Figure 1

<p>Natural variation of partial resistance to <i>Pseudomonas syringae</i> pv. <i>tomato</i> DC3000 among Arabidopsis accessions. <i>In planta</i> bacterial growth was assessed three days post inoculation with a bacterial suspension adjusted to 10⁵ cfu/mL. Means and standard errors were calculated from bacterial densities in at least 4 plants.</p

Figure 5

<p>Expression analysis of the defense marker genes <i>PR1</i> (A), <i>ICS</i> (B) and <i>PR5</i> (C) in HIFs and parental accessions. The transcript levels were determined by Q-RT-PCR with cDNA generated from leaves before (grey) and after (black) inoculation with <i>Pst</i> DC3000 at 5.10⁵ cfu/mL. The expression value of the individual genes was normalized by using the expression level of <i>β-Tubulin4</i> as an internal standard. Mean mRNA levels of three plants are shown with corresponding standard errors. Similar results were obtained from two biological experiments.</p

Figure 2

<p>Distribution of bacterial growth values in the Bay-0×Shahdara recombinant inbred line (RIL) population. The frequency histogram shows the range of <i>in planta</i> bacterial populations observed in RILs three days post inoculation with <i>Pseudomonas syringae</i> pv. <i>tomato</i> DC3000 in one of the greenhouse experiments. The values obtained for the parental accessions, Bay-0 and Shahdara (Sha), and the genetic mean of the population are indicated.</p

Figure 4

<p>Validation of the major QTLs for partial resistance to <i>Pseudomonas syringae</i> pv. <i>tomato</i> DC3000 with heterogeneous inbred families (HIFs). <b>A</b> HIF 102 segregates around MSAT2-38 and HIF 200 segregates for a region of chromosome II around markers MSAT2-5 and MSAT2-38. HIF 214 segregates for a region of chromosome V around MSAT5-14 and NGA139 and HIF 400 segregates around MSAT5-14, NGA139 and MSAT5-22. Regions for which the HIFs segregate are indicated in hatched boxes and the white regions of the chromosomes represent a mix of both parental genomes Bay-0 and Shahdara. <b>B</b> and <b>C</b>  <i>In planta</i> bacterial growth in HIFs fixed for the Bay (grey bar) or Sha (black bar) allele of <i>PRP-Ps1</i> (<b>B</b>) or <i>PRP-Ps2</i> (<b>C</b>). Each value is the average of measurement of <i>in planta</i> bacterial growth in at least 8 plants (means and standard errors) from one experiment. Three independent experiments were performed and showed similar results. Asterisks show significant difference in partial resistance (P<0.05).</p

Statistics for 20 NMR structures of AVR-Pia and AVR1-CO39.

<p>Structures were calculated using CYANA, refined using CNS, and analyzed using PROCHECK.</p><p>(a) Residues in regular secondary structures were derived from the chemical shifts using TALOS+ software.</p><p>(b) PROCHECK was used over the residues 24–85 for AVR-Pia and over the residues 23–83 for AVR1-CO39.</p><p>(c) Main chain atoms (N, Cα, C) over the residues 24–85 for AVR-Pia and over the residues 23–83 for AVR1-CO39.</p><p>Statistics for 20 NMR structures of AVR-Pia and AVR1-CO39.</p

Large numbers of MAX-effectors sharing a characteristic sequence pattern are present in <i>M</i>. <i>oryzae</i> and <i>M</i>. <i>grisea</i>.

<p>A) Sequence pattern of MAX-effectors. The sequence logo was generated using the alignment of MAX-effector candidates identified by a high stringency HMM search (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005228#ppat.1005228.s010" target="_blank">S6 Fig</a>). (B) Numbers of MAX-effector candidates detected by a low stringency HMM sequence pattern search. A database combining 25 pathogenic and non-pathogenic ascomycete fungi and 9 <i>M</i>. <i>oryzae</i> and <i>M</i>. <i>grisea</i> isolates was searched with an HMM pattern based on a structural alignment of AVR-Pia, AVR1-CO39, AVR-Pia and AvrPiz-t.</p

Structure Analysis Uncovers a Highly Diverse but Structurally Conserved Effector Family in Phytopathogenic Fungi

<div><p>Phytopathogenic ascomycete fungi possess huge effector repertoires that are dominated by hundreds of sequence-unrelated small secreted proteins. The molecular function of these effectors and the evolutionary mechanisms that generate this tremendous number of singleton genes are largely unknown. To get a deeper understanding of fungal effectors, we determined by NMR spectroscopy the 3-dimensional structures of the <i>Magnaporthe oryzae</i> effectors AVR1-CO39 and AVR-Pia. Despite a lack of sequence similarity, both proteins have very similar 6 β-sandwich structures that are stabilized in both cases by a disulfide bridge between 2 conserved cysteins located in similar positions of the proteins. Structural similarity searches revealed that AvrPiz-t, another effector from <i>M</i>. <i>oryzae</i>, and ToxB, an effector of the wheat tan spot pathogen <i>Pyrenophora tritici-repentis</i> have the same structures suggesting the existence of a family of sequence-unrelated but structurally conserved fungal effectors that we named MAX-effectors (<u><b><i>M</i></b></u><i>agnaporthe</i><u><b>A</b></u>vrs and To<u><b>x</b></u>B like). Structure-informed pattern searches strengthened this hypothesis by identifying MAX-effector candidates in a broad range of ascomycete phytopathogens. Strong expansion of the MAX-effector family was detected in <i>M</i>. <i>oryzae</i> and <i>M</i>. <i>grisea</i> where they seem to be particularly important since they account for 5–10% of the effector repertoire and 50% of the cloned avirulence effectors. Expression analysis indicated that the majority of <i>M</i>. <i>oryzae</i> MAX-effectors are expressed specifically during early infection suggesting important functions during biotrophic host colonization. We hypothesize that the scenario observed for MAX-effectors can serve as a paradigm for ascomycete effector diversity and that the enormous number of sequence-unrelated ascomycete effectors may in fact belong to a restricted set of structurally conserved effector families.</p></div

The majority of <i>M</i>. <i>oryzae</i> MAX-effectors is expressed specifically during biotrophic infection.

<p>mRNA levels of <i>M</i>. <i>oryzae</i> genes coding for 32 different MAX-effectors (A) and marker genes (B) for appressorium formation and very early infection (<i>ORF3</i> of the <i>ACE1</i> cluster, <i>MGG_0838</i>1), biotrophic infection (<i>BAS3</i>, <i>MGG_11610</i>), late infection <i>(MGG_01147</i>) and constitutive expression (EF1α, <i>MGG_03641</i>) were determined by q-RT-PCR in rice leaf samples harvested 16, 24, 48 and 72 h after inoculation and mycelium grown <i>in vitro</i>. Relative expression levels were calculated by using expression of a constitutively expressed <i>Actin</i> gene (<i>MGG_03982</i>) as a reference and normalized with respect to the highest expression value. Values are means calculated from the relative expression values of three independent biological samples. Individual expression profiles are in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005228#ppat.1005228.s013" target="_blank">S9 Fig</a>.</p

AVR-Pia, AVR1-CO39, AvrPiz-t and ToxB have similar 6 β-sandwich structures.

<p>Topology diagrams (lower row) show that AVR-Pia (A), AVR1-CO39 (B), AvrPiz-t (C) and ToxB (D) possess the same fold. Ribbon diagrams (upper row, generated with PyMOL (<a href="http://www.pymol.org/" target="_blank">http://www.pymol.org</a>)) highlight similarities of their structures. Disulfide bonds are shown in the ribbon diagrams by orange sticks. All four structures were superimposed and a structural alignment was derived using DALI with the ToxB sequence as the reference for numbering (E). Residues not aligned to ToxB are connected by vertical lines and correspond to insertions in loops of AvrPiz-t and AVR-Pia. Triangles over the residues indicate chemical properties (upper-left triangle: yellow for hydrophobic, red for charged, pink for Asn and Gln and blue for other residues) and solvent accessibility (lower-right triangle: from black for buried to white for solvent-exposed). The consensus is defined by at least three similar residues per position. Residues forming β-strands are pink. Disulfide bridges in AVR1-CO39 and ToxB are shown below the consensus by a black line and for AVR-Pia by a grey line. For AvrPiz-t, no disulfide bridge was reported despite presence of the two conserved cysteins [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005228#ppat.1005228.ref042" target="_blank">42</a>].</p

Formy a vliv způsobu výchovy na děti z neúplných rodin

Primers used for this study. (PDF 66 kb