Structural comparison of ScUGPase-1 and AtUGPase.

<p><b>(A)</b> Superposition of monomers of ScUGPase-1 (blue) and AtUGPase (green) (PDB code: 2ICY; RMSD value of 0.552 Å for 369 Cα atoms). UDP-glucose (yellow sticks) is shown in the active site. The arrow indicates β19 and β20 of ScUGPase-1; they are replaced by a unique β-strand in the AtUGPase structure. <b>(B)</b> Comparison of the active site of apo-ScUGPase-1 and AtUGPase containing UDP-glucose. Residues of AtUGPase involved in the stabilization of UDP-glucose are shown as green sticks and residues of ScUGPase-1 likely important for ligand-binding are shown in blue. The active site of the apo-AtUGPase (PDB code: 1Z90) is also included (purple sticks), showing the same arrangement as for AtUGPase containing UDP-glucose.</p

Crystal structure and insights into the oligomeric state of UDP-glucose pyrophosphorylase from sugarcane

<div><p>UDP-glucose pyrophosphorylase (UGPase) is found in all organisms and catalyses the formation of UDP-glucose. In sugarcane, UDP-glucose is a branch-point in the carbon channelling into other carbohydrates, such as sucrose and cellulose, which are the major factors for sugarcane productivity. In most plants, UGPase has been described to be enzymatically active in the monomeric form, while in human and yeast, homo-octamers represent the active form of the protein. Here, we present the crystal structure of UGPase from sugarcane (ScUGPase-1) at resolution of 2.0 Å. The crystals of ScUGPase-1 reveal the presence of two molecules in the asymmetric unit and the multi-angle light scattering analysis shows that ScUGPase-1 forms a mixture of species ranging from monomers to larger oligomers in solution, suggesting similarities with the orthologs from yeast and human.</p></div

Size exclusion chromatography and MALS analysis.

<p><b>(A)</b> Size exclusion chromatogram of ScUGPase-1. Peaks between 40 and 60 mL correspond presumably to higher oligomeric forms of ScUGPase-1, whereas the peak at 75 mL corresponds to the monomeric form. <b>(B)</b> Coomassie-stained gel under reducing conditions showing the purity of ScUGPase-1. <b>(C)</b> SEC-MALS analysis of ScUGPase-1. Blue and green lines indicate the trace from the refractive index detector during SEC for the octamer and monomer, respectively. Black lines on each peak correspond to the averaged molecular weight (Mw; y axis) distribution across the peak.</p

Crystal structure of ScUGPase-1.

<p><b>(A)</b> Cartoon representation of the two molecules of ScUGPase-1 present in the asymmetric unit. Monomer A is coloured in blue and monomer B in orange. <b>(B)</b> The ScUGPase-1 monomer and its domains. The N-terminal domain is shown in purple, catalytic domain in blue and the C-terminal domain in wheat colour. The RFKS⁴¹⁹IPSI motif is shown in red.</p

Multiple sequence alignment of UGPase orthologs.

<p>Proteins were aligned by MUSCLE [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193667#pone.0193667.ref021" target="_blank">21</a>] and the alignment optimized in Jalview [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0193667#pone.0193667.ref022" target="_blank">22</a>]. The aligned sequences from top to bottom with their accession numbers are: ScUGPase-1 from <i>Saccharum</i> spp—sugarcane (A0A075E2Q1); AtUGPase from <i>Arabidopsis thaliana</i> (Q9M9P3); StUGPase from <i>Solanum tuberosum–</i>potato (P19595); SbUGPase from <i>Sorghum bicolor</i> (C5XSC5); HvUGPase from <i>Hordeum vulgare</i>–barley (Q43772); OsUGPase from <i>Oryza sativa–</i>rice (Q93X08); ZmUGPase from <i>Zea mays–</i>maize (B6T4R3); bUGPase from bovine–<i>Bos taurus</i> (Q07130); mUGPase from mouse–<i>Mus musculus</i> (Q91ZJ5-2); hUGPase from human–<i>Homo sapiens</i> (Q16851-2) and yUGPase from yeast–<i>Saccharomyces cerevisiae</i> (P32861). Region coloured in green corresponds to the residues in the C-terminal region involved in the end-to-end interactions in the yeast and human orthologs. Black asterisks (<b>*</b>) indicate the residues important for ligand binding, and red asterisks (<b>*</b>) indicate residues involved in the dimer interface in the crystal of ScUGPase-1. Elements of secondary structure are shown based on the crystal structure of ScUGPase-1. β-sheets and α-helices are shown in green and red, respectively.</p

Evaluation of interactions between the LRR and NB(-ARC) domains of RPP1_NdA.

<p>The association between the NB(-ARC1) subdomain and the LRR is not disrupted by the presence of ATR1 (A) or by mutations at the putative oligomerization interface (B). Constructs were transiently expressed in <i>Nicotiana benthamiana</i> and samples were collected at 48 hours post-infiltration (E = ATR1_Emoy2, C = ATR1_Cala2). Co-immunoprecipitations were performed using α-Flag agarose beads. The expression of ATR1:citrine was detected with an α-GFP antibody. Asterisks indicate non-specific bands. Staining of RuBisCO with Ponceau S provides a loading control. Experiments were performed three times with similar results.</p

Identification of sequences required for RPP1 TIR domain autoactivity.

<p>(A) Schematic overview of the domain architecture of RPP1. Numbers indicate the amino acid position of predicted domain borders for the Niederzenz (NdA) allele of RPP1. (B,C) Determination of the minimal autoactive TIR domain from the NdA allele of RPP1. Both C-terminal (B) and N-terminal (C) truncations were examined for their ability to elicit an effector-independent hypersensitive response (HR). The specific amino acids comprising each construct are indicated in subscript. (D,E) HR phenotypes associated with chimeras or site-directed mutants of N-TIR domains from the NdA and Wassilewskija (WsB) alleles. Site-directed mutagenesis was guided by the amino acid alignment depicted in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005769#ppat.1005769.s002" target="_blank">S2 Fig</a>. Constructs were tested in <i>Nicotiana tabacum</i> via <i>Agrobacterium</i>-mediated transient expression and images were captured at 48 hours post-infiltration. HR phenotypes are scored as negative (-), weak (w), or strong (+). An α-Flag antibody was used to evaluate protein expression, while staining of RuBisCO with Ponceau S provided a loading control. The experiment was performed three times with similar results.</p

Cartoon overview of the IMPα (in ribbon):Prp20 (stick model) complex structure (top), superimposed on the Fo-Fc annealed omit map (green; calculated using Phenix [30], contoured at 2.0 σ).

<p>Figures were produced using PyMOL (DeLano Scientific LLC).</p

Proposed model of cell death activation by RPP1.

<p>In the absence of ATR1, RPP1 is likely maintained in a largely inactive state by a network of N-TIR:NB, NB:LRR, and N-TIR:LRR interactions. Note that the N-TIR:NB interaction occurs at the putative oligomerization interface to prevent effector-independent association. Binding of a recognized allele of ATR1 to the LRR domain may stabilize conformational transitions that reorient the N-TIR domain to expose the oligomerization interface. This allows RPP1 oligomerization via the NB domain, potentially stabilized by LRR:LRR interactions. The reorientation of the N-TIR domain also permits N-TIR domain self-association, which outcompetes N-TIR:NB interactions and ultimately triggers a cell death response (HR). While this model accounts for the effector-independent NB:LRR interaction, the specific interaction interface(s) could differ before and after RPP1 activation.</p

The autoactivity of the RPP1 N-TIR domain is correlated with self-association in solution.

<p>Purified (N-)TIR domain proteins from the Niederzenz (NdA) and Wassilewskija (WsB) alleles of RPP1 were analyzed by size-exclusion chromatography (SEC) coupled with multi-angle laser light scattering (MALS). For each sample, 175 μg of purified protein was separated on a Superdex Increase 200 5/150 GL SEC column and the molecular mass calculated across the elution peak. The colored solid line represents the normalized refractive index trace (arbitrary units) of the protein eluting from the SEC column. At the elution peak, the averaged molecular mass (kDa) of the proteins was calculated from the protein concentration (derived from the refractive index changes) and light scattering data. The averaged molecular masses across the elution peak are represented by dashed lines of the corresponding color. <i>In planta</i> hypersensitive response (HR) phenotypes are indicated for each construct by a “+” (autoactive) or “-”(non-autoactive). These phenotypes are documented in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005769#ppat.1005769.g001" target="_blank">Fig 1</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005769#ppat.1005769.s002" target="_blank">S2</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005769#ppat.1005769.s004" target="_blank">S4</a> Figs. (A) Comparison of the solution properties of RPP1 TIR domains with and without native RPP1 N termini. Numbers in the legend refer to the amino acids that comprise each protein sample. (B) Solution properties of the wild-type WsB N-TIR domain and the gain-of-autoactivity mutants, WsB R230C and WsB K98R I100F. (C) Solution properties of the wild-type NdA N-TIR domain and the loss-of-autoactivity mutants, NdA G229A Y230A and NdA R104A F106A. (D) Comparison of theoretical monomer molecular masses and the measured molecular masses for the proteins analyzed in (A-C).</p