Fig 1.
Rhizoid cells treated with the microtubule-destabilizing drug oryzalin or the microtubule-stabilizing drug Taxol form wavy or branched tubular projections.
Z-maximum projection of propidium iodide-stained mature rhizoids grown on 0.1% DMSO (A), 0.1 μM oryzalin (B) or 3.3 μM Taxol (C), 0.75 μM oryzalin (D) and 5 μM Taxol (E).
Table 1.
Rhizoid phenotypes of T-DNA and UV mutant lines fall in two categories.
The mean values of rhizoid sinuosity and rhizoid diameter were measured in 10 mature rhizoids per genotype and are given +/- SD. Single or double stars indicate a Benjamini-Hochberg adjusted p-value from non-parametric Dunn test lower than 0.05 and 0.01 respectively. Phenotypes significantly different from wild type were highlighted to help visualize the independence of the large rhizoid and wavy rhizoid phenotypes.
Fig 2.
nek mutants develop wavy rhizoids.
A: Schematic representation of MpNEK gene model. UTR regions are represented in dark blue, CDS in light blue. The T-DNA insertion site in VJ72 is indicated with a red triangle. The Right Border position on the T-DNA is indicated with R. The early stop codon is indicated by a green star in UV3.4. B: Mean rhizoid 3D sinuosity from 10 mature rhizoids. Error bars indicate +/- SD. Single or double stars indicate a Benjamini-Hochberg adjusted p-value from non-parametric Dunn test lower than 0.05 and 0.01 respectively. C-H: Mature rhizoids of Tak-1 (C), Tak-2 (D), UV3.4 (E), VJ72 (F). The weakly wavy phenotype of ST47-6 (G) and strong wavy phenotype of ST47-6 (H). I: Phylogenetic tree inferred from full-protein alignment of the NEK orthogroup. The tree is rooted with NEK proteins of chlorophyte algae. Branch support is shown as p-value from SH test. MpNEK is highlighted in a green box. The fully deployed tree can be found in S4 Fig.
Table 2.
Co-segregation test between UV3.4, ST47-6 and VJ72.
Table 3.
Candidate mutations for UV3.4.
Table 4.
Co-segregation analysis of wavy rhizoid and hygromycin resistance phenotypes VJ72xWT F1 population.
HygR: hygromycin resistant. HygS: hygromycin sensitive.
Fig 3.
wdl gain-of-function and loss-of-function mutants develop wavy rhizoids.
A: Schematic representation of MpWDL gene model. UTR regions are represented in dark blue, CDS in light blue. The TPX2 domain spans 3 exons represented in brown. T-DNA insertion sites are indicated with red triangles and the location of right border orientation marked with R. The thin arrows on exon 9 represent the positions of the primers used for qPCR. B: Steady state level of expression of MpWDL normalized to MpEF1a. C: Mean rhizoid 3D sinuosity from 10 mature rhizoids. Error bars indicate +/- SD and two stars indicate a Benjamini-Hochberg adjusted p-value from non-parametric Dunn test lower than 0.01. D-I: Mature rhizoids of Tak-1 (D), Tak-2 (E), wdlGOF-1 (F), wdl-1 (G), wdl-2 (H) and wdl-3 (I). J-L: 2d old gemma of Tak-1 (J), wdl-2 (K) and wdl-2 complemented with proMpWDL::MpWDL-YFP. M: Phylogenetic tree inferred from alignment of the TPX2 domain of TPX2 domain-containing proteins. The tree is rooted with TPX2 proteins of chlorophyte algae. Branch support is shown as p-value from SH test. TPX2 proteins that belong to the MpWDL clade. MpWDL is highlighted in green. The fully deployed tree can be found in S5 Fig.
Table 5.
Co-segregation test between wdl-1, wdl-2, wdl-3 and wdl-4 mutant lines.
Fig 4.
MpWDL-YFP localizes to microtubule bundles of epidermal cells and rhizoids.
A: Dorsal epidermis of a plant transformed with the proMpWDL:MpWDL-YFP gene construct. The red star indicates a mature air pore and the inserted red box frames a developing air pore. B: Meristematic notch of a proMpWDL:MpWDL-YFP. The red arrow points to a mucilage papilla cell directly above the meristematic notch. The yellow arrow points to a phragmoplast, while the red inserted box frames mitotic microtubules during metaphase. C: YFP fluorescence in a midplane optical section (top) and cortical plane optical section (bottom) of a growing rhizoid of a plant transformed with proMpWDL:MpWDL-YFP. D: GFP and YFP fluorescence in a Z-maximum intensity projection of growing rhizoids of a plant transformed with proMpEF1a:MpGFP-MpTUB1 (top) and of a plant transformed with proMpWDL:MpWDL-YFP (bottom). The vertical white lines represent 10, 20 and 30 μm distance from the apex. E: Abundance of MpWDL-YFP signal normalized by GFP-MpTUB1 signal along the longitudinal axis of growing rhizoids (full grey circles, vertical axis on the right hand side). Raw MpWDL-YFP and GFP-MpTUB1 are plotted for reference (orange and blue hollow circles, respectively, vertical axis on the left hand side).
Fig 5.
Microtubules are less bundled and parallel in growing wdl-2 rhizoids.
GFP fluorescence in a Z-maximum intensity projection of a growing rhizoid from a plant transformed with proMpEF1a:GFP-MpTUB1 in wild type (A) and wdl-2 (B) growing rhizoids. Colour code corresponds to a 16 colour scale. C-D: Quantification of the skewness of the signal intensity distribution in pixels corresponding to microtubules (C) and microtubule parallelness (D) in a cortical section from growing rhizoids in wild type and wdl-2 mutants (n = 10). Asterisks indicate the p-value from a Mann-Whitney U test lower than 0.05.