RESUMEN
The plasma membrane-localized receptor kinase FERONIA (FER) plays critical roles in a remarkable variety of biological processes throughout the life cycle of Arabidopsis thaliana. Revealing the molecular connections of FER that underlie these processes starts with identifying the proteins that interact with FER. We applied pupylation-based interaction tagging (PUP-IT) to survey cellular proteins in proximity to FER, encompassing weak and transient interactions that can be difficult to capture for membrane proteins. We reproducibly identified 581, 115, and 736 specific FER-interacting protein candidates in protoplasts, seedlings, and flowers, respectively. We also confirmed fourteen previously characterized FER-interacting proteins. Protoplast transient gene expression expedited the testing of new gene constructs for PUP-IT analyses and the validation of candidate proteins. We verified the proximity labeling of five selected candidates that were not previously characterized as FER-interacting proteins. The PUP-IT method could be a valuable tool to survey and validate protein-protein interactions for targets of interest in diverse subcellular compartments in plants.
RESUMEN
Microbiota benefit their hosts by improving nutrient uptake and pathogen protection. How host immunity restricts microbiota while avoiding autoimmunity is poorly understood. Here we show that the Arabidopsis phytosulfokine receptor 1 (pskr1) mutant displays autoimmunity (plant stunting, defence-gene expression and reduced rhizosphere bacterial growth) in response to growth-promoting Pseudomonas fluorescens. Microbiome profiling and microbiota colonization showed that PSKR1-mediated reduction in bacterial growth and stunting is largely specific to Pseudomonas. Transcriptional profiling demonstrated that PSKR1 regulates the growth-defence trade-off during Pseudomonas colonization: PSKR1 upregulates plant photosynthesis and root growth but suppresses salicylic-acid-mediated defences. Genetic epistasis experiments showed that pskr1 stunting and restriction of bacterial growth are salicylic acid dependent. Finally, we showed that Pseudomonas, but not other bacteria, induces PSKR1 expression in roots, suggesting that Pseudomonas might manipulate plant signalling to promote its colonization. Our data demonstrate a genetic mechanism to coordinate beneficial functions of the microbiome while preventing autoimmunity.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Microbiota , Arabidopsis/metabolismo , Rizosfera , Pseudomonas , Trastornos del Crecimiento , Raíces de Plantas/fisiología , Receptores de Superficie Celular/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismoRESUMEN
Nitrate, the major source of inorganic nitrogen for plants, is a critical signal controlling nutrient transport and assimilation and adaptive growth responses throughout the plant. Understanding how plants perceive nitrate and how this perception is transduced into responses that optimize growth are important for the rational improvement of crop productivity and for mitigating pollution from the use of fertilizers. This review highlights recent findings that reveal key roles of cytosolic-nuclear calcium signalling and dynamic protein phosphorylation via diverse mechanisms in the primary nitrate response (PNR). Nitrate-triggered calcium signatures as well as the critical functions of subgroup III calcium-sensor protein kinases, a specific protein phosphatase 2C, and RNA polymerase II C-terminal domain phosphatase-like 3 are discussed. Moreover, genome-wide meta-analysis of nitrate-regulated genes encoding candidate protein kinases and phosphatases for modulating critical phosphorylation events in the PNR are elaborated. We also consider how phosphoproteomics approaches can contribute to the identification of putative regulatory protein kinases in the PNR. Exploring and integrating experimental strategies, new methodologies, and comprehensive datasets will further advance our understanding of the molecular and cellular mechanisms underlying the complex regulatory processes in the PNR.
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Calcio , Nitratos , Calcio/metabolismo , Nitrógeno , Fosforilación , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
The filamentous fungus Fusarium oxysporum is a soilborne pathogen of many cultivated species and an opportunistic pathogen of humans. F. oxysporum f. sp. matthiolae is one of three formae speciales that are pathogenic to crucifers, including Arabidopsis thaliana, a premier model for plant molecular biology and genetics. Here, we report a genome assembly of F. oxysporum f. sp. matthiolae strain PHW726, generated using a combination of PacBio and Illumina sequencing technologies. The genome assembly presented here should facilitate in-depth investigation of F. oxysporum-Arabidopsis interactions and shed light on the genetics of fungal pathogenesis and plant immunity.
Asunto(s)
Brassicaceae , Fusarium , Genoma Fúngico , Arabidopsis/microbiología , Brassicaceae/microbiología , Fusarium/genética , Genoma Fúngico/genética , Enfermedades de las Plantas/microbiologíaRESUMEN
Three pathogenic forms, or formae speciales (f. spp.), of Fusarium oxysporum infect the roots of Arabidopsis thaliana below ground, instigating symptoms of wilt disease in leaves above ground. In previous reports, Arabidopsis mutants that are deficient in the biosynthesis of abscisic acid or salicylic acid or insensitive to ethylene or jasmonates exhibited either more or less wilt disease, than the wild-type, implicating the involvement of hormones in the normal host response to F. oxysporum. Our analysis of hormone-related mutants finds no evidence that endogenous hormones contribute to infection in roots. Mutants that are deficient in abscisic acid and insensitive to ethylene show no less infection than the wild-type, although they exhibit less disease. Whether a mutant that is insensitive to jasmonates affects infection depends on which forma specialis (f. sp.) is infecting the roots. Insensitivity to jasmonates suppresses infection by F. oxysporum f. sp. conglutinans and F. oxysporum f. sp. matthioli, which produce isoleucine- and leucine-conjugated jasmonate (JA-Ile/Leu), respectively, in culture filtrates, whereas insensitivity to jasmonates has no effect on infection by F. oxysporum f. sp. raphani, which produces no detectable JA-Ile/Leu. Furthermore, insensitivity to jasmonates has no effect on wilt disease of tomato, and the tomato pathogen F. oxysporum f. sp. lycopersici produces no detectable jasmonates. Thus, some, but not all, F. oxysporum pathogens appear to utilize jasmonates as effectors, promoting infection in roots and/or the development of symptoms in shoots. Only when the infection of roots is promoted by jasmonates is wilt disease enhanced in a mutant deficient in salicylic acid biosynthesis.
Asunto(s)
Arabidopsis/metabolismo , Arabidopsis/microbiología , Ciclopentanos/metabolismo , Fusarium/patogenicidad , Oxilipinas/metabolismo , Enfermedades de las Plantas/microbiología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Genes de Plantas , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/fisiología , Isoleucina/análogos & derivados , Isoleucina/metabolismo , Leucina/análogos & derivados , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Solanum lycopersicum/microbiología , Mutación , Enfermedades de las Plantas/genética , Reguladores del Crecimiento de las Plantas/genética , Reguladores del Crecimiento de las Plantas/metabolismo , Raíces de Plantas/microbiologíaRESUMEN
BACKGROUND: Susceptibility to Fusarium wilt disease varies among wild accessions of Arabidopsis thaliana. Six RESISTANCE TO FUSARIUM OXYSPORUM (RFO) quantitative trait loci (QTLs) controlling the resistance of accession Columbia-0 (Col-0) and susceptibility of Taynuilt-0 to Fusarium oxysporum forma specialis matthioli (FOM) are detected in a recombinant population derived from a single backcross of the F1 hybrid (BC1). In particular, the RFO1 QTL appears to interact with three other loci, RFO2, RFO4 and RFO6, and is attributed to the gene At1g79670. RESULTS: When resistance to FOM was mapped in a new BC(1) population, in which the loss-of-function mutant of At1g79670 replaced wild type as the Col-0 parent, RFO1's major effect and RFO1's interaction with RFO2, RFO4 and RFO6 were absent, showing that At1g79670 alone accounts for the RFO1 QTL. Resistance of two QTLs, RFO3 and RFO5, was independent of RFO1 and was reproduced in the new BC(1) population. In analysis of a third BC1 population, resistance to a second pathogen, F. oxysporum forma specialis conglutinans race 1 (FOC1), was mapped and the major effect locus RFO7 was identified. CONCLUSIONS: Natural quantitative resistance to F. oxysporum is largely specific to the infecting forma specialis because different RFO loci were responsible for resistance to FOM and FOC1. The mapping of quantitative disease resistance traits in BC(1) populations, generated from crosses between sequenced Arabidopsis accessions, can be a routine procedure when genome-wide genotyping is efficient, economical and accessible.
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Arabidopsis/inmunología , Arabidopsis/microbiología , Mapeo Cromosómico , Cruzamientos Genéticos , Resistencia a la Enfermedad/inmunología , Fusarium/fisiología , Enfermedades de las Plantas/microbiología , Arabidopsis/genética , Intercambio Genético , Resistencia a la Enfermedad/genética , Ligamiento Genético , Marcadores Genéticos , Genoma de Planta/genética , Técnicas de Genotipaje , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/inmunología , Sitios de Carácter Cuantitativo/genética , Reproducibilidad de los ResultadosRESUMEN
Resistance to wilt fungus Fusarium oxysporum f.sp. matthioli (FOM) is a polygenic trait in Arabidopsis thaliana. RFO3 is one of six quantitative trait loci accounting for the complete resistance of accession Columbia-0 (Col-0) and susceptibility of accession Taynuilt-0 (Ty-0). We find that Col-0 and Ty-0 alleles of RFO3 are representative of two common variants in wild Arabidopsis accessions, that resistance and susceptibility to FOM are ancestral features of the two variants and that resistance from RFO3 is unrivalled by other genes in a genome-wide survey of diversity in accessions. A single receptor-like kinase (RLK) gene in Col-0 is responsible for the resistance of RFO3, although the susceptible Ty-0 allele codes for two RLK homologs. Expression of RFO3 is highest in vascular tissue, which F. oxysporum infects, and root-expressed RFO3 restricts FOM infection of the vascular system. RFO3 confers specific resistance to FOM and provides no resistance to two other crucifer-infecting F. oxysporum pathogens. RFO3's identity, expression and specificity suggest that RFO3 represents diversity in pattern-recognition receptor (PRR) genes. The characteristics of RFO3 and the previously published RFO1 suggest that diversity in RLK PRRs is a major determinant of quantitative resistance in wild plant populations.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Resistencia a la Enfermedad/genética , Fusarium , Genes de Plantas , Enfermedades de las Plantas/genética , Sitios de Carácter Cuantitativo , Alelos , Arabidopsis/metabolismo , Arabidopsis/microbiología , Proteínas de Arabidopsis/metabolismo , Enfermedades de las Plantas/microbiología , Raíces de Plantas , Haz Vascular de Plantas , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Receptores de Reconocimiento de Patrones/genética , Receptores de Reconocimiento de Patrones/metabolismo , Especificidad de la EspecieRESUMEN
In the plant Arabidopsis thaliana, multiple quantitative trait loci (QTLs), including RFO2, account for the strong resistance of accession Columbia-0 (Col-0) and relative susceptibility of Taynuilt-0 (Ty-0) to the vascular wilt fungus Fusarium oxysporum forma specialis matthioli. We find that RFO2 corresponds to diversity in receptor-like protein (RLP) genes. In Col-0, there is a tandem pair of RLP genes: RFO2/At1g17250 confers resistance while RLP2 does not. In Ty-0, the highly diverged RFO2 locus has one RLP gene conferring weaker resistance. While the endogenous RFO2 makes a modest contribution to resistance, transgenic RFO2 provides strong pathogen-specific resistance. The extracellular leucine-rich repeats (eLRRs) in RFO2 and RLP2 are interchangeable for resistance and remarkably similar to eLRRs in the receptor-like kinase PSY1R, which perceives tyrosine-sulfated peptide PSY1. Reduced infection in psy1r and mutants of related phytosulfokine (PSK) receptor genes PSKR1 and PSKR2 shows that tyrosine-sulfated peptide signaling promotes susceptibility. The related eLRRs in RFO2 and PSY1R are not interchangeable; and expression of the RLP nPcR, in which eLRRs in RFO2 are replaced with eLRRs in PSY1R, results in constitutive resistance. Counterintuitively, PSY1 signaling suppresses nPcR because psy1r nPcR is lethal. The fact that PSK signaling does not similarly affect nPcR argues that PSY1 signaling directly downregulates the expression of nPcR. Our results support a speculative but intriguing model to explain RFO2's role in resistance. We propose that F. oxysporum produces an effector that inhibits the normal negative feedback regulation of PSY1R, which stabilizes PSY1 signaling and induces susceptibility. However, RFO2, acting as a decoy receptor for PSY1R, is also stabilized by the effector and instead induces host immunity. Overall, the quantitative resistance of RFO2 is reminiscent of the better-studied monogenic resistance traits.
Asunto(s)
Fusarium/patogenicidad , Péptidos/metabolismo , Enfermedades de las Plantas , Raíces de Plantas/genética , Tirosina/análogos & derivados , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/microbiología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Fusarium/genética , Fusarium/metabolismo , Regulación de la Expresión Génica de las Plantas , Péptidos/química , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Raíces de Plantas/microbiología , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Señales de Clasificación de Proteína/genética , Sitios de Carácter Cuantitativo , Transducción de Señal , Tirosina/química , Tirosina/metabolismoRESUMEN
Host-specific forms of Fusarium oxysporum infect the roots of numerous plant species. I present a novel application of familiar methodology to visualize and quantify F. oxysporum in roots. Infection in the roots of Arabidopsis thaliana, tomato, and cotton was detected with colorimetric reagents that are substrates for Fusarium spp.-derived arabinofuranosidase and N-acetyl-glucosaminidase activities and without the need for genetic modification of either plant host or fungal pathogen. Similar patterns of blue precipitation were produced by treatment with 5-bromo-4-chloro-3-indoxyl-α-l-arabinofuranoside and 5-bromo-4-chloro-3-indoxyl-2-acetamido-2-deoxy-ß-d-glucopyranoside, and these patterns were consistent with prior histological descriptions of F. oxysporum in roots. Infection was quantified in roots of wild-type and mutant Arabidopsis using 4-nitrophenyl-α-l-arabinofuranoside. In keeping with an expectation that disease severity above ground is correlated with F. oxysporum infection below ground, elevated levels of arabinofuranosidase activity were measured in the roots of susceptible agb1 and rfo1 while a reduced level was detected in the resistant eir1. In contrast, disease severity and F. oxysporum infection were uncoupled in tir3. The distribution of staining patterns in roots suggests that AGB1 and RFO1 restrict colonization of the vascular cylinder by F. oxysporum whereas EIR1 promotes colonization of root apices.
Asunto(s)
Arabidopsis/microbiología , Arabinosa/análogos & derivados , Fusarium/aislamiento & purificación , Enfermedades de las Plantas/microbiología , Raíces de Plantas/microbiología , Coloración y Etiquetado/métodos , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabinosa/metabolismo , Proteínas Fúngicas/metabolismo , Fusarium/enzimología , Fusarium/patogenicidad , Subunidades beta de la Proteína de Unión al GTP/genética , Subunidades beta de la Proteína de Unión al GTP/metabolismo , Glicósido Hidrolasas/metabolismo , Gossypium/microbiología , Ácidos Indolacéticos/metabolismo , Solanum lycopersicum/microbiología , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Mutación , Brotes de la Planta/microbiología , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Plantones/microbiología , Especificidad de la Especie , Esporas FúngicasRESUMEN
Fusarium species are among the most important phytopathogenic and toxigenic fungi. To understand the molecular underpinnings of pathogenicity in the genus Fusarium, we compared the genomes of three phenotypically diverse species: Fusarium graminearum, Fusarium verticillioides and Fusarium oxysporum f. sp. lycopersici. Our analysis revealed lineage-specific (LS) genomic regions in F. oxysporum that include four entire chromosomes and account for more than one-quarter of the genome. LS regions are rich in transposons and genes with distinct evolutionary profiles but related to pathogenicity, indicative of horizontal acquisition. Experimentally, we demonstrate the transfer of two LS chromosomes between strains of F. oxysporum, converting a non-pathogenic strain into a pathogen. Transfer of LS chromosomes between otherwise genetically isolated strains explains the polyphyletic origin of host specificity and the emergence of new pathogenic lineages in F. oxysporum. These findings put the evolution of fungal pathogenicity into a new perspective.
Asunto(s)
Cromosomas Fúngicos/genética , Fusarium/genética , Fusarium/patogenicidad , Genoma Fúngico/genética , Genómica , Evolución Molecular , Fusarium/clasificación , Interacciones Huésped-Parásitos/genética , Familia de Multigenes/genética , Fenotipo , Filogenia , Proteoma/genética , Análisis de Secuencia de ADN , Sintenía/genética , Virulencia/genéticaRESUMEN
Arabidopsis thaliana ecotypes differ in their susceptibility to Fusarium wilt diseases. Ecotype Taynuilt-0 (Ty-0) is susceptible to Fusarium oxysporum forma specialis (f.) matthioli whereas Columbia-0 (Col-0) is resistant. Segregation analysis of a cross between Ty-0 and Col-0 revealed six dominant RESISTANCE TO FUSARIUM OXYSPORUM (RFO) loci that significantly contribute to f. matthioli resistance in Col-0 relative to Ty-0. We refer to the locus with the strongest effect as RFO1. Ty-0 plants in which only the Col-0 allele of RFO1 (RFO1(Col-0)) was introduced were resistant to f. matthioli. Surprisingly, RFO1(Col-0) also conferred resistance to f. raphani, demonstrating that RFO1-mediated resistance is not race specific. Expression of resistance by RFO2, RFO4, or RFO6 was dependent on RFO1(Col-0). Map-based cloning of RFO1(Col-0) showed that RFO1 is identical to the previously named Arabidopsis gene WAKL22 (WALL-ASSOCIATED KINASE-LIKE KINASE 22), which encodes a receptor-like kinase that does not contain an extracellular leucine-rich repeat domain. Consistent with these results, a Col-0 rfo1 loss-of-function mutant was more susceptible to f. matthioli, f. conglutinans, and f. raphani. Thus, RFO1 encodes a novel type of dominant disease-resistance protein that confers resistance to a broad spectrum of Fusarium races.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Fusarium/crecimiento & desarrollo , Genes Dominantes/genética , Genes de Plantas/genética , Enfermedades de las Plantas/genética , Proteínas Quinasas/genética , Arabidopsis/efectos de los fármacos , Arabidopsis/microbiología , Mapeo Cromosómico , Cromosomas de las Plantas/genética , ADN Bacteriano/genética , Regulación de la Expresión Génica de las Plantas , Ligamiento Genético , Predisposición Genética a la Enfermedad/genética , Genotipo , Hibridación Genética , Inmunidad Innata/genética , Endogamia , Mutagénesis Insercional , Fenotipo , Enfermedades de las Plantas/microbiología , Plantas Modificadas Genéticamente , Ácido Salicílico/farmacología , Especificidad de la EspecieRESUMEN
Evaluation of Cryptococcus neoformans virulence in a number of nonmammalian hosts suggests that C. neoformans is a nonspecific pathogen. We used the killing of Galleria mellonella (the greater wax moth) caterpillar by C. neoformans to develop an invertebrate host model system that can be used to study cryptococcal virulence, host immune responses to infection, and the effects of antifungal compounds. All varieties of C. neoformans killed G. mellonella. After injection into the insect hemocoel, C. neoformans proliferated and, despite successful phagocytosis by host hemocytes, killed caterpillars both at 37 degrees C and 30 degrees C. The rate and extent of killing depended on the cryptococcal strain and the number of fungal cells injected. The sequenced C. neoformans clinical strain H99 was the most virulent of the strains tested and killed caterpillars with inocula as low as 20 CFU/caterpillar. Several C. neoformans genes previously shown to be involved in mammalian virulence (CAP59, GPA1, RAS1, and PKA1) also played a role in G. mellonella killing. Combination antifungal therapy (amphotericin B plus flucytosine) administered before or after inoculation was more effective than monotherapy in prolonging survival and in decreasing the tissue burden of cryptococci in the hemocoel. The G. mellonella-C. neoformans pathogenicity model may be a substitute for mammalian models of infection with C. neoformans and may facilitate the in vivo study of fungal virulence and efficacy of antifungal therapies.