Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Duplication and neofunctionalization of a horizontally transferred xyloglucanase as a facet of the Red Queen coevolutionary dynamic

doi: 10.1073/pnas.2218927121

Figure Lengend Snippet: Comparative assay of xyloglucanase function across the 11 P. sojae GH12 paralogs. P. sojae GH12 paralogs secreted into S. cerevisiae culture supernatants were incubated with 1% (w/v) xyloglucan at 30 °C, pH 7; an increase in absorbance (OD 544 ) of DNS reagent added to the samples is suggestive of an increase in the reducing sugars released (i.e., from the breakdown of the substrate). ( A ) Significant enzymatic activity toward xyloglucan was detected up to 6 h of incubation for P. sojae _338074, 482953, 520924, and 559651 xyloglucanase variants (Dunnett’s test). ( B ) After 72 h of incubation, significant levels of reducing sugars were also detected for the P. sojae _247788, 260883, 355355, and 520599 enzymes (Dunnett’s test), indicating that eight out of the eleven paralogs display enzymatic activity toward xyloglucan. P. sojae _482953 and P. sojae _559651 appeared to show more rapid degradation than the other active paralogs under these conditions. P. sojae _338074, 482953, 520924, and 559651 enzymatic activities toward xyloglucan were not shown in figure B due to their strong function at 6 h. No significant reducing sugars were detected in the vector-only sample (n = 3, ±SD).

Article Snippet: For xyloglucan oligosaccharide treatment assays, the oligosaccharides XLFG, XLLG, and XXXG were sourced from BOC Sciences (XXLG/XLXG was unable to be synthesized; therefore, it was not possible to test this particular oligosaccharide variant in this assay).

Techniques: Incubation, Activity Assay, Plasmid Preparation

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Duplication and neofunctionalization of a horizontally transferred xyloglucanase as a facet of the Red Queen coevolutionary dynamic

doi: 10.1073/pnas.2218927121

Figure Lengend Snippet: Evolutionary history of oomycete GH12. ( A ) Maximum likelihood tree constructed with IQ-Tree v2.0.3 [WAG+R5 model of evolution selected as the best-fit model by ModelFinder ]. The tree was constructed from an alignment of 161 sequences comprising 211 amino acids to confirm the evolutionary relationships between oomycete GH12. The final tree was visualized with iTOL , and nodes indicate results (%) of nonparametric bootstrap (200 pseudoreplicates) . The tree is rooted with a fungal outgroup because fungi were previously identified as the putative donor group of the GH12 HGT into the oomycetes ( , ). The eleven P. sojae GH12 paralogs are visually identifiable as they have adjacent transcriptome profiles (column B). Symbols indicate the P. sojae paralog with a structurally inferred additional binding site (gray), and paralogs with C-terminal tails 1 and 2 (blue and green, respectively). The numbers of GH12 orthologs across representative Phytophthora spp., and H. arabidopsidis (an obligate pathogen of A. thaliana ) genomes are shown to the Left of the phylogenetic tree, indicating lineage-specific putative local duplications or losses. Some are colored black, indicating they are absent in the tree due to exclusion because of gappy gene models, or they formed uninformative branches, potentially affecting bootstrap resolution (i.e., very long branches or very short indicating little protein variation). All P. sojae paralogs were retained. ( B ) P. sojae life-cycle transcriptome data [FungiDB: ( , )] were used to identify how the eleven P. sojae GH12 paralogs were expressed across (column 1) mycelial, (column 2), cyst and (column 3) 3 d postinfection (soybean hypocotyls infected with P. sojae strain P6497)—expressed in FPKM(Log2). C. Xyloglucanase functional data generated during this study from (column 1) cell culture agar plate-based enzyme assay, (column 2) concentrated supernatant agar-plate based enzyme assay, (column 3) concentrated supernatant DNS reducing sugar assay, and (column 4) concentrated supernatant mass spectrometry of xyloglucan breakdown products (black fill = function detected, white fill = no function detected). ( D ) ROS data in response to P. sojae xyloglucanase variants, presented as percentage ROS generation (%) of the variant that triggered the highest ROS accumulation in N. benthamiana (i.e., P. sojae _482953, which is presented as 100%). ( E ) FungiDB ( , ) was used to locate approximate genomic coordinates of each P. sojae GH12 gene—the relative approximate distances between the paralogs are shown in kilobases (kb), providing additional support for the sister paralog relationships in the phylogenetic tree for: P. sojae _559651 PsXEG1 and P. sojae _360375 PsXLP1 (PHYSO_scaffold_4); and P. sojae_ 482953 (PHYSO_scaffold_2). Scaffold numbers are shown in the figure.

Article Snippet: For xyloglucan oligosaccharide treatment assays, the oligosaccharides XLFG, XLLG, and XXXG were sourced from BOC Sciences (XXLG/XLXG was unable to be synthesized; therefore, it was not possible to test this particular oligosaccharide variant in this assay).

Techniques: Construct, Binding Assay, Infection, Functional Assay, Generated, Cell Culture, Enzymatic Assay, Mass Spectrometry, Variant Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Duplication and neofunctionalization of a horizontally transferred xyloglucanase as a facet of the Red Queen coevolutionary dynamic

doi: 10.1073/pnas.2218927121

Figure Lengend Snippet: P. sojae xyloglucanase paralogs produce variant oligosaccharide break-down products. P. sojae xyloglucanase paralogs secreted into S. cerevisiae culture supernatants were incubated with 1% (w/v) xyloglucan at 30 °C, pH 7, for 72 h. Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS) spectra confirmed the release of xyloglucan oligosaccharides by eight of the 11 variants (corresponding to the functional paralogs identified in ). Four peaks of interest were observed; ions with m/z of ~1,085, 1,247, 1,409, and 1,571—putatively corresponding to the oligosaccharides XXXG, XXLG (or XLXG), XLLG, and XLFG respectively . The Top panel indicates the side chain residues present in each of the oligosaccharide variant types, as described by Fry et al., 1993 . The relative intensities of species identified at these peaks were compared by calculating the ratios between the areas under the peaks, to probe putative differences in preferential binding of the xyloglucan backbone. We find strong evidence that the composition of oligosaccharides varies significantly between the P. sojae xyloglucanase paralogs [Dirichlet likelihood ratio test (randomization approach); P -value < 0.001]. For example, we see almost absence of the XLFG product for three enzymes. Goodness-of-fit testing indicates that the Dirichlet distribution provides an adequate model for these data ( P -value = 0.48).

Article Snippet: For xyloglucan oligosaccharide treatment assays, the oligosaccharides XLFG, XLLG, and XXXG were sourced from BOC Sciences (XXLG/XLXG was unable to be synthesized; therefore, it was not possible to test this particular oligosaccharide variant in this assay).

Techniques: Variant Assay, Incubation, Mass Spectrometry, Functional Assay, Binding Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Duplication and neofunctionalization of a horizontally transferred xyloglucanase as a facet of the Red Queen coevolutionary dynamic

doi: 10.1073/pnas.2218927121

Figure Lengend Snippet: Exploration of function among xyloglucanase paralogs with C-terminal extensions. ( A ) P. sojae _482953 (full-length) and P. sojae _482953 (truncated), and orthologs in P. cactorum and P. nicotianae secreted into S. cerevisiae culture supernatants were incubated with 1% (w/v) xyloglucan at 30 °C, pH 7; an increase in absorbance (OD 544 ) of DNS reagent added to the samples is suggestive of an increase in the reducing sugars released (i.e., from the breakdown of the substrate). Following incubation with xyloglucan for 6 h, we find that removal of the C-terminal extension significantly impairs xyloglucanase function for P. sojae _482953 ( t test; P -value = 0.04) and its ortholog in P. cactorum ( t test; P -value = 0.0001), but this reduction was not found to be significant for the P. nicotianae ortholog at any timepoint. No significant reducing sugars were detected in the vector-only sample (n = 3, ±SD). ( B ) P. sojae _247788 (truncated) and P. sojae _247788 (full-length), and orthologs in P. cactorum and P. nicotianae secreted into S. cerevisiae culture supernatants were incubated with 1% (w/v) xyloglucan at 30 °C, pH 7; an increase in absorbance (OD 544 ) of DNS reagent added to the samples is suggestive of an increase in the reducing sugars released (i.e., from the breakdown of the substrate). P. sojae _247788 (full-length) gave weak enzymatic activity toward xyloglucan by this method, but the orthologous proteins of P. cactorum and P. nicotianae demonstrated significantly higher xyloglucanase function. The truncated orthologs were found to be enzymatically active toward xyloglucan, but with significantly reduced catalysis compared to the full-length proteins after 6 h of incubation with the substrate ( t test; P -value = 0.0002 and 0.01 for P. nicotianae and P. cactorum , respectively). No reducing sugars were detected in the vector-only sample (n = 3, ±SD).

Article Snippet: For xyloglucan oligosaccharide treatment assays, the oligosaccharides XLFG, XLLG, and XXXG were sourced from BOC Sciences (XXLG/XLXG was unable to be synthesized; therefore, it was not possible to test this particular oligosaccharide variant in this assay).

Techniques: Incubation, Plasmid Preparation, Activity Assay

Journal: ACS Sustainable Chemistry & Engineering

Article Title: Crystal Structure of α-Xylosidase from Aspergillus niger in Complex with a Hydrolyzed Xyloglucan Product and New Insights in Accurately Predicting Substrate Specificities of GH31 Family Glycosidases

doi: 10.1021/acssuschemeng.9b07073

Figure Lengend Snippet: Tetrameric structure of AxlA. (A) Two half-tetramers are related by crystal symmetry. Each monomeric subunit (A, B, A′, B′) is color-coded individually. Steric surface is rendered transparently, and secondary structures are shown as ribbon cartoons, the experimentally observed post-translational N-glycans (white) and bioinformatically predicted glycosylation site Asn residues (yellow, with residue numbers labeled for subunit B) are shown as spheres. The active site ligands (hydrolyzed xyloglucan oligosaccharides) are shown as black spheres. (B, C, and D) Tetrameric oligomerization interfaces between different subunits are represented by interfacial residues within 4.5 Å shown as spheres for the AB, AA′ and BB′ interfaces.

Article Snippet: Prior to harvesting, a subset of crystals was soaked with xyloglucan oligosaccharide XFG for 1–5 min. Crystals were either directly flash-frozen in liquid nitrogen or cryoprotected by transferring into MiTeGen’s LV CryoOil (MiTeGen, Ithaca, NY) before being frozen.

Techniques: Labeling

Journal: ACS Sustainable Chemistry & Engineering

Article Title: Crystal Structure of α-Xylosidase from Aspergillus niger in Complex with a Hydrolyzed Xyloglucan Product and New Insights in Accurately Predicting Substrate Specificities of GH31 Family Glycosidases

doi: 10.1021/acssuschemeng.9b07073

Figure Lengend Snippet: Reaction product complex structure of AxlA and proposed catalytic mechanism. (A) The hydrolyzed XFG heptasaccharide catalytic product is shown as sticks (carbon in white) with the corresponding difference omit map contoured at 3.5 σ. The active site residue side chains within 4 Å of the hydrolyzed oligosaccharide are shown as sticks (carbon in cyan) with corresponding 2mFo-dFc map contoured at 2 σ. The hydrogen-bonding interactions between the ligand and active site residues are indicated as black dashes. The conserved nucleophile D395 and general acid D487 aspartate residues and catalytic labile C1 of xylose at −1 site are also labeled. The branched xyloglucan oligosaccharide binding site is connected to a surface pocket of the adjacent subunit (pink), although with no apparent direct interactions with the ligand. (B) shown the same way as part A but in the other active site of the dimer in the asymmetric unit. (C) chemical structures of XFG and d -xylose (atom number labeled). XFG is named according to an existing nomenclature for xyloglucan-derived oligosaccharide (see Abbreviations). (D) Proposed two-step double displacement catalytic mechanism of AxlA leads to conformational retention at the catalytic labile C1 position between the substrate and product.

Article Snippet: Prior to harvesting, a subset of crystals was soaked with xyloglucan oligosaccharide XFG for 1–5 min. Crystals were either directly flash-frozen in liquid nitrogen or cryoprotected by transferring into MiTeGen’s LV CryoOil (MiTeGen, Ithaca, NY) before being frozen.

Techniques: Labeling, Binding Assay, Derivative Assay