ABSTRACT
Plant diseases are a constant and serious threat to agriculture and ecological biodiversity. Plants possess a sophisticated innate immunity system capable of detecting and responding to pathogen infection to prevent disease. Our understanding of this system has grown enormously over the past century. Early genetic descriptions of plant disease resistance and pathogen virulence were embodied in the gene-for-gene hypothesis, while physiological studies identified pathogen-derived elicitors that could trigger defense responses in plant cells and tissues. Molecular studies of these phenomena have now coalesced into an integrated model of plant immunity involving cell surface and intracellular detection of specific pathogen-derived molecules and proteins culminating in the induction of various cellular responses. Extracellular and intracellular receptors engage distinct signaling processes but converge on many similar outputs with substantial evidence now for integration of these pathways into interdependent networks controlling disease outcomes. Many of the molecular details of pathogen recognition and signaling processes are now known, providing opportunities for bioengineering to enhance plant protection from disease. Here we provide an overview of the current understanding of the main principles of plant immunity, with an emphasis on the key scientific milestones leading to these insights.
Subject(s)
Plant Diseases , Plant Immunity , Signal Transduction , Plant Immunity/genetics , Plant Diseases/immunology , Plant Diseases/microbiology , Plant Diseases/genetics , Host-Pathogen Interactions/immunology , Plants/immunology , Plants/microbiology , Plants/genetics , Plant Proteins/genetics , Plant Proteins/metabolismABSTRACT
Crops are constantly exposed to pathogenic microbes. Rust fungi are examples of these harmful microorganisms, which have a major economic impact on wheat production. To protect themselves from pathogens like rust fungi, plants employ a multilayered immune system that includes immunoreceptors encoded by resistance genes. Significant efforts have led to the isolation of numerous resistance genes against rust fungi in cereals, especially in wheat. However, the evolution of virulence of rust fungi hinders the durability of resistance genes as a strategy for crop protection. Rust fungi, like other biotrophic pathogens, secrete an arsenal of effectors to facilitate infection, and these are the molecules that plant immunoreceptors target for pathogen recognition and mounting defense responses. When recognized, these effector proteins are referred to as avirulence (Avr) effectors. Despite the many predicted effectors in wheat rust fungi, only five Avr genes have been identified, all from wheat stem rust. Knowledge of the Avr genes and their variation in the fungal population will inform deployment of the most appropriate wheat disease-resistance genes for breeding and farming. The review provides an overview of methodologies as well as the validation techniques that have been used to characterize Avr effectors from wheat stem rust. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.
Subject(s)
Basidiomycota , Plant Breeding , Basidiomycota/genetics , Virulence/genetics , Disease Resistance/genetics , Crops, Agricultural , Plant Diseases/microbiologyABSTRACT
Puccinia coronata f. sp. avenae (Pca) is an important fungal pathogen causing crown rust that impacts oat production worldwide. Genetic resistance for crop protection against Pca is often overcome by the rapid virulence evolution of the pathogen. This study investigated the factors shaping adaptive evolution of Pca using pathogen populations from distinct geographic regions within the United States and South Africa. Phenotypic and genome-wide sequencing data of these diverse Pca collections, including 217 isolates, uncovered phylogenetic relationships and established distinct genetic composition between populations from northern and southern regions from the United States and South Africa. The population dynamics of Pca involve a bidirectional movement of inoculum between northern and southern regions of the United States and contributions from clonality and sexuality. The population from South Africa is solely clonal. A genome-wide association study (GWAS) employing a haplotype-resolved Pca reference genome was used to define 11 virulence-associated loci corresponding to 25 oat differential lines. These regions were screened to determine candidate Avr effector genes. Overall, the GWAS results allowed us to identify the underlying genetic factors controlling pathogen recognition in an oat differential set used in the United States to assign pathogen races (pathotypes). Key GWAS findings support complex genetic interactions in several oat lines, suggesting allelism among resistance genes or redundancy of genes included in the differential set, multiple resistance genes recognizing genetically linked Avr effector genes, or potentially epistatic relationships. A careful evaluation of the composition of the oat differential set accompanied by the development or implementation of molecular markers is recommended. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
Subject(s)
Basidiomycota , Disease Resistance , Puccinia , Disease Resistance/genetics , Avena/genetics , Avena/microbiology , Virulence/genetics , Genome-Wide Association Study , Phylogeny , Plant Diseases/microbiology , Basidiomycota/genetics , Population DynamicsABSTRACT
Effector proteins are central to the success of plant pathogens, while immunity in host plants is driven by receptor-mediated recognition of these effectors. Understanding the molecular details of effector-receptor interactions is key for the engineering of novel immune receptors. Here, we experimentally determined the crystal structure of the Puccinia graminis f. sp. tritici (Pgt) effector AvrSr27, which was not accurately predicted using AlphaFold2. We characterised the role of the conserved cysteine residues in AvrSr27 using in vitro biochemical assays and examined Sr27-mediated recognition using transient expression in Nicotiana spp. and wheat protoplasts. The AvrSr27 structure contains a novel ß-strand rich modular fold consisting of two structurally similar domains that bind to Zn2+ ions. The N-terminal domain of AvrSr27 is sufficient for interaction with Sr27 and triggering cell death. We identified two Pgt proteins structurally related to AvrSr27 but with low sequence identity that can also associate with Sr27, albeit more weakly. Though only the full-length proteins, trigger Sr27-dependent cell death in transient expression systems. Collectively, our findings have important implications for utilising protein prediction platforms for effector proteins, and those embarking on bespoke engineering of immunity receptors as solutions to plant disease.
Subject(s)
Fungal Proteins , Nicotiana , Triticum , Zinc , Zinc/metabolism , Triticum/immunology , Triticum/microbiology , Nicotiana/immunology , Nicotiana/microbiology , Nicotiana/metabolism , Fungal Proteins/metabolism , Fungal Proteins/chemistry , Puccinia , Plant Immunity , Protein Binding , Amino Acid Sequence , Cell Death , Protein Domains , Models, Molecular , Plant Diseases/microbiology , Plant Diseases/immunologyABSTRACT
Puccinia coronata f. sp. avenae is the causal agent of the disease known as crown rust, which represents a bottleneck in oat production worldwide. Characterization of pathogen populations often involves race (pathotype) assignments using differential sets, which are not uniform across countries. This study compared the virulence profiles of 25 P. coronata f. sp. avenae isolates from Australia using two host differential sets, one from Australia and one from the United States. These differential sets were also genotyped using diversity arrays technology sequencing technology. Phenotypic and genotypic discrepancies were detected on 8 out of 29 common lines between the two sets, indicating that pathogen race assignments based on those lines are not comparable. To further investigate molecular markers that could assist in the stacking of rust resistance genes important for Australia, four published Pc91-linked markers were validated across the differential sets and then screened across a collection of 150 oat cultivars. Drover, Aladdin, and Volta were identified as putative carriers of the Pc91 locus. This is the first report to confirm that the cultivar Volta carries Pc91 and demonstrates the value of implementing molecular markers to characterize materials in breeding pools of oat. Overall, our findings highlight the necessity of examining seed stocks using pedigree and molecular markers to ensure seed uniformity and bring robustness to surveillance methodologies. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
Subject(s)
Avena , Disease Resistance , Genotype , Plant Diseases , Puccinia , Avena/microbiology , Avena/genetics , Plant Diseases/microbiology , Disease Resistance/genetics , Australia , Puccinia/genetics , Phenotype , Virulence/genetics , United States , Genetic Markers/genetics , Basidiomycota/genetics , Basidiomycota/physiologyABSTRACT
Puccinia coronata f. sp. avenae (Pca) is an important foliar pathogen of oat which causes crown rust disease. The virulence profile of 48 Pca isolates derived from different locations in Australia was characterized using a collection of oat lines often utilized in rust surveys in the United States and Australia. This analysis indicates that Pca populations in Eastern Australia are broadly virulent, which contrasts with the population in Western Australia (WA). Several oat lines/Pc genes are effective against all rust samples collected from WA, suggesting they may provide useful resistance in this region if deployed in combination. We identified 19 lines from the United States oat differential set that display disease resistance to Pca in WA, with some in agreement with previous rust survey reports. We adopted the 10-letter nomenclature system to define oat crown rust races in Australia and compare the frequency of those virulence traits to published data from the United States. Based on this nomenclature, 42 unique races were detected among the 48 isolates, reflecting the high diversity of virulence phenotypes for Pca in Australia. Nevertheless, the Pca population in the United States is substantially more broadly virulent than that of Australia. Close examination of resistance profiles for the oat differential set lines after infection with Pca supports hypotheses of allelism or redundancy among Pc genes or the presence of several resistance genes in some oat differential lines. These findings illustrate the need to deconvolute the oat differential set using molecular tools.[Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
Subject(s)
Avena , Plant Diseases , Puccinia , Avena/microbiology , Plant Diseases/microbiology , Australia , Virulence/genetics , Puccinia/pathogenicity , Puccinia/genetics , Disease Resistance/genetics , United States , Basidiomycota/genetics , Basidiomycota/pathogenicity , Basidiomycota/physiologyABSTRACT
The gene-for-gene model proposed by H. H. Flor has been one of the fundamental precepts of plant-pathogen interactions that has underpinned decades of research towards our current concepts of plant immunity. The broad validity of this model as an elegant and accurate genetic description of specific recognition events between the products of plant resistance (R) and pathogen avirulence (Avr) genes has been demonstrated many times over in a wide variety of plant disease systems. In recent years detailed molecular and structural analyses have provided a deep understanding of the principles by which plant immune receptors recognize pathogen effectors, including providing molecular descriptions of many of the genetic loci in flax and flax rust characterized by Flor. Recent advances in molecular and structural understanding of immune receptor recognition and activation mechanisms have brought the field to a new level, where rational design of novel receptors through engineering approaches is becoming a realizable goal. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
Subject(s)
Flax , Flax/genetics , Genetic Loci , Plant Immunity/geneticsABSTRACT
To infect plants, pathogenic fungi secrete small proteins called effectors. Here, we describe the catalytic activity and potential virulence function of the Nudix hydrolase effector AvrM14 from the flax rust fungus (Melampsora lini). We completed extensive in vitro assays to characterise the enzymatic activity of the AvrM14 effector. Additionally, we used in planta transient expression of wild-type and catalytically dead AvrM14 versions followed by biochemical assays, phenotypic analysis and RNA sequencing to unravel how the catalytic activity of AvrM14 impacts plant immunity. AvrM14 is an extremely selective enzyme capable of removing the protective 5' cap from mRNA transcripts in vitro. Homodimerisation of AvrM14 promoted biologically relevant mRNA cap cleavage in vitro and this activity was conserved in related effectors from other Melampsora spp. In planta expression of wild-type AvrM14, but not the catalytically dead version, suppressed immune-related reactive oxygen species production, altered the abundance of some circadian-rhythm-associated mRNA transcripts and reduced the hypersensitive cell-death response triggered by the flax disease resistance protein M1. To date, the decapping of host mRNA as a virulence strategy has not been described beyond viruses. Our results indicate that some fungal pathogens produce Nudix hydrolase effectors with in vitro mRNA-decapping activity capable of interfering with plant immunity.
Subject(s)
Basidiomycota , RNA, Messenger/genetics , RNA, Messenger/metabolism , Basidiomycota/genetics , Fungi/genetics , Pyrophosphatases/metabolism , Virulence/genetics , Plant Diseases/microbiology , Nudix HydrolasesABSTRACT
Plant and animal intracellular nucleotide-binding, leucine-rich repeat (NLR) immune receptors detect pathogen-derived molecules and activate defense. Plant NLRs can be divided into several classes based upon their N-terminal signaling domains, including TIR (Toll-like, Interleukin-1 receptor, Resistance protein)- and CC (coiled-coil)-NLRs. Upon ligand detection, mammalian NAIP and NLRC4 NLRs oligomerize, forming an inflammasome that induces proximity of its N-terminal signaling domains. Recently, a plant CC-NLR was revealed to form an inflammasome-like hetero-oligomer. To further investigate plant NLR signaling mechanisms, we fused the N-terminal TIR domain of several plant NLRs to the N terminus of NLRC4. Inflammasome-dependent induced proximity of the TIR domain in planta initiated defense signaling. Thus, induced proximity of a plant TIR domain imposed by oligomerization of a mammalian inflammasome is sufficient to activate authentic plant defense. Ligand detection and inflammasome formation is maintained when the known components of the NLRC4 inflammasome is transferred across kingdoms, indicating that NLRC4 complex can robustly function without any additional mammalian proteins. Additionally, we found NADase activity of a plant TIR domain is necessary for plant defense activation, but NADase activity of a mammalian or a bacterial TIR is not sufficient to activate defense in plants.
Subject(s)
NLR Proteins , Plant Immunity , Plant Proteins , Recombinant Fusion Proteins , Signal Transduction , Animals , Inflammasomes/genetics , Inflammasomes/immunology , Inflammasomes/metabolism , Mammals , NLR Proteins/chemistry , NLR Proteins/genetics , NLR Proteins/immunology , NLR Proteins/metabolism , Plant Immunity/genetics , Plant Immunity/immunology , Plant Proteins/chemistry , Plant Proteins/genetics , Plant Proteins/immunology , Plant Proteins/metabolism , Protein Domains/genetics , Protein Domains/physiology , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/immunology , Recombinant Fusion Proteins/metabolism , Signal Transduction/genetics , Signal Transduction/immunologyABSTRACT
Pathogen populations are expected to evolve virulence traits in response to resistance deployed in agricultural settings. However, few temporal datasets have been available to characterize this process at the population level. Here, we examined two temporally separated populations of Puccinia coronata f. sp. avenae (Pca), which causes crown rust disease in oat (Avena sativa) sampled from 1990 to 2015. We show that a substantial increase in virulence occurred from 1990 to 2015 and this was associated with a genetic differentiation between populations detected by genome-wide sequencing. We found strong evidence for genetic recombination in these populations, showing the importance of the alternate host in generating genotypic variation through sexual reproduction. However, asexual expansion of some clonal lineages was also observed within years. Genome-wide association analysis identified seven Avr loci associated with virulence towards fifteen Pc resistance genes in oat and suggests that some groups of Pc genes recognize the same pathogen effectors. The temporal shift in virulence patterns in the Pca populations between 1990 and 2015 is associated with changes in allele frequency in these genomic regions. Nucleotide diversity patterns at a single Avr locus corresponding to Pc38, Pc39, Pc55, Pc63, Pc70, and Pc71 showed evidence of a selective sweep associated with the shift to virulence towards these resistance genes in all 2015 collected isolates.
Subject(s)
Gene Frequency , Genes, Fungal , Puccinia/genetics , Avena/microbiology , Polymorphism, Genetic , Puccinia/pathogenicity , Selection, Genetic , Virulence/geneticsABSTRACT
Many fungi and oomycete species are devasting plant pathogens. These eukaryotic filamentous pathogens secrete effector proteins to facilitate plant infection. Fungi and oomycete pathogens have diverse infection strategies and their effectors generally do not share sequence homology. However, they occupy similar host environments, either the plant apoplast or plant cytoplasm, and, therefore, may share some unifying properties based on the requirements of these host compartments. Here, we exploit these biological signals and present the first classifier (EffectorP 3.0) that uses two machine-learning models: one trained on apoplastic effectors and one trained on cytoplasmic effectors. EffectorP 3.0 accurately predicts known apoplastic and cytoplasmic effectors in fungal and oomycete secretomes with low estimated false-positive rates of 3 and 8%, respectively. Cytoplasmic effectors have a higher proportion of positively charged amino acids, whereas apoplastic effectors are enriched for cysteine residues. The combination of fungal and oomycete effectors in training leads to a higher number of predicted cytoplasmic effectors in biotrophic fungi. EffectorP 3.0 expands predicted effector repertoires beyond small, cysteine-rich secreted proteins in fungi and RxLR-motif containing secreted proteins in oomycetes. We show that signal peptide prediction is essential for accurate effector prediction, because EffectorP 3.0 recognizes a cytoplasmic signal also in intracellular, nonsecreted proteins.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
Subject(s)
Fungal Proteins , Oomycetes , Cytoplasm/metabolism , Fungal Proteins/metabolism , Fungi , Oomycetes/metabolism , Plant Diseases/microbiology , Plants/microbiologyABSTRACT
Functional characterization of effector proteins of fungal obligate biotrophic pathogens, especially confirmation of avirulence (Avr) properties, has been notoriously difficult, due to the experimental intractability of many of these organisms. Previous studies in wheat have shown promising data suggesting the type III secretion system (T3SS) of bacteria may be a suitable surrogate for delivery and detection of Avr properties of fungal effectors. However, these delivery systems were tested in the absence of confirmed Avr effectors. Here, we tested two previously described T3SS-mediated delivery systems for their suitability when delivering two confirmed Avr effectors from two fungal pathogens of wheat, Puccinia graminis f. sp. tritici and Magnaporthe oryzae pathotype tritici. We showed that both effectors (AvrSr50 and AvrRmg8) were unable to elicit a hypersensitive response on wheat seedlings with the corresponding resistance gene when expressed by the Pseudomonas fluorescens "Effector to Host Analyser" (EtHAn) system. Furthermore, we found the utility of Burkholderia glumae for screening Avr phenotypes is severely limited, as the wild-type strain elicits nonhost cell death in multiple wheat accessions. These results provide valuable insight into the suitability of these systems for screening fungal effectors for Avr properties that may help guide further development of surrogate bacterial delivery systems in wheat. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
Subject(s)
Bacteria , Triticum , Triticum/microbiology , Plant Diseases/microbiologyABSTRACT
Pathogen effectors are crucial players during plant colonisation and infection. Plant resistance mostly relies on effector recognition to activate defence responses. Understanding how effector proteins escape from plant surveillance is important for plant breeding and resistance deployment. Here we examined the role of genetic diversity of the stem rust (Puccinia graminis f. sp. tritici (Pgt)) AvrSr50 gene in determining recognition by the corresponding wheat Sr50 resistance gene. We solved the crystal structure of a natural variant of AvrSr50 and used site-directed mutagenesis and transient expression assays to dissect the molecular mechanisms explaining gain of virulence. We report that AvrSr50 can escape recognition by Sr50 through different mechanisms including DNA insertion, stop codon loss or by amino-acid variation involving a single substitution of the AvrSr50 surface-exposed residue Q121. We also report structural homology of AvrSr50 to cupin superfamily members and carbohydrate-binding modules indicating a potential role in binding sugar moieties. This study identifies key polymorphic sites present in AvrSr50 alleles from natural stem rust populations that play important roles to escape from Sr50 recognition. This constitutes an important step to better understand Pgt effector evolution and to monitor AvrSr50 variants in natural rust populations.
Subject(s)
Basidiomycota , Disease Resistance , Basidiomycota/physiology , Disease Resistance/genetics , Plant Breeding , Plant Diseases/genetics , Triticum/geneticsABSTRACT
BACKGROUND: Silencing of transposable elements (TEs) is essential for maintaining genome stability. Plants use small RNAs (sRNAs) to direct DNA methylation to TEs (RNA-directed DNA methylation; RdDM). Similar mechanisms of epigenetic silencing in the fungal kingdom have remained elusive. RESULTS: We use sRNA sequencing and methylation data to gain insight into epigenetics in the dikaryotic fungus Puccinia graminis f. sp. tritici (Pgt), which causes the devastating stem rust disease on wheat. We use Hi-C data to define the Pgt centromeres and show that they are repeat-rich regions (~250 kb) that are highly diverse in sequence between haplotypes and, like in plants, are enriched for young TEs. DNA cytosine methylation is particularly active at centromeres but also associated with genome-wide control of young TE insertions. Strikingly, over 90% of Pgt sRNAs and several RNAi genes are differentially expressed during infection. Pgt induces waves of functionally diversified sRNAs during infection. The early wave sRNAs are predominantly 21 nts with a 5' uracil derived from genes. In contrast, the late wave sRNAs are mainly 22-nt sRNAs with a 5' adenine and are strongly induced from centromeric regions. TEs that overlap with late wave sRNAs are more likely to be methylated, both inside and outside the centromeres, and methylated TEs exhibit a silencing effect on nearby genes. CONCLUSIONS: We conclude that rust fungi use an epigenetic silencing pathway that might have similarity with RdDM in plants. The Pgt RNAi machinery and sRNAs are under tight temporal control throughout infection and might ensure genome stability during sporulation.
Subject(s)
Basidiomycota , DNA Methylation , Puccinia , Basidiomycota/genetics , Centromere , DNA Methylation/genetics , DNA Transposable Elements , Genomic Instability , Humans , Plant Diseases/genetics , Puccinia/pathogenicity , RNAABSTRACT
The self-association of Toll/interleukin-1 receptor/resistance protein (TIR) domains has been implicated in signaling in plant and animal immunity receptors. Structure-based studies identified different TIR-domain dimerization interfaces required for signaling of the plant nucleotide-binding oligomerization domain-like receptors (NLRs) L6 from flax and disease resistance protein RPS4 from Arabidopsis Here we show that the crystal structure of the TIR domain from the Arabidopsis NLR suppressor of npr1-1, constitutive 1 (SNC1) contains both an L6-like interface involving helices αD and αE (DE interface) and an RPS4-like interface involving helices αA and αE (AE interface). Mutations in either the AE- or DE-interface region disrupt cell-death signaling activity of SNC1, L6, and RPS4 TIR domains and full-length L6 and RPS4. Self-association of L6 and RPS4 TIR domains is affected by mutations in either region, whereas only AE-interface mutations affect SNC1 TIR-domain self-association. We further show two similar interfaces in the crystal structure of the TIR domain from the Arabidopsis NLR recognition of Peronospora parasitica 1 (RPP1). These data demonstrate that both the AE and DE self-association interfaces are simultaneously required for self-association and cell-death signaling in diverse plant NLRs.
Subject(s)
Arabidopsis Proteins/chemistry , Arabidopsis/genetics , Gene Expression Regulation, Plant , Plant Diseases/genetics , Plant Proteins/chemistry , Amino Acid Sequence , Arabidopsis/immunology , Arabidopsis/microbiology , Arabidopsis Proteins/genetics , Arabidopsis Proteins/immunology , Binding Sites , Cell Death/genetics , Cell Death/immunology , Flax/genetics , Flax/immunology , Flax/microbiology , Host-Pathogen Interactions , Models, Molecular , Mutation , Peronospora/pathogenicity , Peronospora/physiology , Plant Diseases/immunology , Plant Diseases/microbiology , Plant Immunity/genetics , Plant Proteins/genetics , Plant Proteins/immunology , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/immunology , Sequence Alignment , Sequence Homology, Amino Acid , Signal Transduction , Nicotiana/genetics , Nicotiana/immunology , Nicotiana/microbiologyABSTRACT
NOD-like receptors (NLRs) are central components of the plant immune system. L6 is a Toll/interleukin-1 receptor (TIR) domain-containing NLR from flax (Linum usitatissimum) conferring immunity to the flax rust fungus. Comparison of L6 to the weaker allele L7 identified two polymorphic regions in the TIR and the nucleotide binding (NB) domains that regulate both effector ligand-dependent and -independent cell death signaling as well as nucleotide binding to the receptor. This suggests that a negative functional interaction between the TIR and NB domains holds L7 in an inactive/ADP-bound state more tightly than L6, hence decreasing its capacity to adopt the active/ATP-bound state and explaining its weaker activity in planta. L6 and L7 variants with a more stable ADP-bound state failed to bind to AvrL567 in yeast two-hybrid assays, while binding was detected to the signaling active variants. This contrasts with current models predicting that effectors bind to inactive receptors to trigger activation. Based on the correlation between nucleotide binding, effector interaction, and immune signaling properties of L6/L7 variants, we propose that NLRs exist in an equilibrium between ON and OFF states and that effector binding to the ON state stabilizes this conformation, thereby shifting the equilibrium toward the active form of the receptor to trigger defense signaling.
Subject(s)
Flax/metabolism , Models, Biological , Plant Proteins/metabolism , Receptors, Cell Surface/metabolism , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Sequence , Cell Death , Flax/cytology , Molecular Sequence Data , Mutation/genetics , Phenotype , Plant Proteins/chemistry , Polymorphism, Genetic , Protein Binding , Protein Structure, Tertiary , Receptors, Cell Surface/chemistry , Saccharomyces cerevisiae/metabolism , Sequence AlignmentABSTRACT
Plants possess intracellular immune receptors designated "nucleotide-binding domain and leucine-rich repeat" (NLR) proteins that translate pathogen-specific recognition into disease-resistance signaling. The wheat immune receptors Sr33 and Sr50 belong to the class of coiled-coil (CC) NLRs. They confer resistance against a broad spectrum of field isolates of Puccinia graminis f. sp. tritici, including the Ug99 lineage, and are homologs of the barley powdery mildew-resistance protein MLA10. Here, we show that, similarly to MLA10, the Sr33 and Sr50 CC domains are sufficient to induce cell death in Nicotiana benthamiana Autoactive CC domains and full-length Sr33 and Sr50 proteins self-associate in planta In contrast, truncated CC domains equivalent in size to an MLA10 fragment for which a crystal structure was previously determined fail to induce cell death and do not self-associate. Mutations in the truncated region also abolish self-association and cell-death signaling. Analysis of Sr33 and Sr50 CC domains fused to YFP and either nuclear localization or nuclear export signals in N benthamiana showed that cell-death induction occurs in the cytosol. In stable transgenic wheat plants, full-length Sr33 proteins targeted to the cytosol provided rust resistance, whereas nuclear-targeted Sr33 was not functional. These data are consistent with CC-mediated induction of both cell-death signaling and stem rust resistance in the cytosolic compartment, whereas previous research had suggested that MLA10-mediated cell-death and disease resistance signaling occur independently, in the cytosol and nucleus, respectively.
Subject(s)
Disease Resistance/genetics , Edible Grain/immunology , Gene Expression Regulation, Plant , Plant Diseases/immunology , Plant Proteins/immunology , Plant Stems/immunology , Triticum/immunology , Amino Acid Sequence , Basidiomycota/pathogenicity , Basidiomycota/physiology , Cell Nucleus/metabolism , Cell Nucleus/microbiology , Cytosol/immunology , Cytosol/metabolism , Cytosol/microbiology , Edible Grain/genetics , Edible Grain/microbiology , Plant Cells/immunology , Plant Cells/metabolism , Plant Cells/microbiology , Plant Diseases/genetics , Plant Diseases/microbiology , Plant Proteins/genetics , Plant Stems/genetics , Plant Stems/microbiology , Plants, Genetically Modified , Sequence Alignment , Sequence Homology, Amino Acid , Nicotiana/genetics , Nicotiana/immunology , Nicotiana/microbiology , Triticum/genetics , Triticum/microbiologyABSTRACT
Plants use intracellular immunity receptors, known as nucleotide-binding oligomerization domain-like receptors (NLRs), to recognize specific pathogen effector proteins and induce immune responses. These proteins provide resistance to many of the world's most destructive plant pathogens, yet we have a limited understanding of the molecular mechanisms that lead to defense signaling. We examined the wheat NLR protein, Sr33, which is responsible for strain-specific resistance to the wheat stem rust pathogen, Puccinia graminis f. sp. tritici We present the solution structure of a coiled-coil (CC) fragment from Sr33, which adopts a four-helix bundle conformation. Unexpectedly, this structure differs from the published dimeric crystal structure of the equivalent region from the orthologous barley powdery mildew resistance protein, MLA10, but is similar to the structure of the distantly related potato NLR protein, Rx. We demonstrate that these regions are, in fact, largely monomeric and adopt similar folds in solution in all three proteins, suggesting that the CC domains from plant NLRs adopt a conserved fold. However, larger C-terminal fragments of Sr33 and MLA10 can self-associate both in vitro and in planta, and this self-association correlates with their cell death signaling activity. The minimal region of the CC domain required for both cell death signaling and self-association extends to amino acid 142, thus including 22 residues absent from previous biochemical and structural protein studies. These data suggest that self-association of the minimal CC domain is necessary for signaling but is likely to involve a different structural basis than previously suggested by the MLA10 crystallographic dimer.
ABSTRACT
The plant apoplast is integral to intercellular signalling, transport and plant-pathogen interactions. Plant pathogens deliver effectors both into the apoplast and inside host cells, but no computational method currently exists to discriminate between these localizations. We present ApoplastP, the first method for predicting whether an effector or plant protein localizes to the apoplast. ApoplastP uncovers features of apoplastic localization common to both effectors and plant proteins, namely depletion in glutamic acid, acidic amino acids and charged amino acids and enrichment in small amino acids. ApoplastP predicts apoplastic localization in effectors with a sensitivity of 75% and a false positive rate of 5%, improving the accuracy of cysteine-rich classifiers by > 13%. ApoplastP does not depend on the presence of a signal peptide and correctly predicts the localization of unconventionally secreted proteins. The secretomes of fungal saprophytes as well as necrotrophic, hemibiotrophic and extracellular fungal pathogens are enriched for predicted apoplastic proteins. Rust pathogens have low proportions of predicted apoplastic proteins, but these are highly enriched for predicted effectors. ApoplastP pioneers apoplastic localization prediction using machine learning. It will facilitate functional studies and will be valuable for predicting if an effector localizes to the apoplast or if it enters plant cells.
Subject(s)
Machine Learning , Plant Proteins/metabolism , Amino Acid Motifs , Amino Acid Sequence , Conserved Sequence , Cysteine/metabolism , Fungal Proteins/chemistry , Fungal Proteins/metabolism , Oomycetes/metabolism , Plant Proteins/chemistry , Protein Sorting Signals , ProteomicsABSTRACT
BACKGROUND: Rust fungi are an important group of plant pathogens that cause devastating losses in agricultural, silvicultural and natural ecosystems. Plants can be protected from rust disease by resistance genes encoding receptors that trigger a highly effective defence response upon recognition of specific pathogen avirulence proteins. Identifying avirulence genes is crucial for understanding how virulence evolves in the field. RESULTS: To facilitate avirulence gene cloning in the flax rust fungus, Melampsora lini, we constructed a high-density genetic linkage map using single nucleotide polymorphisms detected in restriction site-associated DNA sequencing (RADseq) data. The map comprises 13,412 RADseq markers in 27 linkage groups that together span 5860 cM and contain 2756 recombination bins. The marker sequences were used to anchor 68.9 % of the M. lini genome assembly onto the genetic map. The map and anchored assembly were then used to: 1) show that M. lini has a high overall meiotic recombination rate, but recombination distribution is uneven and large coldspots exist; 2) show that substantial genome rearrangements have occurred in spontaneous loss-of-avirulence mutants; and 3) identify the AvrL2 and AvrM14 avirulence genes by map-based cloning. AvrM14 is a dual-specificity avirulence gene that encodes a predicted nudix hydrolase. AvrL2 is located in the region of the M. lini genome with the lowest recombination rate and encodes a small, highly-charged proline-rich protein. CONCLUSIONS: The M. lini high-density linkage map has greatly advanced our understanding of virulence mechanisms in this pathogen by providing novel insights into genome variability and enabling identification of two new avirulence genes.