Enhanced extracellular production of laccase in Coprinopsis cinerea by silencing chitinase gene.

Laccase, a copper-containing polyphenol oxidase, is an important green biocatalyst. In this study, Laccase Lcc5 was homologous recombinantly expressed in Coprinopsis cinerea and a novel strategy of silencing chitinase gene expression was used to enhance recombinant Lcc5 extracellular yield. Two critical chitinase genes, ChiEn1 and ChiE2, were selected by analyzing the transcriptome data of C. cinerea FA2222, and their silent expression was performed by RNA interference (RNAi). It was found that silencing either ChiEn1 or ChiE2 reduced sporulation and growth rate, and increased cell wall sensitivity, but had no significant effect on mycelial branching. Among them, the extracellular laccase activity of the ChiE2-silenced engineered strain Cclcc5-antiChiE2-5 and the control Cclcc5-13 reached the highest values (38.2 and 25.5 U/mL, respectively) at 250 and 150 rpm agitation speeds, corresponding to productivity of 0.35 and 0.19 U/mL·h, respectively, in a 3-L fermenter culture. Moreover, since Cclcc5-antiChiE2-5 could withstand greater shear forces, its extracellular laccase activity was 2.6-fold higher than that of Cclcc5-13 when the agitation speed was all at 250 rpm. To our knowledge, this is the first report of enhanced recombinant laccase production in C. cinerea by silencing the chitinase gene. This study will pave the way for laccase industrial production and accelerate the development of a C. cinerea high-expression system. KEY POINTS: • ChiEn1 and ChiE2 are critical chitinase genes in C. cinerea FA2222 genome. • Chitinase gene silencing enhanced the tolerance of C. cinerea to shear forces. • High homologous production of Lcc5 is achieved by fermentation in a 3-L fermenter.

Community dynamics and metagenomic analyses reveal Bacteroidota's role in widespread enzymatic Fucus vesiculosus cell wall degradation.

Enzymatic degradation of algae cell wall carbohydrates by microorganisms is under increasing investigation as marine organic matter gains more value as a sustainable resource. The fate of carbon in the marine ecosystem is in part driven by these degradation processes. In this study, we observe the microbiome dynamics of the macroalga Fucus vesiculosus in 25-day-enrichment cultures resulting in partial degradation of the brown algae. Microbial community analyses revealed the phylum Pseudomonadota as the main bacterial fraction dominated by the genera Marinomonas and Vibrio. More importantly, a metagenome-based Hidden Markov model for specific glycosyl hydrolyses and sulphatases identified Bacteroidota as the phylum with the highest potential for cell wall degradation, contrary to their low abundance. For experimental verification, we cloned, expressed, and biochemically characterised two α-L-fucosidases, FUJM18 and FUJM20. While protein structure predictions suggest the highest similarity to a Bacillota origin, protein-protein blasts solely showed weak similarities to defined Bacteroidota proteins. Both enzymes were remarkably active at elevated temperatures and are the basis for a potential synthetic enzyme cocktail for large-scale algal destruction.

A Toxoplasma gondii O-glycosyltransferase that modulates bradyzoite cyst wall rigidity is distinct from host homologues.

Infection with the apicomplexan protozoan Toxoplasma gondii can be life-threatening in immunocompromised hosts. Transmission frequently occurs through the oral ingestion of T. gondii bradyzoite cysts, which transition to tachyzoites, disseminate, and then form cysts containing bradyzoites in the central nervous system, resulting in latent infection. Encapsulation of bradyzoites by a cyst wall is critical for immune evasion, survival, and transmission. O-glycosylation of the protein CST1 by the mucin-type O-glycosyltransferase T. gondii (Txg) GalNAc-T3 influences cyst wall rigidity and stability. Here, we report X-ray crystal structures of TxgGalNAc-T3, revealing multiple features that are strictly conserved among its apicomplexan homologues. This includes a unique 2nd metal that is coupled to substrate binding and enzymatic activity in vitro and cyst wall O-glycosylation in T. gondii. The study illustrates the divergence of pathogenic protozoan GalNAc-Ts from their host homologues and lays the groundwork for studying apicomplexan GalNAc-Ts as therapeutic targets in disease.

Bile promotes Lactobacillus johnsonii N6.2 extracellular vesicle production with conserved immunomodulatory properties.

Recently, Lactobacillus johnsonii N6.2-derived extracellular vesicles (EVs) were shown to reduce apoptosis in human beta cell lines and stimulate insulin secretion in human islets. Our goal was to identify a physiologically relevant environmental condition that induces a hypervesiculation phenotype in L. johnsonii N6.2 and to evaluate if transcriptional changes are involved in this process. Culturing this strain in the presence of 0.2% bovine bile, which mimics a stressor encountered by the bacterium in the small intestine, resulted in approximately a 100-fold increase in EVs relative to cells grown in media without bile. Whole transcriptome analysis of cells grown with bile revealed upregulation of several peptidoglycan hydrolases as well as several genes involved in fatty acid utilization. These results suggest that the hypervesiculation phenotype may be the result of increased cell wall turnover combined with increased accumulation of phospholipids, in agreement with our previous proteomic and lipidomics results. Additionally, EVs isolated from L. johnsonii N6.2 grown in presence of bile maintained their immunomodulatory properties in host-derived ßlox5 pancreatic and THP-1 macrophage cell lines. Our findings suggest that in L. johnsonii N6.2 vesiculogenesis is significantly impacted by the expression of cell wall modifying enzymes and proteins utilized for exogenous fatty acid uptake that are regulated at the transcriptional level. Furthermore, this data suggests that vesiculogenesis could be stimulated in vivo using small molecules thereby maximizing the beneficial interactions between bacteria and their hosts.

Pleiotropic roles of LAMMER kinase, Lkh1 in stress responses and virulence of Cryptococcus neoformans.

Dual-specificity LAMMER kinases are highly evolutionarily conserved in eukaryotes and play pivotal roles in diverse physiological processes, such as growth, differentiation, and stress responses. Although the functions of LAMMER kinase in fungal pathogens in pathogenicity and stress responses have been characterized, its role in Cryptococcus neoformans, a human fungal pathogen and a model yeast of basidiomycetes, remains elusive. In this study, we identified a LKH1 homologous gene and constructed a strain with a deleted LKH1 and a complemented strain. Similar to other fungi, the lkh1Δ mutant showed intrinsic growth defects. We observed that C. neoformans Lkh1 was involved in diverse stress responses, including oxidative stress and cell wall stress. Particularly, Lkh1 regulates DNA damage responses in Rad53-dependent and -independent manners. Furthermore, the absence of LKH1 reduced basidiospore formation. Our observations indicate that Lkh1 becomes hyperphosphorylated upon treatment with rapamycin, a TOR protein inhibitor. Notably, LKH1 deletion led to defects in melanin synthesis and capsule formation. Furthermore, we found that the deletion of LKH1 led to the avirulence of C. neoformans in a systemic cryptococcosis murine model. Taken together, Lkh1 is required for the stress response, sexual differentiation, and virulence of C. neoformans.

Quantification of C. neoformans Capsule Diameter.

The polysaccharide capsule of Cryptococcus neoformans is the primary virulence factor and one of the most commonly studied aspects of this pathogenic yeast. Capsule size varies widely between strains, has the ability to grow rapidly when introduced to stressful or low-nutrient conditions, and has been positively correlated with strain virulence. For these reasons, the size of the capsule is of great interest to C. neoformans researchers. Inducing the growth of the C. neoformans capsule is used during phenotypic testing to help understand the effects of different treatments on the yeast or size differences between strains. Here, we describe one of the standard methods of capsule induction and detail two accepted methods of staining: (i) India ink, a negative stain, used in conjunction with conventional light microscopy and (ii) co-staining with fluorescent dyes of both the cell wall and capsule followed by confocal microscopy. Finally, we outline how to measure capsule diameter manually and offer a protocol for automated diameter measurement of India ink-stained samples using computational image analysis.

Fluorescence and Biochemical Assessment of the Chitin and Chitosan Content of Cryptococcus.

The cell wall of the fungal pathogens Cryptococcus neoformans and C. gattii is critical for cell wall integrity and signaling external threats to the cell, allowing it to adapt and grow in a variety of changing environments. Chitin is a polysaccharide found in the cell walls of fungi that is considered to be essential for fungal survival. Chitosan is a polysaccharide derived from chitin via deacetylation that is also essential for cryptococcal cell wall integrity, fungal pathogenicity, and virulence. Cryptococcus has evolved mechanisms to regulate the amount of chitin and chitosan during growth under laboratory conditions or during mammalian infection. Therefore, levels of chitin and chitosan have been useful phenotypes to define mutant Cryptococcus strains. As a result, we have developed and/or refined various qualitative and quantitative methods for measuring chitin and chitosan. These techniques include those that use fluorescent probes that are known to bind to chitin (e.g., calcofluor white and wheat germ agglutinin), as well as those that preferentially bind to chitosan (e.g., eosin Y and cibacron brilliant red 3B-A). Techniques that enhance the localization and quantification of chitin and chitosan in the cell wall include (i) fluorescence microscopy, (ii) flow cytometry, (iii) and spectrofluorometry. We have also modified two highly selective biochemical methods to measure cellular chitin and chitosan content: the Morgan-Elson and the 3-methyl-2-benzothiazolone hydrazine hydrochloride (MBTH) assays, respectively.

Hypothesis: evidence that the PRS gene products of Saccharomyces cerevisiae support both PRPP synthesis and maintenance of cell wall integrity.

The gene products of PRS1-PRS5 in Saccharomyces cerevisiae are responsible for the production of PRPP (5-phospho-D-ribosyl-α-1-pyrophosphate). However, it has been demonstrated that they are also involved in the cell wall integrity (CWI) signalling pathway as shown by protein-protein interactions (PPIs) with, for example Slt2, the MAP kinase of the CWI pathway. The following databases: SGD, BioGRID and Hit Predict, which collate PPIs from various research papers, have been scrutinized for evidence of PPIs between Prs1-Prs5 and components of the CWI pathway. The level of certainty in PPIs was verified by interaction scores available in the Hit Predict database revealing that well-documented interactions correspond with higher interaction scores and can be graded as high confidence interactions based on a score > 0.28, an annotation score ≥ 0.5 and a method-based high confidence score level of ≥ 0.485. Each of the Prs1-Prs5 polypeptides shows some degree of interaction with the CWI pathway. However, Prs5 has a vital role in the expression of FKS2 and Rlm1, previously only documented by reporter assay studies. This report emphasizes the importance of investigating interactions using more than one approach since every method has its limitations and the use of different methods, as described herein, provides complementary experimental and statistical data, thereby corroborating PPIs. Since the experimental data described so far are consistent with a link between PRPP synthetase and the CWI pathway, our aim was to demonstrate that these data are also supported by high-throughput bioinformatic analyses promoting our hypothesis that two of the five PRS-encoding genes contain information required for the maintenance of CWI by combining data from our targeted approach with relevant, unbiased data from high-throughput analyses.

GhMYB52 Like: A Key Factor That Enhances Lint Yield by Negatively Regulating the Lignin Biosynthesis Pathway in Fibers of Upland Cotton (Gossypium hirsutum L.).

In the context of sustainable agriculture and biomaterial development, understanding and enhancing plant secondary cell wall formation are crucial for improving crop fiber quality and biomass conversion efficiency. This is especially critical for economically important crops like upland cotton (Gossypium hirsutum L.), for which fiber quality and its processing properties are essential. Through comprehensive genome-wide screening and analysis of expression patterns, we identified a particularly high expression of an R2R3 MYB transcription factor, GhMYB52 Like, in the development of the secondary cell wall in cotton fiber cells. Utilizing gene-editing technology to generate a loss-of-function mutant to clarify the role of GhMYB52 Like, we revealed that GhMYB52 Like does not directly contribute to cellulose synthesis in cotton fibers but instead represses a subset of lignin biosynthesis genes, establishing it as a lignin biosynthesis inhibitor. Concurrently, a substantial decrease in the lint index, a critical measure of cotton yield, was noted in parallel with an elevation in lignin levels. This study not only deepens our understanding of the molecular mechanisms underlying cotton fiber development but also offers new perspectives for the molecular improvement of other economically important crops and the enhancement of biomass energy utilization.

Glucosylceramides impact cellulose deposition and cellulose synthase complex motility in Arabidopsis.

Cellulose is an abundant component of plant cell wall matrices, and this para-crystalline polysaccharide is synthesized at the plasma membrane by motile Cellulose Synthase Complexes (CSCs). However, the factors that control CSC activity and motility are not fully resolved. In a targeted chemical screen, we identified the alkylated nojirimycin analog N-Dodecyl Deoxynojirimycin (ND-DNJ) as a small molecule that severely impacts Arabidopsis seedling growth. Previous work suggests that ND-DNJ-related compounds inhibit the biosynthesis of glucosylceramides (GlcCers), a class of glycosphingolipid associated with plant membranes. Our work uncovered major changes in the sphingolipidome of plants treated with ND-DNJ, including reductions in GlcCer abundance and altered acyl chain length distributions. Crystalline cellulose content was also reduced in ND-DNJ-treated plants as well as plants treated with the known GlcCer biosynthesis inhibitor N-[2-hydroxy-1-(4-morpholinylmethyl)-2-phenyl ethyl]-decanamide (PDMP) or plants containing a genetic disruption in GLUCOSYLCERAMIDE SYNTHASE (GCS), the enzyme responsible for sphingolipid glucosylation that results in GlcCer synthesis. Live-cell imaging revealed that CSC speed distributions were reduced upon treatment with ND-DNJ or PDMP, further suggesting an important relationship between glycosylated sphingolipid composition and CSC motility across the plasma membrane. These results indicate that multiple interventions compromising GlcCer biosynthesis disrupt cellulose deposition and CSC motility, suggesting that GlcCers regulate cellulose biosynthesis in plants.

Inhibition of PbeXTH1 and PbeSEOB1 is required for the Valsa canker resistance contributed by Wall-associated kinase gene MbWAK1.

Wall-associated kinases (WAKs) have been determined to recognize pathogenic signals and initiate plant immune responses. However, the roles of the family members in host resistance against Valsa canker, a serious fungal disease of apples and pears, are largely unknown. Here, we identified MbWAK1 in Malus baccata, a resistant germplasm differentially expressed during infection by Valsa mali (Vm). Over-expression of MbWAK1 enhanced the Valsa canker resistance of apple and pear fruits and 'Duli-G03' (Pyrus betulifolia) suspension cells. A large number of phloem, cell wall, and lipid metabolic process-related genes were differentially expressed in overexpressed suspension cell lines in response to Valsa pyri (Vp) signals. Among these, the expression of xyloglucan endotransglucosylase/hydrolase (XTH) gene PbeXTH1 and sieve element occlusion B-like (SEOB) gene PbeSEOB1 were significantly inhibited. Transient expression of PbeXTH1 or PbeSEOB1 compromised the expressional induction of MbWAK1 and the resistance contributed by MbWAK1. In addition, PbeXTH1 and PbeSEOB1 suppressed the immune response induced by MbWAK1. Our results enriched the molecular mechanisms for MbWAK1 against Valsa canker and resistant breeding.

Chitin Deacetylase Homologous Gene cda Contributes to Development and Aflatoxin Synthesis in Aspergillus flavus.

The fungal cell wall serves as the primary interface between fungi and their external environment, providing protection and facilitating interactions with the surroundings. Chitin is a vital structural element in fungal cell wall. Chitin deacetylase (CDA) can transform chitin into chitosan through deacetylation, providing various biological functions across fungal species. Although this modification is widespread in fungi, the biological functions of CDA enzymes in Aspergillus flavus remain largely unexplored. In this study, we aimed to investigate the biofunctions of the CDA family in A. flavus. The A. flavus genome contains six annotated putative chitin deacetylases. We constructed knockout strains targeting each member of the CDA family, including Δcda1, Δcda2, Δcda3, Δcda4, Δcda5, and Δcda6. Functional analyses revealed that the deletion of CDA family members neither significantly affects the chitin content nor exhibits the expected chitin deacetylation function in A. flavus. However, the Δcda6 strain displayed distinct phenotypic characteristics compared to the wild-type (WT), including an increased conidia count, decreased mycelium production, heightened aflatoxin production, and impaired seed colonization. Subcellular localization experiments indicated the cellular localization of CDA6 protein within the cell wall of A. flavus filaments. Moreover, our findings highlight the significance of the CBD1 and CBD2 structural domains in mediating the functional role of the CDA6 protein. Overall, we analyzed the gene functions of CDA family in A. flavus, which contribute to a deeper understanding of the mechanisms underlying aflatoxin contamination and lay the groundwork for potential biocontrol strategies targeting A. flavus.

Adaptation mechanism of three Impatiens species to different habitats based on stem morphology, lignin and MYB4 gene.

BACKGROUND: Impatiens is an important genus with rich species of garden plants, and its distribution is extremely extensive, which is reflected in its diverse ecological environment. However, the specific mechanisms of Impatiens' adaptation to various environments and the mechanism related to lignin remain unclear. RESULTS: Three representative Impatiens species,Impatiens chlorosepala (wet, low degree of lignification), Impatiens uliginosa (aquatic, moderate degree of lignification) and Impatiens rubrostriata (terrestrial, high degree of lignification), were selected and analyzed for their anatomical structures, lignin content and composition, and lignin-related gene expression. There are significant differences in anatomical parameters among the stems of three Impatiens species, and the anatomical structure is consistent with the determination results of lignin content. Furthermore, the thickness of the xylem and cell walls, as well as the ratio of cell wall thickness to stem diameter have a strong correlation with lignin content. The anatomical structure and degree of lignification in Impatiens can be attributed to the plant's growth environment, morphology, and growth rate. Our analysis of lignin-related genes revealed a negative correlation between the MYB4 gene and lignin content. The MYB4 gene may control the lignin synthesis in Impatiens by controlling the structural genes involved in the lignin synthesis pathway, such as HCT, C3H, and COMT. Nonetheless, the regulation pathway differs between species of Impatiens. CONCLUSIONS: This study demonstrated consistency between the stem anatomy of Impatiens and the results obtained from lignin content and composition analyses. It is speculated that MYB4 negatively regulates the lignin synthesis in the stems of three Impatiens species by regulating the expression of structural genes, and its regulation mechanism appears to vary across different Impatiens species. This study analyses the variations among different Impatiens plants in diverse habitats, and can guide further molecular investigations of lignin biosynthesis in Impatiens.

Unveiling a Microexon Switch: Novel Regulation of the Activities of Sugar Assimilation and Plant-Cell-Wall-Degrading Xylanases and Cellulases by Xlr2 in Trichoderma virens.

Functional microexons have not previously been described in filamentous fungi. Here, we describe a novel mechanism of transcriptional regulation in Trichoderma requiring the inclusion of a microexon from the Xlr2 gene. In low-glucose environments, a long mRNA including the microexon encodes a protein with a GAL4-like DNA-binding domain (Xlr2-α), whereas in high-glucose environments, a short mRNA that is produced encodes a protein lacking this DNA-binding domain (Xlr2-ß). Interestingly, the protein isoforms differ in their impact on cellulase and xylanase activity. Deleting the Xlr2 gene reduced both xylanase and cellulase activity and growth on different carbon sources, such as carboxymethylcellulose, xylan, glucose, and arabinose. The overexpression of either Xlr2-α or Xlr2-ß in T. virens showed that the short isoform (Xlr2-ß) caused higher xylanase activity than the wild types or the long isoform (Xlr2-α). Conversely, cellulase activity did not increase when overexpressing Xlr2-ß but was increased with the overexpression of Xlr2-α. This is the first report of a novel transcriptional regulation mechanism of plant-cell-wall-degrading enzyme activity in T. virens. This involves the differential expression of a microexon from a gene encoding a transcriptional regulator.

Homogalacturonan Pectins Tuned as an Effect of Susceptible rbohD, Col-0-Reactions, and Resistance rbohF-, rbohD/F-Reactions to TuMV.

The plant cell wall is an actively reorganized network during plant growth and triggered immunity in response to biotic stress. While the molecular mechanisms managing perception, recognition, and signal transduction in response to pathogens are well studied in the context of damaging intruders, the current understanding of plant cell wall rebuilding and active defense strategies in response to plant virus infections remains poorly characterized. Pectins can act as major elements of the primary cell wall and are dynamic compounds in response to pathogens. Homogalacturonans (HGs), a main component of pectins, have been postulated as defensive molecules in plant-pathogen interactions and linked to resistance responses. This research focused on examining the regulation of selected pectin metabolism components in susceptible (rbohD-, Col-0-TuMV) and resistance (rbohF-, rbohD/F-TuMV) reactions. Regardless of the interaction type, ultrastructural results indicated dynamic cell wall rebuilding. In the susceptible reaction promoted by RbohF, there was upregulation of AtPME3 (pectin methylesterase) but not AtPME17, confirmed by induction of PME3 protein deposition. Moreover, the highest PME activity along with a decrease in cell wall methylesters compared to resistance interactions in rbohD-TuMV were noticed. Consequently, the susceptible reaction of rbohD and Col-0 to TuMV was characterized by a significant domination of low/non-methylesterificated HGs. In contrast, cell wall changes during the resistance response of rbohF and rbohD/F to TuMV were associated with dynamic induction of AtPMEI2, AtPMEI3, AtGAUT1, and AtGAUT7 genes, confirmed by significant induction of PMEI2, PMEI3, and GAUT1 protein deposition. In both resistance reactions, a dynamic decrease in PME activity was documented, which was most intense in rbohD/F-TuMV. This decrease was accompanied by an increase in cell wall methylesters, indicating that the domination of highly methylesterificated HGs was associated with cell wall rebuilding in rbohF and rbohD/F defense responses to TuMV. These findings suggest that selected PME with PMEI enzymes have a diverse impact on the demethylesterification of HGs and metabolism as a result of rboh-TuMV interactions, and are important factors in regulating cell wall changes depending on the type of interaction, especially in resistance responses. Therefore, PMEI2 and PMEI3 could potentially be important signaling resistance factors in the rboh-TuMV pathosystem.

The GPAT4/6/8 clade functions in Arabidopsis root suberization nonredundantly with the GPAT5/7 clade required for suberin lamellae.

Lipid polymers such as cutin and suberin strengthen the diffusion barrier properties of the cell wall in specific cell types and are essential for water relations, mineral nutrition, and stress protection in plants. Land plant-specific glycerol-3-phosphate acyltransferases (GPATs) of different clades are central players in cutin and suberin monomer biosynthesis. Here, we show that the GPAT4/6/8 clade in Arabidopsis thaliana, which is known to mediate cutin formation, is also required for developmentally regulated root suberization, in addition to the established roles of GPAT5/7 in suberization. The GPAT5/7 clade is mainly required for abscisic acid-regulated suberization. In addition, the GPAT5/7 clade is crucial for the formation of the typical lamellated suberin ultrastructure observed by transmission electron microscopy, as distinct amorphous globular polyester structures were deposited in the apoplast of the gpat5 gpat7 double mutant, in contrast to the thinner but still lamellated suberin deposition in the gpat4 gpat6 gpat8 triple mutant. Site-directed mutagenesis revealed that the intrinsic phosphatase activity of GPAT4, GPAT6, and GPAT8, which leads to monoacylglycerol biosynthesis, contributes to suberin formation. GPAT5/7 lack an active phosphatase domain and the amorphous globular polyester structure observed in the gpat5 gpat7 double mutant was partially reverted by treatment with a phosphatase inhibitor or the expression of phosphatase-dead variants of GPAT4/6/8. Thus, GPATs that lack an active phosphatase domain synthetize lysophosphatidic acids that might play a role in the formation of the lamellated structure of suberin. GPATs with active and nonactive phosphatase domains appear to have nonredundant functions and must cooperate to achieve the efficient biosynthesis of correctly structured suberin.

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.

New lactate- and pyruvate-containing polysaccharide and rhamnomannan with xylose residues from the cell wall of Rathayibacter oskolensis VKM Ac-2121T.

The cell wall of endophytic strain Rathayibacter oskolensis VKM Ac-2121T (family Microbacteriaceae, class Actinomycetes) was found to contain neutral and acidic glycopolymers. The neutral polymer is a block-type rhamnomannan partially should be substitutied by xylose residues, [→2)-α-[ß-D-Xylp-(1 â†’ 3)]-D-Manp-(1 â†’ 3)-α-D-Rhap-(1→]∼30 [→2)-α-D-Manp-(1 â†’ 3)-α-D-Rhap-(1→]∼45. The acidic polymer has branched chain, bearing lactate and pyruvate residues, →4)-α-D-[S-Lac-(2-3)-α-L-Rhap-(1 â†’ 3)]-D-Manp-(1 â†’ 3)-α-D-[4,6-R-Pyr]-D-Galp-(1 â†’ 3)-ß-D-Glcp-(1 â†’. The structures of both glycopolymers were not described in the Gram-positive bacteria to date. The glycopolymers were studied by chemical and NMR spectroscopic methods. The results of this study provide new data on diversity of bacterial glycopolymers and may prove useful in the taxonomy of the genus Rathayibacter and for understanding the molecular mechanisms of interaction between plants and plant endophytes.

Mitigating candidiasis with acarbose by targeting Candida albicans α-glucosidase: in-silico, in-vitro and transcriptomic approaches.

Biofilm-associated candidiasis poses a significant challenge in clinical settings due to the limited effectiveness of existing antifungal treatments. The challenges include increased pathogen virulence, multi-drug resistance, and inadequate penetration of antimicrobials into biofilm structures. One potential solution to this problem involves the development of novel drugs that can modulate fungal virulence and biofilm formation, which is essential for pathogenesis. Resistance in Candida albicans is initiated by morphological changes from yeast to hyphal form. This transition triggers a series of events such as cell wall elongation, increased adhesion, invasion of host tissues, pathogenicity, biofilm formation, and the initiation of an immune response. The cell wall is a critical interface for interactions with host cells, primarily through various cell wall proteins, particularly mannoproteins. Thus, cell wall proteins and enzymes are considered potential antifungal targets. In this regard, we explored α-glucosidase as our potential target which plays a crucial role in processing mannoproteins. Previous studies have shown that inhibition of α-glucosidase leads to defects in cell wall integrity, reduced adhesion, diminished secretion of hydrolytic enzymes, alterations in immune recognition, and reduced pathogenicity. Since α-glucosidase, primarily converts carbohydrates, our study focuses on FDA-approved carbohydrate mimic drugs (Glycomimetics) with well-documented applications in various biological contexts. Through virtual screening of 114 FDA-approved carbohydrate-based drugs, a pseudo-sugar Acarbose, emerged as a top hit. Acarbose is known for its pharmacological potential in managing type 2 diabetes mellitus by targeting α-glucosidase. Our preliminary investigations indicate that Acarbose effectively inhibits C. albicans biofilm formation, reduces virulence, impairs morphological switching, and hinders the adhesion and invasion of host cells, all at very low concentrations in the nanomolar range. Furthermore, transcriptomic analysis reveals the mechanism of action of Acarbose, highlighting its role in targeting α-glucosidase.

A MYB-related transcription factor ZmMYBR29 is involved in grain filling.

BACKGROUND: The endosperm serves as the primary source of nutrients for maize (Zea mays L.) kernel embryo development and germination. Positioned at the base of the endosperm, the transfer cells (TCs) of the basal endosperm transfer layer (BETL) generate cell wall ingrowths, which enhance the connectivity between the maternal plant and the developing kernels. These TCs play a crucial role in nutrient transport and defense against pathogens. The molecular mechanism underlying BETL development in maize remains unraveled. RESULTS: This study demonstrated that the MYB-related transcription factor ZmMYBR29, exhibited specific expression in the basal cellularized endosperm, as evidenced by in situ hybridization analysis. Utilizing the CRISPR/Cas9 system, we successfully generated a loss-of-function homozygous zmmybr29 mutant, which presented with smaller kernel size. Observation of histological sections revealed abnormal development and disrupted morphology of the cell wall ingrowths in the BETL. The average grain filling rate decreased significantly by 26.7% in zmmybr29 mutant in comparison to the wild type, which impacted the dry matter accumulation within the kernels and ultimately led to a decrease in grain weight. Analysis of RNA-seq data revealed downregulated expression of genes associated with starch synthesis and carbohydrate metabolism in the mutant. Furthermore, transcriptomic profiling identified 23 genes that expressed specifically in BETL, and the majority of these genes exhibited altered expression patterns in zmmybr29 mutant. CONCLUSIONS: In summary, ZmMYBR29 encodes a MYB-related transcription factor that is expressed specifically in BETL, resulting in the downregulation of genes associated with kernel development. Furthermore, ZmMYBR29 influences kernels weight by affecting the grain filling rate, providing a new perspective for the complementation of the molecular regulatory network in maize endosperm development.