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1.
Proc Natl Acad Sci U S A ; 120(38): e2301003120, 2023 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-37695902

RESUMO

Clustered protocadherin (Pcdh) functions as a cell recognition molecule through the homophilic interaction in the central nervous system. However, its interactions have not yet been visualized in neurons. We previously reported PcdhγB2-Förster resonance energy transfer (FRET) probes to be applicable only to cell lines. Herein, we designed γB2-FRET probes by fusing FRET donor and acceptor fluorescent proteins to a single γB2 molecule and succeeded in visualizing γB2 homophilic interaction in cultured hippocampal neurons. The γB2-FRET probe localized in the soma and neurites, and FRET signals, which were observed at contact sites between neurites, eliminated by ethylene glycol tetraacetic acid (EGTA) addition. Live imaging revealed that the FRET-negative γB2 signals rapidly moved along neurites and soma, whereas the FRET-positive signals remained in place. We observed that the γB2 proteins at synapses rarely interact homophilically. The γB2-FRET probe might allow us to elucidate the function of the homophilic interaction and the cell recognition mechanism.


Assuntos
Neurônios , Protocaderinas , Neuritos , Corpo Celular , Comunicação Celular
2.
J Exp Bot ; 75(13): 3891-3902, 2024 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-38486360

RESUMO

Self-incompatibility (SI) is a crucial mechanism that prevents self-fertilization and inbreeding in flowering plants. Citrus exhibits SI regulated by a polymorphic S-locus containing an S-RNase gene and multiple S-locus F-box (SLF) genes. It has been documented that S-RNase functions as the pistil S determinant, but there is no direct evidence that the SLF genes closely linked with S-RNase function as pollen S determinants in Citrus. This study assembled the genomes of two pummelo (Citrus grandis) plants, obtained three novel complete and well-annotated S-haplotypes, and isolated 36 SLF or SLF-like alleles on the S-loci. Phylogenetic analysis of 138 SLFs revealed that the SLF genes were classified into 12 types, including six types with divergent or missing alleles. Furthermore, transformation experiments verified that the conserved S6-SLF7a protein can lead to the transition of SI to self-compatibility by recognizing non-self S8-RNase in 'Mini-Citrus' plants (S7S8 and S8S29, Fortunella hindsii), a model plant for citrus gene function studies. In vitro assays demonstrated interactions between SLFs of different S haplotypes and the Skp1-Cullin1-F-box subunit CgSSK1 protein. This study provides direct evidence that SLF controls the pollen function in Citrus, demonstrating its role in the 'non-self recognition' SI system.


Assuntos
Citrus , Proteínas F-Box , Filogenia , Proteínas de Plantas , Pólen , Ribonucleases , Autoincompatibilidade em Angiospermas , Citrus/genética , Citrus/fisiologia , Citrus/metabolismo , Autoincompatibilidade em Angiospermas/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Pólen/genética , Pólen/fisiologia , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Ribonucleases/metabolismo , Ribonucleases/genética , Sequência de Aminoácidos
3.
J Struct Biol ; 214(1): 107812, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34800649

RESUMO

In the ciliate Euplotes raikovi, water-borne protein pheromones promote the vegetative cell growth and mating by competitively binding as autocrine and heterologous signals to putative cell receptors represented by membrane-bound pheromone isoforms. A previously determined crystal structure of pheromone Er-1 supported a pheromone/receptor binding model in which strong protein-protein interactions result from the cooperative utilization of two distinct types of contact interfaces that arrange molecules into linear chains, and these into two-dimensional layers. We have now determined the crystal structure of a new pheromone, Er-13, isolated from cultures that are strongly mating reactive withculturessource of pheromone Er-1.The comparison between the Er-1 and Er-13 crystal structuresreinforces the fundamental of the cooperative model of pheromone/receptor binding, in that the molecules arrange into linear chains taking a rigorously alternate opposite orientation reflecting the presumed mutual orientation of pheromone and receptor molecules on the cell surface. In addition, the comparison provides two new lines of evidence for a univocal rationalization of observations on the differentbehaviourbetween the autocrine and heterologous pheromone/receptor complexes. (i) In the Er-13 crystal, chains do not form layers which thus appear to be an over-structureunique tothe Er-1 crystal, not essential for the pheromone signalling mechanisms. (ii) In both crystal structures, the intra-chain interfaces are equally derived from burying amino-acid side-chains mostly residing on helix-3 of the three-helical pheromonefold. This helix is thus identified as the key structural motif underlying the pheromone activity, in line with its tight intra- and interspecificstructuralconservation.


Assuntos
Euplotes , Euplotes/química , Euplotes/metabolismo , Proteínas de Membrana/química , Feromônios/química , Feromônios/metabolismo , Ligação Proteica , Proteínas de Protozoários/metabolismo
4.
Plant Cell Environ ; 45(1): 236-247, 2022 01.
Artigo em Inglês | MEDLINE | ID: mdl-34708407

RESUMO

Upon sensing attack by pathogens and insect herbivores, plants release complex mixtures of volatile compounds. Here, we show that the infection of lima bean (Phaseolus lunatus L.) plants with the non-host bacterial pathogen Pseudomonas syringae pv. tomato led to the production of microbe-induced plant volatiles (MIPVs). Surprisingly, the bacterial type III secretion system, which injects effector proteins directly into the plant cytosol to subvert host functions, was found to prime both intra- and inter-specific defense responses in neighbouring wild tobacco (Nicotiana benthamiana) plants. Screening of each of 16 effectors using the Pseudomonas fluorescens effector-to-host analyser revealed that an effector, HopP1, was responsible for immune activation in receiver tobacco plants. Further study demonstrated that 1-octen-3-ol, 3-octanone and 3-octanol are novel MIPVs emitted by the lima bean plant in a HopP1-dependent manner. Exposure to synthetic 1-octen-3-ol activated immunity in tobacco plants against a virulent pathogen Pseudomonas syringae pv. tabaci. Our results show for the first time that a bacterial type III effector can trigger the emission of C8 plant volatiles that mediate defense priming via plant-plant interactions. These results provide novel insights into the role of airborne chemicals in bacterial pathogen-induced inter-specific plant-plant interactions.


Assuntos
Interações Hospedeiro-Patógeno/fisiologia , Imunidade Vegetal , Pseudomonas syringae/patogenicidade , Sistemas de Secreção Tipo III/fisiologia , Compostos Orgânicos Voláteis/metabolismo , Ar , Capsicum/fisiologia , Cucumis sativus/fisiologia , Regulação da Expressão Gênica de Plantas , Octanóis/farmacologia , Phaseolus/fisiologia , Imunidade Vegetal/efeitos dos fármacos , Transdução de Sinais , Nicotiana/fisiologia , Compostos Orgânicos Voláteis/farmacologia
5.
Proc Natl Acad Sci U S A ; 113(8): 2062-7, 2016 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-26858412

RESUMO

Transmission of mycoviruses that attenuate virulence (hypovirulence) of pathogenic fungi is restricted by allorecognition systems operating in their fungal hosts. We report the use of systematic molecular gene disruption and classical genetics for engineering fungal hosts with superior virus transmission capabilities. Four of five diallelic virus-restricting allorecognition [vegetative incompatibility (vic)] loci were disrupted in the chestnut blight fungus Cryphonectria parasitica using an adapted Cre-loxP recombination system that allowed excision and recycling of selectable marker genes (SMGs). SMG-free, quadruple vic mutant strains representing both allelic backgrounds of the remaining vic locus were then produced through mating. In combination, these super donor strains were able to transmit hypoviruses to strains that were heteroallelic at one or all of the virus-restricting vic loci. These results demonstrate the feasibility of modulating allorecognition to engineer pathogenic fungi for more efficient transmission of virulence-attenuating mycoviruses and enhanced biological control potential.


Assuntos
Micovírus , Engenharia Genética , Loci Gênicos , Sordariales , Aesculus/microbiologia , Micovírus/genética , Micovírus/metabolismo , Micovírus/patogenicidade , Doenças das Plantas/microbiologia , Sordariales/genética , Sordariales/metabolismo , Sordariales/virologia
6.
Development ; 142(20): 3561-70, 2015 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-26395484

RESUMO

The social amoeba Dictyostelium discoideum integrates into a multicellular organism when individual starving cells aggregate and form a mound. The cells then integrate into defined tissues and develop into a fruiting body that consists of a stalk and spores. Aggregation is initially orchestrated by waves of extracellular cyclic adenosine monophosphate (cAMP), and previous theory suggested that cAMP and other field-wide diffusible signals mediate tissue integration and terminal differentiation as well. Cooperation between cells depends on an allorecognition system comprising the polymorphic adhesion proteins TgrB1 and TgrC1. Binding between compatible TgrB1 and TgrC1 variants ensures that non-matching cells segregate into distinct aggregates prior to terminal development. Here, we have embedded a small number of cells with incompatible allotypes within fields of developing cells with compatible allotypes. We found that compatibility of the allotype encoded by the tgrB1 and tgrC1 genes is required for tissue integration, as manifested in cell polarization, coordinated movement and differentiation into prestalk and prespore cells. Our results show that the molecules that mediate allorecognition in D. discoideum also control the integration of individual cells into a unified developing organism, and this acts as a gating step for multicellularity.


Assuntos
Dictyostelium/citologia , Regulação da Expressão Gênica no Desenvolvimento , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Proteínas de Protozoários/metabolismo , Alelos , Animais , Adesão Celular , Comunicação Celular , Diferenciação Celular , Movimento Celular , Quimiotaxia/fisiologia , AMP Cíclico/metabolismo , Dictyostelium/genética , Perfilação da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Morfogênese , Ligação Proteica , Recombinação Genética , Transcrição Gênica
7.
BMC Plant Biol ; 18(1): 357, 2018 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-30558544

RESUMO

BACKGROUND: Pinellia ternata is a Chinese traditional medicinal herb, used to cure diseases including insomnia, eclampsia and cervical carcinoma, for hundreds of years. Non-self-recognition in multicellular organisms can initiate the innate immunity to avoid the invasion of pathogens. A design for pathogen independent, heterosis based, fresh resistance can be generated in F1 hybrid was proposed. RESULTS: By library functional screening, we found that P. ternata genes, named as ptHR375 and ptHR941, were identified with the potential to trigger a hypersensitive response in Nicotiana benthamiana. Significant induction of ROS and Callose deposition in N. benthamiana leaves along with activation of pathogenesis-related genes viz.; PR-1a, PR-5, PDF1.2, NPR1, PAL, RBOHB and ERF1 and antioxidant enzymes was observed. After transformation into N. benthamiana, expression of pathogenesis related genes was significantly up-regulated to generate high level of resistance against Phytophthora capsici without affecting the normal seed germination and morphological characters of the transformed N. benthamiana. UPLC-QTOF-MS analysis of ptHR375 transformed N. benthamiana revealed the induction of Oxytetracycline, Cuelure, Allantoin, Diethylstilbestrol and 1,2-Benzisothiazol-3(2H)-one as bioactive compounds. Here we also proved that F1 hybrids, produced by crossing of the ptHR375 and ptHR941 transformed and non-transformed N. benthamiana, show significant high levels of PR-gene expressions and pathogen resistance. CONCLUSIONS: Heterologous plant genes can activate disease resistance in another plant species and furthermore, by generating F1 hybrids, fresh pathogen independent plant immunity can be obtained. It is also concluded that ptHR375 and ptHR941 play their role in SA and JA/ET defense pathways to activate the resistance against invading pathogens.


Assuntos
Nicotiana/genética , Nicotiana/imunologia , Pinellia/genética , Imunidade Vegetal/genética , Antioxidantes/metabolismo , Regulação da Expressão Gênica de Plantas , Glucanos/genética , Glucanos/metabolismo , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Phytophthora/patogenicidade , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Folhas de Planta/genética , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Espécies Reativas de Oxigênio/metabolismo
8.
Adv Exp Med Biol ; 1003: 3-14, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28667551

RESUMO

The immune system was identified as a protective factor during infectious diseases over a century ago. Current definitions and textbook information are still largely influenced by these early observations, and the immune system is commonly presented as a defence machinery. However, host defence is only one manifestation of the immune system's overall function in the maintenance of tissue homeostasis and system integrity. In fact, the immune system is integral part of fundamental physiological processes such as development, reproduction and wound healing, and a close crosstalk between the immune system and other body systems such as metabolism, the central nervous system and the cardiovascular system is evident. Research and medical professionals in an expanding range of areas start to recognise the implications of the immune system in their respective fields.This chapter provides a brief historical perspective on how our understanding of the immune system has evolved from a defence system to an overarching surveillance machinery to maintain tissue integrity. Current perspectives on the non-defence functions of classical immune cells and factors will also be discussed.


Assuntos
Doenças Transmissíveis/imunologia , Sistema Imunitário/imunologia , Animais , Comunicação Celular , Microambiente Celular , Doenças Transmissíveis/metabolismo , Doenças Transmissíveis/fisiopatologia , Homeostase , Humanos , Sistema Imunitário/metabolismo , Sistema Imunitário/fisiopatologia , Transdução de Sinais
9.
Plant Cell Physiol ; 57(11): 2403-2416, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27565207

RESUMO

Self-incompatibility (SI) in flowering plants is a genetic reproductive barrier to distinguish self- and non-self pollen to promote outbreeding. In Solanaceae, self-pollen is rejected by the ribonucleases expressed in the styles (S-RNases), via its cytotoxic function. On the other side, the male-determinant is the S-locus F-box proteins (SLFs) expressed in pollen. Multiple SLFs collaboratively detoxify non-self S-RNases, therefore, non-self recognition is the mode of self-/non-self discrimination in Solanaceae. It is considered that SLFs function as a substrate-recognition module of the Skp1-Cullin1-F-box (SCF) complex that inactivates non-self S-RNases via their polyubiquitination, which leads to degradation by 26S proteasome. In fact, PhSSK1 (Petunia hybrida SLF-interacting Skp1-like1) was identified as a specific component of SCFSLF and was shown to be essential for detoxification of S-RNase in Petunia However, different molecules are proposed as the candidate Cullin1, another component of SCFSLF, and there is as yet no definite conclusion. Here, we identified five Cullin1s from the expressed sequence tags (ESTs) derived from the male reproductive organ in Petunia Among them, only PhCUL1-P was co-immunoprecipitated with S7-SLF2. In vitro protein-binding assay suggested that PhSSK1 specifically forms a complex with PhCUL1-P in an SLF-dependent manner. Knockdown of PhCUL1-P suppressed fertility of transgenic pollen in cross-compatible pollination in the functional S-RNase-dependent manner. These results suggested that SCFSLF selectively uses PhCUL1-P. Phylogeny of Cullin1s indicates that CUL1-P is recruited into the SI machinery during the evolution of Solanaceae, suggesting that the SI components have evolved differently among species in Solanaceae and Rosaceae, despite both families sharing the S-RNase-based SI.


Assuntos
Proteínas Culina/metabolismo , Petunia/metabolismo , Petunia/fisiologia , Proteínas de Plantas/metabolismo , Autoincompatibilidade em Angiospermas , Regulação da Expressão Gênica de Plantas , Genes de Plantas , MicroRNAs/metabolismo , Especificidade de Órgãos/genética , Penetrância , Petunia/genética , Filogenia , Proteínas de Plantas/genética , Pólen/genética , Polinização , Ligação Proteica , Reprodução , Ribonucleases/metabolismo , Rosaceae/genética , Autoincompatibilidade em Angiospermas/genética , Transgenes
10.
Biochem Biophys Res Commun ; 450(3): 1142-8, 2014 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-24878524

RESUMO

Sexual reproduction is an essential process for generating a genetic variety in the next generation. However, most flowering plants and hermaphroditic animals potentially allow self-fertilization. Approximately 60% of angiosperms possess a self-incompatibility (SI) system to avoid inbreeding. The SI system functions at a process of interaction between pollen (or pollen tube) and the pistil. These SI-responsible factors (S-determinants) in pollen and the pistil are encoded by highly polymorphic multiallelic genes in the S-locus, which are tightly linked making a single haplotype. Different taxonomic families utilize different types of S-determinant proteins. In contrast to the plant system, the mechanisms of SI in simultaneously hermaphroditic animals are largely unknown. Among them, promising candidates for SI in ascidians (primitive chordates) were recently identified. The SI system in the ascidian Cionaintestinalis was found to be very similar to those in flowering plants: The products of sperm- and egg-side multiallelic SI genes, which are tight linked and highly polymorphic, appear to be responsible for the SI system as revealed by genetic analysis. These findings led us to speculate that the SI systems in plants and animals evolved in a manner of convergent evolution. Here, we review the current understanding of the molecular mechanisms of the SI system in flowering plants, particularly Brassicacea, and in ascidians from the viewpoint of common mechanisms shared by plants and animals.


Assuntos
Organismos Hermafroditas/fisiologia , Magnoliopsida/fisiologia , Autoincompatibilidade em Angiospermas/fisiologia , Animais , Ciona intestinalis/genética , Ciona intestinalis/fisiologia , Feminino , Organismos Hermafroditas/genética , Magnoliopsida/genética , Masculino , Modelos Biológicos , Reprodução/genética , Reprodução/fisiologia , Autoincompatibilidade em Angiospermas/genética , Urocordados/genética , Urocordados/fisiologia
11.
Fungal Genet Biol ; 64: 45-57, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24380733

RESUMO

In filamentous fungi a system known as somatic incompatibility (SI) governs self/non-self recognition. SI is controlled by a regulatory signaling network involving proteins encoded at the het (heterokaryon incompatible) loci. Despite the wide occurrence of SI, the molecular identity and structure of only a small number of het genes and their products have been characterized in the model fungi Neurospora crassa and Podospora anserina. Our aim was to identify and study the distribution and evolution of putative het gene homologs in the Basidiomycota. For this purpose we used the information available for the model fungi to identify homologs of het genes in other fungi, especially the Basidiomycota. Putative het-c, het-c2 and un-24 homologs, as well as sequences containing the NACHT, HET or WD40 domains present in the het-e, het-r, het-6 and het-d genes were identified in certain members of the Ascomycota and Basidiomycota. The widespread phylogenetic distribution of certain het genes may reflect the fact that the encoded proteins are involved in fundamental cellular processes other than SI. Although homologs of het-S were previously known only from the Sordariomycetes (Ascomycota), we also identified a putative homolog of this gene in Gymnopus luxurians (Basidiomycota, class Agaricomycetes). Furthermore, with the exception of un-24, all of the putative het genes identified occurred mostly in a multi-copy fashion, some with lineage and species-specific expansions. Overall our results indicated that gene duplication followed by gene loss and/or gene family expansion, as well as multiple events of domain fusion and shuffling played an important role in the evolution of het gene homologs of Basidiomycota and other filamentous fungi.


Assuntos
Basidiomycota/genética , Genes Fúngicos , Ascomicetos/genética , Mapeamento Cromossômico , Evolução Molecular , Genoma Fúngico
12.
Essays Biochem ; 66(5): 447-458, 2022 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-35383834

RESUMO

Perception of non-self molecules known as microbe-associated molecular patterns (MAMPs) by host pattern recognition receptors (PRRs) activates plant pattern-triggered immunity (PTI). Pathogen infections often trigger the release of modified-self molecules, termed damage- or danger-associated molecular patterns (DAMPs), which modulate MAMP-triggered signaling to shape the frontline of plant immune responses against infections. In the context of advances in identifying MAMPs and DAMPs, cognate receptors, and their signaling, here, we focus on the most recent breakthroughs in understanding the perception and role of non-self and modified-self patterns. We highlight the commonalities and differences of MAMPs from diverse microbes, insects, and parasitic plants, as well as the production and perception of DAMPs upon infections. We discuss the interplay between MAMPs and DAMPs for emerging themes of the mutual potentiation and attenuation of PTI signaling upon MAMP and DAMP perception during infections.


Assuntos
Imunidade Vegetal , Receptores de Reconhecimento de Padrão , Imunidade Vegetal/fisiologia , Plantas , Transdução de Sinais
13.
Elife ; 112022 08 30.
Artigo em Inglês | MEDLINE | ID: mdl-36040303

RESUMO

Organisms require the ability to differentiate themselves from organisms of different or even the same species. Allorecognition processes in filamentous fungi are essential to ensure identity of an interconnected syncytial colony to protect it from exploitation and disease. Neurospora crassa has three cell fusion checkpoints controlling formation of an interconnected mycelial network. The locus that controls the second checkpoint, which allows for cell wall dissolution and subsequent fusion between cells/hyphae, cwr (cell wall remodeling), encodes two linked genes, cwr-1 and cwr-2. Previously, it was shown that cwr-1 and cwr-2 show severe linkage disequilibrium with six different haplogroups present in N. crassa populations. Isolates from an identical cwr haplogroup show robust fusion, while somatic cell fusion between isolates of different haplogroups is significantly blocked in cell wall dissolution. The cwr-1 gene encodes a putative polysaccharide monooxygenase (PMO). Herein we confirm that CWR-1 is a C1-oxidizing chitin PMO. We show that the catalytic (PMO) domain of CWR-1 was sufficient for checkpoint function and cell fusion blockage; however, through analysis of active-site, histidine-brace mutants, the catalytic activity of CWR-1 was ruled out as a major factor for allorecognition. Swapping a portion of the PMO domain (V86 to T130) did not switch cwr haplogroup specificity, but rather cells containing this chimera exhibited a novel haplogroup specificity. Allorecognition to mediate cell fusion blockage is likely occurring through a protein-protein interaction between CWR-1 with CWR-2. These data highlight a moonlighting role in allorecognition of the CWR-1 PMO domain.


Assuntos
Neurospora crassa , Quitina , Proteínas Fúngicas/genética , Histidina , Oxigenases de Função Mista/genética , Neurospora crassa/genética
14.
Elife ; 112022 10 07.
Artigo em Inglês | MEDLINE | ID: mdl-36206043

RESUMO

How does a protein at the cell wall determine if a newly encountered fungus is safe to fuse with?


Assuntos
Neurospora crassa , Parede Celular/metabolismo , Neurospora crassa/metabolismo
15.
Methods Mol Biol ; 2132: 669-682, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32306366

RESUMO

Tectonins are conserved defense proteins of innate immune systems featuring a ß-propeller fold. Tectonin 2 from Laccaria bicolor, Lb-Tec2, is the first fungal representative of the tectonin superfamily that has been described. In-depth characterization revealed a specificity for O-methylated glycans and identified a unique sequence motif and binding site architecture underlying this unusual specificity. This chapter provides information on how to produce and purify recombinant Lb-Tec2, characterize its interaction with O-methylated glycans and demonstrate its biological function.


Assuntos
Laccaria/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Motivos de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas de Membrana/genética , Modelos Moleculares , Polissacarídeos , Ligação Proteica , Estrutura Secundária de Proteína
16.
Bio Protoc ; 10(5): e3543, 2020 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-33659517

RESUMO

Many bacteria take part in self recognition and kin discrimination behavior using contact-dependent effectors. Understanding the effects these effectors cause is important to explain bacterial community formation and population dynamics. Typically, kin discrimination effectors are toxins that kill target cells; their effect is therefore obvious and easily measurable. However, many self-recognition effectors, such as the Proteus mirabilis Ids system, are non-lethal and do not cause obvious physiological changes in target cells. Previously, experimental techniques to probe cells experiencing non-lethal kin recognition have been limited. Here we describe a technique to reliably isolate cells deemed self and non-self through Ids self-recognition for downstream phenotypic analysis. Liquid cultures of fluorescently labeled self-recognition mutants are mixed together and inoculated on swarm-permissive agar. Mixed swarms are harvested, and each strain is isolated through fluorescence-activated cell sorting (FACS). The growth rate of each strain is measured on a plate reader. This protocol is adaptable for other bacterial species. We describe briefly how sorted particles can be used for other analyses such as RNA-Seq library preparation.

17.
Front Immunol ; 11: 1911, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33013842

RESUMO

The complement system is part of the innate immune system and plays an important role in the host defense against infectious pathogens. One of the main effects is the opsonization of foreign invaders and subsequent uptake by phagocytosis. Due to the continuous default basal level of active complement molecules, a tight regulation is required to protect the body's own cells (self cells) from opsonization and from complement damage. A major complement regulator is Factor H, which is recruited from the fluid phase and attaches to cell surfaces where it effectively controls complement activation. Besides self cells, pathogens also have the ability to bind Factor H; they can thus escape opsonization and phagocytosis causing severe infections. In order to advance our understanding of the opsonization process at a quantitative level, we developed a mathematical model for the dynamics of the complement system-termed DynaCoSys model-that is based on ordinary differential equations for cell surface-bound molecules and on partial differential equations for concentration profiles of the fluid phase molecules in the environment of cells. This hybrid differential equation approach allows to model the complement cascade focusing on the role of active C3b in the fluid phase and on the cell surface as well as on its inactivation on the cell surface. The DynaCoSys model enables us to quantitatively predict the conditions under which Factor H mediated complement evasion occurs. Furthermore, investigating the quantitative impact of model parameters by a sensitivity analysis, we identify the driving processes of complement activation and regulation in both the self and non-self regime. The two regimes are defined by a critical Factor H concentration on the cell surface and we use the model to investigate the differential impact of complement model parameters on this threshold value. The dynamic modeling on the surface of pathogens are further relevant to understand pathophysiological situations where Factor H mutants and defective Factor H binding to target surfaces results in pathophysiology such as renal and retinal disease. In the future, this DynaCoSys model will be extended to also enable evaluating treatment strategies of complement-related diseases.


Assuntos
Doenças Transmissíveis/imunologia , Ativação do Complemento , Evasão da Resposta Imune , Imunidade Inata , Modelos Imunológicos , Tolerância a Antígenos Próprios , Doenças Transmissíveis/metabolismo , Complemento C3b/imunologia , Complemento C3b/metabolismo , Fator H do Complemento/imunologia , Fator H do Complemento/metabolismo , Humanos , Fagocitose , Ligação Proteica , Transdução de Sinais
18.
Trends Microbiol ; 28(7): 543-553, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32544441

RESUMO

The battle between microbes and their viruses is ancient and ongoing. Clustered regularly interspaced short palindromic repeat (CRISPR) immunity, the first and, to date, only form of adaptive immunity found in prokaryotes, represents a flexible mechanism to recall past infections while also adapting to a changing pathogenic environment. Critical to the role of CRISPR as an adaptive immune mechanism is its capacity for self versus non-self recognition when acquiring novel immune memories. Yet, CRISPR systems vary widely in both how and to what degree they can distinguish foreign from self-derived genetic material. We document known and hypothesized mechanisms that bias the acquisition of immune memory towards non-self targets. We demonstrate that diversity is the rule, with many widespread but no universal mechanisms for self versus non-self recognition.


Assuntos
Bactérias/virologia , Fenômenos Fisiológicos Bacterianos/imunologia , Bacteriófagos/genética , Sistemas CRISPR-Cas/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Bactérias/genética , Imunidade Inata/fisiologia
19.
Genetics ; 211(4): 1255-1267, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30718271

RESUMO

Cells rarely exist alone, which drives the evolution of diverse mechanisms for identifying and responding appropriately to the presence of other nearby cells. Filamentous fungi depend on somatic cell-to-cell communication and fusion for the development and maintenance of a multicellular, interconnected colony that is characteristic of this group of organisms. The filamentous fungus Neurospora crassa is a model for investigating the mechanisms of somatic cell-to-cell communication and fusion. N. crassa cells chemotropically grow toward genetically similar cells, which ultimately make physical contact and undergo cell fusion. Here, we describe the development of a Pprm1-luciferase reporter system that differentiates whether genes function upstream or downstream of a conserved MAP kinase (MAPK) signaling complex, by using a set of mutants required for communication and cell fusion. The vast majority of these mutants are deficient for self-fusion and for fusion when paired with wild-type cells. However, the Δham-11 mutant is unique in that it fails to undergo self-fusion, but chemotropic interactions and cell fusion are restored in Δham-11 + wild-type interactions. In genetically dissimilar cells, chemotropic interactions are regulated by genetic differences at doc-1 and doc-2, which regulate prefusion non-self recognition; cells with dissimilar doc-1 and doc-2 alleles show greatly reduced cell-fusion frequencies. Here, we show that HAM-11 functions in parallel with the DOC-1 and DOC-2 proteins to regulate the activity of the MAPK signaling complex. Together, our data support a model of integrated self and non-self recognition processes that modulate somatic cell-to-cell communication in N. crassa.


Assuntos
Quimiotaxia , Neurospora crassa/fisiologia , Percepção de Quorum , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Sistema de Sinalização das MAP Quinases , Neurospora crassa/genética
20.
Front Plant Sci ; 10: 751, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31263473

RESUMO

Plants can detect the presence of their neighbors belowground, often responding with changes in root growth for resource competition. Recent evidence also implies that perception of neighbors may also elicit defense responses, however, the associated metabolic activities are unclear. We investigated primary and defense-related secondary metabolisms and hormone expressions in tobaccos (Nicotiana rustica) grown either with own roots or roots of another conspecifics in hydroponic condition. The results showed that non-self root interaction significantly reduced photosynthetic activity and assimilate production, leading to a reduction of growth. Non-self interaction also modified plant phenylpropanoids metabolism, yielding higher lignin content (i.e., structural resistance) at whole plant level and higher phenolics accumulation (i.e., chemical defense) in roots. All these metabolic responses were associated with enhanced expressions of phytohormones, particularly jasmonic acid, salicylic acid and cytokinin in roots and abscisic acid in leaves, at the early stage of non-self interaction. Since the presence of neighbors often increase the probability of attacks from, e.g., pathogens and pests, this defense activation may act as an adaptation of plants to these possible upcoming attacks.

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