Relative fold change of genes involved in call wall biosynthesis (GO: 0070592) in top stem samples of arabidopsis transgenic lines.

<p>Relative fold change of genes involved in call wall biosynthesis (GO: 0070592) in top stem samples of arabidopsis transgenic lines.</p

Photosynthesis—antenna protein pathway (KEGG ath00196).

<p>The pathway schematic was modified from the KEGG database with permission [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173094#pone.0173094.ref064" target="_blank">64</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173094#pone.0173094.ref065" target="_blank">65</a>]. The heat maps illustrate differential transcript accumulation in transgenic lines relative to wild-type for both top and mid stem samples. The color corresponds to log₂ fold change in transcript accumulation. Rows in each heat map represent genes and the order of genes corresponds to the order given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173094#pone.0173094.s009" target="_blank">S4B Table</a>.</p

Core set of genes with differential expression in stem tissue of transgenic arabidopsis lines overexpressing fungal carbohydrate-active enzymes.

<p>The heat map represents relative transcript abundance of genes that differ significantly in each of the three transgenic lines (<i>AnAF54</i>, <i>PcGCE-7</i>, and <i>PcGCE-13</i>) relative to the wild-type in both, top stem and mid stem tissue (n = 655, FDR < 0.05). Genes with significantly lower transcript abundance in transgenic lines when compared with non-transformed wild-type are marked with “reduced” (n = 188) and gene with significantly higher transcript abundance are marked with “increased” (n = 467). Data are row normalized.</p

Relative fold change of genes involved in salicylic acid signaling.

<p>Relative fold change of genes involved in salicylic acid signaling.</p

Plant-pathogen interaction pathway (KEGG ath04626).

<p>The pathway schematic was adapted from the KEGG database with permission [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173094#pone.0173094.ref064" target="_blank">64</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173094#pone.0173094.ref065" target="_blank">65</a>]. The heat maps illustrate differential transcript accumulation in transgenic lines relative to wild-type for both top and mid stem samples. The color corresponds to log₂ fold change in transcript accumulation. Rows in each heat map represent genes and the order of genes corresponds to the order given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173094#pone.0173094.s009" target="_blank">S4A Table</a>.</p

Plant cell wall glycosyltransferases: High-throughput recombinant expression screening and general requirements for these challenging enzymes

<div><p>Molecular characterization of plant cell wall glycosyltransferases is a critical step towards understanding the biosynthesis of the complex plant cell wall, and ultimately for efficient engineering of biofuel and agricultural crops. The majority of these enzymes have proven very difficult to obtain in the needed amount and purity for such molecular studies, and recombinant cell wall glycosyltransferase production efforts have largely failed. A daunting number of strategies can be employed to overcome this challenge, including optimization of DNA and protein sequences, choice of expression organism, expression conditions, co-expression partners, purification methods, and optimization of protein solubility and stability. Hence researchers are presented with thousands of potential conditions to test. Ultimately, the subset of conditions that will be sampled depends on practical considerations and prior knowledge of the enzyme(s) being studied. We have developed a rational approach to this process. We devise a pipeline comprising <i>in silico</i> selection of targets and construct design, and high-throughput expression screening, target enrichment, and hit identification. We have applied this pipeline to a test set of <i>Arabidopsis thaliana</i> cell wall glycosyltransferases known to be challenging to obtain in soluble form, as well as to a library of cell wall glycosyltransferases from other plants including agricultural and biofuel crops. The screening results suggest that recombinant cell wall glycosyltransferases in general have a very low soluble:insoluble ratio in lysates from heterologous expression cultures, and that co-expression of chaperones as well as lysis buffer optimization can increase this ratio. We have applied the identified preferred conditions to Reversibly Glycosylated Polypeptide 1 from <i>Arabidopsis thaliana</i>, and processed this enzyme to near-purity in unprecedented milligram amounts. The obtained preparation of Reversibly Glycosylated Polypeptide 1 has the expected arabinopyranose mutase and autoglycosylation activities.</p></div

<i>Arabidopsis thaliana</i> RGP1 scale-up, purification and activity determinations.

<p>(A) Coomassie stained SDS-PAGE of the final chromatographic step yielding almost pure RGP1 (42 kDa). (B) Phosphate-release assay showing autoglycosylating or hydrolytic activity of RGP1 on UDP-glucose. (C) UDP-arabinose mutase activity of RGP1. High-pressure liquid chromatograms of authentic UDP-arabinopyranose (UDP-Ara<i>p</i>) and UDP-arabinofuranose (UDP-Ara<i>f</i>) standards (grey) overlaid with the chromatogram of the reaction mixture of UDP-Ara<i>f</i> with recombinant, purified RGP1 (black).</p

SDS-PAGE of selected samples from the non-<i>Arabidopsis</i> library.

<p>Coomassie-stained gels showing nickel affinity eluates of samples selected from the automated capillary electrophoresis procedure as described. Each lane is marked with the organism and protein name, as well as with the well number for reference to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0177591#pone.0177591.s015" target="_blank">S6 Data</a>. Bands that were excised for mass spectrometry are marked with a green dot (confirmed by mass spectrometry) or a red cross (not confirmed). The lower right gel has samples expressed in the periplasm (pET22DEST vector, no excisions), while the other three gels have samples expressed in the cytoplasm (pET55DEST vector). Sample b8 (upper left gel) has no detectable background on SDS-PAGE, but produces a normal band pattern in the automated capillary electrophoresis, indicating that the absence of bands on SDS-PAGE is likely due to faulty well loading. <i>Ca = Cicer arietinum</i>, <i>Cs = Cucumis sativa</i>, <i>Fv = Fragaria vesca</i>, <i>Gm = Glycine max</i>, <i>Si = Setaria italica</i>, <i>Sl = Solanum lycopersicum</i>, <i>Vv = Vitis vinifera</i>, <i>Zm = Zea mays</i>.</p

Alphabetical list of the CWGTs that were screened.

<p>Alphabetical list of the CWGTs that were screened.</p

Hits from the test library screening.

<p>Hits from the test library screening.</p