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1.
Proc Natl Acad Sci U S A ; 120(15): e2221508120, 2023 04 11.
Article in English | MEDLINE | ID: mdl-37018204

ABSTRACT

Soil-dwelling microbes are the principal inoculum for the root microbiota, but our understanding of microbe-microbe interactions in microbiota establishment remains fragmentary. We tested 39,204 binary interbacterial interactions for inhibitory activities in vitro, allowing us to identify taxonomic signatures in bacterial inhibition profiles. Using genetic and metabolomic approaches, we identified the antimicrobial 2,4-diacetylphloroglucinol (DAPG) and the iron chelator pyoverdine as exometabolites whose combined functions explain most of the inhibitory activity of the strongly antagonistic Pseudomonas brassicacearum R401. Microbiota reconstitution with a core of Arabidopsis thaliana root commensals in the presence of wild-type or mutant strains revealed a root niche-specific cofunction of these exometabolites as root competence determinants and drivers of predictable changes in the root-associated community. In natural environments, both the corresponding biosynthetic operons are enriched in roots, a pattern likely linked to their role as iron sinks, indicating that these cofunctioning exometabolites are adaptive traits contributing to pseudomonad pervasiveness throughout the root microbiota.


Subject(s)
Arabidopsis , Microbiota , Bacteria/genetics , Microbiota/genetics , Symbiosis , Arabidopsis/genetics , Microbial Interactions , Plant Roots/genetics , Soil Microbiology
2.
STAR Protoc ; 1(3): 100226, 2020 12 18.
Article in English | MEDLINE | ID: mdl-33377117

ABSTRACT

We present a gnotobiotic system for microbiota reconstitution on Arabidopsis thaliana under contrasting iron availability. This system induces iron starvation in plants by providing an unavailable form, mimicking conditions in alkaline soils. Inoculation of taxonomically diverse bacteria reconstitutes plants with a synthetic microbiota, allowing observation of nutrient-dependent interactions with commensals. Experimental optimization, including media composition and preparation of seedlings and bacteria, is discussed. This system provides a framework that can be adapted to study plant-microbiota interactions in further nutritional contexts. For complete details on the use and execution of this protocol, please refer to Harbort et al. (2020).


Subject(s)
Cell Culture Techniques/methods , Germ-Free Life/physiology , Plant Roots/growth & development , Arabidopsis/growth & development , Arabidopsis/microbiology , Bacteria , Microbiota , Plant Roots/microbiology , Soil , Soil Microbiology , Symbiosis
3.
Cell Host Microbe ; 28(6): 825-837.e6, 2020 12 09.
Article in English | MEDLINE | ID: mdl-33027611

ABSTRACT

Plants benefit from associations with a diverse community of root-colonizing microbes. Deciphering the mechanisms underpinning these beneficial services are of interest for improving plant productivity. We report a plant-beneficial interaction between Arabidopsis thaliana and the root microbiota under iron deprivation that is dependent on the secretion of plant-derived coumarins. Disrupting this pathway alters the microbiota and impairs plant growth in iron-limiting soil. Furthermore, the microbiota improves iron-limiting plant performance via a mechanism dependent on plant iron import and secretion of the coumarin fraxetin. This beneficial trait is strain specific yet functionally redundant across phylogenetic lineages of the microbiota. Transcriptomic and elemental analyses revealed that this interaction between commensals and coumarins promotes growth by relieving iron starvation. These results show that coumarins improve plant performance by eliciting microbe-assisted iron nutrition. We propose that the bacterial root microbiota, stimulated by secreted coumarins, is an integral mediator of plant adaptation to iron-limiting soils.


Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/microbiology , Arabidopsis/physiology , Coumarins/metabolism , Iron/metabolism , Plant Roots/microbiology , Plant Roots/physiology , Gene Expression Profiling , Microbiota , Phylogeny , Rhizosphere , Secondary Metabolism , Soil/chemistry , Soil Microbiology , Symbiosis
4.
Dev Cell ; 43(4): 449-462.e5, 2017 11 20.
Article in English | MEDLINE | ID: mdl-29103955

ABSTRACT

Neutrophils are essential for immune defense and can respond to infection by releasing chromatin in the form of neutrophil extracellular traps (NETs). Here we show that NETs are induced by mitogens and accompanied by induction of cell-cycle markers, including phosphorylation of the retinoblastoma protein and lamins, nuclear envelope breakdown, and duplication of centrosomes. We identify cyclin-dependent kinases 4 and 6 (CDK4/6) as essential regulators of NETs and show that the response is inhibited by the cell-cycle inhibitor p21Cip. CDK6, in neutrophils, is required for clearance of the fungal pathogen Candida albicans. Our data describe a function for CDK4/6 in immunity.


Subject(s)
Cell Cycle/physiology , Extracellular Traps/metabolism , Neutrophil Activation/physiology , Neutrophils/metabolism , Animals , Cell Cycle/immunology , Cyclin-Dependent Kinase 4/genetics , Cyclin-Dependent Kinase 4/metabolism , Cyclin-Dependent Kinase 6/genetics , Cyclin-Dependent Kinase 6/metabolism , Extracellular Traps/immunology , Mice, Transgenic , Phosphorylation , Retinoblastoma Protein/immunology , Retinoblastoma Protein/metabolism
5.
Methods Mol Biol ; 1124: 307-18, 2014.
Article in English | MEDLINE | ID: mdl-24504961

ABSTRACT

Neutrophil extracellular trap (NET) formation is a recently discovered process in the field of innate immunity. It is important to have consistent standards in inducing and quantifying NET formation to compare data from different labs in this new area of investigation. Here we describe the conditions of neutrophil isolation from peripheral blood and stimulation that we find allow the study of NETosis in vitro. The criteria for conclusively identifying the process of NETosis, and the pros and cons of various quantification methods are discussed.


Subject(s)
Extracellular Space/immunology , Neutrophils/immunology , Cell Separation/methods , Humans , Microscopy, Fluorescence/methods
6.
Science ; 333(6042): 596-601, 2011 Jul 29.
Article in English | MEDLINE | ID: mdl-21798943

ABSTRACT

Plants generate effective responses to infection by recognizing both conserved and variable pathogen-encoded molecules. Pathogens deploy virulence effector proteins into host cells, where they interact physically with host proteins to modulate defense. We generated an interaction network of plant-pathogen effectors from two pathogens spanning the eukaryote-eubacteria divergence, three classes of Arabidopsis immune system proteins, and ~8000 other Arabidopsis proteins. We noted convergence of effectors onto highly interconnected host proteins and indirect, rather than direct, connections between effectors and plant immune receptors. We demonstrated plant immune system functions for 15 of 17 tested host proteins that interact with effectors from both pathogens. Thus, pathogens from different kingdoms deploy independently evolved virulence proteins that interact with a limited set of highly connected cellular hubs to facilitate their diverse life-cycle strategies.


Subject(s)
Arabidopsis/immunology , Arabidopsis/metabolism , Host-Pathogen Interactions , Plant Diseases/immunology , Plant Immunity , Receptors, Immunologic/metabolism , Virulence Factors/metabolism , Arabidopsis/genetics , Arabidopsis/microbiology , Bacterial Proteins/metabolism , Evolution, Molecular , Genes, Plant , Immunity, Innate , Oomycetes/pathogenicity , Protein Interaction Mapping , Pseudomonas syringae/pathogenicity
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