UNASSIGNED: Verticillium dahliae causes a devastating Verticillium wilt disease on hundreds of plant species worldwide, including cotton. Understanding the interaction mechanism between V. dahliae and its hosts is the prerequisite for developing effective strategies for disease prevention.
UNASSIGNED: Here, based on the previous observation of an xylosidase-encoding gene (VdxyL3) in V. dahliae being obviously up-regulated after sensing root exudates from a cotton variety susceptible to this pathogen, we investigated the function of VdxyL3 in the growth and pathogenesis of V. dahliae by generating its deletion-mutant strains (ΔVdxyL3).
UNASSIGNED: Deleting VdxyL3 led to increased colony expansion rate, conidial production, mycelial growth, carbon and nitrogen utilization capacities, and enhanced stress tolerance and pathogenicity of V. dahliae. VdxyL3 is a secretory protein; however, VdxyL3 failed to induce cell death in N. benthamiana based on transient expression experiment. Transcriptomic analysis identified 1300 genes differentially expressed (DEGs) between wild-type (Vd952) and ΔVdxyL3 during infection, including 348 DEGs encoding secretory proteins, among which contained 122 classical secreted proteins and 226 non-classical secreted proteins. It was notable that of the 122 classical secretory proteins, 50 were carbohydrate-active enzymes (CAZymes) and 58 were small cysteine rich proteins (SCRPs), which were required for the pathogenicity of V. dahliae.
UNASSIGNED: The RNA-seq data thus potentially connected the genes encoding these proteins to the pathogenesis of V. dahliae. This study provides an experimental basis for further studies on the interaction between V. dahliae and cotton and the pathogenic mechanism of the fungus.

OBJECTIVE: New characterized carbohydrate-active enzymes are needed for use as tools to discriminate complex carbohydrate structural features. Fungal glycoside hydrolase family 3 (GH3) β-xylosidases have been shown to be useful for the structural elucidation of glucuronic acid (GlcA) and arabinofuranose (Araf) substituted oligoxylosides. A homolog of these GH3 fungal enzymes from the bacterium Segatella baroniae (basonym Prevotella bryantii), Xyl3C, has been previously characterized, but those studies did not address important functional specificity features. In an interest to utilize this enzyme for laboratory methods intended to discriminate the structure of the non-reducing terminus of substituted xylooligosaccharides, we have further characterized this GH3 xylosidase.
RESULTS: In addition to verification of basic functional characteristics of this xylosidase we have determined its mode of action as it relates to non-reducing end xylose release from GlcA and Araf substituted oligoxylosides. Xyl3C cleaves xylose from the non-reducing terminus of β-1,4-xylan until occurrence of a penultimate substituted xylose. If this substitution is O2 linked, then Xyl3C removes the non-reducing xylose to leave the substituted xylose as the new non-reducing terminus. However, if the substitution is O3 linked, Xyl3C does not hydrolyze, thus leaving the substitution one-xylose (penultimate) from the non-reducing terminus. Hence, Xyl3C enables discrimination between O2 and O3 linked substitutions on the xylose penultimate to the non-reducing end. These findings are contrasted using a homologous enzyme also from S. baroniae, Xyl3B, which is found to yield a penultimate substituted nonreducing terminus regardless of which GlcA or Araf substitution exists.

The use of host to secrete several hemicellulase is a cost-effective way for hemicellulose degradation. In this study, the xylose utilization gene xylAB of Escherichia coli BL21 was knocked out, and the xylanase (N20Xyl), β-xylosidase (Xys), and feruloyl esterase (FaeLam) were co-expressed in this strain. By measuring the content of reducing sugars generated by enzymatic hydrolysis of wheat bran in the fermentation supernatant, the order of the three enzymes was screened to obtain the optimal recombinant strain of E. coli BL21/∆xylAB/pDIII-2. Subsequently, fermentation conditions including culture medium, inducer concentration, induction timing, metal ions, and glycine concentration were optimized. Then, different concentrations of wheat bran and xylan were added to the fermentation medium for degradation. The results showed that the extracellular reducing sugars content reached the highest value of 33.70 ± 0.46 g/L when 50 g/L xylan was added. Besides, the scavenging rates of hydroxyl radical by the fermentation supernatant was 81.0 ± 1.41 %, and the total antioxidant capacity reached 2.289 ± 0.55. Furthermore, it showed the growth promotion effect on different lactic acid bacteria. These results provided a basis for constructing E. coli strain to efficiently degrade hemicellulose, and the strain obtained has great potential application to transform hemicellulose into fermentable carbon source.

Plant cell wall-derived oligosaccharides, i.e., damage-associated molecular patterns (DAMPs), could be generated after pathogen attack or during normal plant development, perceived by cell wall receptors, and can alter immunity and cell wall composition. Therefore, we hypothesised that xylo-oligosaccharides (XOS) could act as an elicitor and trigger immune responses. To test this, we treated Arabidopsis with xylobiose (XB) and investigated different parameters. XB-treatment significantly triggered the generation of reactive oxygen species (ROS), activated MAPK protein phosphorylation, and induced callose deposition. The combination of XB (DAMP) and flg22 a microbe-associated molecular pattern (MAMP) further enhanced ROS response and gene expression of PTI marker genes. RNA sequencing analysis revealed that more genes were differentially regulated after 30 min compared to 24 h XB-treated leaves, which correlated with ROS response. Increased xylosidase activity and soluble xylose level after 30 min and 3 h of XB-treatment were observed which might have weakened the DAMP response. However, an increase in total cell wall sugar and a decrease in uronic acid level was observed at both 30 min and 24 h. Additionally, arabinose, rhamnose, and xylose levels were increased in 30 min, and glucose was increased in 24 h compared to mock-treated leaves. The level of jasmonic acid, abscisic acid, auxin, and cytokinin were also affected after XB treatment. Overall, our data revealed that the shortest XOS can act as a DAMP, which triggers the PTI response and alters cell wall composition and hormone level.

Xylanase and β-xylosidase are the key enzymes for hemicellulose hydrolysis. To further improve hydrolysis efficacy, high temperature hydrolysis with thermostable hemicellulases showed promise. In this study, thermostable xylanase (Xyn) and β-xylosidase (XynB) genes from Pseudothermotoga thermarum were cloned and secretory expressed in Bacillu subtilis. Compared with Escherichia coli expression host, B. subtilis resulted in a 1.5 time increase of enzymatic activity for both recombinant enzymes. The optimal temperature and pH were 95°C and 6.5 for Xyn, and 95°C and 6.0 for XynB. Thermostability of both recombinant enzymes was observed between the temperature range of 75-85°C. Molecular docking analysis through AutoDock showed the involvement of Glu525, Asn526, Trp774 and Arg784 in Xyn-ligand interaction, and Val237, Lys238, Val761 and Asn76 in XynB-ligand interaction, respectively. The recombinant Xyn and XynB exhibited synergistic hydrolysis of beechwood xylan and pretreated lignocellulose, where Xyn and XynB pre-hydrolysis achieved a better improvement of pretreated lignocellulose hydrolysis by commercial cellulase. The observed stability of the enzymes at high temperature and the synergistic effect on lignocellulosic substrates suggested possible application of these enzymes in the field of saccharification process.

The Xylo-1 xylosidase, which belongs to the GH43 family, exhibits a high salt tolerance. The present study demonstrated that the catalytic activity of Xylo-1 increased by 195% in the presence of 5 M NaCl. Additionally, the half-life of Xylo-1 increased 25.9-fold in the presence of 1 M NaCl. Through comprehensive analysis including circular dichroism, fluorescence spectroscopy, and molecular dynamics simulations, we elucidated that the presence of Na+ ions increased the contact frequency between the surface acidic amino acids and the surrounding water molecules. This resulted in the stabilization of the surrounding hydration layer of Xylo-1. Additionally, Na+ ions also stabilized the substrate-binding conformation and the fluctuation of water molecules within the active site, which enhanced the catalytic activity of Xylo-1 by increasing the nucleophilic attack by the water molecules. Ultimately, the optimal reaction conditions for the production of xylose by synergistic catalysis with Xylo-1 and xylanase were determined. The results demonstrated that the conversion yield of the method was high for various sources of xylan, indicating the method could have potential industrial applications. This study explored the structure-activity relationship of catalysis in Xylo-1 under high-salt conditions, provides novel insights into the mechanism of halophilic enzymes, and serves as a reference for the industrial application of Xylo-1.

Corn cobs (CCs) are abundant xylan-rich agricultural wastes. Here, we compared CCs XOS yields obtained via two different pretreatment routs, alkali and hydrothermal, using a set of recombinant endo- and exo-acting enzymes from GH10 and GH11 families, which have different restrictions for xylan substitutions. Furthermore, impacts of the pretreatments on chemical composition and physical structure of the CCs samples were evaluated. We demonstrated that alkali pretreatment route rendered 59 mg of XOS per gram of initial biomass, while an overall XOS yield of 115 mg/g was achieved via hydrothermal pretreatment using a combination of GH10 and GH11 enzymes. These results hold a promise of ecologically sustainable enzymatic valorization of CCs via \"green\" and sustainable XOS production.

Commercially available cellulases and amylases can disperse the pathogenic bacteria embedded in biofilms. This suggests that polysaccharide-degrading enzymes would be useful as antibacterial therapies to aid the treatment of biofilm-associated bacteria, e.g., in chronic wounds. Using a published enzyme library, we explored the capacity of 76 diverse recombinant glycoside hydrolases to disperse Staphylococcus aureus biofilms. Four of the 76 recombinant glycoside hydrolases digested purified cellulose, amylose, or pectin. However, these enzymes did not disperse biofilms, indicating that anti-biofilm activity is not general to all glycoside hydrolases and that biofilm activity cannot be predicted from the activity on pure substrates. Only one of the 76 recombinant enzymes was detectably active in biofilm dispersion, an α-xylosidase from Aspergillus nidulans. An α-xylosidase cloned subsequently from Aspergillus thermomutatus likewise demonstrated antibiofilm activity, suggesting that α-xylosidases, in general, can disperse Staphylococcus biofilms. Surprisingly, neither of the two β-xylosidases in the library degraded biofilms. Commercial preparations of amylase and cellulase that are known to be effective in the dispersion of Staphylococcus biofilms were also analyzed. The commercial cellulase contained contaminating proteins with multiple enzymes exhibiting biofilm-dispersing activity. Successfully prospecting for additional antibiofilm enzymes may thus require large libraries and may benefit from purified enzymes. The complexity of biofilms and the diversity of glycoside hydrolases continue to make it difficult to predict or understand the enzymes that could have future therapeutic applications.

Pectin- and hemicellulose-associated structures of plant cell walls participate in defense responses against pathogens of different parasitic lifestyles. The resulting immune responses incorporate phytohormone signaling components associated with salicylic acid (SA) and jasmonic acid (JA). SA plays a pivotal role in systemic acquired resistance (SAR), a form of induced resistance that - after a local immune stimulus - confers long-lasting, systemic protection against a broad range of biotrophic invaders. β-D-XYLOSIDASE 4 (BXL4) protein accumulation is enhanced in the apoplast of plants undergoing SAR. Here, two independent Arabidopsis thaliana mutants of BXL4 displayed compromised systemic defenses, while local resistance responses to Pseudomonas syringae remained largely intact. Because both phloem-mediated and airborne systemic signaling were abrogated in the mutants, the data suggest that BXL4 is a central component in SAR signaling mechanisms. Exogenous xylose, a possible product of BXL4 enzymatic activity in plant cell walls, enhanced systemic defenses. However, GC-MS analysis of SAR-activated plants revealed BXL4-associated changes in the accumulation of certain amino acids and soluble sugars, but not xylose. In contrast, the data suggest a possible role of pectin-associated fucose as well as of the polyamine putrescine as regulatory components of SAR. This is the first evidence of a central role of cell wall metabolic changes in systemic immunity. Additionally, the data reveal a so far unrecognized complexity in the regulation of SAR, which might allow the design of (crop) plant protection measures including SAR-associated cell wall components.

Development of chimeric enzymes by protein engineering can more efficiently contribute toward biomass conversion for bioenergy generation. Therefore, prior to experimental validation, a computational approach by modeling and molecular dynamic simulation can assess the structural and functional behavior of chimeric enzymes. In this study, a bifunctional chimera, CtXyn11A-BoGH43A comprising an efficient endoxylanase (CtXyn11A) from Clostridium thermocellum and xylosidase (BoGH43A) from Bacteroides ovatus was computationally designed and its binding and stability analysis with xylooligosaccharides were performed. The modeled chimera showed β-jellyroll fold for CtXyn11A and 5-bladed β-propeller fold for BoGH43A module. Stereo-chemical properties analyzed by Ramachandran plot showed 98.8% residues in allowed region, validating the modeled chimera. The catalytic residues identified by multiple sequence alignment were Glu94 and Glu184 for CtXyn11A and Asp229 and Glu384 for BoGH43A modules. CtXyn11A followed retaining-type, whereas BoGH43A enforced inverting-type of reaction mechanism during xylan hydrolysis as revealed by superposition and GH11 and GH43 familial analyses. Molecular docking studies showed binding energy, (ΔG) - 4.54 and - 4.18 kcal/mol for CtXyn11A and BoGH43A modules of chimera, respectively, with xylobiose, while - 3.94 and - 3.82 kcal/mol for CtXyn11A and BoGH43A modules of chimera, respectively, with xylotriose. MD simulation of CtXyn11A-BoGH43A complexed with xylobiose and xylotriose till 100 ns displayed stability by RMSD, compactness by R g and conformational stability by SASA analyses. The lowered values of RMSF in active-site residues, Glu94, Glu184, Asp229, Asp335 and Glu384 confirmed the efficient binding of chimera with xylobiose and xylotriose. These results were in agreement with the earlier experimental studies on CtXyn11A releasing xylooligosaccharides from xylan and BoGH43A releasing d-xylose from xylooligosaccharides and xylobiose. The chimera showed stronger affinity in terms of total short-range interaction energy; - 190 and - 121 kJ/mol for with xylobiose and xylotriose, respectively. The bifunctional chimera, CtXyn11A-BoGH43A showed stability and integrity with xylobiose and xylotriose. The designed chimera can be constructed and applied for efficient biomass conversion.