A clone resource of Magnaporthe oryzae effectors that share sequence and structural similarities across host-specific lineages

The blast fungus Magnaporthe oryzae (syn. Pyricularia oryzae) is a destructive plant pathogen that can infect about 50 species of both wild and cultivated grasses, including important crops such as rice and wheat. M. oryzae is composed of genetically differentiated lineages that tend to infect specific host genera. To date, most studies of M. oryzae effectors have focused on the rice-infecting lineage. We describe a clone resource of 195 effectors of Magnaporthe species predicted from all the major host-specific lineages. These clones are freely available as Golden Gate-compatible entry plasmids. Our aim is to provide the community with an open source effector clone library to be used in a variety of functional studies. We hope that this resource will encourage studies of M. oryzae effectors on diverse host species

The neighbouring genes <i>AvrLm10A</i> and <i>AvrLm10B</i> are part of a large multigene family of cooperating effector genes conserved in Dothideomycetes and Sordariomycetes

Fungal effectors (small-secreted proteins) have long been considered as species or even subpopulation-specific. The increasing availability of high-quality fungal genomes and annotations has allowed the identification of trans-species or trans-genera families of effectors. Two avirulence effectors, AvrLm10A and AvrLm10B, of Leptosphaeria maculans, the fungus causing stem canker of oilseed rape, are members of such a large family of effectors. AvrLm10A and AvrLm10B are neighbouring genes, organized in divergent transcriptional orientation. Sequence searches within the L. maculans genome showed that AvrLm10A/AvrLm10B belong to a multigene family comprising five pairs of genes with a similar tail-to-tail organization. The two genes, in a pair, always had the same expression pattern and two expression profiles were distinguished, associated with the biotrophic colonization of cotyledons and/or petioles and stems. Of the two protein pairs further investigated, AvrLm10A_like1/AvrLm10B_like1 and AvrLm10A_like2/AvrLm10B_like2, the second one had the ability to physically interact, similarly to what was previously described for the AvrLm10A/AvrLm10B pair, and cross-interactions were also detected for two pairs. AvrLm10A homologues were identified in more than 30 Dothideomycete and Sordariomycete plant-pathogenic fungi. One of them, SIX5, is an effector from Fusarium oxysporum f. sp. lycopersici physically interacting with the avirulence effector Avr2. We found that AvrLm10A/SIX5 homologues were associated with at least eight distinct putative effector families, suggesting that AvrLm10A/SIX5 is able to cooperate with different effectors. These results point to a general role of the AvrLm10A/SIX5 proteins as “cooperating proteins”, able to interact with diverse families of effectors whose encoding gene is co-regulated with the neighbouring AvrLm10A homologue

A single amino acid polymorphism in a conserved effector of the multihost blast fungus pathogen expands host-target binding spectrum

Accelerated gene evolution is a hallmark of pathogen adaptation and specialization following host-jumps. However, the molecular processes associated with adaptive evolution between host-specific lineages of a multihost plant pathogen remain poorly understood. In the blast fungus Magnaporthe oryzae (Syn. Pyricularia oryzae), host specialization on different grass hosts is generally associated with dynamic patterns of gain and loss of virulence effector genes that tend to define the distinct genetic lineages of this pathogen. Here, we unravelled the biochemical and structural basis of adaptive evolution of APikL2, an exceptionally conserved paralog of the well-studied rice-lineage specific effector AVR-Pik. Whereas AVR-Pik and other members of the six-gene AVR-Pik family show specific patterns of presence/absence polymorphisms between grass-specific lineages of M. oryzae, APikL2 stands out by being ubiquitously present in all blast fungus lineages from 13 different host species. Using biochemical, biophysical and structural biology methods, we show that a single aspartate to asparagine polymorphism expands the binding spectrum of APikL2 to host proteins of the heavy-metal associated (HMA) domain family. This mutation maps to one of the APikL2-HMA binding interfaces and contributes to an altered hydrogen-bonding network. By combining phylogenetic ancestral reconstruction with an analysis of the structural consequences of allelic diversification, we revealed a common mechanism of effector specialization in the AVR-Pik/APikL2 family that involves two major HMA-binding interfaces. Together, our findings provide a detailed molecular evolution and structural biology framework for diversification and adaptation of a fungal pathogen effector family following host-jumps

Complex Interactions between Fungal Avirulence Genes and Their Corresponding Plant Resistance Genes and Consequences for Disease Resistance Management

During infection, pathogens secrete an arsenal of molecules, collectively called effectors, key elements of pathogenesis which modulate innate immunity of the plant and facilitate infection. Some of these effectors can be recognized directly or indirectly by resistance (R) proteins from the plant and are then called avirulence (AVR) proteins. This recognition usually triggers defense responses including the hypersensitive response and results in resistance of the plant. R—AVR gene interactions are frequently exploited in the field to control diseases. Recently, the availability of fungal genomes has accelerated the identification of AVR genes in plant pathogenic fungi, including in fungi infecting agronomically important crops. While single AVR genes recognized by their corresponding R gene were identified, more and more complex interactions between AVR and R genes are reported (e.g., AVR genes recognized by several R genes, R genes recognizing several AVR genes in distinct organisms, one AVR gene suppressing recognition of another AVR gene by its corresponding R gene, two cooperating R genes both necessary to recognize an AVR gene). These complex interactions were particularly reported in pathosystems showing a long co-evolution with their host plant but could also result from the way agronomic crops were obtained and improved (e.g., through interspecific hybridization or introgression of resistance genes from wild related species into cultivated crops). In this review, we describe some complex R—AVR interactions between plants and fungi that were recently reported and discuss their implications for AVR gene evolution and R gene management

Two avirulence genes of Leptosphaeria maculans, displaying suppressive interactions, share a common structural pattern and are part of a larger effector family

International audienceRecognition of a pathogen avirulence (AVR) protein triggers a set of immune responses grouped under the term Effector-Triggered Immunity (ETI). Pathogens can escape ETI by different mechanisms including complete deletion or down-regulation of the AVR genes, point mutations allowing recognition to be evaded while maintaining the virulence function of the AVR protein, and acquisition of new effectors able to suppress recognition. In some cases, the effector that suppresses ETI can itself be recognized by an R gene. A few examples of such mechanism were recently described in fungi but the underlying mechanisms have remained unexplained

A two genes – for – one gene interaction between Leptosphaeria maculans and Brassica napus

International audienceInteractions between Leptosphaeria maculans, causal agent of stem canker of oilseed rape, and its Brassica hosts are models of choice to explore the multiplicity of 'gene-for-gene' complementarities and how they diversified to increased complexity in the course of plant-pathogen co-evolution. Here, we support this postulate by investigating the AvrLm10 avirulence that induces a resistance response when recognized by the Brassica nigra resistance gene Rlm10. Using genome-assisted map-based cloning, we identified and cloned two AvrLm10 candidates as two genes in opposite transcriptional orientation located in a subtelomeric repeat-rich region of the genome. The AvrLm10 genes encode small secreted proteins and show expression profiles in planta similar to those of all L. maculans avirulence genes identified so far. Complementation and silencing assays indicated that both genes are necessary to trigger Rlm10 resistance. Three assays for protein-protein interactions showed that the two AvrLm10 proteins interact physically in vitro and in planta. Some avirulence genes are recognized by two distinct resistance genes and some avirulence genes hide the recognition specificities of another. Our L. maculans model illustrates an additional case where two genes located in opposite transcriptional orientation are necessary to induce resistance. Interestingly, orthologues exist for both L. maculans genes in other phytopathogenic species, with a similar genome organization, which may point to an important conserved effector function linked to heterodimerization of the two proteins

Avirulence proteins of Leptosphaeria maculans involved in suppressive interactions share a common structural pattern and are part of a larger family

National audienceRecognition 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 of the AVR gene, 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 (Plissonneau et al., 2016; Ghanbarnia et al., 2018). The presence of AvrLm4-7 does not impede AvrLm3 and AvrLm5-9 expression, and the three AVR do not physically interact. To decipher the antagonistic interaction between L. maculans AVR effectors, we determined the crystal structure of AvrLm5-9. Surprisingly, despite a lack of sequence similarity, AvrLm5-9 shares structural analogies with AvrLm4-7 (structure previously characterized by Blondeau et al., 2015). Structure-informed searches identified a larger number of putative structural analogues among L. maculans effector candidates (including AvrLm3), as well as among effector candidates from other phytopathogenic fungi (including ECP11-1 from Passalora fulva; Mesarich et al., 2018). We determined the crystal structure of ECP11-1, deduced the 3D structure of AvrLm3, and confirmed that they shared structural analogies with AvrLm4-7 and AvrLm5-9. Remarkably, transformants of L. maculans producing 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

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

Characterization of epistatic interactions within a family of structurally conserved fungal effectors and of their broad-spectrum recognition by a plant resistance protein

International audienceRecognition 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 resistance by different mechanisms including the acquisition of new effectors that suppress AVR recognition. Leptosphaeria maculans, causal agent of oilseed rape stem canker, develops a high diversity of mechanisms to efficiently escape plant resistance including suppression of R protein-mediated recognition by an AVR effector. Indeed, AvrLm4-7 suppresses the recognition of AvrLm3 and AvrLm5-9 by the R proteins Rlm3 and Rlm9, respectively [1,2]. 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 AVR effector from Fulvia fulva. Despite a lack of sequence similarity, AvrLm5-9 and AvrLm3 are structural analogues of AvrLm4-7 (structure previously characterized [3]). Structure-informed sequence database searches identified a larger number of putative structural analogues among L. maculans effector candidates, including AvrLmS-Lep2, as well as among effector candidates from other phytopathogenic fungi [4]. Remarkably, transformants of L. maculans expressing one of these structural analogues, Ecp11-1, triggered Rlm3-mediated immunity. Furthermore, this recognition was suppressed by AvrLm4-7. On these bases, we continued to decipher the interactions between Ecp11-1 / AvrLm3 (and their closely related homologues) and Rlm3 by: (i) performing L. maculans complementation experiments with different homologues; (ii) determining whether AvrLm4-7 could suppress that recognition; (iii) finely characterizing the amino-acids / regions of Ecp11-1 and AvrLm3 which induced recognition by Rlm3. This analysis is a first step towards the understanding of broad-spectrum resistances that may allow multi-pathogen disease management

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

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