ABSTRACT
BACKGROUND: Studying genomic variation in rapidly evolving pathogens potentially enables identification of genes supporting their "core biology", being present, functional and expressed by all strains or "flexible biology", varying between strains. Genes supporting flexible biology may be considered to be "accessory", whilst the "core" gene set is likely to be important for common features of a pathogen species biology, including virulence on all host genotypes. The wheat-pathogenic fungus Zymoseptoria tritici represents one of the most rapidly evolving threats to global food security and was the focus of this study. RESULTS: We constructed a pangenome of 18 European field isolates, with 12 also subjected to RNAseq transcription profiling during infection. Combining this data, we predicted a "core" gene set comprising 9807 sequences which were (1) present in all isolates, (2) lacking inactivating polymorphisms and (3) expressed by all isolates. A large accessory genome, consisting of 45% of the total genes, was also defined. We classified genetic and genomic polymorphism at both chromosomal and individual gene scales. Proteins required for essential functions including virulence had lower-than average sequence variability amongst core genes. Both core and accessory genomes encoded many small, secreted candidate effector proteins that likely interact with plant immunity. Viral vector-mediated transient in planta overexpression of 88 candidates failed to identify any which induced leaf necrosis characteristic of disease. However, functional complementation of a non-pathogenic deletion mutant lacking five core genes demonstrated that full virulence was restored by re-introduction of the single gene exhibiting least sequence polymorphism and highest expression. CONCLUSIONS: These data support the combined use of pangenomics and transcriptomics for defining genes which represent core, and potentially exploitable, weaknesses in rapidly evolving pathogens.
Subject(s)
Gene Expression Profiling , Transcriptome , Virulence/genetics , Genome, Fungal , Genes, Fungal , Plant Diseases/microbiologyABSTRACT
Succinate dehydrogenase inhibitor (SDHI) fungicides are widely used for the control of a broad range of fungal diseases. This has been the most rapidly expanding fungicide group in terms of new molecules discovered and introduced for agricultural use over the past fifteen years. A particular pattern of differential sensitivity (resistance) to the stretched heterocycle amide SDHIs (SHA-SDHIs), a subclass of chemically-related SDHIs, was observed in naïve Zymoseptoria tritici populations not previously exposed to these chemicals. Subclass-specific resistance was confirmed at the enzyme level but did not correlate with the genotypes of the succinate dehydrogenase (SDH) encoding genes. Mapping and characterization of the molecular mechanisms responsible for standing SHA-SDHI resistance in natural field isolates identified a gene paralog of SDHC, termed ZtSDHC3, which encodes for an alternative C subunit of succinate dehydrogenase, named alt-SDHC. Using reverse genetics, we showed that alt-SDHC associates with the three other SDH subunits, leading to a fully functional enzyme and that a unique Qp-site residue within the alt-SDHC protein confers SHA-SDHI resistance. Enzymatic assays, computational modelling and docking simulations for the two SQR enzymes (altC-SQR, WT_SQR) enabled us to describe enzyme-inhibitor interactions at an atomistic level and to propose rational explanations for differential potency and resistance across SHA-SDHIs. European Z. tritici populations displayed a presence (20-30%) / absence polymorphism of ZtSDHC3, as well as differences in ZtSDHC3 expression levels and splicing efficiency. These polymorphisms have a strong impact on SHA-SDHI resistance phenotypes. Characterization of the ZtSDHC3 promoter in European Z. tritici populations suggests that transposon insertions are associated with the strongest resistance phenotypes. These results establish that a dispensable paralogous gene determines SHA-SDHIs fungicide resistance in natural populations of Z. tritici. This study paves the way to an increased awareness of the role of fungicidal target paralogs in resistance to fungicides and demonstrates the paramount importance of population genomics in fungicide discovery.
Subject(s)
Ascomycota/genetics , Drug Resistance, Fungal/genetics , Fungicides, Industrial , Succinate Dehydrogenase/genetics , Ascomycota/drug effects , Ascomycota/enzymology , Plant Diseases/microbiologyABSTRACT
Rapid and cost-effective virus-derived transient expression systems for plants are invaluable in elucidating gene function and are particularly useful in plant species for which transformation-based methods are unavailable or are too time and labor demanding, such as wheat (Triticum aestivum) and maize (Zea mays). The virus-mediated overexpression (VOX) vectors based on Barley stripe mosaic virus and Wheat streak mosaic virus described previously for these species are incapable of expressing free recombinant proteins of more than 150 to 250 amino acids, are not suited for high-throughput screens, and have other limitations. In this study, we report the development of a VOX vector based on a monopartite single-stranded positive sense RNA virus, Foxtail mosaic virus (genus Potexvirus). In this vector, PV101, the gene of interest was inserted downstream of the duplicated subgenomic promoter of the viral coat protein gene, and the corresponding protein was expressed in its free form. The vector allowed the expression of a 239-amino acid-long GFP in both virus-inoculated and upper uninoculated (systemic) leaves of wheat and maize and directed the systemic expression of a larger approximately 600-amino acid protein, GUSPlus, in maize. Moreover, we demonstrated that PV101 can be used for in planta expression and functional analysis of apoplastic pathogen effector proteins such as the host-specific toxin ToxA of Parastagonospora nodorum Therefore, this VOX vector opens possibilities for functional genomics studies in two important cereal crops.
Subject(s)
Genetic Vectors/genetics , Potexvirus/genetics , Recombinant Proteins/genetics , Triticum/genetics , Zea mays/genetics , Ascomycota/genetics , Ascomycota/pathogenicity , Fungal Proteins/genetics , Green Fluorescent Proteins/genetics , Plant Leaves/genetics , Plants, Genetically Modified , Protein Engineering/methods , Recombinant Proteins/metabolismABSTRACT
Novel imidazole-based ketene dithioacetals show impressive in planta activity against the economically important plant pathogens Alternaria solani, Botryotinia fuckeliana, Erysiphe necator and Zymoseptoria tritici. Especially derivatives of the topical antifungal lanoconazole, which bear an alkynyloxy or a heteroaryl group in the para-position of the phenyl ring, exhibit excellent control of the mentioned phytopathogens. These compounds inhibit 14α -demethylase in the sterol biosynthesis pathway of the fungi. Synthesis routes starting from either benzaldehydes or acetophenones as well as structure-activity relationships are discussed in detail.
Subject(s)
Acetals/chemistry , Antifungal Agents/chemical synthesis , Ascomycota/drug effects , Ethylenes/chemistry , Imidazoles/chemistry , Ketones/chemistry , 14-alpha Demethylase Inhibitors/chemistry , 14-alpha Demethylase Inhibitors/metabolism , 14-alpha Demethylase Inhibitors/pharmacology , Acetals/metabolism , Acetals/pharmacology , Alternaria/drug effects , Antifungal Agents/metabolism , Antifungal Agents/pharmacology , Ascomycota/metabolism , Binding Sites , Cytochrome P450 Family 51/chemistry , Cytochrome P450 Family 51/metabolism , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Microbial Sensitivity Tests , Molecular Docking Simulation , Protein Structure, Tertiary , Sterol 14-Demethylase/chemistry , Sterol 14-Demethylase/metabolism , Structure-Activity RelationshipABSTRACT
A novel class of experimental fungicides has been discovered, which consists of special quinolin-6-yloxyacetamides. They are highly active against important phytopathogens, such as Phytophthora infestans (potato and tomato late blight), Mycosphaerella graminicola (wheat leaf blotch) and Uncinula necator (grape powdery mildew). Their fungicidal activity is due to their ability to inhibit fungal tubulin polymerization, leading to microtubule destabilization. An efficient synthesis route has been worked out, which allows the diverse substitution of four identified key positions across the molecular scaffold.
Subject(s)
Acetamides/chemistry , Antifungal Agents/chemical synthesis , Tubulin Modulators/chemical synthesis , Acetamides/chemical synthesis , Acetamides/pharmacology , Antifungal Agents/chemistry , Antifungal Agents/pharmacology , Phytophthora infestans/drug effects , Quinolines/chemistry , Saccharomycetales/drug effects , Structure-Activity Relationship , Tubulin Modulators/chemistry , Tubulin Modulators/pharmacologyABSTRACT
The Mla locus in barley (Hordeum vulgare) conditions isolate-specific immunity to the powdery mildew fungus (Blumeria graminis f. sp. hordei) and encodes intracellular coiled-coil (CC) domain, nucleotide-binding (NB) site, and leucine-rich repeat (LRR)-containing receptor proteins. Over the last decades, genetic studies in breeding material have identified a large number of functional resistance genes at the Mla locus. To study the structural and functional diversity of this locus at the molecular level, we isolated 23 candidate MLA cDNAs from barley accessions that were previously shown by genetic studies to harbor different Mla resistance specificities. Resistance activity was detected for 13 candidate MLA cDNAs in a transient gene-expression assay. Sequence alignment of the deduced MLA proteins improved secondary structure predictions, revealing four additional, previously overlooked LRR. Analysis of nucleotide diversity of the candidate and validated MLA cDNAs revealed 34 sites of positive selection. Recombination or gene conversion events were frequent in the first half of the gene but positive selection was also found when this region was excluded. The positively selected sites are all, except two, located in the LRR domain and cluster in predicted solvent-exposed residues of the repeats 7 to 15 and adjacent turns on the concave side of the predicted solenoid protein structure. This domain-restricted pattern of positively selected sites, together with the length conservation of individual LRR, suggests direct binding of effectors to MLA receptors.
Subject(s)
Hordeum/metabolism , Plant Diseases/immunology , Plant Proteins/genetics , Plant Proteins/metabolism , Alleles , Amino Acid Sequence , Gene Expression Regulation, Plant , Genetic Predisposition to Disease , Genetic Variation , Hordeum/genetics , Hordeum/microbiology , Phenotype , Phylogeny , Plant Diseases/genetics , Selection, GeneticABSTRACT
Nonhost resistance protects plants against attack by the vast majority of potential pathogens, including phytopathogenic fungi. Despite its high biological importance, the molecular architecture of nonhost resistance has remained largely unexplored. Here, we describe the transcriptional responses of one particular genotype of barley (Hordeum vulgare subsp. vulgare 'Ingrid') to three different pairs of adapted (host) and nonadapted (nonhost) isolates of fungal pathogens, which belong to the genera Blumeria (powdery mildew), Puccinia (rust), and Magnaporthe (blast). Nonhost resistance against each of these pathogens was associated with changes in transcript abundance of distinct sets of nonhost-specific genes, although general (not nonhost-associated) transcriptional responses to the different pathogens overlapped considerably. The powdery mildew- and blast-induced differences in transcript abundance between host and nonhost interactions were significantly correlated with differences between a near-isogenic pair of barley lines that carry either the Mlo wild-type allele or the mutated mlo5 allele, which mediates basal resistance to powdery mildew. Moreover, during the interactions of barley with the different host or nonhost pathogens, similar patterns of overrepresented and underrepresented functional categories of genes were found. The results suggest that nonhost resistance and basal host defense of barley are functionally related and that nonhost resistance to different fungal pathogens is associated with more robust regulation of complex but largely nonoverlapping sets of pathogen-responsive genes involved in similar metabolic or signaling pathways.
Subject(s)
Fungi/pathogenicity , Hordeum/microbiology , Transcription, Genetic , Gene Expression Regulation, Plant , Genes, Plant , Hordeum/genetics , Principal Component AnalysisABSTRACT
Plant immune responses are triggered by pattern recognition receptors that detect conserved pathogen-associated molecular patterns (PAMPs) or by resistance (R) proteins recognizing isolate-specific pathogen effectors. We show that in barley, intracellular mildew A (MLA) R proteins function in the nucleus to confer resistance against the powdery mildew fungus. Recognition of the fungal avirulence A10 effector by MLA10 induces nuclear associations between receptor and WRKY transcription factors. The identified WRKY proteins act as repressors of PAMP-triggered basal defense. MLA appears to interfere with the WRKY repressor function, thereby de-repressing PAMP-triggered basal defense. Our findings reveal a mechanism by which these polymorphic immune receptors integrate distinct pathogen signals.
Subject(s)
Arabidopsis/immunology , Ascomycota/immunology , Hordeum/immunology , Plant Diseases/immunology , Plant Proteins/metabolism , Receptors, Immunologic/metabolism , Transcription Factors/metabolism , Amino Acid Sequence , Arabidopsis/genetics , Arabidopsis/metabolism , Arabidopsis/microbiology , Arabidopsis Proteins/genetics , Arabidopsis Proteins/metabolism , Ascomycota/growth & development , Cell Nucleus/metabolism , Cytoplasm/metabolism , Fungal Proteins/metabolism , Hordeum/genetics , Hordeum/metabolism , Hordeum/microbiology , Immunity, Innate , Molecular Sequence Data , Mutation , Plant Diseases/microbiology , Plant Proteins/chemistry , Plant Proteins/genetics , Plants, Genetically Modified , Protein Structure, Tertiary , Receptors, Immunologic/chemistry , Receptors, Immunologic/genetics , Receptors, Pattern Recognition/metabolism , Recombinant Fusion Proteins/metabolism , Transcription Factors/genetics , Two-Hybrid System TechniquesSubject(s)
Arabidopsis/immunology , Bacterial Proteins/metabolism , Fungal Proteins/metabolism , Plant Proteins/metabolism , Solanum lycopersicum/immunology , Arabidopsis/metabolism , Arabidopsis/microbiology , Arabidopsis Proteins/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Carrier Proteins/metabolism , Cladosporium/immunology , Cladosporium/metabolism , Cyclophilins/metabolism , Intracellular Signaling Peptides and Proteins , Leucine , Solanum lycopersicum/metabolism , Solanum lycopersicum/microbiology , Models, Immunological , Mutation , Peptide Hydrolases/genetics , Peptide Hydrolases/metabolism , Plant Diseases , Plant Proteins/chemistry , Plant Proteins/genetics , Protein Conformation , Pseudomonas syringae/immunology , Pseudomonas syringae/metabolism , Recombinant Proteins/metabolism , Repetitive Sequences, Amino AcidABSTRACT
The polymorphic barley (Hordeum vulgare) Mla locus harbors allelic race-specific resistance (R) genes to the powdery mildew fungus Blumeria graminis f sp hordei. The highly sequence-related MLA proteins contain an N-terminal coiled-coil structure, a central nucleotide binding (NB) site, a Leu-rich repeat (LRR) region, and a C-terminal non-LRR region. Using transgenic barley lines expressing epitope-tagged MLA1 and MLA6 derivatives driven by native regulatory sequences, we show a reversible and salt concentration-dependent distribution of the intracellular MLA proteins in soluble and membrane-associated pools. A posttranscriptional process directs fourfold greater accumulation of MLA1 over MLA6. Unexpectedly, in rar1 mutant plants that are compromised for MLA6 but not MLA1 resistance, the steady state level of both MLA isoforms is reduced. Furthermore, differential steady state levels of MLA1/MLA6 hybrid proteins correlate with their requirement for RAR1; the RAR1-independent hybrid protein accumulates to higher levels and the RAR1-dependent one to lower levels. Interestingly, yeast two-hybrid studies reveal that the LRR domains of RAR1-independent but not RAR1-dependent MLA isoforms interact with SGT1, a RAR1 interacting protein required for the function of many NB-LRR type R proteins. Our findings implicate the existence of a conserved mechanism to reach minimal NB-LRR R protein thresholds that are needed to trigger effective resistance responses.
Subject(s)
Carrier Proteins/metabolism , Hordeum/genetics , Hordeum/metabolism , Immunity, Innate/physiology , Plant Proteins/metabolism , Arabidopsis Proteins/metabolism , Carrier Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Membrane/metabolism , Down-Regulation/genetics , Fungi/physiology , Homeostasis/physiology , Hordeum/microbiology , Host-Parasite Interactions/physiology , Immunity, Innate/genetics , Intracellular Signaling Peptides and Proteins , Plant Diseases/genetics , Plant Diseases/microbiology , Plant Proteins/genetics , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Plants, Genetically Modified/microbiology , Protein Isoforms/genetics , Protein Isoforms/metabolism , Protein Structure, Tertiary/physiology , RNA Processing, Post-Transcriptional/physiology , Up-Regulation/geneticsABSTRACT
A large number of resistance specificities to the powdery mildew fungus Blumeria graminis f. sp. hordei map to the barley Mla locus. This complex locus harbors multiple members of three distantly related gene families that encode proteins that contain an N-terminal coiled-coil (CC) structure, a central nucleotide binding (NB) site, a Leu-rich repeat (LRR) region, and a C-terminal non-LRR (CT) region. We identified Mla12, which encodes a CC-NB-LRR-CT protein that shares 89 and 92% identical residues with the known proteins MLA1 and MLA6. Slow Mla12-triggered resistance was altered dramatically to a rapid response by overexpression of Mla12. A series of reciprocal domains swaps between MLA1 and MLA6 identified in each protein recognition domain for cognate powdery mildew fungus avirulence genes (AvrMla1 and AvrMla6). These domains were within different but overlapping LRR regions and the CT part. Unexpectedly, MLA chimeras that confer AvrMla6 recognition exhibited markedly different dependence on Rar1, a gene required for the function of some but not all Mla resistance specificities. Furthermore, uncoupling of MLA6-specific function from RAR1 also uncoupled the response from SGT1, a protein known to associate physically with RAR1. Our findings suggest that differences in the degree of RAR1 dependence of different MLA immunity responses are determined by intrinsic properties of MLA variants and place RAR1/SGT1 activity downstream of and/or coincident with the action of resistance protein-containing recognition complexes.