OsTCP19 coordinates inhibition of lignin biosynthesis and promotion of cellulose biosynthesis to modify lodging resistance in rice.

Lignin and cellulose are two essential elements of plant secondary cell walls that shape the mechanical characteristics of the culm to prevent lodging. However, how the regulation of the lignin and cellulose composition is combined to achieve optimal mechanical characteristics is unclear. Here, we show that increasing OsTCP19 expression in rice coordinately repressed lignin biosynthesis and promoted cellulose biosynthesis, resulting in enhanced lodging resistance. In contrast, repression of OsTCP19 coordinately promoted lignin biosynthesis and inhibited cellulose biosynthesis, leading to greater susceptibility to lodging. We found that OsTCP19 binds to the promoters of both MYB108 and MYB103L to increase their expression, with the former being responsible for repressing lignin biosynthesis and the latter for promoting cellulose biosynthesis. Moreover, up-regulation of OsTCP19 in fibers improved grain yield and lodging resistance. Thus, our results identify the OsTCP19-OsMYB108/OsMYB103L module as a key regulator of lignin and cellulose production in rice, and open up the possibility for precisely manipulating lignin-cellulose composition to improve culm mechanical properties for lodging resistance.

Genome-wide analysis of the cellulose toolbox of Primulina eburnea, a calcium-rich vegetable.

BACKGROUND: Human-guided crop domestication has lasted for more than 10,000 years. In terms of the domestication and breeding of vegetables, cellulose content in edible tissues is one of the most important traits. Primulina eburnea is a recently developed calcium-rich vegetable with a high soluble and bioavailable calcium content in its leaves. However, the high cellulose content in the leaves hampers the taste, and no research has been reported on the genetic basis of cellulose biosynthesis in this calcium-rich vegetable. RESULTS: We identified 36 cellulose biosynthesis-involved genes belonging to eight gene families in the P. eburnea genome. The cellulose accumulated decreasingly throughout leaf development. Nineteen genes were considered core genes in cellulose biosynthesis, which were highly expressed in buds but lowly expressed in mature leaves. In the nitrogen fertilization experiment, exogenous nitrogen decreased the cellulose content in the buds. The expressing pattern of 14 genes were consistent with phenotypic variation in the nitrogen fertilization experiment, and thus they were proposed as cellulose toolbox genes. CONCLUSIONS: The present study provides a strong basis for the subsequent functional research of cellulose biosynthesis-involved genes in P. eburnea, and provides a reference for breeding and/or engineering this calcium-rich vegetable with decreased leaf cellulose content to improve the taste.

Tip growth defective1 interacts with cellulose synthase A3 to regulate cellulose biosynthesis in Arabidopsis.

KEY MESSAGE: AtTIP1 physically and genetically interacts with AtCESA3. AtCESA3 undergoes S-acylation, possibly mediated by AtTIP1, suggesting a specific role of AtTIP1 in cellulose biosynthesis and plant development. S-acylation is a reversible post-translational lipid modification of proteins catalyzed by protein S-acyl transferases (PATs). S-acylation is important for various biological molecular mechanisms including cellulose biosynthesis. Cellulose is synthesized by the cellulose synthase A (CESA) complexes (CSCs) at the plasma membrane. However, specific PAT involving in cellulose biosynthesis has not been identified and the precise mechanism by which PAT regulates the CESAs is largely unknown. Here, we report isolation of tip1-5, an allele of Tip Growth Defective1 (AtTIP1/AtPAT24) with a premature stop codon. tip1-5 genetically interacts with ixr1-2, a point mutant of AtCESA3 which encodes a catalytic subunit of CSC synthesizing primary wall cellulose. We show that AtTIP1 physically interacts with AtCESA3. AtCESA3 undergoes S-acylation, which is possibly mediated by AtTIP1, suggesting a functional relationship between AtTIP1 and AtCESA3. Moreover, the interfascicular fiber cells in the primary inflorescence stems of tip1-5 ixr1-2 double mutant contain thinner cell walls and significantly less crystalline cellulose compared to the single mutants. These results highlight the positive regulation of AtTIP1 in cellulose biosynthesis, and a specific role of AtPAT in plant development.

Overexpression of PnMYB2 from Panax notoginseng induces cellulose and lignin biosynthesis during cell wall formation.

MAIN CONCLUSION: Panax notoginseng PnMYB2 is a transcriptional activator of primary and secondary cell wall formation by promoting the PCW-specific gene CesA3 and key lignin biosynthetic gene CCoAOMT1, respectively. R2R3-MYB transcription factors play important roles in regulation secondary cell wall (SCW) formation. However, there are few reports on the functions of MYB transcription factors which involved in both primary cell wall (PCW) and SCW formation. Here, we isolated an R2R3-MYB transcription factor, PnMYB2, from Panax notoginseng roots which are widely used in Chinese traditional medicines and contain abundant cellulose and lignin. The expression pattern of PnMYB2 was similar to the accumulation pattern of cellulose and lignin contents in different organs. PnMYB2 localized in the nucleus and may function as a transcriptional activator. Overexpression of PnMYB2 in Arabidopsis thaliana enhanced cellulose and lignin biosynthesis, and remarkably increased thickness of PCW and SCW in the stem of transgenic plants compared with wild-type plants. The expression levels of genes associated with PCW-specific cellulose synthase (CesA) genes and key SCW-specific lignin biosynthetic genes were significantly increased in PnMYB2-overexpressing plants compared to the wild type plants. Furthermore, yeast one-hybrid, dual-luciferase reporter assays and electrophoretic mobility shift assays (EMSA) results verified that PnMYB2 could bind and activate the promoters of AtCesA3 and PnCesA3, which are the PCW-specific cellulose biosynthetic genes, and AtCCoAOMT1 and PnCCoAOMT1, which are the key lignin biosynthetic genes. These results demonstrated the central role of PnMYB2 in PCW-specific cellulose formation and SCW-specific lignin biosynthesis.

Deciphering Molecular Mechanism Underlying Self-Flocculation of Zymomonas mobilis for Robust Production.

Zymomonas mobilis metabolizes sugar anaerobically through the Entner-Doudoroff pathway with less ATP generated for lower biomass accumulation to direct more sugar for product formation with improved yield, making it a suitable host to be engineered as microbial cell factories for producing bulk commodities with major costs from feedstock consumption. Self-flocculation of the bacterial cells presents many advantages, such as enhanced tolerance to environmental stresses, a prerequisite for achieving high product titers by using concentrated substrates. ZM401, a self-flocculating mutant developed from ZM4, the unicellular model strain of Z. mobilis, was employed in this work to explore the molecular mechanism underlying this self-flocculating phenotype. Comparative studies between ZM401 and ZM4 indicate that a frameshift caused by a single nucleotide deletion in the poly-T tract of ZMO1082 fused the putative gene with the open reading frame of ZMO1083, encoding the catalytic subunit BcsA of the bacterial cellulose synthase to catalyze cellulose biosynthesis. Furthermore, the single nucleotide polymorphism mutation in the open reading frame of ZMO1055, encoding a bifunctional GGDEF-EAL protein with apparent diguanylate cyclase/phosphodiesterase activities, resulted in the Ala526Val substitution, which consequently compromised in vivo specific phosphodiesterase activity for the degradation of cyclic diguanylic acid, leading to intracellular accumulation of the signaling molecule to activate cellulose biosynthesis. These discoveries are significant for engineering other unicellular strains from Z. mobilis with the self-flocculating phenotype for robust production. IMPORTANCE Stress tolerance is a prerequisite for microbial cell factories to be robust in production, particularly for biorefinery of lignocellulosic biomass to produce biofuels, bioenergy, and bio-based chemicals for sustainable socioeconomic development, since various inhibitors are released during the pretreatment to destroy the recalcitrant lignin-carbohydrate complex for sugar production through enzymatic hydrolysis of the cellulose component, and their detoxification is too costly for producing bulk commodities. Although tolerance to individual stress has been intensively studied, the progress seems less significant since microbial cells are inevitably suffering from multiple stresses simultaneously under production conditions. When self-flocculating, microbial cells are more tolerant to multiple stresses through the general stress response due to enhanced quorum sensing associated with the morphological change for physiological and metabolic advantages. Therefore, elucidation of the molecular mechanism underlying such a self-flocculating phenotype is significant for engineering microbial cells with the unique multicellular morphology through rational design to boost their production performance.

Small but Mighty: An Update on Small Molecule Plant Cellulose Biosynthesis Inhibitors.

Cellulose is one of the most abundant biopolymers on Earth. It provides mechanical support to growing plant cells and important raw materials for paper, textiles and biofuel feedstocks. Cellulose biosynthesis inhibitors (CBIs) are invaluable tools for studying cellulose biosynthesis and can be important herbicides for controlling weed growth. Here, we review CBIs with particular focus on the most widely used CBIs and recently discovered CBIs. We discuss the effects of these CBIs on plant growth and development and plant cell biology and summarize what is known about the mode of action of these different CBIs.

The Mode of Action of Endosidin20 Differs from That of Other Cellulose Biosynthesis Inhibitors.

Endosidin20 (ES20) was recently identified as a cellulose biosynthesis inhibitor (CBI) that targets the catalytic domain of CELLULOSE SYNTHASE 6 (CESA6) and thus inhibits the growth of Arabidopsis thaliana. Here, we characterized the effects of ES20 on the growth of other plant species and found that ES20 is a broad-spectrum plant growth inhibitor. We tested the inhibitory effects of previously characterized CBIs (isoxaben, indaziflam and C17) on the growth of Arabidopsis cesa6 mutants that have reduced sensitivity to ES20. We found that most of these mutants are sensitive to isoxaben, indaziflam and C17, indicating that these tested CBIs have a different mode of action than ES20. ES20 also has a synergistic inhibitory effect on plant growth when jointly applied with other CBIs, further confirming that ES20 has a different mode of action than isoxaben, indaziflam and C17. We demonstrated that plants carrying two missense mutations conferring resistance to ES20 and isoxaben can tolerate the dual inhibitory effects of these CBIs when combined. ES20 inhibits Arabidopsis growth in growth medium and in soil following direct spraying. Therefore, our results pave the way for using ES20 as a broad-spectrum herbicide, and for the use of gene-editing technologies to produce ES20-resistant crop plants.

Cell wall integrity modulates Arabidopsis thaliana cell cycle gene expression in a cytokinin- and nitrate reductase-dependent manner.

During plant growth and defense, cell cycle activity needs to be coordinated with cell wall integrity. Little is known about how this coordination is achieved. Here, we investigated coordination in Arabidopsis thaliana seedlings by studying the impact of cell wall damage (CWD, caused by cellulose biosynthesis inhibition) on cytokinin homeostasis, cell cycle gene expression and cell shape in root tips. CWD inhibited cell cycle gene expression and increased transition zone cell width in an osmosensitive manner. These results were correlated with CWD-induced, osmosensitive changes in cytokinin homeostasis. Expression of CYTOKININ OXIDASE/DEHYDROGENASE 2 and 3 (CKX2, CKX3), which encode cytokinin-degrading enzymes, was induced by CWD and reduced by osmoticum treatment. In nitrate reductase1 nitrate reductase2 (nia1 nia2) seedlings, CKX2 and CKX3 transcript levels were not increased and cell cycle gene expression was not repressed by CWD. Moreover, established CWD-induced responses, such as jasmonic acid, salicylic acid and lignin production, were also absent, implying a central role of NIA1/2-mediated processes in regulation of CWD responses. These results suggest that CWD enhances cytokinin degradation rates through a NIA1/2-mediated process, leading to attenuation of cell cycle gene expression.

Photochemical transformation of a cellulose biosynthesis inhibitor into phytoene desaturase inhibitors.

Mode of action studies showed that 5-methyl-N,N-bis[6-(trifluoromethyl)pyridin-3-yl]pyridin-2-amine (4), a representative from a new class of herbicidal tris-pyridyl amines, is an inhibitor of cellulose biosynthesis (CB). The compound undergoes an oxidative photocyclization, when exposed to UV-B light (300-340 nm) in the presence of oxygen, to give a new class of herbicidal pyrrolodipyridines. These compounds are potent inhibitors of the herbicide target enzyme phytoene desaturase and no longer inhibit CB.

Cellulose synthase complexes display distinct dynamic behaviors during xylem transdifferentiation.

In plants, plasma membrane-embedded CELLULOSE SYNTHASE (CESA) enzyme complexes deposit cellulose polymers into the developing cell wall. Cellulose synthesis requires two different sets of CESA complexes that are active during cell expansion and secondary cell wall thickening, respectively. Hence, developing xylem cells, which first undergo cell expansion and subsequently deposit thick secondary walls, need to completely reorganize their CESA complexes from primary wall- to secondary wall-specific CESAs. Using live-cell imaging, we analyzed the principles underlying this remodeling. At the onset of secondary wall synthesis, the primary wall CESAs ceased to be delivered to the plasma membrane and were gradually removed from both the plasma membrane and the Golgi. For a brief transition period, both primary wall- and secondary wall-specific CESAs coexisted in banded domains of the plasma membrane where secondary wall synthesis is concentrated. During this transition, primary and secondary wall CESAs displayed discrete dynamic behaviors and sensitivities to the inhibitor isoxaben. As secondary wall-specific CESAs were delivered and inserted into the plasma membrane, the primary wall CESAs became concentrated in prevacuolar compartments and lytic vacuoles. This adjustment in localization between the two CESAs was accompanied by concurrent decreased primary wall CESA and increased secondary wall CESA protein abundance. Our data reveal distinct and dynamic subcellular trafficking patterns that underpin the remodeling of the cellulose biosynthetic machinery, resulting in the removal and degradation of the primary wall CESA complex with concurrent production and recycling of the secondary wall CESAs.

Updating Insights into the Catalytic Domain Properties of Plant Cellulose synthase (CesA) and Cellulose synthase-like (Csl) Proteins.

The wall is the last frontier of a plant cell involved in modulating growth, development and defense against biotic stresses. Cellulose and additional polysaccharides of plant cell walls are the most abundant biopolymers on earth, having increased in economic value and thereby attracted significant interest in biotechnology. Cellulose biosynthesis constitutes a highly complicated process relying on the formation of cellulose synthase complexes. Cellulose synthase (CesA) and Cellulose synthase-like (Csl) genes encode enzymes that synthesize cellulose and most hemicellulosic polysaccharides. Arabidopsis and rice are invaluable genetic models and reliable representatives of land plants to comprehend cell wall synthesis. During the past two decades, enormous research progress has been made to understand the mechanisms of cellulose synthesis and construction of the plant cell wall. A plethora of cesa and csl mutants have been characterized, providing functional insights into individual protein isoforms. Recent structural studies have uncovered the mode of CesA assembly and the dynamics of cellulose production. Genetics and structural biology have generated new knowledge and have accelerated the pace of discovery in this field, ultimately opening perspectives towards cellulose synthesis manipulation. This review provides an overview of the major breakthroughs gathering previous and recent genetic and structural advancements, focusing on the function of CesA and Csl catalytic domain in plants.

A novel rice fragile culm 24 mutant encodes a UDP-glucose epimerase that affects cell wall properties and photosynthesis.

UDP-glucose epimerases (UGEs) are essential enzymes for catalysing the conversion of UDP-glucose (UDP-Glc) into UDP-galactose (UDP-Gal). Although UDP-Gal has been well studied as the substrate for the biosynthesis of carbohydrates, glycolipids, and glycoproteins, much remains unknown about the biological function of UGEs in plants. In this study, we selected a novel rice fragile culm 24 (Osfc24) mutant and identified it as a nonsense mutation of the FC24/OsUGE2 gene. The Osfc24 mutant shows a brittleness phenotype with significantly altered cell wall composition and disrupted orientation of the cellulose microfibrils. We found significantly reduced accumulation of arabinogalactan proteins in the cell walls of the mutant, which may consequently affect plant growth and cell wall deposition, and be responsible for the altered cellulose microfibril orientation. The mutant exhibits dwarfism and paler leaves with significantly decreased contents of galactolipids and chlorophyll, resulting in defects in plant photosynthesis. Based on our results, we propose a model for how OsUGE2 participates in two distinct metabolic pathways to co-modulate cellulose biosynthesis and cell wall assembly by dynamically providing UDP-Gal and UDP-Glc substrates.

Effects of exogenous 24-epibrassinolide and brassinazole on negative gravitropism and tension wood formation in hybrid poplar (Populus deltoids × Populus nigra).

MAIN CONCLUSION: Exogenous 24-epibrassinolide (BL) and brassinazole (BRZ) have regulatory roles in G-fiber cell wall development and secondary xylem cell wall carbohydrate biosynthesis during tension wood formation in hybrid poplar. Brassinosteroids (BRs) play important roles in regulating gravitropism and vasculature development. Here, we report the effect of brassinosteroids on negative gravitropism and G-fiber cell wall development of the stem in woody angiosperms. We applied exogenous 24-epibrassinolide (BL) or its biosynthesis inhibitor brassinazole (BRZ) to slanted hybrid poplar trees (Populus deltoids × Populus nigra) and measured the morphology of gravitropic stems, anatomy and chemistry of secondary cell wall. We furthermore analyzed the expression levels of auxin transport and cellulose biosynthetic genes after 24-epibrassinolide (BL) or brassinazole (BRZ) application. The BL-treated seedlings showed no negative gravitropism bending, whereas application of BRZ dramatically enhanced negative gravitropic bending. BL treatment stimulated secondary xylem fiber elongation and G-fiber formation on the upper side of stems but delayed G-fiber maturation. BRZ inhibited xylem fiber elongation but induced the production of more mature G-fibers on the upper side of stems. Wood chemistry analyses and immunolocalization demonstrated that BL and BRZ treatments increased the cellulose content and modified the deposition of cell wall carbohydrates including arabinose, galactose and rhamnose in the secondary xylem. The expression of cellulose biosynthetic genes, especially those related to cellulose microfibril deposition (PtFLA12 and PtCOBL4) was significantly upregulated in BL- and BRZ-treated TW stems compared with control stems. The significant differences of G-fibers development and negative gravitropism bending between 24-epibrassinolide (BL) and brassinazole (BRZ) application suggest that brassinosteroids are important for secondary xylem development during tension wood formation. Our findings provide potential insights into the mechanism by which BRs regulate G-fiber cell wall development to accomplish negative gravitropism in TW formation.

Cellulose biosynthesis inhibitors - a multifunctional toolbox.

In the current review, we examine the growing number of existing Cellulose Biosynthesis Inhibitors (CBIs) and based on those that have been studied with live cell imaging we group their mechanism of action. Attention is paid to the use of CBIs as tools to ask fundamental questions about cellulose biosynthesis.

Elucidation of Xylem-Specific Transcription Factors and Absolute Quantification of Enzymes Regulating Cellulose Biosynthesis in Populus trichocarpa.

Cellulose, the main chemical polymer of wood, is the most abundant polysaccharide in nature.1 The ability to perturb the abundance and structure of cellulose microfibrils is of critical importance to the pulp and paper industry as well as for the textile, wood products, and liquid biofuels industries. Although much has been learned at the transcript level about the biosynthesis of cellulose, a quantitative understanding at the proteome level has yet to be established. The study described herein sought to identify the proteins directly involved in cellulose biosynthesis during wood formation in Populus trichocarpa along with known xylem-specific transcription factors involved in regulating these key proteins. Development of an effective discovery proteomic strategy through a combination of subcellular fractionation of stem differentiating xylem tissue (SDX) with recently optimized FASP digestion protocols, StageTip fractionation, as well as optimized instrument parameters for global proteomic analysis using the quadrupole-orbitrap mass spectrometer resulted in the deepest proteomic coverage of SDX protein from P. trichocarpa with 9,146 protein groups being identified (1% FDR). Of these, 20 cellulosic/hemicellulosic enzymes and 43 xylem-specific transcription factor groups were identified. Finally, selection of surrogate peptides led to an assay for absolute quantification of 14 cellulosic proteins in SDX of P. trichocarpa.

The Arabidopsis COBRA protein facilitates cellulose crystallization at the plasma membrane.

Mutations in the Arabidopsis COBRA gene lead to defects in cellulose synthesis but the function of COBRA is unknown. Here we present evidence that COBRA localizes to discrete particles in the plasma membrane and is sensitive to inhibitors of cellulose synthesis, suggesting that COBRA and the cellulose synthase complex reside in close proximity on the plasma membrane. Live-cell imaging of cellulose synthesis indicated that, once initiated, cellulose synthesis appeared to proceed normally in the cobra mutant. Using isothermal calorimetry, COBRA was found to bind individual ß1-4-linked glucan chains with a KD of 3.2 µm. Competition assays suggests that COBRA binds individual ß1-4-linked glucan chains with higher affinity than crystalline cellulose. Solid-state nuclear magnetic resonance studies of the cell wall of the cobra mutant also indicated that, in addition to decreases in cellulose amount, the properties of the cellulose fibrils and other cell wall polymers differed from wild type by being less crystalline and having an increased number of reducing ends. We interpret the available evidence as suggesting that COBRA facilitates cellulose crystallization from the emerging ß1-4-glucan chains by acting as a "polysaccharide chaperone."

Computational and genetic evidence that different structural conformations of a non-catalytic region affect the function of plant cellulose synthase.

The ß-1,4-glucan chains comprising cellulose are synthesized by cellulose synthases in the plasma membranes of diverse organisms including bacteria and plants. Understanding structure-function relationships in the plant enzymes involved in cellulose synthesis (CESAs) is important because cellulose is the most abundant component in the plant cell wall, a key renewable biomaterial. Here, we explored the structure and function of the region encompassing transmembrane helices (TMHs) 5 and 6 in CESA using computational and genetic tools. Ab initio computational structure prediction revealed novel bi-modal structural conformations of the region between TMH5 and 6 that may affect CESA function. Here we present our computational findings on this region in three CESAs of Arabidopsis thaliana (AtCESA1, 3, and 6), the Atcesa3(ixr1-2) mutant, and a novel missense mutation in AtCESA1. A newly engineered point mutation in AtCESA1 (Atcesa1(F954L) ) that altered the structural conformation in silico resulted in a protein that was not fully functional in the temperature-sensitive Atcesa1(rsw1-1) mutant at the restrictive temperature. The combination of computational and genetic results provides evidence that the ability of the TMH5-6 region to adopt specific structural conformations is important for CESA function.

Breaking-Down Barriers: Proposal of Using Cellulose Biosynthesis Inhibitors and Cellulase Enzyme as a Novel Treatment Modality for Vision Threatening Pythium Insidiosum Keratitis.

Pythium insidiosum, an Oomycete, causes severe keratitis that endangers vision. Its clinical, morphological, and microbiological characteristics are often indistinguishable from those of fungal keratitis, earning it the moniker "parafungus". Distinctive clinical hallmarks that set it apart from other forms of keratitis include radial keratoneuritis, tentacles, marginal infiltration, and a propensity for rapid limbal spread. The therapeutic approach to Pythium keratitis (PK) has long been a subject of debate, and topical and systemic antifungals and antibacterials have been tried with limited success. Approximately 80% of these eyes undergo therapeutic keratoplasty to salvage the eye. Hence, there is a need to innovate for alternative and better medical therapy to safeguard these eyes. The resistance of Pythium to standard antifungal treatments can be attributed to the absence of ergosterol in its cell wall. Cell walls of plants and algae have cellulose as an essential constituent. Cellulose imparts strength and structure and acts as the "skeleton" of the plant. Fungal and animal cell walls typically lack cellulose. The cellular architecture of Pythium shares a similarity with plant and algal cells through the incorporation of cellulose within its cell wall structure. Inhibitors targeting cellulose biosynthesis (CBI), such as Indaziflam, Isoxaben, and Quinoxyphen, serve as critical tools for elucidating the pathways of cellulose synthesis. Furthermore, the enzymatic action of cellulase is instrumental for the extraction of proteins and DNA. To circumvent this issue, we hypothesize that CBI's and cellulase enzymes can act on the Pythium cell wall and may effectively treat PK. The available literature supporting the hypothesis and proof of concept has also been discussed. We have also discussed these drugs' molecular mechanism of action on the Pythium cell wall. We also aim to propose how these drugs can be procured and used as a potential medical management option for this devastating entity.

Cell wall-mediated root development is targeted by a soil-borne bacterial pathogen to promote infection.

Plant pathogens manipulate host development, facilitating colonization and proliferation. Ralstonia solanacearum is a soil-borne bacterial pathogen that penetrates roots and colonizes plants through the vascular system, causing wilting and death. Here, we find that RipAC, an effector protein from R. solanacearum, alters root development in Arabidopsis, promoting the formation of lateral roots and root hairs. RipAC interacts with CELLULOSE SYNTHASE (CESA)-INTERACTIVE PROTEIN 1 (CSI1), which regulates the activity of CESA complexes at the plasma membrane. RipAC disrupts CESA-CSI1 interaction, leading to a reduction in cellulose content, root developmental alterations, and a promotion of bacterial pathogenicity. We find that CSI1 also associates with the receptor kinase FERONIA, forming a complex that negatively regulates immunity in roots; this interaction, however, is not affected by RipAC. Our work reveals a bacterial virulence strategy that selectively affects the activities of a host target, promoting anatomical alterations that facilitate infection without causing activation of immunity.

Insights into substrate coordination and glycosyl transfer of poplar cellulose synthase-8.

Cellulose is an abundant cell wall component of land plants. It is synthesized from UDP-activated glucose molecules by cellulose synthase, a membrane-integrated processive glycosyltransferase. Cellulose synthase couples the elongation of the cellulose polymer with its translocation across the plasma membrane. Here, we present substrate- and product-bound cryogenic electron microscopy structures of the homotrimeric cellulose synthase isoform-8 (CesA8) from hybrid aspen (poplar). UDP-glucose binds to a conserved catalytic pocket adjacent to the entrance to a transmembrane channel. The substrate's glucosyl unit is coordinated by conserved residues of the glycosyltransferase domain and amphipathic interface helices. Site-directed mutagenesis of a conserved gating loop capping the active site reveals its critical function for catalytic activity. Molecular dynamics simulations reveal prolonged interactions of the gating loop with the substrate molecule, particularly across its central conserved region. These transient interactions likely facilitate the proper positioning of the substrate molecule for glycosyl transfer and cellulose translocation.