Endosidin20 Targets the Cellulose Synthase Catalytic Domain to Inhibit Cellulose Biosynthesis

Plant cellulose is synthesized by rosette-structured cellulose synthase (CESA) complexes (CSCs). Each CSC is composed of multiple subunits of CESAs representing three different isoforms. Individual CESA CESAs, the molecular mechanism through which CESA catalyzes cellulose synthesis and whether its catalytic activity influences efficient CSC transport at the subcellular level remain unknown. Here, by performing chemical genetic analyses, biochemical assays, structural modeling, and molecular docking, we demonstrate that ES20 targets the catalytic site of CESA6 in Arabidopsis thaliana . Chemical genetic analysis revealed important amino acids that potentially participate in the catalytic activity of plant CESA6, in addition to previously identified conserved motifs across kingdoms. Using high spatiotemporal resolution live-cell imaging, we found that inhibiting the catalytic activity of CESA6 by ES20 treatment reduced the efficiency of CSC transport to the plasma membrane. Our results demonstrate that ES20 is a chemical inhibitor of CESA activity and trafficking that represents a powerful tool for studying cellulose synthesis in plants. to , ES20 interacts with CESA6 but not CESA6 P595S in a Drug Affinity Responsive Target Stability (DARTS) assay. protein YFP-CESA6 ES20 with ES20


1!
Cellulose is a polymer of β-1,4-D-glucose that serves as an essential cell wall 2! component for controlling the directional growth of plant cells. Cellulose is synthesized 3! at the plasma membrane (PM) by a cellulose synthase complex (CSC) comprising a 25-4! nm diameter rosette of subunits in a hexagonal array that can be observed in numerous 5! plant cell types (Mueller et al., 1976;Giddings et al., 1980;Mueller and Brown, 1980). 6! Each CSC is predicted to contain at least 18 monomeric cellulose synthases (CESAs) 7! representing three different isoforms in a 1:1:1 molar ratio (Pear et al., 1996;Arioli et al., 8! 1998;Doblin et al., 2002;Persson et al., 2007;Fernandes et al., 2011;Newman et al., 9! 2013;Gonneau et al., 2014;Hill et al., 2014). Plant CESAs and cellulose synthases 10! from other kingdoms are Glycosyltransferase family 2 (GT2) proteins, which synthesize 11! β-1,4-glucan using UDP-glucose in the cytosol as substrate (Cantarel et al., 2009;12! Brown et al., 2012;Omadjela et al., 2013). 13! 14! GT2 proteins are thought to share a common GT-A catalytic fold that has been 15! observed in multiple GT2 family proteins (Charnock and Davies, 1999;Cantarel et al., 16! 2009). The conservation of key catalytic motifs between plant and bacterial cellulose 17! synthase allowed the first plant CESA to be cloned from cotton (Gossypium hirsutum) 18! (Pear et al., 1996). Cellulose synthases across kingdoms contain multiple 19! transmembrane domains and a cytoplasmic catalytic domain (McNamara et al., 2015). 20! High-resolution structural analysis of Rhodobacter sphaeroidesin cellulose synthase 21! (RsBcsA) revealed the detailed structural conformation of the catalytic site and showed 22! that cellulose synthase controls both the catalytic synthesis of cellulose and its 23! ! 3! translocation across the PM (Morgan et al., 2013). In silico prediction of the structure of 24! the central cytoplasmic domain of cotton CESA1 using bacterial glycosyltransferases 25! SpsA and K4CP as templates revealed that the catalytic site composition of plant CESA 26! is similar to that of bacterial cellulose synthase (Sethaphong et al., 2013;Slabaugh et 27! al., 2014a). In the crystal structure of RsBcsA and the predicted structure of the 28! cytoplasmic domain of cotton CESA1, the GT-A fold catalytic residues contain 29! conserved DDG, DXD, TED, and QXXRW motifs required for catalytic activity (Morgan 30! et al., 2013;Sethaphong et al., 2013). In RsBcsA, the amino acids at the 31! transmembrane helixes and the interfacial helixes (IF) form a channel that contacts and 32! facilitates the translocation of the glucan (Morgan et al., 2013). Thus, although atomic 33! resolution structures for CSCs and individual CESAs are not yet available, it is 34! reasonable to predict that plant CESAs use similar catalytic motifs for cellulose 35! synthesis and may use a similar glucan-translocating channel for cellulose chain 36! translocation of the PM. However, plant CESAs also contain a plant-conserved 37! sequence (PCR) and class-specific region (CSR) in the cytoplasmic domain that are not 38! present in bacterial cellulose synthases (Pear et al., 1996;Vergara and Carpita, 2001).

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Plant CESAs also form more complicated protein complexes than bacterial cellulose 40! synthase, pointing to possible specific mechanisms for plant cellulose synthesis. 41!

42!
Small molecule inhibitors that disrupt cellulose synthesis have proven useful for 43! understanding the molecular functions and dynamics of CSCs and for identifying new 44! genes involved in cellulose synthesis (Montezinos and Delmer, 1980;Heim et al., 1989;45! Scheible et al., 2001;Desprez et al., 2002;DeBolt et al., 2007;Brabham et al., 2014;46! Worden et al., 2015;Tateno et al., 2016;Tran et al., 2018). Unfortunately, the mode of 47! action of these inhibitors is not well characterized, limiting their value as investigative 48! tools. Here, we report the characterization of the small molecule Endosidin20 (ES20), 49! which inhibits cellulose synthesis by directly targeting Arabidopsis CESA6. We used 50! chemical genetic analyses, structural modeling, molecular docking and biochemical 51! ! 4! assays to show that ES20 targets CESA6 at the catalytic site. Furthermore, we 52! analyzed the cellular localization and trafficking dynamics of CSCs in ES20-treated 53! seedlings and found that CSC delivery to the PM is inhibited by ES20 treatment, which 54! is consistent with the previous finding that the catalytic site of CESA affects the efficient 55! subcellular transport of CSCs (Park et al., 2019). 56! 57!

58!
Endosidin20 inhibits cellulose synthesis 59! Endosidin20 (ES20) ( Figure 1A) was identified through a chemical library screen for 60! small molecules that are active in plants (Drakakaki et al., 2011). When grown in the 61! presence of ES20, roots of Arabidopsis wild type (ecotype Col-0) seedlings became 62! shorter and wider in an ES20 dose-dependent manner ( Figure 1B, 1C). When treated 63! with ES20 overnight, the root tip region was swollen and root elongation was markedly 64! inhibited compared to mock-treated roots ( Figure 1D, 1E). Epidermal cells from the root 65! elongation zone were markedly swollen after ES20 treatment, which was reflected by a 66! significantly decreased cell length and a significantly increased cell width ( Figure 1D-1F, 67! Supplemental Data Set 1). In addition to these root phenotypes, ES20 inhibited etiolated 68! hypocotyl growth in a dose-dependent manner (Supplemental Figure 1A, 1B) and 69! caused epidermal cell swelling (Supplemental Figure 1C-1E). Swollen plant cells and 70! organs are often caused by direct or indirect disruption of cell wall biosynthesis or 71! organization, such as those in CESA-deficient mutants and in wild-type plants treated 72! with inhibitors of cellulose synthesis or microtubule organization (Baskin et al., 1994;73! Arioli et al., 1998;Fagard et al., 2000;Burn et al., 2002;Desprez et al., 2002;Daras et 74! Supplemental Data Set 1). The increased sensitivity of prc1-1 to ES20 suggests that 120! ES20 could target CESA2 and CESA5, which function redundantly with CESA6 121! (Desprez et al., 2007), as well as other CESAs. We hypothesized that missense 122! mutations at conserved amino acids in other CESAs in Arabidopsis might lead to 123! reduced sensitivity to ES20. fra5 (fragile fiber 5) carries a missense mutation at Pro557 124! of Arabidopsis CESA7 (CESA7 P557T ) (Zhong et al., 2003), which is homologous to the 125! conserved Pro595 in CESA6. Another mutant, any1 (anisotropy1), carries a missense 126! mutation at Asp604 of Arabidopsis CESA1 (CESA1 D604N ) (Fujita et al., 2013), which is 127! homologous to the conserved Asp605 in CESA6. We obtained fra5 and any1 and tested 128! their responses to ES20 in growth assays. As reported previously, both fra5 and any1 129! have shorter roots than the wild type ( Figure 1J, 1K), suggesting that CESA7 and 130! CESA1 play a role in normal seedling growth, although CESA7 is thought to be mainly 131! involved in secondary cell wall synthesis. When a lower concentration of ES20 (0.5 µM) 132! was tested, the root growth of fra5 and any1 was inhibited by ES20 at a reduced level 133! compared to wild-type plants ( Figure 1J, 1K and that both the N-and C-termini are located in the cytoplasm (Supplemental Figure  145! 3). Predictions with PredictProtein revealed seven transmembrane regions, with the N-146! terminus of CESA6 located in the cytoplasm and the C-terminus located in the apoplast 147! ( Figure 2A). 148! 149! Next, we aligned the primary sequence of CESA6 with CESA1, CESA3, CESA7, and 150! RsBcsA. We found that 12 of the mutations led to missense mutations in amino acids 151!  Figure 4). Some of the 157! mutations occurred at amino acids that are part of, or very close to, the conserved 158! catalytic motifs. For example, Ser394 is adjacent to the DDG motif, Asp396 is part of the 159! DDG motif, Gly780 is two amino acids away from the TED motif, Thr783 is part of the 160! TED motif, Leu829 is one amino acid away from the QXXRW motif, and Ser818 is four 161! amino acids away from the QXXRW motif ( beyond the central cytoplasmic domain, but these two amino acids are located at the 166! IF3, which is located at the cytoplasm and facilitates glucan translocation in RsBcsA 167! ( Figure 2B) (Morgan et al., 2013). E929K and G935E are also close to the FXVTXK 168! motif, which is part of the gating loop that is also located at the cytoplasm in RsBcsA 169! ( Figure 2) (Morgan et al., 2013). The topology prediction using UniProt places the 170! FXVTXK motif in the apoplast, E929 in the small cytoplasmic loop between the 4 th and 171! the 5 th transmembrane regions, and G935 in the 5 th transmembrane region 172! (Supplemental Figure 3). The topology prediction using PredictProtein places the 173! FXVTXK motif, E929, and G935 in the cytoplasm ( Figure 2). Since the IF3 and the 174! gating loop containing the FXVTXK motif in RsBcsA are located in the cytoplasm, it is 175! more likely that the prediction by PredictProtein (Figure 2) reflects the real topology of 176! plant CESAs. None of the es20r mutations occurred in the PCR or CSR of CESA6.

177!
Altogether, the predicted locations of amino acids that are mutated in the es20r mutants 178! suggest that many of them might affect the catalytic process. 179!

180!
To understand how mutations at conserved amino acids in CESAs affect plant 181! sensitivity to ES20, we used a threading method to model the structure of the central 182! cytoplasmic domain of Arabidopsis CESA6 using the solved crystal structure of RsBcsA 183! as a guide (Morgan et al., 2013;Morgan et al., 2016). The modeled structure of the 184! cytoplasmic domain of CESA6 contains multiple α-helices and a β-sheet folded into a 185! globular structure with a central cavity ( Figure 3A, Supplemental Movie 1). We 186! evaluated the quality of the model with PROCHECK (Laskowski et al., 1993;Laskowski 187! et al., 1996). In the Ramachandran plot, which visualizes energetically allowed regions 188! ! 10! for backbone dihedral angles ψ against φ of amino acid residues, 67.2% of residues 189! were in the most favored regions and 23.4% of residues were in the additional allowed 190! regions. Ligand binding site prediction enabled by COACH identified UDP-glucose 191! phosphonate as a possible ligand for the modeled structure of CESA6 (Yang et al., 192! 2013a). Our modeled CESA6 cytoplasmic domain structure is very similar to that 193! predicted for the cotton CESA1 cytoplasmic domain, and the catalytic core conformation 194! is very similar to that of RsBcsA ( Figure 3A, Supplemental Movie 1) (Morgan et al., 195! 2013;Sethaphong et al., 2013). In our modeled structure, the DDG, DXD, TED, and 196! QLVRW motifs form the catalytic core around the UDP-glucose, and the PCR and CSR 197! extend away from the catalytic core ( Figure 3A, Supplemental Movie 1). We used a 198! molecular docking approach to predict possible binding sites for ES20 on the modeled 199! CESA6 central cytoplasmic domain. We found that ES20 and UDP-glucose 200! phosphonate were docked to the same catalytic core of the modeled CESA6 201! cytoplasmic domain ( Figure 3A, Supplemental Movie 1). 202!

203!
When we examined the 3-D positions of mutated amino acids identified in the es20r 204! mutants, we found that most of these amino acids were either directly located at or very 205! close to the predicted binding site for ES20 and UDP-glucose ( Figure 3A-3C, 206! Supplemental Movie 1, Supplemental Movie 2). After further analysis of the docking 207! results, we found that three amino acids, Ser360, Asp562, and Gln823, were close to 208! ES20 and that hydrogen bonds could form between ES20 and these amino acids 209! ( Figure 3D). The structural modeling and molecular docking data in combination with 210! ! 11! the chemical genetics results suggest that ES20 targets the catalytic sites of CESAs to 211! inhibit plant cellulose synthesis and cell growth. 212!

213!
To further validate our structural model and molecular docking data, we hypothesized 214! that if we mutated other amino acids in the predicted binding site, the plants should 215! have reduced sensitivity to ES20. We selected six amino acids that were located at the 216! predicted ES20 and UDP-glucose binding site on CESA6 and created six YFP-CESA6 217! genomic constructs that each carried a missense mutation in one of these six amino 218! acids ( Figure 2, Figure 3B, 3C, Supplemental Movie 2, amino acids colored blue). The 219! selected amino acids are part of the DXD, TED, and QXXRW motifs, and Asp562 and 220! Gln823 are predicted to be important for the interaction of ES20 with CESA6. We also 221! selected Leu365 and Asp395, which are not part of the predicted UDP-glucose and 222! ES20 binding site, and created YFP-CESA6 genomic constructs that each carried a 223! missense mutation in one of the two amino acids. We then used these constructs to 224! transform prc1-1 and obtained single insertion transgenic lines expressing each of the 225! mutated CESA6 containing a predicted missense mutation. 226!

227!
In the absence of ES20, transgenic plants expressing the wild-type CESA6 construct 228! had normal root and hypocotyl growth compared to the wild-type controls, whereas the 229! transgenic plants expressing the mutated CESA6 had different levels of growth defects, 230! depending on the mutation ( Figure 3E; Supplemental Figure 5A-5E). We analyzed YFP-231! CESA6 protein levels in transgenic lines expressing wild type or mutated YFP-CESA6 232! and found that the severity of growth defects was not correlated with the protein level 233! ! 12! (Supplemental Figure 5F, 5G). In the presence of ES20, transgenic plants expressing 234! wild-type CESA6 constructs had similar sensitivity to ES20 in terms of root and 235! hypocotyl growth compared to wild-type plants ( Figure  5A-5E), suggesting that D395 is required for cell growth but not for the inhibitory activity 251! of ES20. Asp395 and Asp396 are both part of the DDG motif ( Figure 2B). However, 252! Asp396 is within 4 Å of UDP-glucose, and D395 is not within 4 Å of UDP-glucose based 253! on our molecular docking analysis (Supplemental Movie 1). It is interesting that D396N 254! causes reduced sensitivity to ES20 but not D395N. The finding that the six mutations 255! (predicted based on the modeled structure and molecular docking) caused reduced 256! ! 13! sensitivity to ES20 in terms of plant growth provides additional evidence that ES20 257! targets the catalytic site of CESA6. 258!

259!
To determine whether ES20 targets CESA6 directly, we performed several biochemical 260! assays. The Drug Affinity Responsive Target Stability (DARTS) assay detects small 261! molecule and protein interactions by testing whether the small molecule protects the 262! protein from degradation by proteases (Lomenick et al., 2009;Zhang et al., 2016). We 263! isolated total proteins from YFP-CESA6 transgenic seedlings, incubated the proteins 264! with either ES20 or DMSO (as a control), and digested them with pronase. We used 265! anti-GFP antibody to detect the abundance of YFP-CESA6 after pronase digestion. 266! ES20 significantly protected YFP-CESA6 from degradation by pronase ( Figure 4A, 4B, 267! Supplemental Data Set 1), whereas the control molecule, Ampicillin, did not protect 268! YFP-CESA6 from degradation ( Figure 4C, 4D, Supplemental Data Set 1). The finding 269! that ES20 protected YFP-CESA6 from degradation by proteases suggests that ES20 270! and YFP-CESA6 physically interact. To test whether the mutations in our ES20-271! resistant mutants affect the interaction between CESA6 and ES20, we performed a 272! DARTS assay using ES20 and total proteins isolated from YFP-CESA6 P595S seedlings.

351!
To quantify the trafficking and dynamics of CSCs within and between compartments, we 352! performed both static and time-lapse analyses of YFP-CESA6 localization by collecting 353! 3-and 4-D stacks of images from epidermal cells in the root elongation zone by SDCM.

406!
We noticed that mutants in other CESAs might have stronger growth phenotypes than 407! cesa6 mutants; for example, any1 has a stronger root growth phenotype than es20r4 408! (CESA6 D605N ) ( Figure 1H-1J). It is possible that ES20 targets multiple CESAs but we 409! could not identify mutants in other CESAs because the dosage of ES20 (5 µM) we used 410! for the screening was too high to allow us to identify those mutants. CESA7 mainly 411! functions in secondary cell wall synthesis (Gardiner et al., 2003;Taylor et al., 2003;412! Brown et al., 2005). However, we found that fra5 had significantly reduced root growth 413! at the young seedling stage and modestly reduced sensitivity to ES20, indicating that 414! CESA7 functions in young seedling growth as well. However, we cannot rule out the 415! possibility that CESA6 holds a special position in the CSC rosette that allows ES20 to 416! target only CESA6 to affect the entire protein complex during cellulose synthesis. We 417! ! 20! expect that further characterization of the specificity of ES20 for different CESAs in 418! Arabidopsis, other plants, and other kingdoms such as bacteria and oomycetes will be 419! required for better use of ES20 as a general cellulose synthase inhibitor. Based on our 420! current results, ES20 can be used as a CESA6 inhibitor in Arabidopsis to explore the 421! molecular mechanisms of cellulose catalytic synthesis and the integration between 422! cellulose catalytic synthesis and CSC dynamic behaviors.

EMS mutagenesis and mutant screening 501!
To obtain a mutagenized Arabidopsis population, SYP61-CFP and PIN2-GFP seeds 502! were mutagenized following a published protocol (Kim et al., 2006). Mutagenized seeds 503! were sown in soil and the plants were grown under continuous light and allowed to self, 504! yielding M2 seeds. The M2 seeds were collected as pooled populations. Approximately 505! 400,000 seeds from the M2 generation of the SYP61-CFP population and 100,000 506! seeds from the PIN2-GFP M2 population were sterilized and sown on medium 507! containing 5 µM ES20. Individual plants with elongated roots and green leaves were 508! transferred to soil to produce the M3 generation. The M3 plants were examined for 509! ! 24! sensitivity to ES20. Individual M3 lines with reduced sensitivity to ES20 were crossed to 510! the Ler ecotype to generate the mapping population and were also crossed to SYP61-511! CFP or PIN2-GFP to clean up the genetic background. 512!

High-throughput genome sequencing and sequence analysis 514!
The seeds from F2 populations of mutants crossed with Ler were sown on medium 515! containing 5 µM ES20 and the segregation of resistant seedlings was evaluated. The F2 516! populations of the outcrosses segregated for sensitivity to ES20. For each mutant, 517! approximately100 F2 seedlings with longer roots on 5 µM ES20 were pooled for DNA 518! isolation. The genomic DNA was subjected to high-throughput sequencing. The 519! resulting DNA sequence was aligned to the Arabidopsis genome (TAIR10) and single 520! nucleotide polymorphisms (SNPs) were analyzed. Candidate SNPs for ES20 sensitivity 521! were identified using the next-generation EMS mutation mapping tool (Austin et al., 522! 2011). 523!

Crystalline cellulose content measurement 525!
Wild-type Arabidopsis seeds were sown on medium supplemented with 0.1% DMSO or 526! different concentrations of ES20. Following stratification, the plants were grown in the 527! dark for 7 d or in the light for 10 d. 7-day-old dark-grown seedlings or roots from 10-day-528! old light-grown seedlings were used for cell wall preparation. Dark-grown seedlings 529! were washed with ddH 2 O three times to remove seed coats and any residue from the 530! growth medium and ground into a fine powder in liquid nitrogen. The roots from light-531! grown seedlings were cut and washed with ddH 2 O to remove any residue from the 532! ! 25! growth medium and ground into a fine powder in liquid nitrogen. The powder was 533! extracted twice with 80% ethanol, once with 100% ethanol, once with 1:1 (v/v) MeOH-534! CHCl 3 , and once with acetone. The resulting insoluble cell wall fraction was dried in a 535! fume hood for 2 d and weighed. Cellulose content was measured by the Updegraff 536! method (Updegraff, 1969;Foster et al., 2010). Briefly, the cell wall material was 537! hydrolyzed with trifluoroacetic acid (TFA), followed by Updegraff reagent (acetic acid: 538! nitric acid: water, 8:1:2 v/v) to yield crystalline cellulose. Crystalline cellulose was 539! hydrolyzed to glucose using 72% sulfuric acid. Glucose concentration was measured 540! via a colorimetric method by developing color in Anthrone reagent (freshly prepared 2 541! mg/mL anthrone in concentrated sulfuric acid) and reading OD625 nm in a plate reader 542! were verified by DNA sequencing. The verified constructs used to transform prc1-1 565! (CS297), which was obtained from the Arabidopsis Biological Resource Center (ABRC), 566! using Agrobacterium tumefaciens-mediated transformation via the floral dip method 567! (Clough and Bent, 1998). The transformants were selected based on the selection 568! marker. Independent transformant lines were further characterized in the T2 generation 569! for the segregation ratio of the selection marker, and only transformants that contained 570! a single insertion for each construct were selected for further analysis. Homozygous 571! transformants with single CESA6 construct insertions were used for mutant phenotype 572! analyses. 573!

27!
The large cytoplasmic domain of the Arabidopsis CESA6 protein sequence (amino 579! acids 322-868) was sent to i-TASSER server for 3D structure modeling with the 580! threading method (Roy et al., 2010;Yang et al., 2015). The modeled structure was 581! visualized using PyMol software (Alexander et al., 2011). The binding site of UDP-582! glucose on the large cytoplasmic domain model of CESA6 was predicted using the 583! COACH server, and the UDP-glucose phosphonate structure was used for the 584! prediction as per the program suggestion (Yang et al., 2013b, a). The small molecule 585! ES20 was docked with the large cytoplasmic domain model of CESA6 using Autodock 586! Vina in PyRx software (Trott and Olson, 2010;Dallakyan and Olson, 2015). 587!

CESA6c protein expression and purification 589!
To obtain the CESA6 central cytosolic domain protein for the MST assay, we inserted 590! the GFP coding sequence into pRSF-Duet-1 vector using the SacI and PstI restriction 591! sites. The GFP coding sequence was amplified from the pUBN-GFP-DEST vector. The 592! central cytosolic domain of CESA6 (CESA6c) was amplified from Col-0 cDNA into the 593! C-terminus of GFP. Primers used for cloning are listed in Supplemental

DARTS assays 619!
To test for the interaction between CESA6 and ES20 using the DARTS assay, 7-day-old 620! YFP-CESA6 light-grown seedlings were harvested and ground to powder in liquid 621! nitrogen. The ground tissue was homogenized in lysis buffer (50 mM Tris-HCl, pH 7.5, 622! 150 mM NaCl, 0.5% Triton X-100, 2 mM DTT, one tablet/50 mL EDTA free Pierce 623! protease inhibitor (Thermo Fisher)) at a 2:1 ratio (2 mL buffer: 1 g tissue). Homogenized 624! samples were transferred to a 2 mL microcentrifuge tube and centrifuged for 30 min 625! ! 29! (20,000 g, 4°C). The supernatant was collected after centrifugation and saved as total 626! extracted protein. 700 µL extracted total protein was incubated with DMSO (0.1%) or 627! ES20 (300 µM) at room temperature on an orbital shaker for 1 h. The mixture was 628! divided into six small tubes, each containing 100 µL of the mixture, and incubated with 1 629! µL of pronase at a 1:300 dilution at room temperature for 30 min. The proteolysis 630! reaction was terminated by adding SDS loading buffer and boiled at 100 °C for 6 min.

SDCM image processing and quantification 681!
Image analysis was performed using Fiji/ImageJ (Schindelin et al., 2012). For CESA 682! particle density analyses, regions of interest (ROIs) without abundant Golgi signals were 683! chosen using the Freehand selection tool. CESA particles were detected automatically 684! on 8-bit images using the Find Maxima tool with the same noise threshold for all 685! images. CESA particle density for each ROI was calculated by dividing the number of 686! particles by the ROI area. For CESA particle dynamic analyses, 5-min time-lapse series 687! with 5-s intervals were collected. Average intensity projections were generated to 688! identify the trajectories of the CSC particles. Image drift was corrected by the StackReg 689! plugin (Thevenaz et al., 1998)    . ES20 is a cellulose synthesis inhibitor, and cesa6 mutants have reduced sensitivity to ES20. A, Molecular structure of ES20. B and C, ES20 inhibits Arabidopsis root growth in a dose-dependent manner. D to F, ES20 causes root cells to swell. Root cells of 3-day-old light-grown wild-type seedlings treated overnight with 0.1% DMSO (D) or 6 µM ES20 (E). ES20 treatment reduces cell length and increases cell width (F). G, ES20 reduces crystalline cellulose content in cell walls of light-grown roots in a dose-dependent manner. The letters [a-c] indicate statistically significant differences (p < 0.05) determined by one-way ANOVA tests followed by Tukey's multiple comparison tests in different samples. H, cesa6 mutants resulting from a genetic screen of EMS-mutagenized populations have reduced sensitivity to ES20. Representative 7-day-old wild-type seedlings expressing SYP61-CFP or PIN2-GFP, and cesa6 mutant lines grown on medium supplemented with 0.1% DMSO (top) or 1 µM ES20 (bottom). The mutants are listed based in the order of their discovery. I, Genetic complementation of prc1-1/cesa6 growth defects and sensitivity to ES20 by mutated CESA6 constructs. Ten constructs that we tested rescued the root growth defect of prc1-1 to different extents in the absence of ES20 and led to reduced sensitivity to ES20 in transgenic plants. J and K, Mutations in other CESA isoforms (any1/cesa1; fra1/cesa7) also lead to reduced sensitivity to ES20. In F, *** indicates p < 0.001 by two-tailed Student's t test. In K, * indicates p < 0.05 and *** indicates p < 0.001 by two-tailed Student's t test in comparison with Col-0. Error bars represent mean ± SD, with n = 15 for C and F, n = 9 for G, and n = 14 for K. Scale bars in B, H, I and J are 1.0 cm; Scale bars in D and E: 100 µm. A, The predicted topology of CESA6, the location of key motifs for cellulose catalytic synthesis, and the locations of the mutated amino acids that cause reduced sensitivity to ES20. The orientation of the CESA6 N-terminus, the transmembrane regions (TMRs), and the orientation of the cytoplasmic domains are based on predictions by PredictProtein. B, Sequence alignment of CESA6 with RsBcsA. Key motifs, including the plant-conserved region (PCR), class-specific region (CSR), Interface regions (IFs) and