Bioengineering a plant NLR immune receptor with a robust binding interface toward a conserved fungal pathogen effector.

Bioengineering of plant immune receptors has emerged as a key strategy for generating novel disease resistance traits to counteract the expanding threat of plant pathogens to global food security. However, current approaches are limited by rapid evolution of plant pathogens in the field and may lack durability when deployed. Here, we show that the rice nucleotide-binding, leucine-rich repeat (NLR) immune receptor Pik-1 can be engineered to respond to a conserved family of effectors from the multihost blast fungus pathogen Magnaporthe oryzae. We switched the effector binding and response profile of the Pik NLR from its cognate rice blast effector AVR-Pik to the host-determining factor pathogenicity toward weeping lovegrass 2 (Pwl2) by installing a putative host target, OsHIPP43, in place of the native integrated heavy metal-associated domain (generating Pikm-1OsHIPP43). This chimeric receptor also responded to other PWL alleles from diverse blast isolates. The crystal structure of the Pwl2/OsHIPP43 complex revealed a multifaceted, robust interface that cannot be easily disrupted by mutagenesis, and may therefore provide durable, broad resistance to blast isolates carrying PWL effectors in the field. Our findings highlight how the host targets of pathogen effectors can be used to bioengineer recognition specificities that have more robust properties compared to naturally evolved disease resistance genes.

Zinc-finger (ZiF) fold secreted effectors form a functionally diverse family across lineages of the blast fungus Magnaporthe oryzae.

Filamentous plant pathogens deliver effector proteins into host cells to suppress host defence responses and manipulate metabolic processes to support colonization. Understanding the evolution and molecular function of these effectors provides knowledge about pathogenesis and can suggest novel strategies to reduce damage caused by pathogens. However, effector proteins are highly variable, share weak sequence similarity and, although they can be grouped according to their structure, only a few structurally conserved effector families have been functionally characterized to date. Here, we demonstrate that Zinc-finger fold (ZiF) secreted proteins form a functionally diverse effector family in the blast fungus Magnaporthe oryzae. This family relies on the Zinc-finger motif for protein stability and is ubiquitously present in blast fungus lineages infecting 13 different host species, forming different effector tribes. Homologs of the canonical ZiF effector, AVR-Pii, from rice infecting isolates are present in multiple M. oryzae lineages. Wheat infecting strains of the fungus also possess an AVR-Pii like allele that binds host Exo70 proteins and activates the immune receptor Pii. Furthermore, ZiF tribes may vary in the proteins they bind to, indicating functional diversification and an intricate effector/host interactome. Altogether, we uncovered a new effector family with a common protein fold that has functionally diversified in lineages of M. oryzae. This work expands our understanding of the diversity of M. oryzae effectors, the molecular basis of plant pathogenesis and may ultimately facilitate the development of new sources for pathogen resistance.

Disentangling the complex gene interaction networks between rice and the blast fungus identifies a new pathogen effector.

Studies focused solely on single organisms can fail to identify the networks underlying host-pathogen gene-for-gene interactions. Here, we integrate genetic analyses of rice (Oryza sativa, host) and rice blast fungus (Magnaporthe oryzae, pathogen) and uncover a new pathogen recognition specificity of the rice nucleotide-binding domain and leucine-rich repeat protein (NLR) immune receptor Pik, which mediates resistance to M. oryzae expressing the avirulence effector gene AVR-Pik. Rice Piks-1, encoded by an allele of Pik-1, recognizes a previously unidentified effector encoded by the M. oryzae avirulence gene AVR-Mgk1, which is found on a mini-chromosome. AVR-Mgk1 has no sequence similarity to known AVR-Pik effectors and is prone to deletion from the mini-chromosome mediated by repeated Inago2 retrotransposon sequences. AVR-Mgk1 is detected by Piks-1 and by other Pik-1 alleles known to recognize AVR-Pik effectors; recognition is mediated by AVR-Mgk1 binding to the integrated heavy metal-associated (HMA) domain of Piks-1 and other Pik-1 alleles. Our findings highlight how complex gene-for-gene interaction networks can be disentangled by applying forward genetics approaches simultaneously to the host and pathogen. We demonstrate dynamic coevolution between an NLR integrated domain and multiple families of effector proteins.

Genomic surveillance uncovers a pandemic clonal lineage of the wheat blast fungus.

Wheat, one of the most important food crops, is threatened by a blast disease pandemic. Here, we show that a clonal lineage of the wheat blast fungus recently spread to Asia and Africa following two independent introductions from South America. Through a combination of genome analyses and laboratory experiments, we show that the decade-old blast pandemic lineage can be controlled by the Rmg8 disease resistance gene and is sensitive to strobilurin fungicides. However, we also highlight the potential of the pandemic clone to evolve fungicide-insensitive variants and sexually recombine with African lineages. This underscores the urgent need for genomic surveillance to track and mitigate the spread of wheat blast outside of South America and to guide preemptive wheat breeding for blast resistance.

Correction: Genomic surveillance uncovers a pandemic clonal lineage of the wheat blast fungus.

[This corrects the article DOI: 10.1371/journal.pbio.3002052.].

Multiple Horizontal Mini-chromosome Transfers Drive Genome Evolution of Clonal Blast Fungus Lineages.

Crop disease pandemics are often driven by asexually reproducing clonal lineages of plant pathogens that reproduce asexually. How these clonal pathogens continuously adapt to their hosts despite harboring limited genetic variation, and in absence of sexual recombination remains elusive. Here, we reveal multiple instances of horizontal chromosome transfer within pandemic clonal lineages of the blast fungus Magnaporthe (Syn. Pyricularia) oryzae. We identified a horizontally transferred 1.2Mb accessory mini-chromosome which is remarkably conserved between M. oryzae isolates from both the rice blast fungus lineage and the lineage infecting Indian goosegrass (Eleusine indica), a wild grass that often grows in the proximity of cultivated cereal crops. Furthermore, we show that this mini-chromosome was horizontally acquired by clonal rice blast isolates through at least nine distinct transfer events over the past three centuries. These findings establish horizontal mini-chromosome transfer as a mechanism facilitating genetic exchange among different host-associated blast fungus lineages. We propose that blast fungus populations infecting wild grasses act as genetic reservoirs that drive genome evolution of pandemic clonal lineages that afflict cereal crops.

A genetically linked pair of NLR immune receptors shows contrasting patterns of evolution.

Throughout their evolution, plant nucleotide-binding leucine-rich-repeat receptors (NLRs) have acquired widely divergent unconventional integrated domains that enhance their ability to detect pathogen effectors. However, the functional dynamics that drive the evolution of NLRs with integrated domains (NLR-IDs) remain poorly understood. Here, we reconstructed the evolutionary history of an NLR locus prone to unconventional domain integration and experimentally tested hypotheses about the evolution of NLR-IDs. We show that the rice (Oryza sativa) NLR Pias recognizes the effector AVR-Pias of the blast fungal pathogen Magnaporthe oryzae. Pias consists of a functionally specialized NLR pair, the helper Pias-1 and the sensor Pias-2, that is allelic to the previously characterized Pia pair of NLRs: the helper RGA4 and the sensor RGA5. Remarkably, Pias-2 carries a C-terminal DUF761 domain at a similar position to the heavy metal-associated (HMA) domain of RGA5. Phylogenomic analysis showed that Pias-2/RGA5 sensor NLRs have undergone recurrent genomic recombination within the genus Oryza, resulting in up to six sequence-divergent domain integrations. Allelic NLRs with divergent functions have been maintained transspecies in different Oryza lineages to detect sequence-divergent pathogen effectors. By contrast, Pias-1 has retained its NLR helper activity throughout evolution and is capable of functioning together with the divergent sensor-NLR RGA5 to respond to AVR-Pia. These results suggest that opposite selective forces have driven the evolution of paired NLRs: highly dynamic domain integration events maintained by balancing selection for sensor NLRs, in sharp contrast to purifying selection and functional conservation of immune signaling for helper NLRs.

Genomic rearrangements generate hypervariable mini-chromosomes in host-specific isolates of the blast fungus.

Supernumerary mini-chromosomes-a unique type of genomic structural variation-have been implicated in the emergence of virulence traits in plant pathogenic fungi. However, the mechanisms that facilitate the emergence and maintenance of mini-chromosomes across fungi remain poorly understood. In the blast fungus Magnaporthe oryzae (Syn. Pyricularia oryzae), mini-chromosomes have been first described in the early 1990s but, until very recently, have been overlooked in genomic studies. Here we investigated structural variation in four isolates of the blast fungus M. oryzae from different grass hosts and analyzed the sequences of mini-chromosomes in the rice, foxtail millet and goosegrass isolates. The mini-chromosomes of these isolates turned out to be highly diverse with distinct sequence composition. They are enriched in repetitive elements and have lower gene density than core-chromosomes. We identified several virulence-related genes in the mini-chromosome of the rice isolate, including the virulence-related polyketide synthase Ace1 and two variants of the effector gene AVR-Pik. Macrosynteny analyses around these loci revealed structural rearrangements, including inter-chromosomal translocations between core- and mini-chromosomes. Our findings provide evidence that mini-chromosomes emerge from structural rearrangements and segmental duplication of core-chromosomes and might contribute to adaptive evolution of the blast fungus.

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.

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.

Lessons in Effector and NLR Biology of Plant-Microbe Systems.

A diversity of plant-associated organisms secrete effectors-proteins and metabolites that modulate plant physiology to favor host infection and colonization. However, effectors can also activate plant immune receptors, notably nucleotide-binding domain and leucine-rich repeat region (NLR)-containing proteins, enabling plants to fight off invading organisms. This interplay between effectors, their host targets, and the matching immune receptors is shaped by intricate molecular mechanisms and exceptionally dynamic coevolution. In this article, we focus on three effectors, AVR-Pik, AVR-Pia, and AVR-Pii, from the rice blast fungus Magnaporthe oryzae (syn. Pyricularia oryzae), and their corresponding rice NLR immune receptors, Pik, Pia, and Pii, to highlight general concepts of plant-microbe interactions. We draw 12 lessons in effector and NLR biology that have emerged from studying these three little effectors and are broadly applicable to other plant-microbe systems.

Fungal chitinases: function, regulation, and potential roles in plant/pathogen interactions.

In the past decades our knowledge about fungal cell wall architecture increased tremendously and led to the identification of many enzymes involved in polysaccharide synthesis and remodeling, which are also of biotechnological interest. Fungal cell walls play an important role in conferring mechanic stability during cell division and polar growth. Additionally, in phytopathogenic fungi the cell wall is the first structure that gets into intimate contact with the host plant. A major constituent of fungal cell walls is chitin, a homopolymer of N-acetylglucosamine units. To ensure plasticity, polymeric chitin needs continuous remodeling which is maintained by chitinolytic enzymes, including lytic polysaccharide monooxygenases N-acetylglucosaminidases, and chitinases. Depending on the species and lifestyle of fungi, there is great variation in the number of encoded chitinases and their function. Chitinases can have housekeeping function in plasticizing the cell wall or can act more specifically during cell separation, nutritional chitin acquisition, or competitive interaction with other fungi. Although chitinase research made huge progress in the last decades, our knowledge about their role in phytopathogenic fungi is still scarce. Recent findings in the dimorphic basidiomycete Ustilago maydis show that chitinases play different physiological functions throughout the life cycle and raise questions about their role during plant-fungus interactions. In this work we summarize these functions, mechanisms of chitinase regulation and their putative role during pathogen/host interactions.

Chitinases Are Essential for Cell Separation in Ustilago maydis.

Chitin is an essential component of the fungal cell wall, providing rigidity and stability. Its degradation is mediated by chitinases and supposedly ensures the dynamic plasticity of the cell wall during growth and morphogenesis. Hence, chitinases should be particularly important for fungi with dramatic morphological changes, such as Ustilago maydis. This smut fungus switches from yeast to filamentous growth for plant infection, proliferates as a mycelium in planta, and forms teliospores for spreading. Here, we investigate the contribution of its four chitinolytic enzymes to the different morphological changes during the complete life cycle in a comprehensive study of deletion strains combined with biochemical and cell biological approaches. Interestingly, two chitinases act redundantly in cell separation during yeast growth. They mediate the degradation of remnant chitin in the fragmentation zone between mother and daughter cell. In contrast, even the complete lack of chitinolytic activity does not affect formation of the infectious filament, infection, biotrophic growth, or teliospore germination. Thus, unexpectedly we can exclude a major role for chitinolytic enzymes in morphogenesis or pathogenicity of U. maydis. Nevertheless, redundant activity of even two chitinases is essential for cell separation during saprophytic growth, possibly to improve nutrient access or spreading of yeast cells by wind or rain.

Alternative splicing regulation in plants by SP7-like effectors from symbiotic arbuscular mycorrhizal fungi.

Most plants in natural ecosystems associate with arbuscular mycorrhizal (AM) fungi to survive soil nutrient limitations. To engage in symbiosis, AM fungi secrete effector molecules that, similar to pathogenic effectors, reprogram plant cells. Here we show that the Glomeromycotina-specific SP7 effector family impacts on the alternative splicing program of their hosts. SP7-like effectors localize at nuclear condensates and interact with the plant mRNA processing machinery, most prominently with the splicing factor SR45 and the core splicing proteins U1-70K and U2AF35. Ectopic expression of these effectors in the crop plant potato and in Arabidopsis induced developmental changes that paralleled to the alternative splicing modulation of a specific subset of genes. We propose that SP7-like proteins act as negative regulators of SR45 to modulate the fate of specific mRNAs in arbuscule-containing cells. Unraveling the communication mechanisms between symbiotic fungi and their host plants will help to identify targets to improve plant nutrition.

Deletion of the ATP20 gene in Ustilago maydis produces an unstable dimer of F1FO-ATP synthase associated with a decrease in mitochondrial ATP synthesis and a high H2O2 production.

The F1FO-ATP synthase uses the energy stored in the electrochemical proton gradient to synthesize ATP. This complex is found in the inner mitochondrial membrane as a monomer and dimer. The dimer shows higher ATPase activity than the monomer and is essential for cristae folding. The monomer-monomer interface is constituted by subunits a, i/j, e, g, and k. The role of the subunit g in a strict respiratory organism is unknown. A gene knockout was generated in Ustilago maydis to study the role of subunit g on mitochondrial metabolism and cristae architecture. Deletion of the ATP20 gene, encoding the g subunit, did not affect cell growth or glucose consumption, but biomass production was lower in the mutant strain (gΔ strain). Ultrastructure observations showed that mitochondrial size and cristae shape were similar in wild-type and gΔ strains. The mitochondrial membrane potential in both strains had a similar magnitude, but oxygen consumption was higher in the WT strain. ATP synthesis was 20 % lower in the gΔ strain. Additionally, the mutant strain expressed the alternative oxidase in the early stages of growth (exponential phase), probably as a response to ROS stress. Dimer from mutant strain was unstable to digitonin solubilization, avoiding its isolation and kinetic characterization. The isolated monomeric state activated by n-dodecyl-ß-D-maltopyranoside showed similar kinetic constants to the monomer from the WT strain. A decrease in mitochondrial ATP synthesis and the presence of the AOX during the exponential growth phase suggests that deletion of the g gene induces ROS stress.

Two NLR immune receptors acquired high-affinity binding to a fungal effector through convergent evolution of their integrated domain.

A subset of plant NLR immune receptors carry unconventional integrated domains in addition to their canonical domain architecture. One example is rice Pik-1 that comprises an integrated heavy metal-associated (HMA) domain. Here, we reconstructed the evolutionary history of Pik-1 and its NLR partner, Pik-2, and tested hypotheses about adaptive evolution of the HMA domain. Phylogenetic analyses revealed that the HMA domain integrated into Pik-1 before Oryzinae speciation over 15 million years ago and has been under diversifying selection. Ancestral sequence reconstruction coupled with functional studies showed that two Pik-1 allelic variants independently evolved from a weakly binding ancestral state to high-affinity binding of the blast fungus effector AVR-PikD. We conclude that for most of its evolutionary history the Pik-1 HMA domain did not sense AVR-PikD, and that different Pik-1 receptors have recently evolved through distinct biochemical paths to produce similar phenotypic outcomes. These findings highlight the dynamic nature of the evolutionary mechanisms underpinning NLR adaptation to plant pathogens.

The germinal centre kinase Don3 is crucial for unconventional secretion of chitinase Cts1 in Ustilago maydis.

Unconventional secretion has emerged as an increasingly important cellular process in eukaryotic cells. The underlying translocation mechanisms are diverse and often little understood. We study unconventional secretion of chitinase Cts1 in the corn smut fungus Ustilago maydis. This protein participates in the cytokinesis of yeast cells. During budding it localizes to the septated fragmentation zone where it presumably functions in the degradation of remnant chitin to allow separation of mother and daughter cell. However, the mechanistic details of Cts1 export remain unclear. Here we investigated the mechanism of unconventional Cts1 secretion with a focus on cytokinesis. Cell-cycle inhibition experiments supported the hypothesis that Cts1 export is connected to cytokinesis. To substantiate this finding we analysed gene deletion mutants impaired in cell separation and discovered that strains defective in secondary septum formation were affected in Cts1 export. The germinal centre kinase Don3 had a particularly strong influence on unconventional secretion. Using a synthetic switch, we unambiguously verified an essential role of Don3 for cytokinesis-dependent Cts1 export via the fragmentation zone. Thus, we gained novel insights into the mechanism of unconventional secretion and discovered the first regulatory component of this process.

The Blast Fungus Decoded: Genomes in Flux.

Plant disease outbreaks caused by fungi are a chronic threat to global food security. A prime case is blast disease, which is caused by the ascomycete fungus Magnaporthe oryzae (syn. Pyricularia oryzae), which is infamous as the most destructive disease of the staple crop rice. However, despite its Linnaean binomial name, M. oryzae is a multihost pathogen that infects more than 50 species of grasses. A timely study by P. Gladieux and colleagues (mBio 9:e01219-17, 2018, https://doi.org/10.1128/mBio.01219-17) reports the most extensive population genomic analysis of the blast fungus thus far. M. oryzae consists of an assemblage of differentiated lineages that tend to be associated with particular host genera. Nonetheless, there is clear evidence of gene flow between lineages consistent with maintaining M. oryzae as a single species. Here, we discuss these findings with an emphasis on the ecologic and genetic mechanisms underpinning gene flow. This work also bears practical implications for diagnostics, surveillance, and management of blast diseases.

CRISPR Crops: Plant Genome Editing Toward Disease Resistance.

Genome editing by sequence-specific nucleases (SSNs) has revolutionized biology by enabling targeted modifications of genomes. Although routine plant genome editing emerged only a few years ago, we are already witnessing the first applications to improve disease resistance. In particular, CRISPR-Cas9 has democratized the use of genome editing in plants thanks to the ease and robustness of this method. Here, we review the recent developments in plant genome editing and its application to enhancing disease resistance against plant pathogens. In the future, bioedited disease resistant crops will become a standard tool in plant breeding.