NKS1/ELMO4 is an integral protein of a pectin synthesis protein complex and maintains Golgi morphology and cell adhesion in Arabidopsis.

Adjacent plant cells are connected by specialized cell wall regions, called middle lamellae, which influence critical agricultural characteristics, including fruit ripening and organ abscission. Middle lamellae are enriched in pectin polysaccharides, specifically homogalacturonan (HG). Here, we identify a plant-specific Arabidopsis DUF1068 protein, called NKS1/ELMO4, that is required for middle lamellae integrity and cell adhesion. NKS1 localizes to the Golgi apparatus and loss of NKS1 results in changes to Golgi structure and function. The nks1 mutants also display HG deficient phenotypes, including reduced seedling growth, changes to cell wall composition, and tissue integrity defects. These phenotypes are comparable to qua1 and qua2 mutants, which are defective in HG biosynthesis. Notably, genetic interactions indicate that NKS1 and the QUAs work in a common pathway. Protein interaction analyses and modeling corroborate that they work together in a stable protein complex with other pectin-related proteins. We propose that NKS1 is an integral part of a large pectin synthesis protein complex and that proper function of this complex is important to support Golgi structure and function.

Insights into the cell-wall dynamics in grapevine berries during ripening and in response to biotic and abiotic stresses.

The cell wall (CW) is the dynamic structure of a plant cell, acting as a barrier against biotic and abiotic stresses. In grape berries, the modifications of pulp and skin CW during softening ensure flexibility during cell expansion and determine the final berry texture. In addition, the CW of grape berry skin is of fundamental importance for winemaking, controlling secondary metabolite extractability. Grapevine varieties with contrasting CW characteristics generally respond differently to biotic and abiotic stresses. In the context of climate change, it is important to investigate the CW dynamics occurring upon different stresses, to define new adaptation strategies. This review summarizes the molecular mechanisms underlying CW modifications during grapevine berry fruit ripening, plant-pathogen interaction, or in response to environmental stresses, also considering the most recently published transcriptomic data. Furthermore, perspectives of new biotechnological approaches aiming at modifying the CW properties based on other crops' examples are also presented.

A rice GT61 glycosyltransferase possesses dual activities mediating 2-O-xylosyl and 2-O-arabinosyl substitutions of xylan.

MAIN CONCLUSION: A member of the rice GT61 clade B is capable of transferring both 2-O-xylosyl and 2-O-arabinosyl residues onto xylan and another member specifically catalyses addition of 2-O-xylosyl residue onto xylan. Grass xylan is substituted predominantly with 3-O-arabinofuranose (Araf) as well as with some minor side chains, such as 2-O-Araf and 2-O-(methyl)glucuronic acid [(Me)GlcA]. 3-O-Arabinosylation of grass xylan has been shown to be catalysed by grass-expanded clade A members of the glycosyltransferase family 61. However, glycosyltransferases mediating 2-O-arabinosylation of grass xylan remain elusive. Here, we performed biochemical studies of two rice GT61 clade B members and found that one of them was capable of transferring both xylosyl (Xyl) and Araf residues from UDP-Xyl and UDP-Araf, respectively, onto xylooligomer acceptors, whereas the other specifically catalysed Xyl transfer onto xylooligomers, indicating that the former is a xylan xylosyl/arabinosyl transferase (named OsXXAT1 herein) and the latter is a xylan xylosyltransferase (named OsXYXT2). Structural analysis of the OsXXAT1- and OsXYXT2-catalysed reaction products revealed that the Xyl and Araf residues were transferred onto O-2 positions of xylooligomers. Furthermore, we demonstrated that OsXXAT1 and OsXYXT2 were able to substitute acetylated xylooligomers, but only OsXXAT1 could xylosylate GlcA-substituted xylooligomers. OsXXAT1 and OsXYXT2 were predicted to adopt a GT-B fold structure and molecular docking revealed candidate amino acid residues at the predicted active site involved in binding of the nucleotide sugar donor and the xylohexaose acceptor substrates. Together, our results establish that OsXXAT1 is a xylan 2-O-xylosyl/2-O-arabinosyl transferase and OsXYXT2 is a xylan 2-O-xylosyltransferase, which expands our knowledge of roles of the GT61 family in grass xylan synthesis.

Construction and characterization of a hypervesiculation strain of Escherichia coli Nissle 1917.

Outer membrane vesicles (OMVs) are produced by Gram-negative bacteria and deliver microbial molecules to distant target cells in a host. OMVs secreted by probiotic probiotic strain Escherichia coli Nissle 1917 (EcN) have been reported to induce an immune response. In this study, we aimed to increase the OMV production of EcN. The double gene knockout of mlaE and nlpI was conducted in EcN because the ΔmlaEΔnlpI of experimental strain E. coli K12 showed the highest OMV production in our previous report. The ΔmlaEΔnlpI of EcN showed approximately 8 times higher OMV production compared with the parental (wild-type) strain. Quick-freeze, deep-etch replica electron microscopy revealed that plasmolysis occurred in the elongated ΔmlaEΔnlpI cells and the peptidoglycan (PG) had numerous holes. While these phenomena are similar to the findings for the ΔmlaEΔnlpI of K12, there were more PG holes in the ΔmlaEΔnlpI of EcN than the K12 strain, which were observed not only at the tip of the long axis but also in the whole PG structure. Further analysis clarified that the viability of ΔmlaEΔnlpI of EcN decreased compared with that of the wild-type. Although the amount of PG in ΔmlaEΔnlpI cells was about half of that in wild-type, the components of amino acids in PG did not change in ΔmlaEΔnlpI. Although the viability decreased compared to the wild-type, the ΔmlaEΔnlpI grew in normal culture conditions. The hypervesiculation strain constructed here is expected to be used as an enhanced probiotic strain.

Advances and prospects of analytic methods for bacterial transglycosylation and inhibitor discovery.

The cell wall is essential for bacteria to maintain structural rigidity and withstand external osmotic pressure. In bacteria, the cell wall is composed of peptidoglycan. Lipid II is the basic unit for constructing highly cross-linked peptidoglycan scaffolds. Transglycosylase (TGase) is the initiating enzyme in peptidoglycan synthesis that catalyzes the ligation of lipid II moieties into repeating GlcNAc-MurNAc polysaccharides, followed by transpeptidation to generate cross-linked structures. In addition to the transglycosylases in the class-A penicillin-binding proteins (aPBPs), SEDS (shape, elongation, division and sporulation) proteins are also present in most bacteria and play vital roles in cell wall renewal, elongation, and division. In this review, we focus on the latest analytical methods including the use of radioactive labeling, gel electrophoresis, mass spectrometry, fluorescence labeling, probing undecaprenyl pyrophosphate, fluorescence anisotropy, ligand-binding-induced tryptophan fluorescence quenching, and surface plasmon resonance to evaluate TGase activity in cell wall formation. This review also covers the discovery of TGase inhibitors as potential antibacterial agents. We hope that this review will give readers a better understanding of the chemistry and basic research for the development of novel antibiotics.

Cell wall as a barrier for protein extraction from tomato leaves: A biochemical study.

Solanum lycopersicum (Tomato) leaves and stems are considered waste. Valorization of this waste can be achieved by for example the extraction of proteins. This prospect is promising but currently not feasible, since protein extraction yields from tomato leaves are low, amongst other due to the (physical) barrier formed by the plant cell walls. However, the molecular aspects of the relationship between cell wall properties and protein extractability from tomato leaves are currently not clear and thus objective of this study. To fill this knowledge gap the biochemical composition of plant cell walls was measured and related to protein extraction yields at different plant ages, leaf positions, and across different tomato accessions, including two Solanum lycopersicum cultivars and the wildtype species S. pimpinellifolium and S. pennellii. For all genotypes, protein extraction yields from tomato leaves were the highest in young tissues, with a decreasing trend towards older plant material. This decrease of protein extraction yield was accompanied by a significant increase of arabinose and galacturonic acid content and a decrease of galactose content in the cell walls of old-vs-young tissues. This resulted in strong negative correlations between protein extraction yield and the content of arabinose and galacturonic acid in the cell wall, and a positive correlation between the content of galactose and protein extraction yield. Overall, these results point to the importance of the pectin network on protein extractability, making pectin a potential breeding target for enhancing protein extractability from tomato leaves.

Peptidoglycan from Bacillus anthracis Inhibits Human Macrophage Efferocytosis in Part by Reducing Cell Surface Expression of MERTK and TIM-3.

Bacillus anthracis peptidoglycan (PGN) is a major component of the bacterial cell wall and a key pathogen-associated molecular pattern contributing to anthrax pathology, including organ dysfunction and coagulopathy. Increases in apoptotic leukocytes are a late-stage feature of anthrax and sepsis, suggesting there is a defect in apoptotic clearance. In this study, we tested the hypothesis that B. anthracis PGN inhibits the capacity of human monocyte-derived macrophages (MΦ) to efferocytose apoptotic cells. Exposure of CD163+CD206+ MΦ to PGN for 24 h impaired efferocytosis in a manner dependent on human serum opsonins but independent of complement component C3. PGN treatment reduced cell surface expression of the proefferocytic signaling receptors MERTK, TYRO3, AXL, integrin αVß5, CD36, and TIM-3, whereas TIM-1, αVß3, CD300b, CD300f, STABILIN-1, and STABILIN-2 were unaffected. ADAM17 is a major membrane-bound protease implicated in mediating efferocytotic receptor cleavage. We found multiple ADAM17-mediated substrates increased in PGN-treated supernatant, suggesting involvement of membrane-bound proteases. ADAM17 inhibitors TAPI-0 and Marimastat prevented TNF release, indicating effective protease inhibition, and modestly increased cell-surface levels of MerTK and TIM-3 but only partially restored efferocytic capacity by PGN-treated MΦ. We conclude that human serum factors are required for optimal recognition of PGN by human MΦ and that B. anthracis PGN inhibits efferocytosis in part by reducing cell surface expression of MERTK and TIM-3.

Phosphorus deficiency-induced cell wall pectin demethylesterification enhances cadmium accumulation in roots of Salix caprea.

Regions affected by heavy metal contamination frequently encounter phosphorus (P) deficiency. Numerous studies highlight crucial role of P in facilitating cadmium (Cd) accumulation in woody plants. However, the regulatory mechanism by which P affects Cd accumulation in roots remains ambiguous. This study aims to investigate the effects of phosphorus (P) deficiency on Cd accumulation, Cd subcellular distribution, and cell wall components in the roots of Salix caprea under Cd stress. The results revealed that under P deficiency conditions, there was a 35.4% elevation in Cd content in roots, coupled with a 60.1% reduction in Cd content in shoots, compared to the P sufficiency conditions. Under deficient P conditions, the predominant response of roots to Cd exposure was the increased sequestration of Cd in root cell walls. The sequestration of Cd in root cell walls increased from 37.1% under sufficient P conditions to 66.7% under P deficiency, with pectin identified as the primary Cd binding site under both P conditions. Among cell wall components, P deficiency led to a significant 31.7% increase in Cd content within pectin compared to P sufficiency conditions, but did not change the pectin content. Notably, P deficiency significantly increased pectin methylesterase (PME) activity by regulating the expression of PME and PMEI genes, leading to a 10.4% reduction in the degree of pectin methylesterification. This may elucidate the absence of significant changes in pectin content under P deficiency conditions and the concurrent increase in Cd accumulation in pectin. Fourier transform infrared spectroscopy (FTIR) results indicated an increase in carboxyl groups in the root cell walls under P deficiency compared to sufficient P treatment. The results provide deep insights into the mechanisms of higher Cd accumulation in root mediated by P deficiency.

Reduced peptidoglycan synthesis capacity impairs growth of E. coli at high salt concentration.

Gram-negative bacteria have a thin peptidoglycan layer between the cytoplasmic and outer membranes protecting the cell from osmotic challenges. Hydrolases of this structure are needed to cleave bonds to allow the newly synthesized peptidoglycan strands to be inserted by synthases. These enzymes need to be tightly regulated and their activities coordinated to prevent cell lysis. To better understand this process in Escherichia coli, we probed the genetic interactions of mrcA (encodes PBP1A) and mrcB (encodes PBP1B) with genes encoding peptidoglycan amidases and endopeptidases in envelope stress conditions. Our extensive genetic interaction network analysis revealed relatively few combinations of hydrolase gene deletions with reduced fitness in the absence of PBP1A or PBP1B, showing that none of the amidases or endopeptidases is strictly required for the functioning of one of the class A PBPs. This illustrates the robustness of the peptidoglycan growth mechanism. However, we discovered that the fitness of ∆mrcB cells is significantly reduced under high salt stress and in vitro activity assays suggest that this phenotype is caused by a reduced peptidoglycan synthesis activity of PBP1A at high salt concentration.IMPORTANCEEscherichia coli and many other bacteria have a surprisingly high number of peptidoglycan hydrolases. These enzymes function in concert with synthases to facilitate the expansion of the peptidoglycan sacculus under a range of growth and stress conditions. The synthases PBP1A and PBP1B both contribute to peptidoglycan expansion during cell division and growth. Our genetic interaction analysis revealed that these two penicillin-binding proteins (PBPs) do not need specific amidases, endopeptidases, or lytic transglycosylases for function. We show that PBP1A and PBP1B do not work equally well when cells encounter high salt stress and demonstrate that PBP1A alone cannot provide sufficient PG synthesis activity under this condition. These results show how the two class A PBPs and peptidoglycan hydrolases govern cell envelope integrity in E. coli in response to environmental challenges and particularly highlight the importance of PBP1B in maintaining cell fitness under high salt conditions.

Wall teichoic acid substitution with glucose governs phage susceptibility of Staphylococcus epidermidis.

The species- and clone-specific susceptibility of Staphylococcus cells for bacteriophages is governed by the structures and glycosylation patterns of wall teichoic acid (WTA) glycopolymers. The glycosylation-dependent phage-WTA interactions in the opportunistic pathogen Staphylococcus epidermidis and in other coagulase-negative staphylococci (CoNS) have remained unknown. We report a new S. epidermidis WTA glycosyltransferase TagE whose deletion confers resistance to siphoviruses such as ΦE72 but enables binding of otherwise unbound podoviruses. S. epidermidis glycerolphosphate WTA was found to be modified with glucose in a tagE-dependent manner. TagE is encoded together with the enzymes PgcA and GtaB providing uridine diphosphate-activated glucose. ΦE72 transduced several other CoNS species encoding TagE homologs, suggesting that WTA glycosylation via TagE is a frequent trait among CoNS that permits interspecies horizontal gene transfer. Our study unravels a crucial mechanism of phage-Staphylococcus interaction and horizontal gene transfer, and it will help in the design of anti-staphylococcal phage therapies.IMPORTANCEPhages are highly specific for certain bacterial hosts, and some can transduce DNA even across species boundaries. How phages recognize cognate host cells remains incompletely understood. Phages infecting members of the genus Staphylococcus bind to wall teichoic acid (WTA) glycopolymers with highly variable structures and glycosylation patterns. How WTA is glycosylated in the opportunistic pathogen Staphylococcus epidermidis and in other coagulase-negative staphylococci (CoNS) species has remained unknown. We describe that S. epidermidis glycosylates its WTA backbone with glucose, and we identify a cluster of three genes responsible for glucose activation and transfer to WTA. Their inactivation strongly alters phage susceptibility patterns, yielding resistance to siphoviruses but susceptibility to podoviruses. Many different CoNS species with related glycosylation genes can exchange DNA via siphovirus ΦE72, suggesting that glucose-modified WTA is crucial for interspecies horizontal gene transfer. Our finding will help to develop antibacterial phage therapies and unravel routes of genetic exchange.

Monitoring the Interaction of the Peptidoglycan with the Bacterial ß-Barrel Assembly Machinery.

Gram-negative bacteria coordinate the biosynthesis of their different cell envelope components. Growth of the outer membrane (OM) requires the essential ß-barrel assembly machine (BAM), which inserts OM proteins (OMPs) into the OM. The underlying peptidoglycan (PG) sacculus grows by the insertion of nascent glycan chains. We have previously identified interactions between BAM and PG in E. coli and showed that these interactions coordinate OM biogenesis with PG growth. BAM responds to the maturation state of the PG, and this mechanism activates preferentially BAM complexes at sites of active PG synthesis. Here we present protocols to purify soluble Bam proteins and full-length BamABCDE, isolate PG and soluble PG fragments, and study BAM-PG interactions with the isolated components. We also describe the protocol to detect interactions between Bam proteins and PG in cells.

Importance of Non-Covalent Interactions in Yeast Cell Wall Molecular Organization.

This review covers a group of non-covalently associated molecules, particularly proteins (NCAp), incorporated in the yeast cell wall (CW) with neither disulfide bridges with proteins covalently attached to polysaccharides nor other covalent bonds. Most NCAp, particularly Bgl2, are polysaccharide-remodeling enzymes. Either directly contacting their substrate or appearing as CW lipid-associated molecules, such as in vesicles, they represent the most movable enzymes and may play a central role in CW biogenesis. The absence of the covalent anchoring of NCAp allows them to be there where and when it is necessary. Another group of non-covalently attached to CW molecules are polyphosphates (polyP), the universal regulators of the activity of many enzymes. These anionic polymers are able to form complexes with metal ions and increase the diversity of non-covalent interactions through charged functional groups with both proteins and polysaccharides. The mechanism of regulation of polysaccharide-remodeling enzyme activity in the CW is unknown. We hypothesize that polyP content in the CW is regulated by another NCAp of the CW-acid phosphatase-which, along with post-translational modifications, may thus affect the activity, conformation and compartmentalization of Bgl2 and, possibly, some other polysaccharide-remodeling enzymes.

Sound waves alter the viability of tobacco cells via changes in cytosolic calcium, membrane integrity, and cell wall composition.

The effect of sound waves (SWs) on plant cells can be considered as important as other mechanical stimuli like touch, wind, rain, and gravity, causing certain responses associated with the downstream signaling pathways on the whole plant. The objective of the present study was to elucidate the response of suspension-cultured tobacco cells (Nicotiana tabacum L. cv Burley 21) to SW at different intensities. The sinusoidal SW (1,000 Hz) was produced through a signal generator, amplified, and beamed to the one layer floating tobacco cells inside a soundproof chamber at intensities of 60, 75, and 90 dB at the plate level for 15, 30, 45, and 60 min. Calibration of the applied SW intensities, accuracy, and uniformity of SW was performed by a sound level meter, and the cells were treated. The effect of SW on tobacco cells was monitored by quantitation of cytosolic calcium, redox status, membrane integrity, wall components, and the activity of wall modifying enzymes. Cytosolic calcium ions increased as a function of sound intensity with a maximum level of 90 dB. Exposure to 90 dB was also accompanied by a significant increase of H2O2 and membrane lipid peroxidation rate but the reduction of total antioxidant and radical scavenging capacities. The increase of wall rigidity in these cells was attributed to an increase in wall-bound phenolic acids and lignin and the activities of phenylalanine ammonia-lyase and covalently bound peroxidase. In comparison, in 60- and 75 dB, radical scavenging capacity increased, and the activity of wall stiffening enzymes reduced, but cell viability showed no changes. The outcome of the current study reveals that the impact of SW on plant cells is started by an increase in cytosolic calcium. However, upon calcium signaling, downstream events, including alteration of H2O2 and cell redox status and the activities of wall modifying enzymes, determined the extent of SW effects on tobacco cells.

Characterization of the fiber-like cortical cells in moss gametophytes.

MAIN CONCLUSION: Fiber-like cells with thickened cell walls of specific structure and polymer composition that includes (1 → 4)-ß-galactans develop in the outer stem cortex of several moss species gametophytes. The early land plants evolved several specialized cell types and tissues that did not exist in their aquatic ancestors. Of these, water-conducting elements and reproductive organs have received most of the research attention. The evolution of tissues specialized to fulfill a mechanical function is by far less studied despite their wide distribution in land plants. For vascular plants following a homoiohydric trajectory, the evolutionary emergence of mechanical tissues is mainly discussed starting with the fern-like plants with their hypodermal sterome or sclerified fibers that have xylan and lignin-based cell walls. However, mechanical challenges were also faced by bryophytes, which lack lignified cell-walls. To characterize mechanical tissues in the bryophyte lineage, following a poikilohydric trajectory, we used six wild moss species (Polytrichum juniperinum, Dicranum sp., Rhodobryum roseum, Eurhynchiadelphus sp., Climacium dendroides, and Hylocomium splendens) and analyzed the structure and composition of their cell walls. In all of them, the outer stem cortex of the leafy gametophytic generation had fiber-like cells with a thickened but non-lignified cell wall. Such cells have a spindle-like shape with pointed tips. The additional thick cell wall layer in those fiber-like cells is composed of sublayers with structural evidence for different cellulose microfibril orientation, and with specific polymer composition that includes (1 → 4)-ß-galactans. Thus, the basic cellular characters of the cells that provide mechanical support in vascular plant taxa (elongated cell shape, location at the periphery of a primary organ, the thickened cell wall and its peculiar composition and structure) also exist in mosses.

Physiological characterization of single-gene lysis proteins.

Single-strand RNA (ssRNA) and single-strand DNA phages elicit host lysis using a single gene, in each case designated as sgl. Of the 11 identified Sgls, three have been shown to be specific inhibitors of different steps in the pathway that supplies lipid II to the peptidoglycan (PG) biosynthesis machinery. These Sgls have been called "protein antibiotics" because the lytic event is a septal catastrophe indistinguishable from that caused by cell wall antibiotics. Here, we designate these as type I Sgls. In this formalism, the other eight Sgls are assigned to type II, the best-studied of which is protein L of the paradigm F-specific ssRNA phage MS2. Comparisons have suggested that type II Sgls have four sequence elements distinguished by hydrophobic and polar character. Environmental metatranscriptomics has revealed thousands of new ssRNA phage genomes, each of which presumably has an Sgl. Here, we describe methods to distinguish type I and type II Sgls. Using phase contrast microscopy, we show that both classes of Sgls cause the formation of blebs prior to lysis, but the location of the blebs differs significantly. In addition, we show that L and other type II Sgls do not inhibit the net synthesis of PG, as measured by radio-labeling of PG. Finally, we provide direct evidence that the Sgl from Pseudomonas phage PP7 is a type I Sgl, in support of a recent report based on a genetic selection. This shows that the putative four-element sequence structure suggested for L is not a reliable discriminator for the operational characterization of Sgls. IMPORTANCE: The ssRNA phage world has recently undergone a metagenomic expansion upward of a thousandfold. Each genome likely carries at least one single-gene lysis (sgl) cistron encoding a protein that single-handedly induces host autolysis. Here, we initiate an approach to segregate the Sgls into operational types based on physiological analysis, as a first step toward the alluring goal of finding many new ways to induce bacterial death and the attendant expectations for new antibiotic development.

Defects in the cell wall and its deposition caused by loss-of-function of three RLKs alter root hydrotropism in Arabidopsis thaliana.

Root tips can sense moisture gradients and grow into environments with higher water potential. This process is called root hydrotropism. Here, we report three closely related receptor-like kinases (RLKs) that play critical roles in root hydrotropism: ALTERED ROOT HYDROTROPIC RESPONSE 1 (ARH1), FEI1, and FEI2. Overexpression of these RLKs strongly reduce root hydrotropism, but corresponding loss-of-function mutants exhibit an increased hydrotropic response in their roots. All these RLKs show polar localization at the plasma membrane regions in root tips. The biosynthesis of the cell wall, cutin, and wax (CCW) is significantly impaired in root tips of arh1-2 fei1-C fei2-C. A series of known CCW mutants also exhibit increased root hydrotropism and reduced osmotic tolerance, similar to the characteristics of the triple mutant. Our results demonstrat that the integrity of the cell wall, cutin, and root cap wax mediate a trade-off between root hydrotropism and osmotic tolerance.

Overexpression of the PtrNF-YA6 gene inhibits secondary cell wall thickening in poplar.

The NF-Y gene family in plants plays a crucial role in numerous biological processes, encompassing hormone response, stress response, as well as growth and development. In this study, we first used bioinformatics techniques to identify members of the NF-YA family that may function in wood formation. We then used molecular biology techniques to investigate the role and molecular mechanism of PtrNF-YA6 in secondary cell wall (SCW) formation in Populus trichocarpa. We found that PtrNF-YA6 protein was localized in the nucleus and had no transcriptional activating activity. Overexpression of PtrNF-YA6 had an inhibitory effect on plant growth and development and significantly suppressed hemicellulose synthesis and SCW thickening in transgenic plants. Yeast one-hybrid and ChIP-PCR assays revealed that PtrNF-YA6 directly regulated the expression of hemicellulose synthesis genes (PtrGT47A-1, PtrGT8C, PtrGT8F, PtrGT43B, PtrGT47C, PtrGT8A and PtrGT8B). In conclusion, PtrNF-YA6 can inhibit plant hemicellulose synthesis and SCW thickening by regulating the expression of downstream SCW formation-related target genes.

Exploring the role of the LkABCG36 transporter in lignin accumulation.

Lignin is a complex biopolymer formed through the condensation of three monomeric precursors known as monolignols. However, the mechanism underlying lignin precursor transport remains elusive, with uncertainty over whether it occurs through passive diffusion or an active energized process. ATP-binding cassette 36 (ABCG36) plays important roles in abiotic stress resistance. In this study, we investigated the transport functions of LkABCG36 (Larix kaempferi) for lignin precursors and the potential effects of LkABCG36 overexpression in plants. LkABCG36 enhanced the ability of tobacco (Nicotiana tabacum) bright yellow-2 (BY-2) cells to resist monolignol alcohol stress. Furthermore, LkABCG36 overexpression promoted lignin deposition in tobacco plant stem tissue. To understand the underlying mechanism, we measured the BY-2 cell ability to export lignin monomers and the uptake of monolignol precursors in inside-out (inverted) plasma membrane vesicles. We found that the transport of coniferyl and sinapyl alcohols is an ATP-dependent process. Our data suggest that LkABCG36 contributes to lignin accumulation in tobacco stem tissues through a mechanism involving the active transport of lignin precursors to the cell wall. These findings shed light on the lignin biosynthesis process, with important implications for enhancing lignin deposition in plants, potentially leading to improved stress tolerance and biomass production.

Histone acetyltransferase Sas3 contributes to fungal development, cell wall integrity, and virulence in Aspergillus fumigatus.

Histone acetyltransferase (HAT)-mediated epigenetic modification is essential for diverse cellular processes in eukaryotes. However, the functions of HATs in the human pathogen Aspergillus fumigatus remain poorly understood. In this study, we characterized the functions of MOZ, Ybf2/Sas3, Sas2, and Tip60 (MYST)-family histone acetyltransferase something about silencing (Sas3) in A. fumigatus. Phenotypic analysis revealed that loss of Sas3 results in significant impairments in colony growth, conidiation, and virulence in the Galleria mellonella model. Subcellular localization and Western blot analysis demonstrated that Sas3 localizes to nuclei and is capable of acetylating lysine 9 and 14 of histone H3 in vivo. Importantly, we found that Sas3 is critical for the cell wall integrity (CWI) pathway in A. fumigatus as evidenced by hypersensitivity to cell wall-perturbing agents, altered cell wall thickness, and abnormal phosphorylation levels of CWI protein kinase MpkA. Furthermore, site-directed mutagenesis studies revealed that the conserved glycine residues G641 and G643 and glutamate residue E664 are crucial for the acetylation activity of Sas3. Unexpectedly, only triple mutations of Sas3 (G641A/G643A/E664A) displayed defective phenotypes similar to the Δsas3 mutant, while double or single mutations did not. This result implies that the role of Sas3 may extend beyond histone acetylation. Collectively, our findings demonstrate that MYST-family HAT Sas3 plays an important role in the fungal development, virulence, and cell wall integrity in A. fumigatus. IMPORTANCE: Epigenetic modification governed by HATs is indispensable for various cellular processes in eukaryotes. Nonetheless, the precise functions of HATs in the human pathogen Aspergillus fumigatus remain elusive. In this study, we unveil the roles of MYST-family HAT Sas3 in colony growth, conidiation, virulence, and cell wall stress response in A. fumigatus. Particularly, our findings demonstrate that Sas3 can function through mechanisms unrelated to histone acetylation, as evidenced by site-directed mutagenesis experiments. Overall, this study broadens our understanding of the regulatory mechanism of HATs in fungal pathogens.

A self-regulatory cell-wall-sensing module at cell edges controls plant growth.

Morphogenesis of multicellular organs requires coordination of cellular growth. In plants, cell growth is determined by turgor pressure and the mechanical properties of the cell wall, which also glues cells together. Because plants have to integrate tissue-scale mechanical stresses arising through growth in a fixed tissue topology, they need to monitor cell wall mechanical status and adapt growth accordingly. Molecular factors have been identified, but whether cell geometry contributes to wall sensing is unknown. Here we propose that plant cell edges act as cell-wall-sensing domains during growth. We describe two Receptor-Like Proteins, RLP4 and RLP4-L1, which occupy a unique polarity domain at cell edges established through a targeted secretory transport pathway. We show that RLP4s associate with the cell wall at edges via their extracellular domain, respond to changes in cell wall mechanics and contribute to directional growth control in Arabidopsis.