ROP genes are essential for polarized growth. (A) Gene models of the Physcomitrella patens ROP family are shown with exons indicated by boxes and introns by thin black lines. Coding and untranslated regions are denoted by thick and thin boxes, respectively. The lines underneath the gene models represent sequence regions that are targeted by ROP RNAi constructs. Solid lines indicate that the denoted sequence was used in the RNAi construct, whereas dashed lines indicate highly similar sequence regions that are targeted by the RNAi constructs ROP4cds and ROP-3′Utr. Scale bar is 500 bp. Representative chlorophyll autofluorescence images of 7-day-old NLS4 plants regenerated from protoplasts expressing the indicated RNAi constructs are shown below the gene models. Scale bar: 100 µm. (B) Quantification of plant area (dark gray) and solidity (light gray) for control RNAi (n=450 plants), ROP4cds (n=200 plants) and ROP-3′Utr (n=490 plants). The area is based on chlorophyll autofluorescence and is presented having been normalized to that of control plants. Solidity is defined as convex hull area divided by area. (C) Relative expression levels of the ROP genes normalized to that of UBIQUITIN10 in 8-day-old plants expressing control (dark gray) and ROP4cds (light gray) RNAi constructs. E is the amplification efficiency. Inset in C, the relative expression levels of ROP1 and ROP2 normalized to that of UBIQUITIN10. Error bars represent s.e.m.

ROP RNAi plants have a cell adhesion defect. (A) Micrographs of chlorophyll autofluorescence of 8-day-old NLS4 plants expressing control, ROP4cds or MyoXI RNAi constructs before and after mild sonication. Scale bar: 100 µm. (B) Frequency of whole (dark gray) and broken (light gray) plants after mild sonication of 30 8-day-old NLS4 plants expressing control, ROP4cds or MyoXI RNAi constructs.

Cell wall deposition is altered in ROP RNAi plants. (A) Micrographs of 7-day-old NLS4 plants expressing control, ROP4cds or MyoXI RNAi constructs that had been stained with Calcofluor, Aniline Blue and Fast Scarlet 4B or imaged with polarized light. Scale bars: 50 µm. (B) Fluorescence intensity of Calcofluor (CW, gray), Aniline Blue (AB, white) and Fast Scarlet 4B (FS, dark gray) staining in plants expressing the indicated RNAi constructs normalized to that of 7-day-old NLS4 plants expressing the control construct [n=60 (CW), n=15 (AB) and n=23 (FS) plants], ROP4cds [n=61 (CW), n=15 (AB), n=18 (FS) plants] and MyoXI [n=25 (CW), n=19 (AB), n=22 (FS) plants]. (C) Light retardance of cell walls based on polarized-light images using three external cell walls from eight to ten images for each RNAi construct. Error bars represent s.e.m., and the letters above the bars indicate statistical groups with α=0.05 using ANOVA.

A single ROP gene is sufficient for polarized growth. (A) Immunoblot using an antibody against A. thaliana ROP2 shows a large increase in ROP protein level in protoplasts overexpressing ROP4 or ROP3. Arrow indicates position where moss ROP proteins migrate, at ∼21 kDa. Upper band is a cross-reacting protein, demonstrating equal protein load on the gel. (B) Diagram illustrating the homologous-recombination-mediated replacement of the 3′UTR in the ROP4 locus targeted by the ROP-3′Utr RNAi construct, generating a stable RNAi-insensitive line. Exons are indicated in light gray, introns in black, untranslated regions in dark gray and the hygromycin-resistance cassette in white. Scale bar: 500 bp. Below, micrographs of chlorophyll autofluorescence of 7-day-old NLS4/ROP4Δ3′Utr plants regenerated from protoplasts expressing the indicated RNAi constructs. Scale bar: 100 µm. (C) Quantification of plant area (dark gray) and solidity (light gray) for control (n=75 plants), ROP4cds (n=75 plants) and ROP-3′Utr (n=75 plants). (D) Relative expression levels of ROP genes normalized to that of UBIQUITIN10 in 8-day-old NLS4/ROP4Δ3′Utr plants expressing control (dark gray) and ROP-3′Utr (light gray) RNAi constructs. E is the amplification efficiency. Error bars represent s.e.m. Inset in D, relative expression levels of ROP1 and ROP2 normalized to that of UBIQUITIN10.

ROP RNAi impacts on cortical actin dynamics and organization. (A) Confocal images of single cortical planes through 6-day-old NLS4/Lifeact–mEGFP plants expressing either control or ROP4cds RNAi constructs. (B) Eccentricity measurements for 6-day-old NLS4/Lifeact–mEGFP plants expressing either control (n=6 cells) or ROP4cds (n=9 cells) RNAi constructs as a measurement of actin filament order. Error bars represent s.e.m., and letters above bars indicate statistical groups with α=0.05 using ANOVA. (C) Spinning-disc confocal images of 6-day-old NLS4/Lifeact–mEGFP plants expressing either control or ROP4cds RNAi constructs. Cells were imaged at the cortical plane every second. Also see supplementary material Movie 1. (D) The correlation coefficient between two images was calculated at all possible temporal spacings (time interval) for time-lapse sequence acquisitions of 6-day-old NLS4/Lifeact–mEGFP plants expressing either control (n=6 cells) or ROP4cds (n=8 cells) RNAi constructs. Error bars represent s.e.m. Scale bars: 5 µm.