Microsatellite genotyping of Botrytis cinerea grey mold and noble rot populations in France

Microsatellite genotyping using 8 markers. Populations of B. cinerea collected in the field, in 3 wine-producing French regions, from grey mold and nobel rot symptoms. Data are given in allele size in bp (haploid data). 0 indicate missing values

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

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

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

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

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

Dataset Saleh et al

Dataset Saleh et a

Sequenced isolates.

(XLSX)</p

Genomic Analysis of the Necrotrophic Fungal Pathogens <i>Sclerotinia sclerotiorum</i> and <i>Botrytis cinerea</i>

<div><p><i>Sclerotinia sclerotiorum</i> and <i>Botrytis cinerea</i> are closely related necrotrophic plant pathogenic fungi notable for their wide host ranges and environmental persistence. These attributes have made these species models for understanding the complexity of necrotrophic, broad host-range pathogenicity. Despite their similarities, the two species differ in mating behaviour and the ability to produce asexual spores. We have sequenced the genomes of one strain of <i>S. sclerotiorum</i> and two strains of <i>B. cinerea</i>. The comparative analysis of these genomes relative to one another and to other sequenced fungal genomes is provided here. Their 38–39 Mb genomes include 11,860–14,270 predicted genes, which share 83% amino acid identity on average between the two species. We have mapped the <i>S. sclerotiorum</i> assembly to 16 chromosomes and found large-scale co-linearity with the <i>B. cinerea</i> genomes. Seven percent of the <i>S. sclerotiorum</i> genome comprises transposable elements compared to <1% of <i>B. cinerea</i>. The arsenal of genes associated with necrotrophic processes is similar between the species, including genes involved in plant cell wall degradation and oxalic acid production. Analysis of secondary metabolism gene clusters revealed an expansion in number and diversity of <i>B. cinerea</i>–specific secondary metabolites relative to <i>S. sclerotiorum</i>. The potential diversity in secondary metabolism might be involved in adaptation to specific ecological niches. Comparative genome analysis revealed the basis of differing sexual mating compatibility systems between <i>S. sclerotiorum</i> and <i>B. cinerea</i>. The organization of the mating-type loci differs, and their structures provide evidence for the evolution of heterothallism from homothallism. These data shed light on the evolutionary and mechanistic bases of the genetically complex traits of necrotrophic pathogenicity and sexual mating. This resource should facilitate the functional studies designed to better understand what makes these fungi such successful and persistent pathogens of agronomic crops.</p></div