RESUMEN
FLOWERING LOCUS T (FT) protein, physiologically florigen, has been identified as a system integrator of numerous flowering time pathways in many studies, and its homologs are found throughout the plant lineage. It is important to uncover how precisely florigenic homologs contribute to flowering initiation and how these factors interact genetically. Here we dissected the function of Brachypodium FT orthologs BdFTL1 and BdFTL2 using overexpression and gene-editing experiments. Transgenic assays showed that both BdFTL1 and BdFTL2 could promote flowering, whereas BdFTL2 was essential for flowering initiation. Notably, BdFTL1 is subject to alternative splicing (AS), and its transcriptional level and AS are significantly affected by BdFTL2. Additionally, BdFTL2 could bind with the PHD-containing protein BdES43, an H3K4me3 reader. Furthermore, BdES43 was antagonistic to BdFTL2 in flowering initiation in a transcription-dependent manner and significantly affected BdFTL1 expression. BdFTL2, BdES43 and H3K4me3 also had highly similar distribution patterns within the BdFTL1 locus, indicating their interplay in regulating target genes. Taken together, florigen BdFTL2 functions as a potential epigenetic effector of BdFTL1 by interacting with a BdES43-H3K4me3 complex. This finding provides an additional insight for the regulatory mechanism underlying the multifaceted roles of florigen.
Asunto(s)
Brachypodium/genética , Florigena/metabolismo , Histonas/metabolismo , Brachypodium/crecimiento & desarrollo , Flores/genética , Flores/crecimiento & desarrollo , Histonas/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Factores de TiempoRESUMEN
Nuclear Factor Y (NF-Y) is a heterotrimeric transcription factor that binds CCAAT elements. The NF-Y trimer is composed of a Histone Fold Domain (HFD) dimer (NF-YB/NF-YC) and NF-YA, which confers DNA sequence specificity. NF-YA shares a conserved domain with the CONSTANS, CONSTANS-LIKE, TOC1 (CCT) proteins. We show that CONSTANS (CO/B-BOX PROTEIN1 BBX1), a master flowering regulator, forms a trimer with Arabidopsis thaliana NF-YB2/NF-YC3 to efficiently bind the CORE element of the FLOWERING LOCUS T promoter. We term this complex NF-CO. Using saturation mutagenesis, electrophoretic mobility shift assays, and RNA-sequencing profiling of co, nf-yb, and nf-yc mutants, we identify CCACA elements as the core NF-CO binding site. CO physically interacts with the same HFD surface required for NF-YA association, as determined by mutations in NF-YB2 and NF-YC9, and tested in vitro and in vivo. The co-7 mutation in the CCT domain, corresponding to an NF-YA arginine directly involved in CCAAT recognition, abolishes NF-CO binding to DNA. In summary, a unifying molecular mechanism of CO function relates it to the NF-YA paradigm, as part of a trimeric complex imparting sequence specificity to HFD/DNA interactions. It is likely that members of the large CCT family participate in similar complexes with At-NF-YB and At-NF-YC, broadening HFD combinatorial possibilities in terms of trimerization, DNA binding specificities, and transcriptional regulation.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , ADN de Plantas/genética , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/metabolismo , Proteínas de Arabidopsis/genética , Factor de Unión a CCAAT/genética , Factor de Unión a CCAAT/metabolismo , Monóxido de Carbono/metabolismo , Proteínas de Unión al ADN/genética , Unión Proteica , Factores de Transcripción/genéticaRESUMEN
Photoperiod dependent flowering is one of several mechanisms used by plants to initiate the developmental transition from vegetative growth to reproductive growth. The NUCLEAR FACTOR Y (NF-Y) transcription factors are heterotrimeric complexes composed of NF-YA and histone-fold domain (HFD) containing NF-YB/NF-YC, that initiate photoperiod-dependent flowering by cooperatively interacting with CONSTANS (CO) to drive the expression of FLOWERING LOCUS T (FT). This involves NF-Y and CO binding at distal CCAAT and proximal "CORE" elements, respectively, in the FT promoter. While this is well established for the HFD subunits, there remains some question over the potential role of NF-YA as either positive or negative regulators of this process. Here we provide strong support, in the form of genetic and biochemical analyses, that NF-YA, in complex with NF-YB/NF-YC proteins, can directly bind the distal CCAAT box in the FT promoter and are positive regulators of flowering in an FT-dependent manner.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Factor de Unión a CCAAT/genética , Flores/genética , Secuencia de Aminoácidos/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/biosíntesis , Factor de Unión a CCAAT/biosíntesis , Flores/crecimiento & desarrollo , Regulación de la Expresión Génica de las Plantas , Regiones Promotoras Genéticas , Unión ProteicaRESUMEN
For many plant species, reproductive success relies on the proper timing of flowering, and photoperiod provides a key environmental input. Photoperiod-dependent flowering depends on timely expression of FLOWERING LOCUS T (FT); however, the coordination of various cis-regulatory elements in the FT promoter is not well understood. Here, we provide evidence that long-distance chromatin loops bring distal enhancer elements into close association with the proximal promoter elements bound by CONSTANS (CO). Additionally, we show that NUCLEAR FACTOR Y (NF-Y) binds a CCAAT box in the distal enhancer element and that CCAAT disruption dramatically reduces FT promoter activity. Thus, we propose the recruitment model of photoperiod-dependent flowering where NF-Y complexes, bound at the FT distal enhancer element, help recruit CO to proximal cis-regulatory elements and initiate the transition to reproductive growth.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Factor de Unión a CCAAT/genética , Cromatina/genética , Flores , Regiones Promotoras GenéticasRESUMEN
The CCAAT box is one of the most common cis-elements present in eukaryotic promoters and is bound by the transcription factor NUCLEAR FACTOR Y (NF-Y). NF-Y is composed of three subunits, NF-YA, NF-YB, and NF-YC. Unlike animals and fungi, plants have significantly expanded the number of genes encoding NF-Y subunits. We provide a comprehensive classification of NF-Y genes, with a separation of closely related, but distinct, histone fold domain proteins. We additionally review recent experiments that have placed NF-Y at the center of many developmental stress-responsive processes in the plant lineage.
Asunto(s)
Factor de Unión a CCAAT/metabolismo , Proteínas de Plantas/metabolismo , Factor de Unión a CCAAT/química , Factor de Unión a CCAAT/clasificación , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/clasificaciónRESUMEN
In the plant kingdom, each of the NUCLEAR FACTOR-Y (NF-Y) transcription factor families, NF-YA, NF-YB, and NF-YC, has undergone a great expansion compared to the animal kingdom. For example, Arabidopsis thaliana has 10 members of each gene family compared to only one in humans. Progress towards understanding the significance of this expansion is limited due to a lack of studies looking at the complete gene family during plant development. In the current study, transgenic overexpression lines were created for all 10 Arabidopsis NF-YA genes and examined for general development and alterations in abscisic acid (ABA)-mediated seed germination. NF-YA overexpression typically led to severe growth retardation and developmental defects, which extended from embryogenesis through to adult plants. Although overexpression of all NF-YA family members consistently led to growth retardation, some transgenic lines were hypersensitive to ABA during germination while others were hyposensitive. The opposing germination phenotypes were associated with the phylogenetic relationships between the NF-YA members. In addition, ABA marker genes were misregulated and ABA induction of gene expression was reduced in the overexpressors. Collectively, this study demonstrates that although NF-Ys have retained high degrees of similarity, they have evolved unique and sometimes opposing roles during plant development.
RESUMEN
Gene duplication is an evolutionary mechanism that provides new genetic material. Since gene duplication is a major driver for molecular evolution, examining the fate of duplicated genes is an area of active research. The fate of duplicated genes can include loss, subfunctionalization, and neofunctionalization. In this manuscript, we chose to experimentally study the fate of duplicated genes using the Arabidopsis NUCLEAR FACTOR Y (NF-Y) transcription factor family. NF-Y transcription factors are heterotrimeric complexes, composed of NF-YA, NF-YB, and NF-YC. NF-YA subunits are responsible for nucleotide-specific binding to a CCAAT cis-regulatory element. NF-YB and NF-YC subunits make less specific, but essential complex-stabilizing contacts with the DNA flanking the core CCAAT pentamer. While ubiquitous in eukaryotes, each NF-Y family has expanded by duplication in the plant lineage. For example, the model plant Arabidopsis contains 10 each of the NF-Y subunits. Here we examine the fate of duplicated NF-YB proteins in Arabidopsis, which are composed of central histone fold domains (HFD) and less conserved flanking regions (N- and C-termini). Specifically, the principal question we wished to address in this manuscript was to what extent can the 10 Arabidopsis NF-YB paralogs functionally substitute the genes NF-YB2 and NF-YB3 in the promotion of photoperiodic flowering? Our results demonstrate that the conserved histone fold domains (HFD) may be under pressure for purifying (negative) selection, while the non-conserved N- and C-termini may be under pressure for diversifying (positive) selection, which explained each paralog's ability to substitute. In conclusion, our data demonstrate that the N- and C-termini may have allowed the duplicated genes to undergo functional diversification, allowing the retention of the duplicated genes.
Asunto(s)
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Duplicación de Gen , Histonas/metabolismo , Factor de Unión a CCAAT/genética , Factor de Unión a CCAAT/metabolismo , Factores de Transcripción/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismoRESUMEN
NF-Y transcription factors represent a complex of three proteins called NF-YA, NF-YB and NF-YC. Each protein is highly conserved in eukaryotes, and in the plant lineage has undergone numerous rounds of duplication. Individual NF-Y are emerging as important regulators of several essential plant processes, including embryogenesis, drought resistance, maintenance of meristems in nitrogen-fixing nodules and photoperiod-dependent flowering time. Building on the recent finding that NF-YB2 and NF-YB3 have overlapping functionality in Arabidopsis photoperiod-dependent flowering (Kumimoto et al., 2008), we have identified three NF-YC (NF-YC3, NF-YC4, and NF-YC9) that are also required for flowering, and physically interact in vivo with both NF-YB2 and NF-YB3. Furthermore, NF-YC3, NF-YC4 and NF-YC9 can physically interact with full-length CONSTANS (CO), and are genetically required for CO-mediated floral promotion. Collectively, the present data greatly strengthens and extends the argument that CO utilizes NF-Y transcription factor complexes for the activation of FLOWERING LOCUS T (FT) during photoperiod-dependent floral initiation.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/fisiología , Flores/crecimiento & desarrollo , Fotoperiodo , Proteínas de Arabidopsis/clasificación , Proteínas de Arabidopsis/genética , Filogenia , ARN Mensajero/genéticaRESUMEN
BACKGROUND: Monocots, especially the temperate grasses, represent some of the most agriculturally important crops for both current food needs and future biofuel development. Because most of the agriculturally important grass species are difficult to study (e.g., they often have large, repetitive genomes and can be difficult to grow in laboratory settings), developing genetically tractable model systems is essential. Brachypodium distachyon (hereafter Brachypodium) is an emerging model system for the temperate grasses. To fully realize the potential of this model system, publicly accessible discovery tools are essential. High quality cDNA libraries that can be readily adapted for multiple downstream purposes are a needed resource. Additionally, yeast two-hybrid (Y2H) libraries are an important discovery tool for protein-protein interactions and are not currently available for Brachypodium. RESULTS: We describe the creation of two high quality, publicly available Gateway™ cDNA entry libraries and their derived Y2H libraries for Brachypodium. The first entry library represents cloned cDNA populations from both short day (SD, 8/16-h light/dark) and long day (LD, 20/4-h light/dark) grown plants, while the second library was generated from hormone treated tissues. Both libraries have extensive genome coverage (~5 × 107 primary clones each) and average clone lengths of ~1.5 Kb. These entry libraries were then used to create two recombination-derived Y2H libraries. Initial proof-of-concept screens demonstrated that a protein with known interaction partners could readily re-isolate those partners, as well as novel interactors. CONCLUSIONS: Accessible community resources are a hallmark of successful biological model systems. Brachypodium has the potential to be a broadly useful model system for the grasses, but still requires many of these resources. The Gateway™ compatible entry libraries created here will facilitate studies for multiple user-defined purposes and the derived Y2H libraries can be immediately applied to large scale screening and discovery of novel protein-protein interactions. All libraries are freely available for distribution to the research community.
Asunto(s)
Brachypodium/genética , Biblioteca de Genes , Técnicas del Sistema de Dos Híbridos , Clonación Molecular , Hormonas/farmacología , Modelos Biológicos , ARN de Planta/metabolismoRESUMEN
Plants recognize many pathogens through the action of a diverse family of proteins called disease resistance (R) genes. The Arabidopsis R gene RPM1 encodes resistance to specific Pseudomonas syringae strains. We describe an RPM1-interacting protein that is an ortholog of TIP49a, previously shown to interact with the TATA binding protein (TBP) complex and to modulate c-myc- and beta-catenin-mediated signaling in animals. Reduction of Arabidopsis TIP49a (AtTIP49a) mRNA levels results in measurable increases of two R-dependent responses without constitutively activating defense responses, suggesting that AtTIP49a can act as a negative regulator of at least some R functions. Further, AtTIP49a is essential for both sporophyte and female gametophyte viability. Thus, regulators of R function overlap with essential modulators of plant development.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/embriología , Proteínas Portadoras/metabolismo , Inmunidad Innata/genética , Enfermedades de las Plantas/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas Portadoras/genética , Células Clonales , Secuencia Conservada , ADN Helicasas , Evolución Molecular , Eliminación de Gen , Regulación de la Expresión Génica , Genes de Plantas , Proteínas de Plantas/metabolismo , Pseudomonas/patogenicidad , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Supresión GenéticaRESUMEN
The NUCLEAR FACTOR-Y (NF-Y) families of transcription factors are important regulators of plant development and physiology. Though NF-Y regulatory roles have recently been suggested for numerous aspects of plant biology, their roles in flowering time, early seedling development, stress responses, hormone signaling, and nodulation are the best characterized. The past few years have also seen significant advances in our understanding of the mechanistic function of the NF-Y, and as such, increasingly complex and interesting questions are now more approachable. This review will primarily focus on these developmental, physiological, and mechanistic roles of the NF-Y in recent research.
Asunto(s)
Factor de Unión a CCAAT/metabolismo , Proteínas de Plantas/metabolismo , Factores de Transcripción/metabolismo , Factor de Unión a CCAAT/genética , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Proteínas de Plantas/genética , Factores de Transcripción/genéticaRESUMEN
NUCLEAR FACTOR-Y (NF-Y) is a heterotrimeric transcription factor (TF) consisting of evolutionarily distinct NF-YA, NF-YB and NF-YC subunits. The functional NF-Y heterotrimer binds to CCAAT elements in eukaryotic gene promoters and influences their expression. The genome of the model organism Arabidopsis thaliana encodes 10 distinct NF-YA, NF-YB, and NF-YC proteins, allowing for enormous combinatorial and functional diversity. Two decades of research have elucidated the importance of NF-Ys in plant growth, development and stress responses; however, the molecular mechanisms of action remain largely unexplored. Intriguingly, recent evidence suggests that NF-Ys are frequently associated with other groups of TFs, expanding the potential NF-Y combinatorial complexity. Further, information regarding the regulation of individual NF-Y subunits at the transcriptional and post-transcriptional level is beginning to emerge. In this review, we will identify developing trends within the NF-Y field and discuss recent progress towards a better understanding of NF-Y function, molecular action, and regulation in the context of Arabidopsis. This article is part of a Special Issue entitled: Nuclear Factor Y in Development and Disease, edited by Prof. Roberto Mantovani.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Factor de Unión a CCAAT/metabolismo , Regulación de la Expresión Génica de las Plantas/fisiología , Estrés Fisiológico/fisiología , Transcripción Genética/fisiología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Factor de Unión a CCAAT/genéticaRESUMEN
In the model organism Arabidopsis thaliana the heterotrimeric transcription factor NUCLEAR FACTOR Y (NF-Y) has been shown to play multiple roles in facilitating plant growth and development. Although NF-Y itself represents a multi-protein transcriptional complex, recent studies have shown important interactions with other transcription factors, especially those in the bZIP family. Here we add to the growing evidence that NF-Y and bZIP form common complexes to affect many processes. We carried out transcriptional profiling on nf-yc mutants and through subsequent analyses found an enrichment of bZIP binding sites in the promoter elements of misregulated genes. Using NF-Y as bait, yeast two hybrid assays yielded interactions with bZIP proteins that are known to control ABA signaling. Accordingly, we find that plants mutant for several NF-Y subunits show characteristic phenotypes associated with the disruption of ABA signaling. While previous reports have shown additive roles for NF-YC family members in photoperiodic flowering, we found that they can have opposing roles in ABA signaling. Collectively, these results demonstrated the importance and complexity of NF-Y in the integration of environmental and hormone signals.
Asunto(s)
Ácido Abscísico/metabolismo , Arabidopsis/fisiología , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Factor de Unión a CCAAT/metabolismo , Germinación/fisiología , Complejos Multiproteicos/metabolismo , Transducción de Señal/fisiología , Biología Computacional , Perfilación de la Expresión Génica , Análisis por Micromatrices , Mapeo de Interacción de Proteínas , Transducción de Señal/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
BACKGROUND: Nuclear Factor Y (NF-Y) is a heterotrimeric transcription factor composed of NF-YA, NF-YB and NF-YC proteins. Using the dicot plant model system Arabidopsis thaliana (Arabidopsis), NF-Y were previously shown to control a variety of agronomically important traits, including drought tolerance, flowering time, and seed development. The aim of the current research was to identify and characterize NF-Y families in the emerging monocot model plant Brachypodium distachyon (Brachypodium) with the long term goal of assisting in the translation of known dicot NF-Y functions to the grasses. METHODOLOGY/PRINCIPAL FINDINGS: We identified, annotated, and further characterized 7 NF-YA, 17 NF-YB, and 12 NF-YC proteins in Brachypodium (BdNF-Y). By examining phylogenetic relationships, orthology predictions, and tissue-specific expression patterns for all 36 BdNF-Y, we proposed numerous examples of likely functional conservation between dicots and monocots. To test one of these orthology predictions, we demonstrated that a BdNF-YB with predicted orthology to Arabidopsis floral-promoting NF-Y proteins can rescue a late flowering Arabidopsis mutant. CONCLUSIONS/SIGNIFICANCE: The Brachypodium genome encodes a similar complement of NF-Y to other sequenced angiosperms. Information regarding NF-Y phylogenetic relationships, predicted orthologies, and expression patterns can facilitate their study in the grasses. The current data serves as an entry point for translating many NF-Y functions from dicots to the genetically tractable monocot model system Brachypodium. In turn, studies of NF-Y function in Brachypodium promise to be more readily translatable to the agriculturally important grasses.
Asunto(s)
Brachypodium/metabolismo , Proteínas de Plantas/metabolismo , Factores de Transcripción/metabolismo , Filogenia , Proteínas de Plantas/clasificaciónRESUMEN
All aspects of plant and animal development are controlled by complex networks of transcription factors. Transcription factors are essential for converting signaling inputs, such as changes in daylength, into complex gene regulatory outputs. While some transcription factors control gene expression by binding to cis-regulatory elements as individual subunits, others function in a combinatorial fashion. How individual subunits of combinatorial transcription factors are spatially and temporally deployed (e.g. expression-level, posttranslational modifications and subcellular localization) has profound effects on their control of gene expression. In the model plant Arabidopsis (Arabidopsis thaliana), we have identified 36 Nuclear Factor Y (NF-Y) transcription factor subunits (10 NF-YA, 13 NF-YB, and 13 NF-YC subunits) that can theoretically combine to form 1,690 unique complexes. Individual plant subunits have functions in flowering time, embryo maturation, and meristem development, but how they combine to control these processes is unknown. To assist in the process of defining unique NF-Y complexes, we have created promoter:beta-glucuronidase fusion lines for all 36 Arabidopsis genes. Here, we show NF-Y expression patterns inferred from these promoter:beta-glucuronidase lines for roots, light- versus dark-grown seedlings, rosettes, and flowers. Additionally, we review the phylogenetic relationships and examine protein alignments for each NF-Y subunit family. The results are discussed with a special emphasis on potential roles for NF-Y subunits in photoperiod-controlled flowering time.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Factor de Unión a CCAAT/genética , Regulación de la Expresión Génica de las Plantas , Recombinación Genética , Factores de Transcripción/genética , Arabidopsis/clasificación , Proteínas de Arabidopsis/clasificación , Clonación Molecular , Técnicas Químicas Combinatorias/métodos , Familia de Multigenes , FilogeniaRESUMEN
Plants use sophisticated strategies to balance responses to oxidative stress. Programmed cell death, including the hypersensitive response (HR) associated with successful pathogen recognition, is one cellular response regulated by reactive oxygen in various cellular contexts. The Arabidopsis basic leucine zipper (bZIP) transcription factor AtbZIP10 shuttles between the nucleus and the cytoplasm and binds consensus G- and C-box DNA sequences. Surprisingly, AtbZIP10 can be retained outside the nucleus by LSD1, a protein that protects Arabidopsis cells from death in the face of oxidative stress signals. We demonstrate that AtbZIP10 is a positive mediator of the uncontrolled cell death observed in lsd1 mutants. AtbZIP10 and LSD1 act antagonistically in both pathogen-induced HR and basal defense responses. LSD1 likely functions as a cellular hub, where its interaction with AtbZIP10 and additional, as yet unidentified, proteins contributes significantly to plant oxidative stress responses.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citología , Arabidopsis/metabolismo , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/metabolismo , Apoptosis , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/genética , Citoplasma/metabolismo , Proteínas de Unión al ADN/genética , Genes de Plantas , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Mutación , Oomicetos/patogenicidad , Estrés Oxidativo , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Plantas Modificadas Genéticamente , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/genética , Técnicas del Sistema de Dos HíbridosRESUMEN
Pathogen recognition by the plant immune system is governed by structurally related, polymorphic products of disease resistance (R) genes. RAR1 and/or SGT1b mediate the function of many R proteins. RAR1 controls preactivation R protein accumulation by an unknown mechanism. We demonstrate that Arabidopsis SGT1b has two distinct, genetically separable functions in the plant immune system: SGT1b antagonizes RAR1 to negatively regulate R protein accumulation before infection, and SGT1b has a RAR1-independent function that regulates programmed cell death during infection. The balanced activities of RAR1 and SGT1, in concert with cytosolic HSP90, modulate preactivation R protein accumulation and signaling competence.
Asunto(s)
Proteínas de Arabidopsis/fisiología , Arabidopsis/inmunología , Proteínas Portadoras/fisiología , Proteínas de Ciclo Celular/fisiología , Proteínas de Plantas/fisiología , Apoptosis , Arabidopsis/microbiología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/inmunología , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/inmunología , Proteínas de Ciclo Celular/inmunología , Genes de Plantas , Proteínas HSP90 de Choque Térmico/inmunología , Proteínas HSP90 de Choque Térmico/fisiología , Péptidos y Proteínas de Señalización Intracelular , Mutación , Peronospora/fisiología , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/antagonistas & inhibidores , Proteínas de Plantas/inmunología , Plantas Modificadas Genéticamente , Transducción de SeñalRESUMEN
The Arabidopsis RPM1 protein confers resistance to disease caused by Pseudomonas syringae strains delivering either the AvrRpm1 or AvrB type III effector proteins into host cells. We characterized two closely related RPM1-interacting proteins, RIN2 and RIN3. RIN2 and RIN3 encode RING-finger type ubiquitin ligases with six apparent transmembrane domains and an ubiquitin-binding CUE domain. RIN2 and RIN3 are orthologs of the mammalian autocrine motility factor receptor, a cytokine receptor localized in both plasma membrane caveolae and the endoplasmic reticulum. RIN2 is predominantly localized to the plasma membrane, as are RPM1 and RPS2. The C-terminal regions of RIN2 and RIN3, including the CUE domain, interact strongly with an RPM1 N-terminal fragment and weakly with a similar domain from the Arabidopsis RPS2 protein. RIN2 and RIN3 can dimerize through their C-terminal regions. The RING-finger domains of RIN2 and RIN3 encode ubiquitin ligases. Inoculation with P. syringae DC3000(avrRpm1) or P. syringae DC3000(avrRpt2) induces differential decreases of RIN2 mobility in SDS-PAGE and disappearance of the majority of RIN2. A rin2 rin3 double mutant expresses diminished RPM1- and RPS2-dependent hypersensitive response (HR), but no alteration of pathogen growth. Thus, the RIN2/RIN3 RING E3 ligases apparently act on a substrate that regulates RPM1- and RPS2-dependent HR.