Extracellular pectin-RALF phase separation mediates FERONIA global signaling function.

The FERONIA (FER)-LLG1 co-receptor and its peptide ligand RALF regulate myriad processes for plant growth and survival. Focusing on signal-induced cell surface responses, we discovered that intrinsically disordered RALF triggers clustering and endocytosis of its cognate receptors and FER- and LLG1-dependent endocytosis of non-cognate regulators of diverse processes, thus capable of broadly impacting downstream responses. RALF, however, remains extracellular. We demonstrate that RALF binds the cell wall polysaccharide pectin. They phase separate and recruit FER and LLG1 into pectin-RALF-FER-LLG1 condensates to initiate RALF-triggered cell surface responses. We show further that two frequently encountered environmental challenges, elevated salt and temperature, trigger RALF-pectin phase separation, promiscuous receptor clustering and massive endocytosis, and that this process is crucial for recovery from stress-induced growth attenuation. Our results support that RALF-pectin phase separation mediates an exoskeletal mechanism to broadly activate FER-LLG1-dependent cell surface responses to mediate the global role of FER in plant growth and survival.

An antibiotic from an uncultured bacterium binds to an immutable target.

Antimicrobial resistance is a leading mortality factor worldwide. Here, we report the discovery of clovibactin, an antibiotic isolated from uncultured soil bacteria. Clovibactin efficiently kills drug-resistant Gram-positive bacterial pathogens without detectable resistance. Using biochemical assays, solid-state nuclear magnetic resonance, and atomic force microscopy, we dissect its mode of action. Clovibactin blocks cell wall synthesis by targeting pyrophosphate of multiple essential peptidoglycan precursors (C55PP, lipid II, and lipid IIIWTA). Clovibactin uses an unusual hydrophobic interface to tightly wrap around pyrophosphate but bypasses the variable structural elements of precursors, accounting for the lack of resistance. Selective and efficient target binding is achieved by the sequestration of precursors into supramolecular fibrils that only form on bacterial membranes that contain lipid-anchored pyrophosphate groups. This potent antibiotic holds the promise of enabling the design of improved therapeutics that kill bacterial pathogens without resistance development.

Plant Cell Wall Loosening by Expansins.

Expansins comprise an ancient group of cell wall proteins ubiquitous in land plants and their algal ancestors. During cell growth, they facilitate passive yielding of the wall's cellulose networks to turgor-generated tensile stresses, without evidence of enzymatic activity. Expansins are also implicated in fruit softening and other developmental processes and in adaptive responses to environmental stresses and pathogens. The major expansin families in plants include α-expansins (EXPAs), which act on cellulose-cellulose junctions, and ß-expansins, which can act on xylans. EXPAs mediate acid growth, which contributes to wall enlargement by auxin and other growth agents. The genomes of diverse microbes, including many plant pathogens, also encode expansins designated expansin-like X. Expansins are proposed to disrupt noncovalent bonding between laterally aligned polysaccharides (notably cellulose), facilitating wall loosening for a variety of biological roles.

Structure and Mechanism of the Lipid Flippase MurJ.

Biosynthesis of many important polysaccharides (including peptidoglycan, lipopolysaccharide, and N-linked glycans) necessitates the transport of lipid-linked oligosaccharides (LLO) across membranes from their cytosolic site of synthesis to their sites of utilization. Much of our current understanding of LLO transport comes from genetic, biochemical, and structural studies of the multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) superfamily protein MurJ, which flips the peptidoglycan precursor lipid II. MurJ plays a pivotal role in bacterial cell wall synthesis and is an emerging antibiotic target. Here, we review the mechanism of LLO flipping by MurJ, including the structural basis for lipid II flipping and ion coupling. We then discuss inhibition of MurJ by antibacterials, including humimycins and the phage M lysis protein, as well as how studies on MurJ could provide insight into other flippases, both within and beyond the MOP superfamily.

The Penium margaritaceum Genome: Hallmarks of the Origins of Land Plants.

The evolutionary features and molecular innovations that enabled plants to first colonize land are not well understood. Here, insights are provided through our report of the genome sequence of the unicellular alga Penium margaritaceum, a member of the Zygnematophyceae, the sister lineage to land plants. The genome has a high proportion of repeat sequences that are associated with massive segmental gene duplications, likely facilitating neofunctionalization. Compared with representatives of earlier diverging algal lineages, P. margaritaceum has expanded repertoires of gene families, signaling networks, and adaptive responses that highlight the evolutionary trajectory toward terrestrialization. These encompass a broad range of physiological processes and protective cellular features, such as flavonoid compounds and large families of modifying enzymes involved in cell wall biosynthesis, assembly, and remodeling. Transcriptome profiling further elucidated adaptations, responses, and selective pressures associated with the semi-terrestrial ecosystems of P. margaritaceum, where a simple body plan would be an advantage.

The Root Cap Cuticle: A Cell Wall Structure for Seedling Establishment and Lateral Root Formation.

The root cap surrounding the tip of plant roots is thought to protect the delicate stem cells in the root meristem. We discovered that the first layer of root cap cells is covered by an electron-opaque cell wall modification resembling a plant cuticle. Cuticles are polyester-based protective structures considered exclusive to aerial plant organs. Mutations in cutin biosynthesis genes affect the composition and ultrastructure of this cuticular structure, confirming its cutin-like characteristics. Strikingly, targeted degradation of the root cap cuticle causes a hypersensitivity to abiotic stresses during seedling establishment. Furthermore, lateral root primordia also display a cuticle that, when defective, causes delayed outgrowth and organ deformations, suggesting that it facilitates lateral root emergence. Our results show that the previously unrecognized root cap cuticle protects the root meristem during the critical phase of seedling establishment and promotes the efficient formation of lateral roots.

Pollen Cell Wall Patterns Form from Modulated Phases.

The ornately geometric walls of pollen grains have inspired scientists for decades. We show that the evolved diversity of these patterns is entirely recapitulated by a biophysical model in which an initially uniform polysaccharide layer in the extracellular space, mechanically coupled to the cell membrane, phase separates to a spatially modulated state. Experiments reveal this process occurring in living cells. We observe that in ∼10% of extant species, this phase separation reaches equilibrium during development such that individual pollen grains are identical and perfectly reproducible. About 90% of species undergo an arrest of this process prior to equilibrium such that individual grains are similar but inexact copies. Equilibrium patterns have appeared multiple times during the evolution of seed plants, but selection does not favor these states. This framework for pattern development provides a route to rationalizing the surface textures of other secreted structures, such as cell walls and insect cuticle.

Imaging Bacterial Cell Wall Biosynthesis.

Peptidoglycan is an essential component of the cell wall that protects bacteria from environmental stress. A carefully coordinated biosynthesis of peptidoglycan during cell elongation and division is required for cell viability. This biosynthesis involves sophisticated enzyme machineries that dynamically synthesize, remodel, and degrade peptidoglycan. However, when and where bacteria build peptidoglycan, and how this is coordinated with cell growth, have been long-standing questions in the field. The improvement of microscopy techniques has provided powerful approaches to study peptidoglycan biosynthesis with high spatiotemporal resolution. Recent development of molecular probes further accelerated the growth of the field, which has advanced our knowledge of peptidoglycan biosynthesis dynamics and mechanisms. Here, we review the technologies for imaging the bacterial cell wall and its biosynthesis activity. We focus on the applications of fluorescent d-amino acids, a newly developed type of probe, to visualize and study peptidoglycan synthesis and dynamics, and we provide direction for prospective research.

A Lignin Molecular Brace Controls Precision Processing of Cell Walls Critical for Surface Integrity in Arabidopsis.

The cell wall, a defining feature of plants, provides a rigid structure critical for bonding cells together. To overcome this physical constraint, plants must process cell wall linkages during growth and development. However, little is known about the mechanism guiding cell-cell detachment and cell wall remodeling. Here, we identify two neighboring cell types in Arabidopsis that coordinate their activities to control cell wall processing, thereby ensuring precise abscission to discard organs. One cell type produces a honeycomb structure of lignin, which acts as a mechanical "brace" to localize cell wall breakdown and spatially limit abscising cells. The second cell type undergoes transdifferentiation into epidermal cells, forming protective cuticle, demonstrating de novo specification of epidermal cells, previously thought to be restricted to embryogenesis. Loss of the lignin brace leads to inadequate cuticle formation, resulting in surface barrier defects and susceptible to infection. Together, we show how plants precisely accomplish abscission.

Unveiling the Role of the Most Impactful Cardiovascular Risk Locus through Haplotype Editing.

The 9p21.3 cardiovascular disease locus is the most influential common genetic risk factor for coronary artery disease (CAD), accounting for ∼10%-15% of disease in non-African populations. The ∼60 kb risk haplotype is human-specific and lacks coding genes, hindering efforts to decipher its function. Here, we produce induced pluripotent stem cells (iPSCs) from risk and non-risk individuals, delete each haplotype using genome editing, and generate vascular smooth muscle cells (VSMCs). Risk VSMCs exhibit globally altered transcriptional networks that intersect with previously identified CAD risk genes and pathways, concomitant with aberrant adhesion, contraction, and proliferation. Unexpectedly, deleting the risk haplotype rescues VSMC stability, while expressing the 9p21.3-associated long non-coding RNA ANRIL induces risk phenotypes in non-risk VSMCs. This study shows that the risk haplotype selectively predisposes VSMCs to adopt a cell state associated with CAD phenotypes, defines new VSMC-based networks of CAD risk genes, and establishes haplotype-edited iPSCs as powerful tools for functionally annotating the human genome.

Lysozyme Counteracts ß-Lactam Antibiotics by Promoting the Emergence of L-Form Bacteria.

ß-lactam antibiotics inhibit bacterial cell wall assembly and, under classical microbiological culture conditions that are generally hypotonic, induce explosive cell death. Here, we show that under more physiological, osmoprotective conditions, for various Gram-positive bacteria, lysis is delayed or abolished, apparently because inhibition of class A penicillin-binding protein leads to a block in autolytic activity. Although these cells still then die by other mechanisms, exogenous lytic enzymes, such as lysozyme, can rescue viability by enabling the escape of cell wall-deficient "L-form" bacteria. This protective L-form conversion was also observed in macrophages and in an animal model, presumably due to the production of host lytic activities, including lysozyme. Our results demonstrate the potential for L-form switching in the host environment and highlight the unexpected effects of innate immune effectors, such as lysozyme, on antibiotic activity. Unlike previously described dormant persisters, L-forms can continue to proliferate in the presence of antibiotic.

P. aeruginosa CtpA protease adopts a novel activation mechanism to initiate the proteolytic process.

During bacterial cell growth, hydrolases cleave peptide cross-links between strands of the peptidoglycan sacculus to allow new strand insertion. The Pseudomonas aeruginosa carboxyl-terminal processing protease (CTP) CtpA regulates some of these hydrolases by degrading them. CtpA assembles as an inactive hexamer composed of a trimer-of-dimers, but its lipoprotein binding partner LbcA activates CtpA by an unknown mechanism. Here, we report the cryo-EM structures of the CtpA-LbcA complex. LbcA has an N-terminal adaptor domain that binds to CtpA, and a C-terminal superhelical tetratricopeptide repeat domain. One LbcA molecule attaches to each of the three vertices of a CtpA hexamer. LbcA triggers relocation of the CtpA PDZ domain, remodeling of the substrate binding pocket, and realignment of the catalytic residues. Surprisingly, only one CtpA molecule in a CtpA dimer is activated upon LbcA binding. Also, a long loop from one CtpA dimer inserts into a neighboring dimer to facilitate the proteolytic activity. This work has revealed an activation mechanism for a bacterial CTP that is strikingly different from other CTPs that have been characterized structurally.

Candida auris Genetics and Emergence.

Candida auris is a multidrug-resistant fungal pathogen that presents a serious threat to global human health. Since the first reported case in 2009 in Japan, C. auris infections have been reported in more than 40 countries, with mortality rates between 30% and 60%. In addition, C. auris has the potential to cause outbreaks in health care settings, especially in nursing homes for elderly patients, owing to its efficient transmission via skin-to-skin contact. Most importantly, C. auris is the first fungal pathogen to show pronounced and sometimes untreatable clinical drug resistance to all known antifungal classes, including azoles, amphotericin B, and echinocandins. In this review, we explore the causes of the rapid spread of C. auris. We also highlight its genome organization and drug resistance mechanisms and propose future research directions that should be undertaken to curb the spread of this multidrug-resistant pathogen.

Cardiomyocyte orientation recovery at micrometer scale reveals long-axis fiber continuum in heart walls.

Coordinated cardiomyocyte contraction drives the mammalian heart to beat and circulate blood. No consensus model of cardiomyocyte geometrical arrangement exists, due to the limited spatial resolution of whole heart imaging methods and the piecemeal nature of studies based on histological sections. By combining microscopy and computer vision, we produced the first-ever three-dimensional cardiomyocyte orientation reconstruction across mouse ventricular walls at the micrometer scale, representing a gain of three orders of magnitude in spatial resolution. We recovered a cardiomyocyte arrangement aligned to the long-axis direction of the outer ventricular walls. This cellular network lies in a thin shell and forms a continuum with longitudinally arranged cardiomyocytes in the inner walls, with a complex geometry at the apex. Our reconstruction methods can be applied at fine spatial scales to further understanding of heart wall electrical function and mechanics, and set the stage for the study of micron-scale fiber remodeling in heart disease.

The DYRKP1 kinase regulates cell wall degradation in Chlamydomonas by inducing matrix metalloproteinase expression.

The cell wall of plants and algae is an important cell structure that protects cells from changes in the external physical and chemical environment. This extracellular matrix, composed of polysaccharides and glycoproteins, must be constantly remodeled throughout the life cycle. However, compared to matrix polysaccharides, little is known about the mechanisms regulating the formation and degradation of matrix glycoproteins. We report here that a plant kinase belonging to the DUAL-SPECIFICITY TYROSINE PHOSPHORYLATION-REGULATED KINASE (DYRK) family present in all eukaryotes regulates cell wall degradation after mitosis of Chlamydomonas reinhardtii by inducing the expression of matrix metalloproteinases (MMPs). Without the plant DYRK kinase (DYRKP1), daughter cells cannot disassemble parental cell walls and remain trapped inside for more than 10 days. On the other hand, the DYRKP1 complementation line shows normal degradation of the parental cell wall. Transcriptomic and proteomic analyses indicate a marked down-regulation of MMP gene expression and accumulation, respectively, in the dyrkp1 mutants. The mutants deficient in MMPs retain palmelloid structures for a longer time than the background strain, like dyrkp1 mutants. Our findings show that DYRKP1, by ensuring timely MMP expression, enables the successful execution of the cell cycle. Altogether, this study provides insight into the life cycle regulation in plants and algae.

Acceleration is the key to drag reduction in turbulent flow.

A turbulent pipe flow experiment was conducted where the surface of the pipe was oscillated azimuthally over a wide range of frequencies, amplitudes, and Reynolds numbers. The drag was reduced by as much as 35%. Past work has suggested that the drag reduction scales with the velocity amplitude of the motion, its period, and/or the Reynolds number. Here, we find that the key parameter is the acceleration, which greatly simplifies the complexity of the phenomenon. This result is shown to apply to channel flows with spanwise surface oscillation as well. This insight opens potential avenues for reducing fuel consumption by large vehicles and for reducing energy costs in large piping systems.

The nonlinear mechanics of highly extensible plant epidermal cell walls.

Plant epidermal cell walls maintain the mechanical integrity of plants and restrict organ growth. Mechanical analyses can give insights into wall structure and are inputs for mechanobiology models of plant growth. To better understand the intrinsic mechanics of epidermal cell walls and how they may accommodate large deformations during growth, we analyzed a geometrically simple material, onion epidermal strips consisting of only the outer (periclinal) cell wall, ~7 µm thick. With uniaxial stretching by >40%, the wall showed complex three-phase stress-strain responses while cyclic stretching revealed reversible and irreversible deformations and elastic hysteresis. Stretching at varying strain rates and temperatures indicated the wall behaved more like a network of flexible cellulose fibers capable of sliding than a viscoelastic composite with pectin viscosity. We developed an analytic framework to quantify nonlinear wall mechanics in terms of stiffness, deformation, and energy dissipation, finding that the wall stretches by combined elastic and plastic deformation without compromising its stiffness. We also analyzed mechanical changes in slightly dehydrated walls. Their extension became stiffer and more irreversible, highlighting the influence of water on cellulose stiffness and sliding. This study offers insights into the structure and deformation modes of primary cell walls and presents a framework that is also applicable to tissues and whole organs.

Hemodynamic regulation allows stable growth of microvascular networks.

How do vessels find optimal radii? Capillaries are known to adapt their radii to maintain the shear stress of blood flow at the vessel wall at a set point, yet models of adaptation purely based on average shear stress have not been able to produce complex loopy networks that resemble real microvascular systems. For narrow vessels where red blood cells travel in a single file, the shear stress on vessel endothelium peaks sharply when a red blood cell passes through. We show that stable shear-stress-based adaptation is possible if vessel shear stress set points are cued to the stress peaks. Model networks that respond to peak stresses alone can quantitatively reproduce the observed zebrafish trunk microcirculation, including its adaptive trajectory when hematocrit changes or parts of the network are amputated. Our work reveals the potential for mechanotransduction alone to generate stable hydraulically tuned microvascular networks.

A fungal protein organizes both glycogen and cell wall glucans.

Glycogen is a glucose storage molecule composed of branched α-1,4-glucan chains, best known as an energy reserve that can be broken down to fuel central metabolism. Because fungal cells have a specialized need for glucose in building cell wall glucans, we investigated whether glycogen is used for this process. For these studies, we focused on the pathogenic yeast Cryptococcus neoformans, which causes ~150,000 deaths per year worldwide. We identified two proteins that influence formation of both glycogen and the cell wall: glycogenin (Glg1), which initiates glycogen synthesis, and a protein that we call Glucan organizing enzyme 1 (Goe1). We found that cells missing Glg1 lack α-1,4-glucan in their walls, indicating that this material is derived from glycogen. Without Goe1, glycogen rosettes are mislocalized and ß-1,3-glucan in the cell wall is reduced. Altogether, our results provide mechanisms for a close association between glycogen and cell wall.

The disordered C-terminal tail of fungal LPMOs from phytopathogens mediates protein dimerization and impacts plant penetration.

Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that oxidatively degrade various polysaccharides, such as cellulose. Despite extensive research on this class of enzymes, the role played by their C-terminal regions predicted to be intrinsically disordered (dCTR) has been overlooked. Here, we investigated the function of the dCTR of an LPMO, called CoAA9A, up-regulated during plant infection by Colletotrichum orbiculare, the causative agent of anthracnose. After recombinant production of the full-length protein, we found that the dCTR mediates CoAA9A dimerization in vitro, via a disulfide bridge, a hitherto-never-reported property that positively affects both binding and activity on cellulose. Using SAXS experiments, we show that the homodimer is in an extended conformation. In vivo, we demonstrate that gene deletion impairs formation of the infection-specialized cell called appressorium and delays penetration of the plant. Using immunochemistry, we show that the protein is a dimer not only in vitro but also in vivo when secreted by the appressorium. As these peculiar LPMOs are also found in other plant pathogens, our findings open up broad avenues for crop protection.