The tomato Prf complex is a molecular trap for bacterial effectors based on Pto transphosphorylation

The bacteria Pseudomonas syringae is a pathogen of many crop species and one of the model pathogens for studying plant and bacterial arms race coevolution. In the current model, plants perceive bacteria pathogens via plasma membrane receptors, and recognition leads to the activation of general defenses. In turn, bacteria inject proteins called effectors into the plant cell to prevent the activation of immune responses. AvrPto and AvrPtoB are two such proteins that inhibit multiple plant kinases. The tomato plant has reacted to these effectors by the evolution of a cytoplasmic resistance complex. This complex is compromised of two proteins, Prf and Pto kinase, and is capable of recognizing the effector proteins. How the Pto kinase is able to avoid inhibition by the effector proteins is currently unknown. Our data shows how the tomato plant utilizes dimerization of resistance proteins to gain advantage over the faster evolving bacterial pathogen. Here we illustrate that oligomerisation of Prf brings into proximity two Pto kinases allowing them to avoid inhibition by the effectors by transphosphorylation and to activate immune responses

Microstructure and precipitation in Al-Li-Cu-Mg-(Mn, Zr) alloys

Hot rolled Al-6Li-1Cu-1Mg-0.2Mn (at.%) (Al-1.6Li-2.2Cu-0.9Mg-0.4Mn, wt.%) and Al-6Li-1Cu-1Mg-0.03Zr (at.%) (Al-1.6Li-2.3Cu-1Mg-0.1Zr, wt.%) alloys developed for age forming were studied by tensile testing, electron backscatter diffraction (EBSD), three-dimensional atom probe (3DAP), transmission electron microscopy (TEM) and differential scanning calorimetry (DSC). For both alloys, DSC analysis shows that ageing at 150°C leads initially to formation of zones/clusters, which are later gradually replaced by S phase. On ageing at 190°C, S phase formation is completed within 12 h. The precipitates identified by 3D atom probe and TEM can be classified into (a) Li-rich clusters containing Cu and Mg, (b) a plate-shaped metastable precipitate (similar to GPB2 zones/S''), (c) S phase and (d) delta' spherical particles rich in Li. The Zr containing alloy also contains beta' (Al3Zr) precipitates and composite beta'/delta' particles. The beta' precipitates reduces recrystallisation and grain growth leading to fine grains and subgrains

Gene gain and loss during evolution of obligate parasitism in the white rust pathogen of Arabidopsis thaliana

Biotrophic eukaryotic plant pathogens require a living host for their growth and form an intimate haustorial interface with parasitized cells. Evolution to biotrophy occurred independently in fungal rusts and powdery mildews, and in oomycete white rusts and downy mildews. Biotroph evolution and molecular mechanisms of biotrophy are poorly understood. It has been proposed, but not shown, that obligate biotrophy results from (i) reduced selection for maintenance of biosynthetic pathways and (ii) gain of mechanisms to evade host recognition or suppress host defence. Here we use Illumina sequencing to define the genome, transcriptome, and gene models for the obligate biotroph oomycete and Arabidopsis parasite, Albugo laibachii. A. laibachii is a member of the Chromalveolata, which incorporates Heterokonts (containing the oomycetes), Apicomplexa (which includes human parasites like Plasmodium falciparum and Toxoplasma gondii), and four other taxa. From comparisons with other oomycete plant pathogens and other chromalveolates, we reveal independent loss of molybdenum-cofactor-requiring enzymes in downy mildews, white rusts, and the malaria parasite P. falciparum. Biotrophy also requires ‘‘effectors’’ to suppress host defence; we reveal RXLR and Crinkler effectors shared with other oomycetes, and also discover and verify a novel class of effectors, the ‘‘CHXCs’’, by showing effector delivery and effector functionality. Our findings suggest that evolution to progressively more intimate association between host and parasite results in reduced selection for retention of certain biosynthetic pathways, and particularly reduced selection for retention of molybdopterinrequiring biosynthetic pathways. These mechanisms are not only relevant to plant pathogenic oomycetes but also to human pathogens within the Chromalveolata

Transphosphorylation is required for Ser-198 and Thr-199 phosphorylation.

<p>(<b>A</b>) <i>In trans</i> inhibition of Pto S198 and T199 phosphorylation. The slower migrating from of Pto is suppressed <i>in trans</i> by prf^K1128A and pto^D164N, but not pto^S189A/T199A, pto^S189A and pto^T199A. Pto-5Myc, Pto-FLAG, pto mutant-FLAG, AvrPto, Prf-3HA, prf^K1128A-3HA constructs were transiently expressed in wild-type <i>N. benthamiana</i> as indicated, Prf-3HA and prf^K1128A-3HA were immunoprecipitated (IP) using anti-HA antibodies. Immunoblots (IB) were performed with the antibodies indicated on the left. (<b>B</b>) The slower migrating from of Pto was suppressed <i>in trans</i> by prf^K1128A and pto^D164N, but not by pto^S189A pto^T199A and pto^S189A/T199A. Pto-5Myc, Pto-FLAG, pto mutant-FLAG, AvrPto, Prf-3HA, prf^K1128A-3HA constructs were transiently expressed in wild-type <i>N. benthamiana</i> as indicated. Prf-3HA and prf^K1128A-3HA were immunoprecipitated using anti-HA antibodies. The relative abundance of slow- and fast-migrating forms of Pto-5Myc after AvrPto recognition was quantified two days post infiltration, from anti-Myc immunoblots using Quantity One, Bio-Rad (adjusted volume = [CNT*mm2] data counts/mm²).</p

Double phosphorylation of Pto peptide 187–202 and 188–202 upon activation of signalling.

<p>Prf-3HA, prf^K1128A –3HA, prf^D1416V –3HA, N-term -3HA (prf^1–546 –3HA), Pto-FLAG AvrPto and AvrPtoB were expressed transiently in <i>N. benthamiana</i> under the control of 35S promoter; the total amount of Pto-FLAG was immunoprecipitated using anti-FLAG antibodies. The number of peptides identified with 0, 1, and 2 phosphorylation events is indicated.</p>*<p>ligand-independent HR.</p

Phosphorylation on Pto residues S198 and T199 is required for signalling.

<p>(<b>A</b>) Trypan blue staining of cell death in <i>N. benthamiana</i> leaves. Pto-FLAG, pto mutant-FLAG, AvrPto, AvrPtoB, and prf^D1416V-3HA constructs were transiently expressed in Pro_Prf:<i>Prf-5Myc</i> or wild-type <i>N. benthamiana</i> as indicated and the tissue was stained 2 days post infiltration. The bar indicates 0.5 mm. Dead cells stain dark blue in this qualitative assay. Each row is derived from a single leaf, within which relative amounts of cell death were comparable, and is representative of six replicates. (<b>B</b>) The slow-migrating form of Pto requires kinase activity and double phosphorylation. Pto-FLAG, pto mutant-FLAG, AvrPto, and Prf-3HA constructs were transiently expressed in wild-type <i>N. benthamiana</i> as indicated, Prf-3HA was immunoprecipitated (IP) using anti-HA antibodies. Immunoblots (IB) were performed with the antibodies indicated on the left. (<b>C</b>) Quantification of the relative abundance of slow- and fast-migrating forms of Pto under elicitation conditions as described in B with Quantity One, Bio-Rad (adjusted volume = [CNT*mm2] data counts/mm²). Error bars are standard deviation of relative abundance between the same samples in independent immunoblots, probed with anti-Pto antibody.</p

Transphosphorylation is required for signalling.

<p>(<b>A</b>) The phospho-mimic mutant pto^S198D/T199D induced cell death after AvrPto and AvrPtoB recognition. pto^S198D/T199D -FLAG, pto^{D164N/S198D/T199D}-FLAG, AvrPto, and AvrPtoB constructs were transiently expressed in Pro_Prf:<i>Prf-5Myc N. benthamiana</i> as indicated. Cell death was visualized with trypan blue staining two days post infiltration. Relative accumulation of pto^S198D/T199D and pto^{D164N/S198D/T199D} was detected with immunoblot (IB) with aντι-Pto antibody. Coomassie Brilliant Blue (CBB) staining of the IB membrane verified equal protein loading. (<b>B</b>) AvrPto-induced signalling by the phospho-mimic mutant pto^S198D/T199D is not suppressed <i>in trans</i> by pto^D164N. Pto-FLAG, pto^S198D/T199D-FLAG, pto^D164N-HA and AvrPto constructs were transiently expressed in Pro_Prf:<i>Prf-5Myc N. benthamiana</i> as indicated. Cell death was visualized as in A and relative accumulation of proteins was detected with IB with the indicated antibodies. CBB staining of the IB membranes verified equal protein loading. (<b>C</b>) Phosphorylation of the kinase-inactive, constitutive gain-of-function (CGF) mutant pto^L205D at Ser-198 and Thr-199 is required for its hypersensitive cell death response inducing ability. pto^L205D-FLAG, pto^{S198A/T199A/L205D}-FLAG and pto^S198A/T199A-FLAG constructs were transiently expressed in Pro_Prf:<i>Prf-5Myc N. benthamiana</i> as indicated. Cell death was visualized as in A and relative accumulation of proteins was detected with IB with anti-Pto antibody. CBB staining of the IB membranes verified equal protein loading. (<b>D</b>) Signalling by the kinase-inactive, CGF mutant pto^L205D mutant is suppressed <i>in trans</i> by pto^D164N. pto^L205D-HA, pto^D164N-FLAG, pto^S198D/T199D-FLAG, pto^S198A/T199A-FLAG and Pto-FLAG constructs were transiently expressed in Pro_Prf:<i>Prf-5Myc N. benthamiana</i> as indicated. Cell death was visualized as in A and relative accumulation of proteins was detected with IB with the indicated antibodies. CBB staining of the IB membranes verified equal protein loading. For all images, the bar indicates 0.5 mm. Each row of trypan blue staining is derived from a single leaf, within which relative amounts of cell death were comparable, and representative of six replicates.</p

Phosphorylation of Pto upon activation of signalling.

<p>(<b>A</b>) Slow migration of Prf-associated Pto after effector recognition. The indicated Prf, AvrPto and AvrPtoB constructs were transiently expressed in stable transgenic 35S:<i>Pto N. benthamiana</i> plants. Three days post infiltration, Prf-3HA and prf^K1128A-3HA proteins were immunoprecipitated (IP) using anti-HA antibodies. Immunoblots (IB) for Prf and Pto were performed with the antibodies indicated on the left. (<b>B</b>) A functional Prf molecule is required to generate the slow-migrating Pto form. The indicated Prf constructs were transiently expressed in stable transgenic 35S:<i>Pto N. benthamiana</i> plants. Three days post infiltration, Prf-3HA, prf^D1416V-3HA and N-term-3HA (prf^1–546-3HA) proteins were immunoprecipitated using anti-HA antibodies. Immunoblots were performed with the antibodies indicated on the left. Equal protein loading was verified by Coomassie Brilliant Blue (CBB) staining of the membranes. The experiment was repeated six times and typical results are shown.</p

Summary of Pto signalling activities.

1<p>From <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003123#ppat.1003123.s006" target="_blank">Figure S6</a>.</p>2<p>From <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003123#ppat-1003123-g002" target="_blank">Figure 2A</a>.</p>3<p>From <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003123#ppat.1003123.s007" target="_blank">Figure S7</a>.</p