Host cell wall composition and localized microenvironment implicated in resistance to basal stem degradation by lettuce drop (Sclerotinia minor).

BACKGROUND: Sclerotinia spp. are generalist fungal pathogens, infecting over 700 plant hosts worldwide, including major crops. While host resistance is the most sustainable and cost-effective method for disease management, complete resistance to Sclerotinia diseases is rare. We recently identified soft basal stem as a potential susceptibility factor to Sclerotinia minor infection in lettuce (Lactuca sativa) under greenhouse conditions. RESULTS: Analysis of stem and root cell wall composition in five L. sativa and one L. serriola accessions with varying growth habits and S. minor resistance levels revealed strong association between hemicellulose constituents, lignin polymers, disease phenotypes, and basal stem mechanical strength. Accessions resistant to basal stem degradation consistently exhibited higher levels of syringyl, guaiacyl, and xylose, but lower levels of fucose in stems. These findings suggest that stem cell wall polymers recalcitrant to breakdown by lignocellulolytic enzymes may contribute to stem strength-mediated resistance against S. minor. CONCLUSIONS: The lignin content, particularly guaiacyl and syringyl, along with xylose could potentially serve as biomarkers for identifying more resistant lettuce accessions and breeding lines. Basal stem degradation by S. minor was influenced by localized microenvironment conditions around the stem base of the plants.

Characterization of Subcellular Dynamics of Sterol Methyltransferases Clarifies Defective Cell Division in smt2 smt3, a C-24 Ethyl Sterol-Deficient Mutant of Arabidopsis.

An Arabidopsis sterol mutant, smt2 smt3, defective in sterolmethyltransferase2 (SMT2), exhibits severe growth abnormalities. The loss of C-24 ethyl sterols, maintaining the biosynthesis of C-24 methyl sterols and brassinosteroids, suggests specific roles of C-24 ethyl sterols. We characterized the subcellular localizations of fluorescent protein-fused sterol biosynthetic enzymes, such as SMT2-GFP, and found these enzymes in the endoplasmic reticulum during interphase and identified their movement to the division plane during cytokinesis. The mobilization of endoplasmic reticulum-localized SMT2-GFP was independent of the polarized transport of cytokinetic vesicles to the division plane. In smt2 smt3, SMT2-GFP moved to the abnormal division plane, and unclear cell plate ends were surrounded by hazy structures from SMT2-GFP fluorescent signals and unincorporated cellulose debris. Unusual cortical microtubule organization and impaired cytoskeletal function accompanied the failure to determine the cortical division site and division plane formation. These results indicated that both endoplasmic reticulum membrane remodeling and cytokinetic vesicle transport during cytokinesis were impaired, resulting in the defects of cell wall generation. The cell wall integrity was compromised in the daughter cells, preventing the correct determination of the subsequent cell division site. We discuss the possible roles of C-24 ethyl sterols in the interaction between the cytoskeletal network and the plasma membrane.

Melatonin Increases Root Cell Wall Phosphorus Reutilization via an NO Dependent Pathway in Rice (Oryza sativa).

Melatonin (MT) has been implicated in the plant response to phosphorus (P) stress; however, the precise molecular mechanisms involved remain unclear. This study investigated whether MT controls internal P distribution and root cell wall P remobilization in rice. Rice was treated with varying MT and P levels and analyzed using biochemical and molecular techniques to study phosphorus utilization. The results demonstrated that low P levels lead to a rapid increase in endogenous MT levels in rice roots. Furthermore, the exogenous application of MT significantly improved rice tolerance to P deficiency, as evidenced by the increased biomass and reduced proportion of roots to shoots under P-deficient conditions. MT application also mitigated the decrease in P content regardless in both the roots and shoots. Mechanistically, MT accelerated the reutilization of P, particularly in the root pectin fraction, leading to increased soluble P liberation. In addition, MT enhanced the expression of OsPT8, a gene involved in root-to-shoot P translocation. Furthermore, we observed that MT induced the production of nitric oxide (NO) in P-deficient rice roots and that the mitigating effect of MT on P deficiency was compromised in the presence of the NO inhibitor, c-PTIO, implying that NO is involved in the MT-facilitated mitigation of P deficiency in rice. Overall, our findings highlight the potential of MT as a promising strategy for enhancing rice tolerance to P deficiency and improving P use efficiency in agricultural practices.

Bacteriophage-encoded protein utilization in bacterial ghost production: a mini-review.

Bacterial ghosts (BGs) are described as bacterial cell envelopes that retain their structure but lack cytoplasmic contents. The study of BGs spans multiple disciplinary domains, and the development of BG production techniques to obtain ample and stable BG samples holds significant implications for probing the biological characteristics of BGs, devising novel disease treatment strategies, and leveraging their industrial applications. Numerous products encoded within bacteriophage (phage) genomes possess the capability to lyse bacteria, thereby inducing BG formation primarily via disruption of bacterial cell wall integrity. This review comprehensively surveys the utilization of phage-encoded proteins in BG production techniques, encompassing methodologies such as phage E protein-mediated lysis, perforin protein-induced lysis, and strategies combining E protein with holin-endolysin systems. Additionally, discussions and summaries are provided on the current applications, challenges, and modification strategies associated with different techniques. Through a focused exploration of BG production techniques, with an emphasis on precise manipulation of BG formation using phage-encoded protein technologies, this study aims to furnish robust tools and methodologies for delving into the mechanisms underlying BG formation, as well as for the development of novel therapeutic strategies and applications based on BGs.

Acidothermus cellulolyticus E1 endoglucanase expressed in planta undergoes extensive hydroxyproline-O-glycosylation and exhibits enhanced impact on biomass digestibility.

KEY MESSAGE: E1 holoenzyme was extensively Hyp-O-glycosylated at the proline rich linker region in plants, which substantially increased the molecular size and improved the enzymatic digestibility of the biomass of transgenic plants. Thermophilic E1 endo-1,4-ß-glucanase derived from Acidothermus cellulolyticus has been frequently expressed in planta to reconstruct the plant cell wall to overcome biomass recalcitrance. However, the expressed holoenzyme exhibited a larger molecular size (~ 100 kDa) than the theoretical one (57 kDa), possibly due to posttranslational modifications in the recombinant enzyme within plant cells. This study investigates the glycosylation of the E1 holoenzyme expressed in tobacco plants and determines its impact on enzyme activity and biomass digestibility. The E1 holoenzyme, E1 catalytic domain (E1cd) and E1 linker (E1Lk) were each expressed in tobacco plants and suspension cells. The accumulation of holoenzyme was 2.0- to 2.3- times higher than that of E1cd. The proline-rich E1Lk region was extensively hydroxyproline-O-glycosylated with arabinogalactan polysaccharides. Compared with E1cd, the holoenzyme displayed a broader optimal temperature range (70 to 85 ºC). When grown in greenhouse, the expression of E1 holoenzyme induced notable phenotypic changes in plants, including delayed flowering and leaf variegation post-flowering. However, the final yield of plant biomass was not significantly affected. Finally, plant biomass engineering with E1 holoenzyme showed 1.7- to 1.8-fold higher saccharification efficiency than the E1cd lines and 2.4- to 2.7-fold higher than the wild-type lines, which was ascribed to the synergetic action of the E1Lk and cellulose binding module in reducing cell wall recalcitrance.

Synthesis of a Borrelia burgdorferi-Derived Muropeptide Standard Fragment Library.

The interplay between the human innate immune system and bacterial cell wall components is pivotal in understanding diseases such as Crohn's disease and Lyme arthritis. Lyme disease, caused by Borrelia burgdorferi, is the most prevalent tick-borne illness in the United States, with a substantial number of cases reported annually. While antibiotic treatments are generally effective, approximately 10% of Lyme disease cases develop persistent arthritis, suggesting a dysregulated host immune response. We have previously identified a link between the immunogenic B. burgdorferi peptidoglycan (PG) and Lyme arthritis and showed that this pathogen sheds significant amounts of PG fragments during growth. Here, we synthesize these PG fragments, including ornithine-containing monosaccharides and disaccharides, to mimic the unique composition of Borrelia cell walls, using reproducible and rigorous synthetic methods. This synthetic approach allows for the modular preparation of PG derivatives, providing a diverse library of well-defined fragments. These fragments will serve as valuable tools for investigating the role of PG-mediated innate immune response in Lyme disease and aid in the development of improved diagnostic methods and treatment strategies.

Transcription factor FoAce2 regulates virulence, vegetative growth, conidiation, and cell wall homeostasis in Fusarium oxysporum f. sp. cubense.

Fusarium wilt of banana, caused by the fungus Fusarium oxysporum f. sp. cubense (Foc), is a serious fungal disease that affects banana plants globally. To explore the virulence mechanisms of this pathogen, we created a null mutation of the transcription factor gene FoAce2 (encoding F. oxysporum angiotensin converting enzyme 2). Deletion of FoAce2 resulted in slower growth, decreased aerial mycelia and conidiation, and a significant decrease in fungal virulence against banana hosts relative to those of the wild-type (WT) fungus. Additionally, transmission electron microscopy showed that the cell wall was thicker in the FoAce2 deletion mutants. Consistent with this finding, the cell wall glucose level was decreased in the ΔFoAce2 mutants compared with that in the WT and complemented strain, ΔFoAce2-C1. Complementation with the WT FoAce2 gene fully reversed the mutant phenotypes. Analysis of the transcriptome of ΔFoAce2 and the WT strain showed alterations in the expression levels of many genes associated with virulence and growth. Thus, FoAce2 appears to be essential for Foc virulence, cell wall homeostasis, conidiation, and vegetative growth.

Cell wall polysaccharide enhances Lacticaseibacillus paracasei strain Shirota growth in milk and contributes to acid and bile tolerance.

LCPS-1, a cell wall polysaccharide (CWPS), is bound to the cell wall of the probiotic Lacticaseibacillus paracasei (formerly known as Lactobacillus casei) strain Shirota (LcS). Generally, the role of CWPS in the viability and survivability of bacteria is yet to be fully understood. This study aimed to elucidate the role of LCPS-1 in the viability and survivability of LcS. A mutant strain completely lacking LCPS-1 was constructed and evaluated for growth in bovine and soy milk and susceptibility to acid and bile. The growth of the mutant in bovine and soy milk temporarily stalled after the late logarithmic phase while wild-type LcS continued growing, resulting in a significantly lower number of viable cells for the mutant strain (p < 0.01). Significantly higher cell death relative to that of the wild-type strain was observed for the mutant strain following acid treatment at pH 3.0 (p < 0.01), with 60 and 92 % survival, respectively. The absence of LCPS-1 also reduced the survival rate of LcS cells from 3.3 to 0.8 % following 0.2 % bile treatment. The survival rate of the mutant after consecutive treatment with acid and bile was 19 %, while 73 % of the wild-type LcS survived. These results indicate that LCPS-1 leads to higher LcS growth in milk and improves tolerance to acid and bile. This study reveals the contribution of probiotic bacterial CWPS to acidic and gastrointestinal stress tolerance. Based on these findings, characterizing and modifying CWPS in probiotic strains could enhance manufacturing yields and improve gastrointestinal stress tolerance after consumption by hosts, ultimately advancing the development of more effective probiotics.

Toxic eburicol accumulation drives the antifungal activity of azoles against Aspergillus fumigatus.

Azole antifungals inhibit the sterol C14-demethylase (CYP51/Erg11) of the ergosterol biosynthesis pathway. Here we show that the azole-induced synthesis of fungicidal cell wall carbohydrate patches in the pathogenic mold Aspergillus fumigatus strictly correlates with the accumulation of the CYP51 substrate eburicol. A lack of other essential ergosterol biosynthesis enzymes, such as sterol C24-methyltransferase (Erg6A), squalene synthase (Erg9) or squalene epoxidase (Erg1) does not trigger comparable cell wall alterations. Partial repression of Erg6A, which converts lanosterol into eburicol, increases azole resistance. The sterol C5-desaturase (ERG3)-dependent conversion of eburicol into 14-methylergosta-8,24(28)-dien-3ß,6α-diol, the "toxic diol" responsible for the fungistatic activity against yeasts, is not required for the fungicidal effects in A. fumigatus. While ERG3-lacking yeasts are azole resistant, ERG3-lacking A. fumigatus becomes more susceptible. Mutants lacking mitochondrial complex III functionality, which are much less effectively killed, but strongly inhibited in growth by azoles, convert eburicol more efficiently into the supposedly "toxic diol". We propose that the mode of action of azoles against A. fumigatus relies on accumulation of eburicol which exerts fungicidal effects by triggering cell wall carbohydrate patch formation.

Exploring the plasmodesmata callose-binding protein gene family in upland cotton: unraveling insights for enhancing fiber length.

Plasmodesmata are transmembrane channels embedded within the cell wall that can facilitate the intercellular communication in plants. Plasmodesmata callose-binding (PDCB) protein that associates with the plasmodesmata contributes to cell wall extension. Given that the elongation of cotton fiber cells correlates with the dynamics of the cell wall, this protein can be related to the cotton fiber elongation. This study sought to identify PDCB family members within the Gossypium. hirsutum genome and to elucidate their expression profiles. A total of 45 distinct family members were observed through the identification and screening processes. The analysis of their physicochemical properties revealed the similarity in the amino acid composition and molecular weight across most members. The phylogenetic analysis facilitated the construction of an evolutionary tree, categorizing these members into five groups mainly distributed on 20 chromosomes. The fine mapping results facilitated a tissue-specific examination of group V, revealing that the expression level of GhPDCB9 peaked five days after flowering. The VIGS experiments resulted in a marked decrease in the gene expression level and a significant reduction in the mature fiber length, averaging a shortening of 1.43-4.77 mm. The results indicated that GhPDCB9 played a pivotal role in the cotton fiber development and served as a candidate for enhancing cotton yield.

Cell Wall Binding Strategies Based on Cu3SbS3 Nanoparticles for Selective Bacterial Elimination and Promotion of Infected Wound Healing.

Utilizing nanomaterials as an alternative to antibiotics, with a focus on maintaining high biosafety, has emerged as a promising strategy to combat antibiotic resistance. Nevertheless, the challenge lies in the indiscriminate attack of nanomaterials on both bacterial and mammalian cells, which limits their practicality. Herein, Cu3SbS3 nanoparticles (NPs) capable of generating reactive oxygen species (ROS) are discovered to selectively adsorb and eliminate bacteria without causing obvious harm to mammalian cells, thanks to the interaction between O of N-acetylmuramic acid in bacterial cell walls and Cu of the NPs. Coupled with the short diffusion distance of ROS in the surrounding medium, a selective antibacterial effect is achieved. Additionally, the antibacterial mechanism is then identified: Cu3SbS3 NPs catalyze the generation of O2•-, which has subsequently been conversed by superoxide dismutase to H2O2. The latter is secondary catalyzed by the NPs to form •OH and 1O2, initiating an in situ attack on bacteria. This process depletes bacterial glutathione in conjunction with the disruption of the antioxidant defense system of bacteria. Notably, Cu3SbS3 NPs are demonstrated to efficiently impede biofilm formation; thus, a healing of MRSA-infected wounds was promoted. The bacterial cell wall-binding nanoantibacterial agents can be widely expanded through diversified design.

Long-term cardiovascular inflammation and fibrosis in a murine model of vasculitis induced by Lactobacillus casei cell wall extract.

Background: Kawasaki disease (KD), an acute febrile illness and systemic vasculitis, is the leading cause of acquired heart disease in children in industrialized countries. KD leads to the development of coronary artery aneurysms (CAA) in affected children, which may persist for months and even years after the acute phase of the disease. There is an unmet need to characterize the immune and pathological mechanisms of the long-term complications of KD. Methods: We examined cardiovascular complications in the Lactobacillus casei cell wall extract (LCWE) mouse model of KD-like vasculitis over 4 months. The long-term immune, pathological, and functional changes occurring in cardiovascular lesions were characterized by histological examination, flow cytometric analysis, immunofluorescent staining of cardiovascular tissues, and transthoracic echocardiogram. Results: CAA and abdominal aorta dilations were detected up to 16 weeks following LCWE injection and initiation of acute vasculitis. We observed alterations in the composition of circulating immune cell profiles, such as increased monocyte frequencies in the acute phase of the disease and higher counts of neutrophils. We determined a positive correlation between circulating neutrophil and inflammatory monocyte counts and the severity of cardiovascular lesions early after LCWE injection. LCWE-induced KD-like vasculitis was associated with myocarditis and myocardial dysfunction, characterized by diminished ejection fraction and left ventricular remodeling, which worsened over time. We observed extensive fibrosis within the inflamed cardiac tissue early in the disease and myocardial fibrosis in later stages. Conclusion: Our findings indicate that increased circulating neutrophil counts in the acute phase are a reliable predictor of cardiovascular inflammation severity in LCWE-injected mice. Furthermore, long-term cardiac complications stemming from inflammatory cell infiltrations in the aortic root and coronary arteries, myocardial dysfunction, and myocardial fibrosis persist over long periods and are still detected up to 16 weeks after LCWE injection.

Salt stress alters the cell wall components and structure in Miscanthus sinensis stems.

Miscanthus is a perennial grass suitable for the production of lignocellulosic biomass on marginal lands. The effects of salt stress on Miscanthus cell wall composition and its consequences on biomass quality have nonetheless received relatively little attention. In this study, we investigated how exposure to moderate (100 mM NaCl) or severe (200 mM NaCl) saline growing conditions altered the composition of both primary and secondary cell wall components in the stems of 15 Miscanthus sinensis genotypes. The exposure to stress drastically impacted biomass yield and cell wall composition in terms of content and structural features. In general, the observed compositional changes were more pronounced under severe stress conditions and were more apparent in genotypes with a higher sensitivity towards stress. Besides a severely reduced cellulose content, salt stress led to increased pectin content, presumably in the form of highly branched rhamnogalacturonan type I. Although salt stress had a limited effect on the total lignin content, the acid-soluble lignin content was strongly increased in the most sensitive genotypes. This effect was also reflected in substantially altered lignin structures and led to a markedly reduced incorporation of syringyl subunits and p-coumaric acid moieties. Interestingly, plants that were allowed a recovery period after stress ultimately had a reduced lignin content compared to those continuously grown under control conditions. In addition, the salt stress-induced cell wall alterations contributed to an improved enzymatic saccharification efficiency.

Lysinibacillus pinottii sp. nov., a novel species with anti-mosquito and anti-mollusk activity.

An isolate of a Gram-positive, strictly aerobic, motile, rod-shaped, endospore forming bacterium was originally isolated from soil when screening and bioprospecting for plant beneficial microorganisms. Phylogenetic analysis of the 16S rRNA gene sequences indicated that this strain was closely related to Lysinibacillus fusiformis NRRL NRS-350T (99.7%) and Lysinibacillus sphaericus NRRL B-23268T (99.2%). In phenotypic characterization, the novel strain was found to grow between 10 and 45 °C and tolerate up to 8% (w/v) NaCl. Furthermore, the strain grew in media with pH 5 to 10 (optimal growth at pH 7.0). The predominant cellular fatty acids were observed to be iso-C15: 0 (52.3%), anteiso-C15: 0 (14.8%), C16:1ω7C alcohol (11.2%), and C16: 0 (9.5%). The cell-wall peptidoglycan contained lysine-aspartic acid, the same as congeners. A draft genome was assembled and the DNA G+C content was determined to be 37.1% (mol content). A phylogenomic analysis on the core genome of the new strain and 5 closest type strains of Lysinibacillus revealed this strain formed a distinct monophyletic clade with the nearest neighbor being Lysinibacillus fusiformis. DNA-DNA relatedness studies using in silico DNA-DNA hybridizations (DDH) showed this species was below the species threshold of 70%. Based upon the consensus of phylogenetic and phenotypic analyses, we conclude that this strain represents a novel species within the genus Lysinibacillus, for which the name Lysinibacillus pinottii sp. nov. is proposed, with type strain PB211T (= NRRL B-65672T, = CCUG 77181T).

Metabolic remodeling by RNA polymerase gene mutations is associated with reduced ß-lactam susceptibility in oxacillin-susceptible MRSA.

The emergence of oxacillin-susceptible methicillin-resistant Staphylococcus aureus (OS-MRSA) has imposed further challenges to the clinical management of MRSA infections. When exposed to ß-lactam antibiotics, these strains can easily acquire reduced ß-lactam susceptibility through chromosomal mutations, including those in RNA polymerase (RNAP) genes such as rpoBC, which may then lead to treatment failure. Despite the increasing prevalence of such strains and the apparent challenges they pose for diagnosis and treatment, there is limited information available on the actual mechanisms underlying such chromosomal mutation-related transitions to reduced ß-lactam susceptibility, as it does not directly associate with the expression of mecA. This study investigated the cellular physiology and metabolism of six missense mutants with reduced oxacillin susceptibility, each carrying respective mutations on RpoBH929P, RpoBQ645H, RpoCG950R, RpoCG498D, RpiAA64E, and FruBA211E, using capillary electrophoresis-mass spectrometry-based metabolomics analysis. Our results showed that rpoBC mutations caused RNAP transcription dysfunction, leading to an intracellular accumulation of ribonucleotides. These mutations also led to the accumulation of UDP-Glc/Gal and UDP-GlcNAc, which are precursors of UTP-associated peptidoglycan and wall teichoic acid. Excessive amounts of building blocks then contributed to the cell wall thickening of mutant strains, as observed in transmission electron microscopy, and ultimately resulted in decreased susceptibility to ß-lactam in OS-MRSA. IMPORTANCE: The emergence of oxacillin-susceptible methicillin-resistant Staphylococcus aureus (OS-MRSA) strains has created new challenges for treating MRSA infections. These strains can become resistant to ß-lactam antibiotics through chromosomal mutations, including those in the RNA polymerase (RNAP) genes such as rpoBC, leading to treatment failure. This study investigated the mechanisms underlying reduced ß-lactam susceptibility in four rpoBC mutants of OS-MRSA. The results showed that rpoBC mutations caused RNAP transcription dysfunction, leading to an intracellular accumulation of ribonucleotides and precursors of peptidoglycan as well as wall teichoic acid. This, in turn, caused thickening of the cell wall and ultimately resulted in decreased susceptibility to ß-lactam in OS-MRSA. These findings provide insights into the mechanisms of antibiotic resistance in OS-MRSA and highlight the importance of continued research in developing effective treatments to combat antibiotic resistance.

Natural variation of TBR confers plant zinc toxicity tolerance through root cell wall pectin methylesterification.

Zinc (Zn) is an essential micronutrient but can be cytotoxic when present in excess. Plants have evolved mechanisms to tolerate Zn toxicity. To identify genetic loci responsible for natural variation of plant tolerance to Zn toxicity, we conduct genome-wide association studies for root growth responses to high Zn and identify 21 significant associated loci. Among these loci, we identify Trichome Birefringence (TBR) allelic variation determining root growth variation in high Zn conditions. Natural alleles of TBR determine TBR transcript and protein levels which affect pectin methylesterification in root cell walls. Together with previously published data showing that pectin methylesterification increase goes along with decreased Zn binding to cell walls in TBR mutants, our findings lead to a model in which TBR allelic variation enables Zn tolerance through modulating root cell wall pectin methylesterification. The role of TBR in Zn tolerance is conserved across dicot and monocot plant species.

Expression profile analysis of cotton fiber secondary cell wall thickening stage.

To determine the genes associated with the fiber strength trait in cotton, three different cotton cultivars were selected: Sea Island cotton (Xinhai 32, with hyper-long fibers labeled as HL), and upland cotton (17-24, with long fibers labeled as L, and 62-33, with short fibers labeled as S). These cultivars were chosen to assess fiber samples with varying qualities. RNA-seq technology was used to analyze the expression profiles of cotton fibers at the secondary cell wall (SCW) thickening stage (20, 25, and 30 days post-anthesis (DPA)). The results showed that a large number of differentially expressed genes (DEGs) were obtained from the three assessed cotton cultivars at different stages of SCW development. For instance, at 20 DPA, Sea Island cotton (HL) had 6,215 and 5,364 DEGs compared to upland cotton 17-24 (L) and 62-33 (S), respectively. Meanwhile, there were 1,236 DEGs between two upland cotton cultivars, 17-24 (L) and 62-33 (S). Gene Ontology (GO) term enrichment identified 42 functions, including 20 biological processes, 11 cellular components, and 11 molecular functions. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis identified several pathways involved in SCW synthesis and thickening, such as glycolysis/gluconeogenesis, galactose metabolism, propanoate metabolism, biosynthesis of unsaturated fatty acids pathway, valine, leucine and isoleucine degradation, fatty acid elongation pathways, and plant hormone signal transduction. Through the identification of shared DEGs, 46 DEGs were found to exhibit considerable expressional differences at different fiber stages from the three cotton cultivars. These shared DEGs have functions including REDOX enzymes, binding proteins, hydrolases (such as GDSL thioesterase), transferases, metalloproteins (cytochromatin-like genes), kinases, carbohydrates, and transcription factors (MYB and WRKY). Therefore, RT-qPCR was performed to verify the expression levels of nine of the 46 identified DEGs, an approach which demonstrated the reliability of RNA-seq data. Our results provided valuable molecular resources for clarifying the cell biology of SCW biosynthesis during fiber development in cotton.

Confocal Imaging of Seeds.

In flowering plants, proper seed development is achieved through the constant interplay of fertilization products, embryo and endosperm, and maternal tissues. Understanding such a complex biological process requires microscopy techniques able to unveil the seed internal morphological structure. Seed thickness and relatively low permeability make conventional tissue staining techniques impractical unless combined with time-consuming dissecting methods. Here, we describe two techniques to imaging the three-dimensional structure of Arabidopsis seeds by confocal laser scanning microscopy. Both procedures, while differing in their time of execution and resolution, are based on cell wall staining of seed tissues with fluorescent dyes.

Alteration of ß-glucan in the emerging fungal pathogen Candida auris leads to immune evasion and increased virulence.

Candida auris is an emerging pathogenic yeast that has been categorized as a global public health threat and a critical priority among fungal pathogens. Despite this, the immune response against C. auris infection is still not well understood. Hosts fight Candida infections through the immune system that recognizes pathogen-associated molecular patterns such as ß-glucan, mannan, and chitin on the fungal cell wall. In this study, levels of ß-glucan and mannan exposures in C. auris grown under different physiologically relevant stimuli were quantified by flow cytometry-based analysis. Lactate, hypoxia, and sublethal concentration of fluconazole trigger a decrease in surface ß-glucan while low pH triggers an increase in ß-glucan. There is no inverse pattern between exposure levels of ß-glucan and mannan in the cell wall architecture among the three clades. To determine the effect of cell wall remodeling on the immune response, a phagocytosis assay was performed, followed by quantification of released cytokines by ELISA. Lactate-induced decrease in ß-glucan leads to reduced uptake of C. auris by PMA-differentiated THP-1 and RAW 264.7 macrophages. Furthermore, reduced production of CCL3/MIP-1⍺ but not TNF-⍺ and IL-10 were observed. An in vivo infection analysis using silkworms reveals that a reduction in ß-glucan triggers an increase in the virulence of C. auris. This study demonstrates that ß-glucan alteration occurs in C. auris and serves as an escape mechanism from immune cells leading to increased virulence.

Microalgae protein digestibility: How to crack open the black box?

Microalgae are booming as a sustainable protein source for human nutrition and animal feed. Nevertheless, certain strains were reported to have robust cell walls limiting protein digestibility. There are several disruption approaches to break down the cell integrity and increase digestive enzyme accessibility. This review's intent is to discuss the digestibility of microalgae proteins in intact cells and after their disruption. In intact single cells, the extent of protein digestibility is chiefly related to cell wall structural properties (differing among strains) as well as digestion method and when added to food or feed protein digestibility changes depending on the matrix's composition. The degree of effectiveness of the disruption method varies among studies, and it is complicated to compare them due to variabilities in digestibility models, strains, disruption method/conditions and their consequent impact on the microalgae cell structure. More exhaustive studies are still required to fill knowledge gaps on the structure of microalgal cell walls and to find efficient and cost-effective disruption technologies to increase proteins availability without hindering their quality.