RESUMO
Defense against pathogens in multicellular eukaryotes depends on intracellular immune receptors, yet surveillance by these receptors is poorly understood. Several plant nucleotide-binding, leucine-rich repeat (NB-LRR) immune receptors carry fusions with other protein domains. The Arabidopsis RRS1-R NB-LRR protein carries a C-terminal WRKY DNA binding domain and forms a receptor complex with RPS4, another NB-LRR protein. This complex detects the bacterial effectors AvrRps4 or PopP2 and then activates defense. Both bacterial proteins interact with the RRS1 WRKY domain, and PopP2 acetylates lysines to block DNA binding. PopP2 and AvrRps4 interact with other WRKY domain-containing proteins, suggesting these effectors interfere with WRKY transcription factor-dependent defense, and RPS4/RRS1 has integrated a "decoy" domain that enables detection of effectors that target WRKY proteins. We propose that NB-LRR receptor pairs, one member of which carries an additional protein domain, enable perception of pathogen effectors whose function is to target that domain.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Arabidopsis/microbiologia , Proteínas de Arabidopsis/química , Proteínas de Bactérias/imunologia , Imunidade Inata , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Pseudomonas fluorescens/metabolismo , Pseudomonas fluorescens/patogenicidade , Pseudomonas syringae/imunologia , Pseudomonas syringae/metabolismo , Nicotiana/imunologia , Nicotiana/microbiologiaRESUMO
IMPORTIN-α3/MOS6 (MODIFIER OF SNC1, 6) is one of nine importin-α isoforms in Arabidopsis that recruit nuclear localization signal-containing cargo proteins to the nuclear import machinery. IMP-α3/MOS6 is required genetically for full autoimmunity of the nucleotide-binding leucine-rich repeat immune receptor mutant snc1 (suppressor of npr1-1, constitutive 1) and MOS6 also contributes to basal disease resistance. Here, we investigated the contribution of the other importin-α genes to both types of immune responses, and we analyzed potential interactions of all importin-α isoforms with SNC1. By using reverse-genetic analyses in Arabidopsis and protein-protein interaction assays in Nicotiana benthamiana, we provide evidence that among the nine α-importins in Arabidopsis, IMP-α3/MOS6 is the main nuclear transport receptor of SNC1, and that IMP-α3/MOS6 is required selectively for autoimmunity of snc1 and basal resistance to mildly virulent Pseudomonas syringae in Arabidopsis.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/imunologia , Resistência à Doença/fisiologia , Carioferinas/fisiologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Autoimunidade/fisiologia , Carioferinas/metabolismo , Filogenia , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Pseudomonas syringaeAssuntos
Proteínas de Plantas , Poli(ADP-Ribose) Polimerases , Tolerância ao Sal , Triticum , Adenosina Difosfato Ribose/metabolismo , Pão , Regulação da Expressão Gênica de Plantas , Poli(ADP-Ribose) Polimerases/química , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Triticum/enzimologia , Triticum/genética , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Domínios ProteicosRESUMO
The oomycete pathogen Hyaloperonospora arabidopsidis (Hpa) causes downy mildew disease on Arabidopsis. To colonize its host, Hpa translocates effector proteins that suppress plant immunity into infected host cells. Here, we investigate the relevance of the interaction between one of these effectors, HaRxL106, and Arabidopsis RADICAL-INDUCED CELL DEATH1 (RCD1). We use pathogen infection assays as well as molecular and biochemical analyses to test the hypothesis that HaRxL106 manipulates RCD1 to attenuate transcriptional activation of defense genes. We report that HaRxL106 suppresses transcriptional activation of salicylic acid (SA)-induced defense genes and alters plant growth responses to light. HaRxL106-mediated suppression of immunity is abolished in RCD1 loss-of-function mutants. We report that RCD1-type proteins are phosphorylated, and we identified Mut9-like kinases (MLKs), which function as phosphoregulatory nodes at the level of photoreceptors, as RCD1-interacting proteins. An mlk1,3,4 triple mutant exhibits stronger SA-induced defense marker gene expression compared with wild-type plants, suggesting that MLKs also affect transcriptional regulation of SA signaling. Based on the combined evidence, we hypothesize that nuclear RCD1/MLK complexes act as signaling nodes that integrate information from environmental cues and pathogen sensors, and that the Arabidopsis downy mildew pathogen targets RCD1 to prevent activation of plant immunity.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Arabidopsis/microbiologia , Proteínas Nucleares/metabolismo , Oomicetos/metabolismo , Imunidade Vegetal , Proteínas/metabolismo , ADP Ribose Transferases/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Mutação/genética , Proteínas Nucleares/genética , Oomicetos/efeitos dos fármacos , Oomicetos/isolamento & purificação , Oomicetos/patogenicidade , Doenças das Plantas/microbiologia , Imunidade Vegetal/efeitos dos fármacos , Plantas Geneticamente Modificadas , Domínios Proteicos , Multimerização Proteica/efeitos dos fármacos , Ácido Salicílico/farmacologia , Transdução de Sinais/efeitos da radiação , Transcrição Gênica/efeitos dos fármacos , Virulência/efeitos dos fármacosRESUMO
Pathogen-responsive mitogen-activated protein kinase (MAPK or MPK) cascades relay signals from activated immune receptors across the nuclear envelope to intranuclear targets. However, in plants, little is known about the spatial control of MAPK signaling. Here, we report that the Arabidopsis (Arabidopsis thaliana) nuclear pore complex protein Nup88/MOS7 is essential for immunity to the necrotrophic fungus Botrytis cinerea The mos7-1 mutation, causing a four-amino acid deletion, compromises B. cinerea-induced activation of the key immunoregulatory MAPKs MPK3/MPK6 and reduces MPK3 protein levels posttranscriptionally. Furthermore, MOS7 contributes to retaining a sufficient MPK3 abundance in the nucleus, which is required for full immunity to B. cinerea Finally, we present a structural model of MOS7 and show that the mos7-1 mutation compromises interactions with Nup98a/b, two phenylalanine-glycine repeat nucleoporins implicated in maintaining the selective nuclear pore complex permeability barrier. Together, our analysis uncovered MOS7 and Nup98 as novel components of plant immunity toward a necrotrophic pathogen and provides mechanistic insights into how these nucleoporins coordinate nucleocytoplasmic transport to mount a robust immune response.
Assuntos
Arabidopsis/genética , Sistema de Sinalização das MAP Quinases/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Doenças das Plantas/genética , Transporte Ativo do Núcleo Celular/genética , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Botrytis/imunologia , Botrytis/fisiologia , Resistência à Doença/genética , Resistência à Doença/imunologia , Regulação da Expressão Gênica de Plantas , Interações Hospedeiro-Patógeno/imunologia , Immunoblotting , Microscopia Confocal , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Doenças das Plantas/microbiologia , Imunidade Vegetal/genética , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Importin-αs are essential adapter proteins that recruit cytoplasmic proteins destined for active nuclear import to the nuclear transport machinery. Cargo proteins interact with the importin-α armadillo repeat domain via nuclear localization sequences (NLSs), short amino acids motifs enriched in Lys and Arg residues. Plant genomes typically encode several importin-α paralogs that can have both specific and partially redundant functions. Although some cargos are preferentially imported by a distinct importin-α it remains unknown how this specificity is generated and to what extent cargos compete for binding to nuclear transport receptors. Here we report that the effector protein HaRxL106 from the oomycete pathogen Hyaloperonospora arabidopsidis co-opts the host cell's nuclear import machinery. We use HaRxL106 as a probe to determine redundant and specific functions of importin-α paralogs from Arabidopsis thaliana. A crystal structure of the importin-α3/MOS6 armadillo repeat domain suggests that five of the six Arabidopsis importin-αs expressed in rosette leaves have an almost identical NLS-binding site. Comparison of the importin-α binding affinities of HaRxL106 and other cargos in vitro and in plant cells suggests that relatively small affinity differences in vitro affect the rate of transport complex formation in vivo. Our results suggest that cargo affinity for importin-α, sequence variation at the importin-α NLS-binding sites and tissue-specific expression levels of importin-αs determine formation of cargo/importin-α transport complexes in plant cells.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , Carioferinas/fisiologia , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/microbiologia , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sequência Conservada , Escherichia coli/genética , Interações Hospedeiro-Patógeno , Carioferinas/química , Carioferinas/genética , Carioferinas/metabolismo , Modelos Moleculares , Oomicetos/genética , Estrutura Terciária de ProteínaRESUMO
BACKGROUND: Plants have evolved complex mechanisms to adapt growth and development to the light environment. The COP1/SPA complex is a key repressor of photomorphogenesis in dark-grown Arabidopsis plants and acts as an E3 ubiquitin ligase to ubiquitinate transcription factors involved in the light response. In the light, COP1/SPA activity is inhibited by photoreceptors, thereby allowing accumulation of these transcription factors and a subsequent light response. Previous results have shown that the four members of the SPA family exhibit partially divergent functions. In particular, SPA1 and SPA2 strongly differ in their responsiveness to light, while they have indistinguishable activities in darkness. The much higher light-responsiveness of SPA2 is partially explained by the much stronger light-induced degradation of SPA2 when compared to SPA1. Here, we have conducted SPA1/SPA2 domain swap experiments to identify the protein domain(s) responsible for the functional divergence between SPA1 and SPA2. RESULTS: We have individually swapped the three domains between SPA1 and SPA2 - the N-terminal kinase-like domain, the coiled-coil domain and the WD-repeat domain - and expressed them in spa mutant Arabidopsis plants. The phenotypes of transgenic seedlings show that the respective N-terminal kinase-like domain is primarily responsible for the respective light-responsiveness of SPA1 and SPA2. Furthermore, the most divergent part of the N-terminal domain was sufficient to confer a SPA1- or SPA2-like activity to the respective SPA protein. The stronger light-induced degradation of SPA2 when compared to SPA1 was also primarily conferred by the SPA2 N-terminal domain. At last, the different affinities of SPA1 and SPA2 for cryptochrome 2 are defined by the N-terminal domain of the respective SPA protein. In contrast, both SPA1 and SPA2 similarly interacted with COP1 in light-grown seedlings. CONCLUSIONS: Our results show that the distinct activities and protein stabilities of SPA1 and SPA2 in light-grown seedlings are primarily encoded by their N-terminal kinase-like domains. Similarly, the different affinities of SPA1 and SPA2 for cry2 are explained by their respective N-terminal domain. Hence, after a duplication event during evolution, the N-terminal domains of SPA1 and SPA2 underwent subfunctionalization, possibly to allow optimal adaptation of growth and development to a changing light environment.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Arabidopsis/genética , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Evolução Biológica , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Luz , Domínios Proteicos , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/metabolismo , Plântula/efeitos da radiaçãoRESUMO
The downy mildew pathogen Hyaloperonospora arabidopsidis (Hpa) is a filamentous oomycete that invades plant cells via sophisticated but poorly understood structures called haustoria. Haustoria are separated from the host cell cytoplasm and surrounded by an extrahaustorial membrane (EHM) of unknown origin. In some interactions, including Hpa-Arabidopsis, haustoria are progressively encased by host-derived, callose-rich materials but the molecular mechanisms by which callose accumulates around haustoria remain unclear. Here, we report that PLASMODESMATA-LOCATED PROTEIN 1 (PDLP1) is expressed at high levels in Hpa infected cells. Unlike other plasma membrane proteins, which are often excluded from the EHM, PDLP1 is located at the EHM in Hpa-infected cells prior to encasement. The transmembrane domain and cytoplasmic tail of PDLP1 are sufficient to convey this localization. PDLP1 also associates with the developing encasement but this association is lost when encasements are fully mature. We found that the pdlp1,2,3 triple mutant is more susceptible to Hpa while overexpression of PDLP1 enhances plant resistance, suggesting that PDLPs enhance basal immunity against Hpa. Haustorial encasements are depleted in callose in pdlp1,2,3 mutant plants whereas PDLP1 over-expression elevates callose deposition around haustoria and across the cell surface. These data indicate that PDLPs contribute to callose encasement of Hpa haustoria and suggests that the deposition of callose at haustoria may involve similar mechanisms to callose deposition at plasmodesmata.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Transporte/metabolismo , Regulação da Expressão Gênica de Plantas , Glucanos/metabolismo , Oomicetos/fisiologia , Doenças das Plantas/imunologia , Arabidopsis/genética , Arabidopsis/imunologia , Arabidopsis/microbiologia , Proteínas de Arabidopsis/genética , Proteínas de Transporte/genética , Interações Hospedeiro-Patógeno , Peptídeos e Proteínas de Sinalização Intracelular , Mutação , Doenças das Plantas/microbiologia , Plantas Geneticamente Modificadas , Plasmodesmos/metabolismoRESUMO
Plants have evolved strong innate immunity mechanisms, but successful pathogens evade or suppress plant immunity via effectors delivered into the plant cell. Hyaloperonospora arabidopsidis (Hpa) causes downy mildew on Arabidopsis thaliana, and a genome sequence is available for isolate Emoy2. Here, we exploit the availability of genome sequences for Hpa and Arabidopsis to measure gene-expression changes in both Hpa and Arabidopsis simultaneously during infection. Using a high-throughput cDNA tag sequencing method, we reveal expression patterns of Hpa predicted effectors and Arabidopsis genes in compatible and incompatible interactions, and promoter elements associated with Hpa genes expressed during infection. By resequencing Hpa isolate Waco9, we found it evades Arabidopsis resistance gene RPP1 through deletion of the cognate recognized effector ATR1. Arabidopsis salicylic acid (SA)-responsive genes including PR1 were activated not only at early time points in the incompatible interaction but also at late time points in the compatible interaction. By histochemical analysis, we found that Hpa suppresses SA-inducible PR1 expression, specifically in the haustoriated cells into which host-translocated effectors are delivered, but not in non-haustoriated adjacent cells. Finally, we found a highly-expressed Hpa effector candidate that suppresses responsiveness to SA. As this approach can be easily applied to host-pathogen interactions for which both host and pathogen genome sequences are available, this work opens the door towards transcriptome studies in infection biology that should help unravel pathogen infection strategies and the mechanisms by which host defense responses are overcome.
Assuntos
Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Interações Hospedeiro-Patógeno/imunologia , Oomicetos/efeitos dos fármacos , Doenças das Plantas/imunologia , Imunidade Vegetal/imunologia , Ácido Salicílico/farmacologia , Proteínas de Arabidopsis/genética , Sequência de Bases/genética , Regulação da Expressão Gênica de Plantas , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Oomicetos/genética , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Ácido Salicílico/metabolismoRESUMO
Poly(ADP-ribosyl)ation (PARylation) is a reversible post-translational protein modification that has profound regulatory functions in metabolism, development and immunity, and is conserved throughout the eukaryotic lineage. Contrary to metazoa, many components and mechanistic details of PARylation have remained unidentified in plants. Here we present the transcriptional co-regulator RADICAL-INDUCED CELL DEATH1 (RCD1) as a plant PAR-reader. RCD1 is a multidomain protein with intrinsically disordered regions (IDRs) separating its domains. We have reported earlier that RCD1 regulates plant development and stress-tolerance by interacting with numerous transcription factors (TFs) through its C-terminal RST domain. This study suggests that the N-terminal WWE and PARP-like domains, as well as the connecting IDR play an important regulatory role for RCD1 function. We show that RCD1 binds PAR in vitro via its WWE domain and that PAR-binding determines RCD1 localization to nuclear bodies (NBs) in vivo. Additionally, we found that RCD1 function and stability is controlled by Photoregulatory Protein Kinases (PPKs). PPKs localize with RCD1 in NBs and phosphorylate RCD1 at multiple sites affecting its stability. This work proposes a mechanism for negative transcriptional regulation in plants, in which RCD1 localizes to NBs, binds TFs with its RST domain and is degraded after phosphorylation by PPKs.
Assuntos
Poli ADP Ribosilação , Poli Adenosina Difosfato Ribose , Poli Adenosina Difosfato Ribose/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação da Expressão GênicaRESUMO
The COP1/SPA complex acts as an E3 ubiquitin ligase to repress photomorphogenesis by targeting activators of the light response for degradation. Genetic analysis has shown that the four members of the SPA gene family (SPA1-SPA4) have overlapping but distinct functions. In particular, SPA1 and SPA2 differ in that SPA1 encodes a potent repressor in light- and dark-grown seedlings, but SPA2 fully loses its function when seedlings are exposed to light, indicating that SPA2 function is hyper-inactivated by light. Here, we have used chimeric SPA1/SPA2 constructs to show that the distinct functions of SPA1 and SPA2 genes in light-grown seedlings are due to the SPA protein sequences and independent of the SPA promoter sequences. Biochemical analysis of SPA1 and SPA2 protein levels shows that light exposure leads to rapid proteasomal degradation of SPA2, and, more weakly, of SPA1, but not of COP1. This suggests that light inactivates the COP1/SPA complex partly by reducing SPA protein levels. Although SPA2 was more strongly degraded than SPA1, this was not the sole reason for the lack of SPA2 function in the light. We found that the SPA2 protein is inherently incapable of repressing photomorphogenesis in light-grown seedlings. The data therefore indicate that light inactivates the function of SPA2 through a post-translational mechanism that eliminates the activity of the remaining SPA2 protein in the cell.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Luz , Proteínas Quinases/metabolismo , Plântula/efeitos da radiação , Arabidopsis/metabolismo , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/efeitos da radiação , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/efeitos da radiação , Flores/genética , Flores/crescimento & desenvolvimento , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/efeitos da radiação , Regiões Promotoras Genéticas , Proteínas Quinases/genética , Proteínas Quinases/efeitos da radiação , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases , RNA de Plantas/genética , Plântula/genética , Plântula/metabolismoRESUMO
Manipulating defence responses in infected host cells is a prerequisite for filamentous plant pathogens to complete their life cycle on infected host plants. During infection of its host Arabidopsis thaliana, the oomycete pathogen Hyaloperonospora arabidopsidis secretes numerous RXLR-type effector proteins, some of which are translocated into host cells. RXLR-type effectors share conserved N-terminal translocation motifs but show high diversity in their C-terminal `effector domains' that manipulate host defence mechanisms. Therefore, obtaining structural information on the effector domains of RXLR-type effectors will contribute to elucidating their molecular-virulence functions in infected host cells. Here, the expression, purification and crystallization of the effector domain of RXLR3 from H. arabidopsidis isolate Waco9 are reported. The crystals belonged to space group P2(1)2(1)2, with unit-cell parameters a = 61.49, b = 27.99, c = 37.59 Å. X-ray data were collected to a resolution of 1.8 Å from a single crystal using synchrotron radiation.
Assuntos
Oomicetos/química , Fatores de Virulência/química , Cristalização , Cristalografia por Raios XRESUMO
Recognition of specific pathogen molecules inside the cell by nucleotide-binding domain and leucine-rich repeat (NB-LRR) receptors constitutes an important layer of innate immunity in plants. Receptor activation triggers host cellular reprogramming involving transcriptional potentiation of basal defenses and localized programmed cell death. The sites and modes of action of NB-LRR receptors are, however, poorly understood. Arabidopsis Toll/Interleukin-1 (TIR) type NB-LRR receptor RPS4 recognizes the bacterial type III effector AvrRps4. We show that epitope-tagged RPS4 expressed under its native regulatory sequences distributes between endomembranes and nuclei in healthy and AvrRps4-triggered tissues. RPS4 accumulation in the nucleus, mediated by a bipartite nuclear localization sequence (NLS) at its C terminus, is necessary for triggering immunity through authentic activation by AvrRps4 in Arabidopsis or as an effector-independent "deregulated" receptor in tobacco. A strikingly conserved feature of TIR-NB-LRR receptors is their recruitment of the nucleocytoplasmic basal-defense regulator EDS1 in resistance to diverse pathogens. We find that EDS1 is an indispensable component of RPS4 signaling and that it functions downstream of RPS4 activation but upstream of RPS4-mediated transcriptional reprogramming in the nucleus.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica de Plantas , Doenças das Plantas/imunologia , Proteínas de Plantas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Ligação a DNA/genética , Sinais de Localização Nuclear , Doenças das Plantas/microbiologia , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Pseudomonas syringae/genética , Pseudomonas syringae/metabolismo , Rhizobium/patogenicidade , Nicotiana/genética , Nicotiana/metabolismo , Nicotiana/microbiologiaRESUMO
Proteins of the Poly(ADP-Ribose) Polymerase (PARP) family modify target proteins by covalent attachment of ADP-ribose moieties onto amino acid side chains. In Arabidopsis, PARP proteins contribute to repair of DNA lesions and modulate plant responses to various abiotic and biotic stressors. Arabidopsis PARP1 and PARP2 are nuclear proteins and given that their molecular weights exceed the diffusion limit of nuclear pore complexes, an active import mechanism into the nucleus is likely. Here we use confocal microscopy of fluorescent protein-tagged Arabidopsis PARP2 and PARP2 deletion constructs in combination with site-directed mutagenesis to identify a nuclear localization sequence in PARP2 that is required for nuclear import. We report that in co-immunoprecipitation assays PARP2 interacts with several isoforms of the importin-α group of nuclear transport adapters and that PARP2 binding to IMPORTIN-α2 is mediated by the identified nuclear localization sequence. Our results demonstrate that PARP2 is a cargo protein of the canonical importin-α/ß nuclear import pathway.
RESUMO
Although a plethora of nuclear envelope (NE) transmembrane proteins (NETs) have been identified in opisthokonts, plant NETs are largely unknown. The only known NET homologues in plants are Sad1/UNC-84 (SUN) proteins, which bind Klarsicht/ANC-1/Syne-1 homology (KASH) proteins. Therefore, de novo identification of plant NETs is necessary. Based on similarities between opisthokont KASH proteins and the only known plant KASH proteins, WPP domain-interacting proteins, we used a computational method to identify the KASH subset of plant NETs. Ten potential plant KASH protein families were identified, and five candidates from four of these families were verified for their NE localization, depending on SUN domain interaction. Of those, Arabidopsis thaliana SINE1 is involved in actin-dependent nuclear positioning in guard cells, whereas its paralogue SINE2 contributes to innate immunity against an oomycete pathogen. This study dramatically expands our knowledge of plant KASH proteins and suggests that plants and opisthokonts have recruited different KASH proteins to perform NE regulatory functions.
Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/genética , Membrana Nuclear/metabolismo , Proteínas Nucleares/química , Actinas/metabolismo , Agrobacterium tumefaciens , Sequência de Aminoácidos , Proteínas de Arabidopsis/genética , Núcleo Celular/metabolismo , Biologia Computacional , Genoma de Planta , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Proteômica , Proteínas de Ligação a RNA/genética , Homologia de Sequência de Aminoácidos , NicotianaRESUMO
The plasma membrane-localised FLAGELLIN SENSING 2 (FLS2) receptor is an important component of plant immunity against potentially pathogenic bacteria, acting to recognise the conserved flg22 peptide of flagellin. FLS2 shares the common structure of transmembrane receptor kinases with a receptor-like ectodomain composed of leucine-rich repeats (LRR) and an active intracellular kinase domain. Upon ligand binding, FLS2 dimerises with the regulatory LRR-receptor kinase BRI1-associated kinase 1, which in turn triggers downstream signalling cascades. Although lacking crystal structure data, recent advances have been made in our understanding of flg22 recognition based on structural and functional analyses of FLS2. These studies have revealed critical regions/residues of FLS2 and post-translational modifications that regulate the abundance and activity of this receptor. In this review, we present the current knowledge on the structural mechanism of the FLS2-flg22 interaction and subsequent receptor-mediated signalling.
Assuntos
Proteínas de Arabidopsis/química , Proteínas Quinases/química , Receptores de Reconhecimento de Padrão/química , Sequência de Aminoácidos , Animais , Arabidopsis/imunologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiologia , Domínio Catalítico , Flagelina/imunologia , Glicosilação , Modelos Moleculares , Dados de Sequência Molecular , Doenças das Plantas/microbiologia , Imunidade Vegetal , Ligação Proteica , Proteínas Quinases/fisiologia , Processamento de Proteína Pós-Traducional , Estrutura Secundária de Proteína , Receptores de Reconhecimento de Padrão/fisiologia , Transdução de Sinais/imunologiaRESUMO
Over the past decade, considerable advances have been made in understanding the molecular mechanisms that underpin the arms race between plant pathogens and their hosts. Alongside genomic, bioinformatic, proteomic, biochemical and cell biological analyses of plant-pathogen interactions, three-dimensional structural studies of virulence proteins deployed by pathogens to promote infection, in some cases complexed with their plant cell targets, have uncovered key insights into the functions of these molecules. Structural information on plant immune receptors, which regulate the response to pathogen attack, is also starting to emerge. Structural studies of bacterial plant pathogen-host systems have been leading the way, but studies of filamentous plant pathogens are gathering pace. In this Review, we summarize the key developments in the structural biology of plant pathogen-host interactions.
Assuntos
Doenças das Plantas/microbiologia , Plantas/imunologia , Plantas/microbiologia , Bactérias/genética , Bactérias/metabolismo , Proteínas de Bactérias/química , Proteínas Fúngicas/química , Regulação da Expressão Gênica , Interações Hospedeiro-Patógeno , Fatores de Virulência/químicaRESUMO
Nuclear translocation of immune regulatory proteins and signal transducers is an essential process in animal and plant defense signaling against pathogenic microbes. Import of proteins containing a nuclear localization signal (NLS) into the nucleus is mediated by nuclear transport receptors termed importins, typically dimers of a cargo-binding α-subunit and a ß-subunit that mediates translocation through the nuclear pore complex. Here, we review recent reports of importin-α cargo specificity and mutant phenotypes in plant- and animal-microbe interactions. Using homology modeling of the NLS-binding cleft of nine predicted Arabidopsis α-importins and analyses of their gene expression patterns, we discuss functional redundancy and specialization within this transport receptor family. In addition, we consider how pathogen effector proteins that promote infection by manipulating host cell nuclear processes might compete with endogenous cargo proteins for nuclear uptake.
RESUMO
In plant effector-triggered immunity (ETI), intracellular nucleotide binding-leucine rich repeat (NLR) receptors are activated by specific pathogen effectors. The Arabidopsis TIR (Toll-Interleukin-1 receptor domain)-NLR (denoted TNL) gene pair, RPS4 and RRS1, confers resistance to Pseudomonas syringae pv tomato (Pst) strain DC3000 expressing the Type III-secreted effector, AvrRps4. Nuclear accumulation of AvrRps4, RPS4, and the TNL resistance regulator EDS1 is necessary for ETI. RRS1 possesses a C-terminal "WRKY" transcription factor DNA binding domain suggesting that important RPS4/RRS1 recognition and/or resistance signaling events occur at the nuclear chromatin. In Arabidopsis accession Ws-0, the RPS4(Ws) /RRS1(Ws) allelic pair governs resistance to Pst/AvrRps4 accompanied by host programed cell death (pcd). In accession Col-0, RPS4(Col) /RRS1(Col) effectively limits Pst/AvrRps4 growth without pcd. Constitutive expression of HA-StrepII tagged RPS4(Col) (in a 35S:RPS4-HS line) confers temperature-conditioned EDS1-dependent auto-immunity. Here we show that a high (28°C, non-permissive) to moderate (19°C, permissive) temperature shift of 35S:RPS4-HS plants can be used to follow defense-related transcriptional dynamics without a pathogen effector trigger. By comparing responses of 35S:RPS4-HS with 35S:RPS4-HS rrs1-11 and 35S:RPS4-HS eds1-2 mutants, we establish that RPS4(Col) auto-immunity depends entirely on EDS1 and partially on RRS1(Col) . Examination of gene expression microarray data over 24 h after temperature shift reveals a mainly quantitative RRS1(Col) contribution to up- or down-regulation of a small subset of RPS4(Col) -reprogramed, EDS1-dependent genes. We find significant over-representation of WRKY transcription factor binding W-box cis-elements within the promoters of these genes. Our data show that RRS1(Col) contributes to temperature-conditioned RPS4(Col) auto-immunity and are consistent with activated RPS4(Col) engaging RRS1(Col) for resistance signaling.