RESUMO
Plant-associated microbiota can extend plant immune system function, improve nutrient acquisition and availability, and alleviate abiotic stresses. Thus, naturally beneficial microbial therapeutics are enticing tools to improve plant productivity. The basic definition of plant microbiota across species and ecosystems, combined with the development of reductionist experimental models and the manipulation of plant phenotypes with microbes, has fueled interest in its translation to agriculture. However, the great majority of microbes exhibiting plant-productivity traits in the lab and greenhouse fail in the field. Therapeutic microbes must reach détente, the establishment of uneasy homeostasis, with the plant immune system, invade heterogeneous pre-established plant-associated communities, and persist in a new and potentially remodeled community. Environmental conditions can alter community structure and thus impact the engraftment of therapeutic microbes. We survey recent breakthroughs, challenges, and opportunities in translating beneficial microbes from the lab to the field.
Assuntos
Microbiota , Plantas , Agricultura , Fenótipo , Plantas/microbiologia , Microbiologia do Solo , Estresse Fisiológico , EcossistemaRESUMO
Plants have an innate immune system to fight off potential invaders that is based on the perception of nonself or modified-self molecules. Microbe-associated molecular patterns (MAMPs) are evolutionarily conserved microbial molecules whose extracellular detection by specific cell surface receptors initiates an array of biochemical responses collectively known as MAMP-triggered immunity (MTI). Well-characterized MAMPs include chitin, peptidoglycan, and flg22, a 22-amino acid epitope found in the major building block of the bacterial flagellum, FliC. The importance of MAMP detection by the plant immune system is underscored by the large diversity of strategies used by pathogens to interfere with MTI and that failure to do so is often associated with loss of virulence. Yet, whether or how MTI functions beyond pathogenic interactions is not well understood. Here we demonstrate that a community of root commensal bacteria modulates a specific and evolutionarily conserved sector of the Arabidopsis immune system. We identify a set of robust, taxonomically diverse MTI suppressor strains that are efficient root colonizers and, notably, can enhance the colonization capacity of other tested commensal bacteria. We highlight the importance of extracellular strategies for MTI suppression by showing that the type 2, not the type 3, secretion system is required for the immunomodulatory activity of one robust MTI suppressor. Our findings reveal that root colonization by commensals is controlled by MTI, which, in turn, can be selectively modulated by specific members of a representative bacterial root microbiota.
Assuntos
Microbiota/fisiologia , Imunidade Vegetal/imunologia , Raízes de Plantas/microbiologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Bactérias/metabolismo , Expressão Gênica/genética , Regulação da Expressão Gênica de Plantas/genética , Imunidade , Microbiota/imunologia , Doenças das Plantas/microbiologia , Raízes de Plantas/imunologia , Plantas/microbiologia , Microbiologia do Solo , Simbiose/imunologia , VirulênciaRESUMO
Witches' broom disease of cacao is caused by the pathogenic fungus Moniliophthora perniciosa. By using tomato (Solanum lycopersicum) cultivar Micro-Tom (MT) as a model system, we investigated the physiological and metabolic consequences of M. perniciosa infection to determine whether symptoms result from sink establishment during infection. Infection of MT by M. perniciosa caused reductions in root biomass and fruit yield, a decrease in leaf gas exchange, and down-regulation of photosynthesis-related genes. The total leaf area and water potential decreased, while ABA levels, water conductance/conductivity, and ABA-related gene expression increased. Genes related to sugar metabolism and those involved in secondary cell wall deposition were up-regulated upon infection, and the concentrations of sugars, fumarate, and amino acids increased. 14C-glucose was mobilized towards infected MT stems, but not in inoculated stems of the MT line overexpressing CYTOKININ OXIDASE-2 (35S::AtCKX2), suggesting a role for cytokinin in establishing a sugar sink. The up-regulation of genes involved in cell wall deposition and phenylpropanoid metabolism in infected MT, but not in 35S::AtCKX2 plants, suggests establishment of a cytokinin-mediated sink that promotes tissue overgrowth with an increase in lignin. Possibly, M. perniciosa could benefit from the accumulation of secondary cell walls during its saprotrophic phase of infection.
Assuntos
Agaricales , Cacau , Solanum lycopersicum , Agaricales/genética , Cacau/genética , Parede Celular , Citocininas , Solanum lycopersicum/genética , Solanum lycopersicum/microbiologia , Doenças das Plantas/microbiologia , Açúcares , ÁguaRESUMO
Moniliophthora perniciosa causes witches' broom disease of cacao and inflicts symptoms suggestive of hormonal imbalance. We investigated whether infection of the tomato (Solanum lycopersicum) model system Micro-Tom (MT) by the Solanaceae (S)-biotype of Moniliophthora perniciosa, which causes stem swelling and hypertrophic growth of axillary shoots, results from changes in host cytokinin metabolism. Inoculation of an MT-transgenic line that overexpresses the Arabidopsis CYTOKININ OXIDASE-2 gene (35S::AtCKX2) resulted in a reduction in disease incidence and stem diameter. RNA-sequencing analysis of infected MT and 35S::AtCKX2 revealed the activation of cytokinin-responsive marker genes when symptoms were conspicuous. The expression of an Moniliophthora perniciosa tRNA-ISOPENTENYL-TRANSFERASE suggests the production of isopentenyladenine (iP), detected in mycelia grown in vitro. Inoculated MT stems showed higher levels of dihydrozeatin and trans-zeatin but not iP. The application of benzyladenine induced symptoms similar to infection, whereas applying the cytokinin receptor inhibitors LGR-991 and PI55 decreased symptoms. Moniliophthora perniciosa produces iP that might contribute to cytokinin synthesis by the host, which results in vascular and cortex enlargement, axillary shoot outgrowth, reduction in root biomass and an increase in fruit locule number. This strategy may be associated with the manipulation of sink establishment to favour infection by the fungus.
Assuntos
Agaricales , Cacau , Solanum lycopersicum , Citocininas , Solanum lycopersicum/genética , Doenças por Fitoplasmas , Doenças das PlantasRESUMO
Moniliophthora perniciosa is the causative agent of witches' broom disease, which devastates cacao cultures in South America. This pathogenic fungus infects meristematic tissues and derives nutrients from the plant apoplast during an unusually long-lasting biotrophic stage. To survive, the fungus produces proteins to suppress the plant immune response. Proteins of the PR-1 (pathogenesis-related 1)/CAP superfamily have been implicated in fungal virulence and immune suppression. The genome of M. perniciosa encodes 11 homologues of plant PR-1 proteins, designated MpPR-1 proteins, but their precise mode of action is poorly understood. In this study, we expressed MpPR-1 proteins in a yeast model lacking endogenous CAP proteins. We show that some members of the MpPR-1 family bind and promote secretion of sterols, whereas others bind and promote secretion of fatty acids. Lipid binding by purified MpPR-1 occurs with micromolar affinity and is saturable in vitro Sterol binding by MpPR-1 requires the presence of a flexible loop region containing aromatic amino acids, the caveolin-binding motif. Remarkably, MpPR-1 family members that do not bind sterols can be converted to sterol binders by a single point mutation in the caveolin-binding motif. We discuss the possible implications of the lipid-binding activity of MpPR-1 family members with regard to the mode of action of these proteins during M. perniciosa infections.
Assuntos
Agaricales/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Proteínas Fúngicas/metabolismo , Esteróis/metabolismo , Agaricales/química , Agaricales/patogenicidade , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Ligação Competitiva , Cacau/microbiologia , Colesterol/química , Colesterol/metabolismo , Ácidos Graxos não Esterificados/química , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Deleção de Genes , Cinética , Ligantes , Mutagênese Sítio-Dirigida , Ácido Palmítico/química , Ácido Palmítico/metabolismo , Mutação Puntual , Conformação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Esteróis/químicaRESUMO
BACKGROUND: The bioethanol production system used in Brazil is based on the fermentation of sucrose from sugarcane feedstock by highly adapted strains of the yeast Saccharomyces cerevisiae. Bacterial contaminants present in the distillery environment often produce yeast-bacteria cellular co-aggregation particles that resemble yeast-yeast cell adhesion (flocculation). The formation of such particles is undesirable because it slows the fermentation kinetics and reduces the overall bioethanol yield. RESULTS: In this study, we investigated the molecular physiology of one of the main S. cerevisiae strains used in Brazilian bioethanol production, PE-2, under two contrasting conditions: typical fermentation, when most yeast cells are in suspension, and co-aggregated fermentation. The transcriptional profile of PE-2 was assessed by RNA-seq during industrial scale fed-batch fermentation. Comparative analysis between the two conditions revealed transcriptional profiles that were differentiated primarily by a deep gene repression in the co-aggregated samples. The data also indicated that Lactobacillus fermentum was likely the main bacterial species responsible for cellular co-aggregation and for the high levels of organic acids detected in the samples. CONCLUSIONS: Here, we report the high-resolution gene expression profiling of strain PE-2 during industrial-scale fermentations and the transcriptional reprograming observed under co-aggregation conditions. This dataset constitutes an important resource that can provide support for further development of this key yeast biocatalyst.
Assuntos
Bactérias/genética , Etanol/metabolismo , Perfilação da Expressão Gênica , Saccharomyces cerevisiae/genética , Bactérias/crescimento & desenvolvimento , Bactérias/metabolismo , Biomassa , Brasil , Fermentação , Floculação , Ontologia Genética , Genótipo , Microbiologia Industrial/métodos , Cinética , Interações Microbianas , Mutação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Saccharum/metabolismoRESUMO
Cerato-platanins (CP) are small, cysteine-rich fungal-secreted proteins involved in the various stages of the host-fungus interaction process, acting as phytotoxins, elicitors, and allergens. We identified 12 CP genes (MpCP1 to MpCP12) in the genome of Moniliophthora perniciosa, the causal agent of witches' broom disease in cacao, and showed that they present distinct expression profiles throughout fungal development and infection. We determined the X-ray crystal structures of MpCP1, MpCP2, MpCP3, and MpCP5, representative of different branches of a phylogenetic tree and expressed at different stages of the disease. Structure-based biochemistry, in combination with nuclear magnetic resonance and mass spectrometry, allowed us to define specialized capabilities regarding self-assembling and the direct binding to chitin and N-acetylglucosamine (NAG) tetramers, a fungal cell wall building block, and to map a previously unknown binding region in MpCP5. Moreover, fibers of MpCP2 were shown to act as expansin and facilitate basidiospore germination whereas soluble MpCP5 blocked NAG6-induced defense response. The correlation between these roles, the fungus life cycle, and its tug-of-war interaction with cacao plants is discussed.
Assuntos
Agaricales/genética , Cacau/microbiologia , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Genoma Fúngico/genética , Doenças das Plantas/microbiologia , Acetilglucosamina/metabolismo , Agaricales/efeitos dos fármacos , Agaricales/crescimento & desenvolvimento , Agaricales/metabolismo , Sequência de Bases , Parede Celular/metabolismo , Quitina/metabolismo , Cristalografia por Raios X , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Expressão Gênica , Interações Hospedeiro-Patógeno , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Filogenia , Ligação Proteica , Análise de Sequência de DNA , Análise de Sequência de RNA , Esporos FúngicosRESUMO
Plants harbor a complex immune system to fight off invaders and prevent diseases. For decades, the interactions between plants and pathogens have been investigated primarily through the lens of binary interactions, largely neglecting the diversity of microbes that naturally inhabit plant tissues. Recent research, however, demonstrates that resident microbes are more than mere spectators. Instead, the plant microbiome extends host immune function and influences the outcome of a pathogen infection. Both plants and the interacting microbes produce a large diversity of metabolites that form an intricate chemical network of nutrients, signals, and antimicrobial molecules. In this review, we discuss the involvement of the plant microbiome in disease development, focusing on the biochemical conversation that occurs between plants and their associated microbiota before, during and after infection. We also highlight outstanding questions and possible directions for future research.
Assuntos
Microbiota , Plantas/metabolismoRESUMO
BACKGROUND: Synthetic biology allows the development of new biochemical pathways for the production of chemicals from renewable sources. One major challenge is the identification of suitable microorganisms to hold these pathways with sufficient robustness and high yield. In this work we analyzed the genome of the propionic acid producer Actinobacteria Propionibacterium acidipropionici (ATCC 4875). RESULTS: The assembled P. acidipropionici genome has 3,656,170 base pairs (bp) with 68.8% G + C content and a low-copy plasmid of 6,868 bp. We identified 3,336 protein coding genes, approximately 1000 more than P. freudenreichii and P. acnes, with an increase in the number of genes putatively involved in maintenance of genome integrity, as well as the presence of an invertase and genes putatively involved in carbon catabolite repression. In addition, we made an experimental confirmation of the ability of P. acidipropionici to fix CO2, but no phosphoenolpyruvate carboxylase coding gene was found in the genome. Instead, we identified the pyruvate carboxylase gene and confirmed the presence of the corresponding enzyme in proteome analysis as a potential candidate for this activity. Similarly, the phosphate acetyltransferase and acetate kinase genes, which are considered responsible for acetate formation, were not present in the genome. In P. acidipropionici, a similar function seems to be performed by an ADP forming acetate-CoA ligase gene and its corresponding enzyme was confirmed in the proteome analysis. CONCLUSIONS: Our data shows that P. acidipropionici has several of the desired features that are required to become a platform for the production of chemical commodities: multiple pathways for efficient feedstock utilization, ability to fix CO2, robustness, and efficient production of propionic acid, a potential precursor for valuable 3-carbon compounds.
Assuntos
Proteínas de Bactérias/genética , Genoma Bacteriano , Microbiologia Industrial , Propionatos/metabolismo , Propionibacterium/genética , Propionibacterium/metabolismo , Acetato-CoA Ligase/genética , Acetato-CoA Ligase/metabolismo , Proteínas de Bactérias/metabolismo , Composição de Bases , Sequência de Bases , Dióxido de Carbono/metabolismo , Redes e Vias Metabólicas , Dados de Sequência Molecular , Plasmídeos , Piruvato Carboxilase/genética , Piruvato Carboxilase/metabolismo , beta-Frutofuranosidase/genética , beta-Frutofuranosidase/metabolismoRESUMO
BACKGROUND: Plant pathogenesis related-1 (PR-1) proteins belong to the CAP superfamily and have been characterized as markers of induced defense against pathogens. Moniliophthora perniciosa and Moniliophthora roreri are hemibiotrophic fungi that respectively cause the witches' broom disease and frosty pod rot in Theobroma cacao. Interestingly, a large number of plant PR-1-like genes are present in the genomes of both species and many are up-regulated during the biotrophic interaction. In this study, we investigated the evolution of PR-1 proteins from 22 genomes of Moniliophthora isolates and 16 other Agaricales species, performing genomic investigation, phylogenetic reconstruction, positive selection search and gene expression analysis. RESULTS: Phylogenetic analysis revealed conserved PR-1 genes (PR-1a, b, d, j), shared by many Agaricales saprotrophic species, that have diversified in new PR-1 genes putatively related to pathogenicity in Moniliophthora (PR-1f, g, h, i), as well as in recent specialization cases within M. perniciosa biotypes (PR-1c, k, l) and M. roreri (PR-1n). PR-1 families in Moniliophthora with higher evolutionary rates exhibit induced expression in the biotrophic interaction and positive selection clues, supporting the hypothesis that these proteins accumulated adaptive changes in response to host-pathogen arms race. Furthermore, although previous work showed that MpPR-1 can detoxify plant antifungal compounds in yeast, we found that in the presence of eugenol M. perniciosa differentially expresses only MpPR-1e, k, d, of which two are not linked to pathogenicity, suggesting that detoxification might not be the main function of most MpPR-1. CONCLUSIONS: Based on analyses of genomic and expression data, we provided evidence that the evolution of PR-1 in Moniliophthora was adaptive and potentially related to the emergence of the parasitic lifestyle in this genus. Additionally, we also discuss how fungal PR-1 proteins could have adapted from basal conserved functions to possible roles in fungal pathogenesis.
Assuntos
Agaricales , Doenças das Plantas , Agaricales/genética , Humanos , Estilo de Vida , FilogeniaRESUMO
Plants recognize a wide variety of microbial molecules to detect and respond to potential invaders. Recognition of Microbe-Associated Molecular Patterns (MAMPs) by cell surface receptors initiate a cascade of biochemical responses that include, among others, ion fluxes across the plasma membrane. A consequence of such event is a decrease in the concentration of extracellular H+ ions, which can be experimentally detected in plant cell suspensions as a shift in the pH of the medium. Thus, similarly to reactive oxygen species (ROS) accumulation, phosphorylation of MAP kinases and induction of defense-related genes, MAMP-induced medium alkalinization can be used as a proxy for the activation of plant immune responses. Here, we describe a detailed protocol for the measurement of medium alkalinization of tobacco BY-2 cell suspensions upon treatment with two different MAMPs: chitohexamers derived from fungal cell walls (NAG6; N-acetylglucosamine) and the flagellin epitope flg22, found in the bacterial flagellum. This method provides a reliable and fast platform to access MAMP-Triggered Immunity (MTI) in tobacco cell suspensions and can be easily adapted to other plant species as well as to other MAMPs.
RESUMO
Assimilate partitioning to the root system is a desirable developmental trait to control but little is known of the signaling pathway underlying partitioning. A null mutation in the gene encoding the Gß subunit of the heterotrimeric G protein complex, a nexus for a variety of signaling pathways, confers altered sugar partitioning in roots. While fixed carbon rapidly reached the roots of wild type and agb1-2 mutant seedlings, agb1 roots had more of this fixed carbon in the form of glucose, fructose, and sucrose which manifested as a higher lateral root density. Upon glucose treatment, the agb1-2 mutant had abnormal gene expression in the root tip validated by transcriptome analysis. In addition, PIN2 membrane localization was altered in the agb1-2 mutant. The heterotrimeric G protein complex integrates photosynthesis-derived sugar signaling incorporating both membrane-and transcriptional-based mechanisms. The time constants for these signaling mechanisms are in the same range as photosynthate delivery to the root, raising the possibility that root cells are able to use changes in carbon fixation in real time to adjust growth behavior.
RESUMO
Simultaneous multiplex mutation of large gene families using Cas9 has the potential to revolutionize agriculture and plant sciences. The targeting of multiple genomic sites at once raises concerns about the efficiency and specificity in targeting. The model Arabidopsis thaliana is widely used in basic plant research. Previous work has suggested that the Cas9 off-target rate in Arabidopsis is undetectable. Here we use deep sequencing on pooled plants simultaneously targeting 14 distinct genomic loci to demonstrate that multiplex targeting in Arabidopsis is highly specific to on-target sites with no detectable off-target events. In addition, chromosomal translocations are extremely rare. The high specificity of Cas9 in Arabidopsis makes this a reliable method for clean mutant generation with no need to enhance specificity or adopt alternate Cas9 variants.
Assuntos
Arabidopsis/genética , Sistemas CRISPR-Cas/genética , Marcação de Genes/métodos , Loci Gênicos/genética , Genoma de Planta/genética , Estudo de Associação Genômica Ampla , Translocação Genética/genéticaRESUMO
We identified loci responsible for natural variation in Arabidopsis thaliana (Arabidopsis) responses to a bacterial pathogen virulence factor, HopAM1. HopAM1 is a type III effector protein secreted by the virulent Pseudomonas syringae strain Pto DC3000. Delivery of HopAM1 from disarmed Pseudomonas strains leads to local cell death, meristem chlorosis, or both, with varying intensities in different Arabidopsis accessions. These phenotypes are not associated with differences in bacterial growth restriction. We treated the two phenotypes as quantitative traits to identify host loci controlling responses to HopAM1. Genome-wide association (GWA) of 64 Arabidopsis accessions identified independent variants highly correlated with response to each phenotype. Quantitative trait locus (QTL) mapping in a recombinant inbred population between Bur-0 and Col-0 accessions revealed genetic linkage to regions distinct from the top GWA hits. Two major QTL associated with HopAM1-induced cell death were also associated with HopAM1-induced chlorosis. HopAM1-induced changes in Arabidopsis gene expression showed that rapid HopAM1-dependent cell death in Bur-0 is correlated with effector-triggered immune responses. Studies of the effect of mutations in known plant immune system genes showed, surprisingly, that both cell death and chlorosis phenotypes are enhanced by loss of EDS1, a regulatory hub in the plant immune-signaling network. Our results reveal complex genetic architecture for response to this particular type III virulence effector, in contrast to the typical monogenic control of cell death and disease resistance triggered by most type III effectors.
Assuntos
Arabidopsis/genética , Arabidopsis/imunologia , Locos de Características Quantitativas/imunologia , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/imunologia , Genes de Plantas , Estudo de Associação Genômica Ampla , Fenótipo , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Pseudomonas syringae/genética , Pseudomonas syringae/imunologia , Fatores de Virulência/metabolismoRESUMO
The widespread SCP/TAPS superfamily (SCP/Tpx-1/Ag5/PR-1/Sc7) has multiple biological functions, including roles in the immune response of plants and animals, development of male reproductive tract in mammals, venom activity in insects and reptiles and host invasion by parasitic worms. Plant Pathogenesis Related 1 (PR-1) proteins belong to this superfamily and have been characterized as markers of induced defense against pathogens. This work presents the characterization of eleven genes homologous to plant PR-1 genes, designated as MpPR-1, which were identified in the genome of Moniliophthora perniciosa, a basidiomycete fungus responsible for causing the devastating witches' broom disease in cacao. We describe gene structure, protein alignment and modeling analyses of the MpPR-1 family. Additionally, the expression profiles of MpPR-1 genes were assessed by qPCR in different stages throughout the fungal life cycle. A specific expression pattern was verified for each member of the MpPR-1 family in the conditions analyzed. Interestingly, some of them were highly and specifically expressed during the interaction of the fungus with cacao, suggesting a role for the MpPR-1 proteins in the infective process of this pathogen. Hypothetical functions assigned to members of the MpPR-1 family include neutralization of plant defenses, antimicrobial activity to avoid competitors and fruiting body physiology. This study provides strong evidence on the importance of PR-1-like genes for fungal virulence on plants.