of RABE1C and RABE1CΔ51–56 with the Golgi marker G-rb.
(A) Transiently transformed Arabidopsis thaliana Col-0 cells. Top two rows from left to right: pUBI10:YFP-RABE1C, Golgi Marker G-rb, merged YFP and mCherry signals; bottom two rows from left to right: pUBI10:YFP- RABE1CΔ51–56, Golgi Marker G-rb, merged YFP and mCherry signals. The scale of the overview pictures (rows 1 and 3) displays 25 μm. The scales of the detailed pictures display 10 μm (row 2) and 5 μm (row 4). (B) Co-localization analysis. Co-localization was quantitatively analyzed using the Colocalization Threshold function in ImageJ. The Pearson coefficient r was determined for 10 cells. Depending on cell size, five to ten regions of interest were defined manually in areas that contained punctate structures and minimal diffuse fluorescence signals. One asterisk (*) indicates a significance level of p<0.05, three asterisks (***) indicate a significance level of p<0.001; significance was determined using a two-tailed T-test.
Fig 7.
Localization of RABE1C and RABE1CΔ51–56 co-expressed with CLC2-mCHERRY.
(A) Transiently transformed Arabidopsis thaliana Col-0 cells. Top two rows from left to right: pUBI10:YFP-RABE1C, CLC2:mCherry, merged YFP and mCherry signals; bottom two rows from left to right: pUBI10:YFP- RABE1CΔ51–56, CLC2:mCherry, merged YFP and mCherry signals. The scale of the overview pictures (rows 1 and 3) displays 25 μm. The scale of the detailed pictures displays 10 μm (row 2 and 4). (B) Boxplot comparing the density of punctate structures of RABE1C and RABE1CΔ51–56 when co-expressed with CLC2-mCherry. The means of the data are not significantly different in a two-tailed T-test at a significance level of p<0.05.
Fig 8.
Proteomic analysis of excretion in spi-4, its suppressor line M20, and Col-0.
(A) Principal component analysis of the proteomic results (n = 4). (B) Protein numbers and filter strategy for selecting candidate proteins showing significantly differential abundance in the excretions of spi-4 and Col-0, but not between Col-0 and the suppressor line M20 (FDR: 0.05). (C) Volcano plot of the filtered data set, visualizing significantly increased (red) and decreased (blue) candidate proteins (FDR: 0.05). (D) Distribution of subcellular localization of differentially excreted proteins. All depictions are based on data from S2 Table.