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

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

A new family of structurally conserved fungal effectors displays epistatic interactions with plant resistance proteins

Abstract Recognition of a pathogen avirulence (AVR) effector protein by a cognate plant resistance (R) protein triggers a set of immune responses that render the plant resistant. Pathogens can escape this so-called Effector-Triggered Immunity (ETI) by different mechanisms including the deletion or loss-of-function mutation of the AVR gene, the incorporation of point mutations that allow recognition to be evaded while maintaining virulence function, and the acquisition of new effectors that suppress AVR recognition. The Dothideomycete Leptosphaeria maculans , causal agent of oilseed rape stem canker, is one of the few fungal pathogens where suppression of ETI by an AVR effector has been demonstrated. Indeed, AvrLm4-7 suppresses the recognition of AvrLm3 and AvrLm5-9 by the R proteins Rlm3 and Rlm9, respectively. The presence of AvrLm4-7 does not impede AvrLm3 and AvrLm5-9 expression, and the three AVR proteins do not appear to physically interact. To decipher the epistatic interaction between these L. maculans AVR effectors, we determined the crystal structure of AvrLm5-9 and obtained a 3D model of AvrLm3, based on the crystal structure of Ecp11-1, a homologous avirulence effector candidate from Fulvia fulva . Despite a lack of sequence similarity, AvrLm5-9 and AvrLm3 are structural analogues of AvrLm4-7 (structure previously characterized). Structure-informed sequence database searches identified a larger number of putative structural analogues among L. maculans effector candidates, including the avirulence effector AvrLmS-Lep2, all produced during the early stages of oilseed rape infection, as well as among effector candidates from other phytopathogenic fungi. These structural analogues were called LARS (for Leptosphaeria AviRulence-Suppressing) effectors. Remarkably, transformants of L. maculans expressing one of these structural analogues, Ecp11-1, triggered Rlm3-mediated immunity. Furthermore, this recognition could be suppressed by AvrLm4-7. These results suggest that Ecp11-1 has the same function as AvrLm3, or that the Ecp11-1 structure is sufficiently close to that of AvrLm3 to be recognized by Rlm3. Author summary An efficient strategy to control fungal diseases in the field is genetic control using resistant crop cultivars. Crop resistance mainly relies on gene-for-gene relationships between plant resistance ( R ) genes and pathogen avirulence ( AVR ) genes, as defined by Flor in the 1940s. However, gene-for-gene relationships diversify to increased complexity in the course of plant-pathogen co-evolution. Resistance against the plant-pathogenic fungus Leptosphaeria maculans by Brassica napus and other Brassica species relies on recognition of effector (AVR) proteins by R proteins; however, L. maculans is able to produce an effector that suppresses a subset of these specific recognitions. Using a protein structure approach, we revealed structural analogy between different effectors within L. maculans and other plant-pathogenic species in the Dothideomycetes and Sordariomycetes classes, defining a new family of effectors called LARS, and providing a clue to the mechanisms behind recognition suppression. Cross-species expression of one LARS effector from Fulvia fulva , a pathogen of tomato, in L. maculans triggers recognition by an oilseed rape resistant cultivar. These results highlight the need to integrate knowledge on effector structures to improve resistance management and to develop broad-spectrum resistances for multi-pathogen control of diseases