RESUMO
Tomato (Solanum lycopersicum) is one of the world's most important food crops, and as such, its production needs to be protected from infectious diseases that can significantly reduce yield and quality. Here, we survey the effector-triggered immunity (ETI) landscape of tomato against the bacterial pathogen Pseudomonas syringae. We perform comprehensive ETI screens in five cultivated tomato varieties and two wild relatives, as well as an immunodiversity screen on a collection of 149 tomato varieties that includes both wild and cultivated varieties. The screens reveal a tomato ETI landscape that is more limited than what was previously found in the model plant Arabidopsis thaliana. We also demonstrate that ETI eliciting effectors can protect tomato against P. syringae infection when the effector is delivered by a non-virulent strain either prior to or simultaneously with a virulent strain. Overall, our findings provide a snapshot of the ETI landscape of tomatoes and demonstrate that ETI can be used as a biocontrol treatment to protect crop plants.
Assuntos
Doenças das Plantas , Imunidade Vegetal , Pseudomonas syringae , Solanum lycopersicum , Solanum lycopersicum/microbiologia , Solanum lycopersicum/imunologia , Pseudomonas syringae/imunologia , Pseudomonas syringae/patogenicidade , Doenças das Plantas/microbiologia , Doenças das Plantas/imunologia , Arabidopsis/imunologia , Arabidopsis/microbiologia , Proteínas de Plantas/imunologia , Virulência , Regulação da Expressão Gênica de Plantas , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/imunologiaRESUMO
The emergence of new pathogens is an ongoing threat to human health and agriculture. While zoonotic spillovers received considerable attention, the emergence of crop diseases is less well studied. Here, we identify genomic factors associated with the emergence of Pseudomonas syringae bacterial blight of coffee. Fifty-three P. syringae strains from diseased Brazilian coffee plants were sequenced. Comparative and evolutionary analyses were used to identify loci associated with coffee blight. Growth and symptomology assays were performed to validate the findings. Coffee isolates clustered in three lineages, including primary phylogroups PG3 and PG4, and secondary phylogroup PG11. Genome-wide association study of the primary PG strains identified 37 loci, including five effectors, most of which were encoded on a plasmid unique to the PG3 and PG4 coffee strains. Evolutionary analyses support the emergence of coffee blight in PG4 when the coffee-associated plasmid and associated effectors derived from a divergent plasmid carried by strains associated with other hosts. This plasmid was only recently transferred into PG3. Natural diversity and CRISPR-Cas9 plasmid curing were used to show that strains with the coffee-associated plasmid grow to higher densities and cause more severe disease symptoms in coffee. This work identifies possible evolutionary mechanisms underlying the emergence of a new lineage of coffee pathogens.
Assuntos
Genoma Bacteriano , Pseudomonas syringae , Humanos , Pseudomonas syringae/genética , Café , Estudo de Associação Genômica Ampla , Plasmídeos/genética , Doenças das Plantas/microbiologiaRESUMO
The field of plant pathology has revealed many of the mechanisms underlying the arms race, providing crucial knowledge and genetic resources for improving plant health. Although the host-microbe interaction seemingly favors rapidly evolving pathogens, it has also generated a vast evolutionary history of largely unexplored plant immunodiversity. We review studies that characterize the scope and distribution of genetic and ecological diversity in model and non-model systems with specific reference to pathogen effector diversity, plant immunodiversity in both cultivated species and their wild relatives, and diversity in the plant-associated microbiota. We show how the study of evolutionary and ecological processes can reveal patterns of genetic convergence, conservation, and diversification, and that this diversity is increasingly tractable in both experimental and translational systems. Perhaps most importantly, these patterns of diversity provide largely untapped resources that can be deployed for the rational engineering of durable resistance for sustainable agriculture.
Assuntos
Patologia Vegetal , Plantas/genética , Evolução BiológicaRESUMO
Although virulence is typically attributed to single pathogenic strains, here we investigated whether effectors secreted by a population of non-virulent strains could function as public goods to enable the emergence of collective virulence. We disaggregated the 36 type III effectors of the phytopathogenic bacterium Pseudomonas syringae strain PtoDC3000 into a 'metaclone' of 36 coisogenic strains, each carrying a single effector in an effectorless background. Each coisogenic strain was individually unfit, but the metaclone was collectively as virulent as the wild-type strain on Arabidopsis thaliana, suggesting that effectors can drive the emergence of cooperation-based virulence through their public action. We show that independently evolved effector suits can equally drive this cooperative behaviour by transferring the effector alleles native to the strain PmaES4326 into the conspecific but divergent strain PtoDC3000. Finally, we transferred the disaggregated PtoDC3000 effector arsenal into Pseudomonas fluorescens and show that their cooperative action was sufficient to convert this rhizosphere-inhabiting beneficial bacterium into a phyllosphere pathogen. These results emphasize the importance of microbial community interactions and expand the ecological scale at which disease may be attributed.
Assuntos
Arabidopsis , Proteínas de Bactérias , Virulência , Proteínas de Bactérias/genética , Pseudomonas syringae/genética , Bactérias , Arabidopsis/microbiologiaRESUMO
A key facet of innate immunity in plants entails the recognition of pathogen "effector" virulence proteins by host Nucleotide-Binding Leucine-Rich Repeat Receptors (NLRs). Among characterized NLRs, the broadly conserved ZAR1 NLR is particularly remarkable due to its capacity to recognize at least six distinct families of effectors from at least two bacterial genera. This expanded recognition spectrum is conferred through interactions between ZAR1 and a dynamic network of two families of Receptor-Like Cytoplasmic Kinases (RLCKs): ZED1-Related Kinases (ZRKs) and PBS1-Like Kinases (PBLs). In this review, we survey the history of functional studies on ZAR1, with an emphasis on how the ZAR1-RLCK network functions to trap diverse effectors. We discuss 1) the dynamics of the ZAR1-associated RLCK network; 2) the specificity between ZRKs and PBLs; and 3) the specificity between effectors and the RLCK network. We posit that the shared protein fold of kinases and the switch-like properties of their interactions make them ideal effector sensors, enabling ZAR1 to act as a broad spectrum guardian of host kinases.
RESUMO
Pseudomonas syringae is a genetically diverse bacterial species complex responsible for numerous agronomically important crop diseases. Individual P. syringae isolates are assigned pathovar designations based on their host of isolation and the associated disease symptoms, and these pathovar designations are often assumed to reflect host specificity although this assumption has rarely been rigorously tested. Here we developed a rapid seed infection assay to measure the virulence of 121 diverse P. syringae isolates on common bean (Phaseolus vulgaris). This collection includes P. syringae phylogroup 2 (PG2) bean isolates (pathovar syringae) that cause bacterial spot disease and P. syringae phylogroup 3 (PG3) bean isolates (pathovar phaseolicola) that cause the more serious halo blight disease. We found that bean isolates in general were significantly more virulent on bean than non-bean isolates and observed no significant virulence difference between the PG2 and PG3 bean isolates. However, when we compared virulence within PGs we found that PG3 bean isolates were significantly more virulent than PG3 non-bean isolates, while there was no significant difference in virulence between PG2 bean and non-bean isolates. These results indicate that PG3 strains have a higher level of host specificity than PG2 strains. We then used gradient boosting machine learning to predict each strain's virulence on bean based on whole genome k-mers, type III secreted effector k-mers, and the presence/absence of type III effectors and phytotoxins. Our model performed best using whole genome data and was able to predict virulence with high accuracy (mean absolute error = 0.05). Finally, we functionally validated the model by predicting virulence for 16 strains and found that 15 (94%) had virulence levels within the bounds of estimated predictions. This study strengthens the hypothesis that P. syringae PG2 strains have evolved a different lifestyle than other P. syringae strains as reflected in their lower level of host specificity. It also acts as a proof-of-principle to demonstrate the power of machine learning for predicting host specific adaptation.
Assuntos
Phaseolus , Pseudomonas syringae , Árvores de Decisões , Especificidade de Hospedeiro , Phaseolus/microbiologia , Doenças das Plantas/microbiologia , VirulênciaRESUMO
Pseudomonas syringae is an evolutionarily diverse bacterial species complex and a preeminent model for the study of plant-pathogen interactions due in part to its remarkably broad host range. A critical feature of P. syringae virulence is the employment of suites of type III secreted effector (T3SE) proteins, which vary widely in composition and function. These effectors act on a variety of plant intracellular targets to promote pathogenesis but can also be avirulence factors when detected by host immune complexes. In this review, we survey the phylogenetic diversity (PD) of the P. syringae effectorome, comprising 70 distinct T3SE families identified to date, and highlight how avoidance of host immune detection has shaped effectorome diversity through functional redundancy, diversification, and horizontal transfer. We present emerging avenues for research and novel insights that can be gained via future investigations of plant-pathogen interactions through the fusion of large-scale interaction screens and phylogenomic approaches.
Assuntos
Proteínas de Bactérias , Pseudomonas syringae , Filogenia , VirulênciaRESUMO
The bacterial plant pathogen Pseudomonas syringae requires type III secreted effectors (T3SEs) for pathogenesis. However, a major facet of plant immunity entails the recognition of a subset of P. syringae's T3SEs by intracellular host receptors in a process called Effector-Triggered Immunity (ETI). Prior work has shown that ETI-eliciting T3SEs are pervasive in the P. syringae species complex raising the question of how P. syringae mitigates its ETI load to become a successful pathogen. While pathogens can evade ETI by T3SE mutation, recombination, or loss, there is increasing evidence that effector-effector (a.k.a., metaeffector) interactions can suppress ETI. To study the ETI-suppression potential of P. syringae T3SE repertoires, we compared the ETI-elicitation profiles of two genetically divergent strains: P. syringae pv. tomato DC3000 (PtoDC3000) and P. syringae pv. maculicola ES4326 (PmaES4326), which are both virulent on Arabidopsis thaliana but harbour largely distinct effector repertoires. Of the 529 T3SE alleles screened on A. thaliana Col-0 from the P. syringae T3SE compendium (PsyTEC), 69 alleles from 21 T3SE families elicited ETI in at least one of the two strain backgrounds, while 50 elicited ETI in both backgrounds, resulting in 19 differential ETI responses including two novel ETI-eliciting families: AvrPto1 and HopT1. Although most of these differences were quantitative, three ETI responses were completely absent in one of the pathogenic backgrounds. We performed ETI suppression screens to test if metaeffector interactions contributed to these ETI differences, and found that HopQ1a suppressed AvrPto1m-mediated ETI, while HopG1c and HopF1g suppressed HopT1b-mediated ETI. Overall, these results show that P. syringae strains leverage metaeffector interactions and ETI suppression to overcome the ETI load associated with their native T3SE repertoires.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/genética , Proteínas de Bactérias/genética , Humanos , Doenças das Plantas/microbiologia , Imunidade Vegetal , Pseudomonas syringaeRESUMO
The bacterial phytopathogen Pseudomonas syringae causes disease on a wide array of plants, including the model plant Arabidopsis thaliana and its agronomically important relatives in the Brassicaceae family. To cause disease, P. syringae delivers effector proteins into plant cells through a type III secretion system. In response, plant nucleotide-binding leucine-rich repeat proteins recognize specific effectors and mount effector-triggered immunity (ETI). While ETI is pervasive across A. thaliana, with at least 19 families of P. syringae effectors recognized in this model species, the ETI landscapes of crop species have yet to be systematically studied. Here, we investigated the conservation of the A. thaliana ETI landscape in two closely related oilseed crops, Brassica napus (canola) and Camelina sativa (false flax). We show that the level of immune conservation is inversely related to the degree of evolutionary divergence from A. thaliana, with the more closely related C. sativa losing ETI responses to only one of the 19 P. syringae effectors tested, while the more distantly related B. napus loses ETI responses to four effectors. In contrast to the qualitative conservation of immune response, the quantitative rank order is not as well-maintained across the three species and diverges increasingly with evolutionary distance from A. thaliana. Overall, our results indicate that the A. thaliana ETI profile is qualitatively conserved in oilseed crops, but quantitatively distinct.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Bactérias/metabolismo , Produtos Agrícolas/metabolismo , Doenças das Plantas/microbiologia , Imunidade Vegetal/genética , Pseudomonas syringaeRESUMO
A critical step in studying biological features (e.g., genetic variants, gene families, metabolic capabilities, or taxa) is assessing their diversity and distribution among a sample of individuals. Accurate assessments of these patterns are essential for linking features to traits or outcomes of interest and understanding their functional impact. Consequently, it is of crucial importance that the measures employed for quantifying feature diversity can perform robustly under any evolutionary scenario. However, the standard measures used for quantifying and comparing the distribution of features, such as prevalence, phylogenetic diversity, and related approaches, either do not take into consideration evolutionary history, or assume strictly vertical patterns of inheritance. Consequently, these approaches cannot accurately assess diversity for features that have undergone recombination or horizontal transfer. To address this issue, we have devised RecPD, a novel recombination-aware phylogenetic-diversity statistic for measuring the distribution and diversity of features under all evolutionary scenarios. RecPD utilizes ancestral-state reconstruction to map the presence / absence of features onto ancestral nodes in a species tree, and then identifies potential recombination events in the evolutionary history of the feature. We also derive several related measures from RecPD that can be used to assess and quantify evolutionary dynamics and correlation of feature evolutionary histories. We used simulation studies to show that RecPD reliably reconstructs feature evolutionary histories under diverse recombination and loss scenarios. We then applied RecPD in two diverse real-world scenarios including a preliminary study type III effector protein families secreted by the plant pathogenic bacterium Pseudomonas syringae and growth phenotypes of the Pseudomonas genus and demonstrate that prevalence is an inadequate measure that obscures the potential impact of recombination. We believe RecPD will have broad utility for revealing and quantifying complex evolutionary processes for features at any biological level.
Assuntos
Evolução Biológica , Recombinação Genética , Evolução Molecular , Filogenia , Recombinação Genética/genéticaRESUMO
Pathogenic effector proteins use a variety of enzymatic activities to manipulate host cellular proteins and favor the infection process. However, these perturbations can be sensed by nucleotide-binding leucine-rich-repeat (NLR) proteins to activate effector-triggered immunity (ETI). Here we have identified a small molecule (Zaractin) that mimics the immune eliciting activity of the Pseudomonas syringae type III secreted effector (T3SE) HopF1r and show that both HopF1r and Zaractin activate the same NLR-mediated immune pathway in Arabidopsis Our results demonstrate that the ETI-inducing action of pathogenic effectors can be harnessed to identify synthetic activators of the eukaryotic immune system.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Imunidade Vegetal/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Arabidopsis/microbiologia , Proteínas de Bactérias/metabolismo , Proteínas NLR/metabolismo , Doenças das Plantas/microbiologia , Ligação Proteica/efeitos dos fármacos , Pseudomonas syringae/patogenicidadeRESUMO
The natural diversity of pathogen effectors and host immune components represents a snapshot of the underlying evolutionary processes driving the host-pathogen arms race. In plants, this arms race is manifested by an ongoing cycle of disease and resistance driven by pathogenic effectors that promote disease (effector-triggered susceptibility; ETS) and plant resistance proteins that recognize effector activity to trigger immunity (effector-triggered immunity; ETI). Here we discuss how this ongoing ETS-ETI cycle has shaped the natural diversity of both plant resistance proteins and pathogen effectors. We focus on the evolutionary forces that drive the diversification of the molecules that determine the outcome of plant-pathogen interactions and introduce the concept of metapopulation dynamics (i.e., the introduction of genetic variation from conspecific organisms in different populations) as an alternative mechanism that can introduce and maintain diversity in both host and pathogen populations.
Assuntos
Interações Hospedeiro-Patógeno , Doenças das Plantas , Imunidade Vegetal , Interações Hospedeiro-Patógeno/genética , Doenças das Plantas/genética , Imunidade Vegetal/genética , Proteínas de Plantas , Plantas/genéticaRESUMO
Proteome networks are a crucial facet of biological systems that mediate cellular functions and responses to the environment. However, a main limitation of traditional approaches to study protein interactions, such as yeast-2-hybrid and affinity purification-coupled with mass spectrometry (AP-MS), is their restricted ability to identify interactions for membrane-bound and/or insoluble protein complexes. These types of interactions include many of the protein complexes that mediate the perception and response to cellular stimuli and are therefore of great research interest. Proximity-dependent biotinylation (PDB) coupled to mass spectrometry provides a powerful approach to survey proximal protein interactions in living cells, including membrane bound and insoluble complexes. One PDB method, BioID, translationally fuses a promiscuous biotin ligase to a bait protein of interest, allowing covalent biotinylation of proximal proteins (within ~10 nm). Modified proteins can be purified from cells without the need to maintain protein interactions, and subsequently identified by mass spectrometry. Although BioID has revolutionized the study of proteomes in numerous organisms, its application to plant systems has only recently been realized. In this chapter, we outline a protocol for BioID in tissues of the model plant Arabidopsis thaliana.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Biotina/química , Complexos Multiproteicos , Mapeamento de Interação de Proteínas , Proteômica , Arabidopsis/química , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Biotinilação , Cromatografia de Afinidade , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Ligação ProteicaAssuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Arabidopsis/microbiologia , Fosfotransferases/genética , Doenças das Plantas/genética , Imunidade Vegetal/imunologia , Virulência/genética , Análise por Conglomerados , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Imunidade Vegetal/genética , Pseudomonas syringae/imunologia , Xanthomonas campestris/imunologiaRESUMO
The Arabidopsis nucleotide-binding leucine-rich repeat protein ZAR1 can recognize at least six distinct families of pathogenic effector proteins to mount an effector-triggered immune response. This remarkable immunodiversity appears to be conveyed by receptor-like cytoplasmic kinase (RLCK) complexes, which associate with ZAR1 to sense several effector-induced kinase perturbations. Here we show that the recently identified ZAR1-mediated immune responses against the HopX1, HopO1, and HopBA1 effector families of Pseudomonas syringae rely on an expanded diversity of RLCK sensors. We show that individual sensors can recognize distinct effector families, thereby contributing to the expanded surveillance potential of ZAR1 and supporting its role as a guardian of the plant kinome.
RESUMO
Effector-triggered immunity (ETI), induced by host immune receptors in response to microbial effectors, protects plants against virulent pathogens. However, a systematic study of ETI prevalence against species-wide pathogen diversity is lacking. We constructed the Pseudomonas syringae Type III Effector Compendium (PsyTEC) to reduce the pan-genome complexity of 5127 unique effector proteins, distributed among 70 families from 494 strains, to 529 representative alleles. We screened PsyTEC on the model plant Arabidopsis thaliana and identified 59 ETI-eliciting alleles (11.2%) from 19 families (27.1%), with orthologs distributed among 96.8% of P. syringae strains. We also identified two previously undescribed host immune receptors, including CAR1, which recognizes the conserved effectors AvrE and HopAA1, and found that 94.7% of strains harbor alleles predicted to be recognized by either CAR1 or ZAR1.
Assuntos
Arabidopsis/imunologia , Arabidopsis/microbiologia , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata/genética , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Imunidade Vegetal/genética , Pseudomonas syringae/patogenicidade , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Genoma de Planta , Interações Hospedeiro-Patógeno/genética , Doenças das Plantas/genética , Pseudomonas syringae/genéticaRESUMO
An understanding of how biological diversity affects plant-microbe interactions is becoming increasingly important, particularly with respect to components of the pathogen effector arsenal and the plant immune system. Although technological improvements have greatly advanced our ability to examine molecular sequences and interactions, relatively few advances have been made that facilitate high-throughput, in vivo pathology screens. Here, we present a high-throughput, microplate-based, nondestructive seedling pathology assay, and apply it to identify Arabidopsis thaliana effector-triggered immunity (ETI) responses against Pseudomonas syringae type III secreted effectors. The assay was carried out in a 48-well microplate format with spray inoculation, and disease symptoms were quantitatively recorded in a semiautomated manner, thereby greatly reducing both time and costs. The assay requires only slight modifications of common labware and uses no proprietary software. We validated the assay by recapitulating known ETI responses induced by P. syringae in Arabidopsis. We also demonstrated that we can quantitatively differentiate responses from a diversity of plant genotypes grown in the same microplate. Finally, we showed that the results obtained from our assay can be used to perform genome-wide association studies to identify host immunity genes, recapitulating results that have been independently obtained with mature plants.
Assuntos
Arabidopsis/imunologia , Ensaios de Triagem em Larga Escala , Imunidade Vegetal , Pseudomonas syringae/patogenicidade , Plântula/imunologia , Proteínas de Bactérias , Doenças das Plantas/microbiologiaRESUMO
A broad range of Gram-negative bacteria employ a type III secretion system (T3SS) to deliver virulence proteins termed type III secreted effectors directly into the cytoplasm of eukaryotic host cells. While effectors can contribute to the colonization of eukaryotic hosts by bacterial symbionts and pathogens, they can also elicit host immune responses that restrict bacterial growth. These opposing selective pressures have shaped the evolution of effector families and may be responsible for their incredible diversity in biochemical function, mechanism of action, and taxonomic distribution. In this chapter, we focus on three distinct effector families whose members are distributed among both plant and animal pathogens. We first discuss the LRR-NEL and YopJ families of effectors. These two effector families possess ubiquitin ligase and acetyltransferase activity, respectively, which in both cases can be directed against host innate immune signal transduction pathways to promote infection. Finally, we discuss the TALE family of transcription activator-like effectors that serve to reprogram host immunity transcriptional responses. This chapter aims to highlight the diversity within these three effector families that results from the strong and dynamic evolutionary forces shaping the interface between host and bacterium.
Assuntos
Evolução Molecular , Interações Hospedeiro-Patógeno , Sistemas de Secreção Tipo III/classificação , Sistemas de Secreção Tipo III/metabolismo , Animais , Proteínas de Bactérias , Bactérias Gram-Negativas/patogenicidade , Interações Hospedeiro-Patógeno/imunologia , Imunidade Inata , Sistemas de Secreção Tipo III/imunologia , Fatores de VirulênciaRESUMO
The Pseudomonas syringae acetyltransferase HopZ1a is delivered into host cells by the type III secretion system to promote bacterial growth. However, in the model plant host Arabidopsis thaliana, HopZ1a activity results in an effector-triggered immune response (ETI) that limits bacterial proliferation. HopZ1a-triggered immunity requires the nucleotide-binding, leucine-rich repeat domain (NLR) protein, ZAR1, and the pseudokinase, ZED1. Here we demonstrate that HopZ1a can acetylate members of a family of 'receptor-like cytoplasmic kinases' (RLCK family VII; also known as PBS1-like kinases, or PBLs) and promote their interaction with ZED1 and ZAR1 to form a ZAR1-ZED1-PBL ternary complex. Interactions between ZED1 and PBL kinases are determined by the pseudokinase features of ZED1, and mutants designed to restore ZED1 kinase motifs can (1) bind to PBLs, (2) recruit ZAR1, and (3) trigger ZAR1-dependent immunity in planta, all independently of HopZ1a. A ZED1 mutant that mimics acetylation by HopZ1a also triggers immunity in planta, providing evidence that effector-induced perturbations of ZED1 also activate ZAR1. Overall, our results suggest that interactions between these two RLCK families are promoted by perturbations of structural features that distinguish active from inactive kinase domain conformations. We propose that effector-induced interactions between ZED1/ZRK pseudokinases (RLCK family XII) and PBL kinases (RLCK family VII) provide a sensitive mechanism for detecting perturbations of either kinase family to activate ZAR1-mediated ETI.
Assuntos
Proteínas de Arabidopsis/imunologia , Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Arabidopsis/metabolismo , Fosfotransferases/imunologia , Fosfotransferases/metabolismo , Imunidade Vegetal , Acetilação , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/imunologia , Proteínas de Transporte/metabolismo , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/imunologia , Modelos Imunológicos , Mutação , Fosfotransferases/genética , Domínios e Motivos de Interação entre Proteínas , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Pseudomonas syringae/imunologia , Pseudomonas syringae/metabolismo , Pseudomonas syringae/patogenicidadeRESUMO
Antofine, a phenanthroindolizidine alkaloid, is a bioactive natural product isolated from milkweeds that exhibits numerous biological activities, including anticancer, antimicrobial, antiviral, and anti-inflammatory properties. However, the direct targets and mode of action of antofine have not been determined. In this report, we show that antofine displays antifungal properties against the phytopathogen Fusarium graminearum, the cause of Fusarium head blight disease (FHB). FHB does devastating damage to agriculture, causing billions of dollars in economic losses annually. We therefore sought to understand the mode of action of antofine in F. graminearum using insights from yeast chemical genomic screens. We used haploinsufficiency profiling (HIP) to identify putative targets of antofine in yeast and identified three candidate targets, two of which had homologs in F. graminearum The Fusarium homologues of two targets, glutamate dehydrogenase (FgGDH) and resistance to rapamycin deletion 2 (FgRRD2), can bind antofine. Of the two genes, only the Fgrrd2 knockout displayed a loss of virulence in wheat, indicating that RRD2 is an antivirulence target of antofine in F. graminearum Mechanistically, we demonstrate that antofine disrupts the interaction between FgRRD2 and FgTap42, which is part of the Tap42-phosphatase complex in the target of rapamycin (TOR) signaling pathway, a central regulator of cell growth in eukaryotes and a pathway of extensive study for controlling numerous pathologies.IMPORTANCEFusarium head blight caused by the fungal pathogen Fusarium graminearum is a devastating disease of cereal crops worldwide, with limited effective chemical treatments available. Here we show that the natural alkaloid compound antofine can inhibit fusarium head blight in wheat. Using yeast genomic screening, we identified the TOR pathway component RRD2 as a target of antofine that is also required for F. graminearum pathogenicity.