A Fully Functional ROP Fluorescent Fusion Protein Reveals Roles for This GTPase in Subcellular and Tissue-Level Patterning.

Rho of Plants (ROPs) are GTPases that regulate polarity and patterned wall deposition in plants. As these small, globular proteins have many interactors, it has been difficult to ensure that methods to visualize ROP in live cells do not affect ROP function. Here, motivated by work in fission yeast (Schizosaccharomyces pombe), we generated a fluorescent moss (Physcomitrium [Physcomitrella] patens) ROP4 fusion protein by inserting mNeonGreen after Gly-134. Plants harboring tagged ROP4 and no other ROP genes were phenotypically normal. Plants lacking all four ROP genes comprised an unpatterned clump of spherical cells that were unable to form gametophores, demonstrating that ROP is essentially for spatial patterning at the cellular and tissue levels. The functional ROP fusion protein formed a steep gradient at the apical plasma membranes of growing tip cells. ROP also predicted the site of branch formation in the apical cell at the onset of mitosis, which occurs one to two cell cycles before a branch cell emerges. While fluorescence recovery after photobleaching studies demonstrated that ROP dynamics do not depend on the cytoskeleton, acute depolymerization of the cytoskeleton removed ROP from the membrane only in recently divided cells, pointing to a feedback mechanism between the cell cycle, cytoskeleton, and ROP.

Root hairs are important for nutrient uptake (Gilroy and Jones, 2000), while pollen tubes 12 are critical for sexual reproduction (Chen et al., 2018). In non-flowering plants, polarized 13 cell expansion, also known as tip growth, generates protonemata and rhizoids. 14 Protonemata in mosses establish the plant, as this is the tissue that emerges from the 15 spore, while rhizoids help to anchor the tissue to the soil in both mosses and liverworts 16 (Rounds and Bezanilla, 2013;Shimamura, 2016). controlling polarized growth (Burkart et al., 2015). 45 Like the majority of small G-proteins, many ROPs (including all four in P. patens) 46 possess a C-terminal CAAX motif that is important for membrane targeting (Yang, 2002;47 Yalovsky, 2015;Lavy et al., 2002). The cysteine in the CAAX motif serves as a site for 48 prenylation, creating a lipid anchor that can insert into the plasma membrane (Geyer 49 and Wittinghofer, 1997). ROPs also bind to numerous effectors and are regulated by 50 many interacting proteins. For example, guanine exchange factors (GEFs) trigger ROP 51 to release GDP and bind to GTP. GTPase-activating proteins (GAPs) bind to GTP-ROP 52 and activate its intrinsic GTPase activity. Guanine dissociation inhibitors (GDI) bind to 53 GDP-ROP, interacting with the covalent lipid modification, thereby removing GDP-ROP 54 from the plasma membrane. These proteins interact with ROP directly to precisely 55 regulate its activation both spatially and temporally (Feiguelman et  on the cytoskeleton. We discovered that ROP polarization in growing cells is affected by 127 changes in the cytoskeleton in a cell cycle-dependent manner, revealing a potential 128 feedback mechanism between growth, polarity, and the cell cycle. 129

N-terminally tagged ROP4 is not functional 132
Previously, homologous recombination was used to replace the genomic region 133 containing the ROP4 open reading frame with either a 5' GFP-tagged cDNA or an 134 untagged cDNA (Burkart et al., 2015). ROP4 was chosen because it encodes the same 135 protein as ROP1. Furthermore, ROP4 and ROP3 exhibit similar expression levels and 136 are the two most highly expressed ROP genes in protonemata. Even though the GFP-137 tagged protein was expressed, neither the GFP-tagged nor the untagged cDNA was 138 able to replace the function of the wild-type gene, suggesting that the genomic context 139 of the coding sequence is critical for ROP4 function. To minimally alter gene structure,  RNAi, we could specifically silence the other three ROP genes that are functionally 147 redundant with ROP4 in the tagged line (Burkart et al., 2015). 148 Transforming the stably tagged lines with an RNAi construct targeting the coding 149 sequence regions of all ROP genes (CDS RNAi) and the nuclear GFP:GUS reporter 150 resulted in plants lacking nuclear GFP that were composed of small spherical cells (Fig.  151 1A). Transformation with a construct that targets the 3' untranslated regions of all ROP 152 genes (UTR RNAi) and the nuclear GFP:GUS reporter should not affect ROP4 153 expression, since the targeting sequence has been deleted from the ROP4 gene. Thus, 154 we would predict that if the tagged ROP4 is functional, plants transformed with the UTR-155 RNAi construct should exhibit polarized growth. In contrast to this prediction, plants 156 transformed with the UTR RNAi construct were unpolarized and indistinguishable from 157 the CDS RNAi transformed plants (Fig. 1A). These data demonstrate that the N-158 terminally tagged ROP proteins are not functional, as neither can maintain polarized 159 growth in moss protonemata in the absence of all other ROP proteins. 160

161
Internally tagged ROP4 fully rescues polarized growth 162 Inspired by previous work in fission yeast (Bendezú et al., 2015) where the ROP 163 homolog CDC42 was functionally tagged in the middle of the protein (sandwich tag), we 164 set out to test whether a similar approach might work for ROP. The fission yeast CDC42 165 sandwich tag was inserted in a small stretch of amino acids after the α3' helix. As a 166 result, the fluorescent protein looped out of the protein away from interfaces known to 167 mediate interactions with CDC42. We identified a small stretch of non-conserved amino 168 acids in ROP4 at the analogous CDC42 insertion site and speculated that this region 169 may not be essential for ROP protein function. To test this notion, we generated a 170 sandwich tag for P. patens ROP4 by inserting the coding sequence of mNeonGreen 171 (mNG) after Glycine 134 (Supplemental Figure 1B). 172 Using CRISPR-mediated HDR, we introduced sequences encoding mNG into the 173 ROP4-∆3'UTR reporter line, rendering the sandwich-tagged ROP4 locus insensitive to 174 the UTR RNAi construct. Remarkably, in two independently generated sandwich-tagged 175 lines, silencing with the UTR RNAi construct resulted in polarized plants (Fig. 1B) 176 indistinguishable from the parental untagged line. Notably, plant size was reduced to the 177 same extent in sandwich-tagged and untagged control lines (Fig. 1C), and solidity, a 178 measure of plant polarity, was similar in all lines (Fig. 1D). These data demonstrate that 179 the untagged and sandwich-tagged ROP4 (hereafter referred to as ROP4-swmNG) 180 contribute equally to ROP function. As expected, silencing with the CDS RNAi construct 181 in all lines resulted in unpolarized plants composed of circular cells with no 182 developmental organization (Fig. 1B). The finding that the sandwich-tagged lines 183 transformed with the control RNAi constructs were smaller than the untagged control 184 parental line might reflect differences in expression and/or stability of the sandwich-185 tagged fusion protein due to the deletion of the 3'UTR. 186 To control for possible expression effects resulting from deletion of the 3' UTR 187 and to ensure that incomplete silencing of ROP did not account for the observed rescue 188 of polarized growth, we isolated several lines where the ROP4 locus with an intact 3' 189 UTR was tagged appropriately with swmNG and carried null mutations in ROP1, 2 and reached 5-10 µm back from the tip ( Fig. 2A). 229 To quantify the differences between the plasma membrane localizations of the N-230 terminal and sandwich fusion proteins, we drew a line on the plasma membrane of 231 medial sections of apical cells with normalized fluorescence. We obtained fluorescence 232 intensity profiles of the plasma membrane and measured the width of the peak at the 233 same intensity value for all cells (Fig. 2B). We discovered that GFP-ROP was spread 234 out over a larger area of the cell apex compared to ROP4-swmNG ( Fig. 2C), which is 235 consistent with the maximum projections of confocal Z-stacks (Fig. 1A). We also fit a 236 line to both the increase and decrease in fluorescence intensity and calculated the 237 absolute value of the slope (Fig. 2B). ROP4-swmNG had significantly larger slopes than 238 GFP-ROP (Fig. 2D), demonstrating that the sandwich tagged ROP also formed a 239 steeper gradient on the plasma membrane. Together, these data demonstrate that the 240 functional ROP4-swmNG localizes to a more restricted region of the cell apex than the 241 N-terminal fusion protein.  To investigate if ROP dynamics at the cell cortex depend on the cytoskeleton, we 303 used FRAP (fluorescence recovery after photobleaching) to examine ROP turnover in 304 control cells and in cells that maintained the apical ROP gradient in the absence of the 305 cytoskeleton. In the same 5-60 min time window after drug addition, cells that 306 maintained normal ROP localization were photobleached at the cell tip (Supplemental 307 Figure 3B) backgrounds, the ROP signal readily recovered within 40 seconds 309 (Supplemental Figure 3A, B black lines). Treatment with oryzalin (green lines) or 310 latrunculin B (magenta lines) did not cause substantial changes in the rate or level of 311 fluorescence recovery. Only latrunculin B treatment in ∆rop1/2/3, but not in wild type, 312 slightly decreased the rate of ROP recovery. As a whole, the FRAP data indicate that 313 ROP4-swmNG mobility is not dependent on microtubules or actin in the wild type 314 arrowheads). Similar to cells that naturally pause (Fig. 3A), these data demonstrate that 334 in cells that stop growing due to the disruption of actin, the ROP signal disappears from 335 the tip, suggesting that there is a feedback mechanism between growth and ROP 336 localization to help maintain ROP at the site of polarized growth.  their cells grew isotropically with no obvious branching pattern (Fig. 4, Supplemental  345 Movies 6, 7). While cell division still generated a flat cell plate between daughter cells, 346 the combination of subsequent isotropic expansion coupled with weak cell adhesion 347 resulted in daughter cells that eventually became loosely associated spheres (Fig. 4  348 orange arrowheads) Furthermore, the ∆rop1/2/3/4 mutants did not form gametophores. 349 We also isolated a triple mutant, ∆rop1/3/4 (Supplemental Figure 2), with an intact 350 Supplemental Movie 10). ROP was also found at the cell cortex across from the branch 376 site marking the site of emergence of a future second branch (Fig. 5A cyan arrows). 377 These data demonstrate that ROP predicts the site of branch formation long before cell 378 expansion occurs at the site. 379 Finally, we wondered whether ROP localization is associated with cell wall 380 patterning prior to expansion. We reasoned that areas with the weakest cell wall would 381 be the most sensitive to enzymatic digestion. Thus, we treated growing protonemal Here, we showed that the most commonly used fusion protein, an N-terminally tagged 413 ROP, is not functional, as it does not support growth when it is the only ROP present.  Here, we found that ROP localization to the cell apex depends on actin and 428 microtubules in cells that have just completed cell division. However, in cells that are 429 closer to cell division, the acute loss of either microtubules or actin (5-60 min) does not 430 affect ROP localization at the plasma membrane (Fig. 3). Additionally, our FRAP studies 431 showed that, similar to findings in root hairs (Molendijk, 2001), which are fully 432 differentiated, the turnover of ROP at the plasma membrane is largely independent of 433 the cytoskeleton. These results support the finding that in yeast, rather than the 434 cytoskeleton, differences in the diffusion rates of the active and inactive forms of G-  that site. Often when ROP localization is diffuse, the area of expansion is larger, but as 457 the expansion focuses and forms a tip, ROP localization also focuses, demonstrating 458 that membrane domains containing ROP strongly correlate with active growth sites (Fig.  459 3). A reduction in cytoplasmic microtubule levels disrupts growth directionality, but the 460 actual growth machinery, which depends on actin, is not inhibited. 461 During branch formation, a physiologically relevant growth initiation process, we 462 found that ROP marks the future expansion site several hours before tip growth occurs 463 (Fig. 5A, Supplemental Movie 10). In fact, based on this timing, ROP is recruited to the 464 cell cortex in the apical cell just prior to mitosis, implying that branch patterning is 465 established one to two cell cycles before branch emergence. During division of the 466 apical cell, ROP moves from the cell cortex to the phragmoplast. After division is 467 complete, ROP again is enriched at the cortex adjacent to the new cell plate, but only 468 on the subapical cell side. Cell wall remodeling likely occurs here, as we discovered that 469 the cell wall is frequently weakest at these sites (Fig. 5B, Supplemental Movie 11).  Table 1) were used to amplify 800-1000 bp of genomic 504 sequence up-and downstream of the protospacer target site (5' and 3' homology arms). 505 The 2 homology arms were cloned into pDONR vectors and recombined with mEGFP 506 or 3XmEGFP (Vidali et al., 2009) into a plasmid backbone derived from pGEM-T Easy 507 (Promega), using a 3-way recombination reaction (Invitrogen) as described in (Mallett et  508 al., 2019). To generate the homology plasmid for the sandwich tag, primers DC773-778 509 were designed to amplify 5' and 3' homology arms. A fragment of the mNG coding 510 sequence was amplified by DC763,764. For the sandwich tag, we incorporated a linker 511 as described previously for CDC42 (Bendezú et al., 2015). The homology plasmid was 512 assembled by fusing the 5' homology arm, mNG, and the 3' homology arm into a 513 plasmid backbone derived from pGEM-T Easy (Promega), using NEB HiFi assembly 514 (New England Biolabs). Because the protospacer-targeted sequence was present in the 515 homology plasmid, we introduced a point mutation at the third nucleotide of the 516 protospacer adjacent motif (PAM) sequence in the homology arm using primers 517

DC771,772. 518
To generate null mutations in ROP1, 2, and 3, we constructed a pMH-Cas9-gate 519 plasmid with three protospacers targeting the three genes specifically, according to 520 (Mallett et al., 2019). First, the three protospacers were synthesized as complimentary 521 oligos and annealed to each other. They were ligated into three different entry clones 522 and recombined into pMH-Cas9 using a 3-way recombination reaction (Invitrogen) to 523 create pMH-∆ROP123. A plasmid with two protospacers targeting a single ROP1 at two 524 different positions (pMH-ROP1-ps23) was constructed in a similar manner. 525 Primers DC421-486 were used to genotype N-terminal tagged ROP4. 526 Genotyping of ROP4-swmNG was done using primers DC634,635,767,768. For ROP 527 knockout plants, genotyping was done using competition PCR (Harayama and Riezman, 528 2017) with primers DC1195-1434 and DC1185,1186. We were able to isolate 529 transformed plants in which all three loci had incorporated the oligo sequences by HDR 530 at very low frequency. However, at high frequency, we isolated mutants with one or two 531 loci edited. To generate more mutants, we either transformed pMH-ROP123 again into 532 single or double mutants or pMH-ROP1-ps23 into lines with the ROP1 and 2 loci 533 mutated and selected plants with a deletion between the two protospacers. Hoagland's liquid medium. Wild type and ∆rop1/3/4 plants were homogenized before 600 loading as described in (Bascom et al., 2016). However, because ∆rop1/2/3/4 cells were 601 not tightly adhered to one another, no homogenization was required before loading.

FRAP assay 658
Actively growing apical cells were identified in a PDMS device using a Nikon A1R 659 laser-scanning confocal microscope with a 1.49 NA 60× oil immersion objective (Nikon). 660 A circular stimulation ROI with a diameter of 2.4 µm was placed at the apical plasma 661 membrane where the ROP signal was the strongest. The 408 nm laser at 50% power 662 was used for stimulation. Before stimulation, images were taken every 1 s for 5 s, 663 followed by stimulation for 1 s, and the imaging continued after stimulation every 1 s for 664 a total of 1.5 min. The mean fluorescence intensity of the ROP-swmNG signal was 665 measured within the stimulation ROI. For each cell, the mean intensity measurements 666 for the first five time points before stimulation were averaged to generate the reference 667 intensity, and the mean intensity value of every time point in the whole movie was 668 divided by the reference intensity value to create a normalized mean intensity. Tukey HSD test. P values smaller than 0.05 in the Tukey HSD test were reported as 678 significantly different groups (Supplemental Tables 2-5). To compare the quantification 679 of the tip gradient signal peak width and slope (Figure 2), Student t Test for unpaired 680 data with equal variance was used, and P values smaller than 0.05 were determined as 681 significant (Supplemental Tables 6-7).  Table 1. Primers used in this study. 696 Supplemental Table 2. One-way ANOVA for Figure 1C. 697 Supplemental Table 3. One-way ANOVA for Figure 1D. 698 Supplemental Table 4. One-way ANOVA for Figure 1F. 699 Supplemental Table 5. One-way ANOVA for Figure 1G.