RESUMO
NLR family proteins act as intracellular receptors. Gene duplication amplifies the number of NLR genes, and subsequent mutations occasionally provide modifications to the second gene that benefits immunity. However, evolutionary processes after gene duplication and functional relationships between duplicated NLRs remain largely unclear. Here, we report that the rice NLR protein Pit1 is associated with its paralogue Pit2. The two are required for the resistance to rice blast fungus but have different functions: Pit1 induces cell death, while Pit2 competitively suppresses Pit1-mediated cell death. During evolution, the suppression of Pit1 by Pit2 was probably generated through positive selection on two fate-determining residues in the NB-ARC domain of Pit2, which account for functional differences between Pit1 and Pit2. Consequently, Pit2 lost its plasma membrane localization but acquired a new function to interfere with Pit1 in the cytosol. These findings illuminate the evolutionary trajectory of tandemly duplicated NLR genes after gene duplication.
Assuntos
Duplicação Gênica , Proteínas NLR , Oryza , Proteínas de Plantas , Proteínas NLR/genética , Proteínas NLR/metabolismo , Oryza/genética , Oryza/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Evolução Molecular , Doenças das Plantas/microbiologia , Doenças das Plantas/genética , Doenças das Plantas/imunologia , Resistência à Doença/genética , Morte Celular , Filogenia , Regulação da Expressão Gênica de PlantasRESUMO
Plants employ two different types of immune receptors, cell surface pattern recognition receptors (PRRs) and intracellular nucleotide-binding and leucine-rich repeat-containing proteins (NLRs), to cope with pathogen invasion. Both immune receptors often share similar downstream components and responses but it remains unknown whether a PRR and an NLR assemble into the same protein complex or two distinct receptor complexes. We have previously found that the small GTPase OsRac1 plays key roles in the signaling of OsCERK1, a PRR for fungal chitin, and of Pit, an NLR for rice blast fungus, and associates directly and indirectly with both of these immune receptors. In this study, using biochemical and bioimaging approaches, we revealed that OsRac1 formed two distinct receptor complexes with OsCERK1 and with Pit. Supporting this result, OsCERK1 and Pit utilized different transport systems for anchorage to the plasma membrane (PM). Activation of OsCERK1 and Pit led to OsRac1 activation and, concomitantly, OsRac1 shifted from a small to a large protein complex fraction. We also found that the chaperone Hsp90 contributed to the proper transport of Pit to the PM and the immune induction of Pit. These findings illuminate how the PRR OsCERK1 and the NLR Pit orchestrate rice immunity through the small GTPase OsRac1.
Assuntos
GTP Fosfo-Hidrolases/genética , Proteínas NLR/genética , Oryza/genética , Imunidade Vegetal/genética , Proteínas de Plantas/genética , Receptores de Reconhecimento de Padrão/genética , GTP Fosfo-Hidrolases/metabolismo , Proteínas NLR/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Receptores de Reconhecimento de Padrão/metabolismoRESUMO
DNA methylation is an epigenetic modification that specifies the basic state of pluripotent stem cells and regulates the developmental transition from stem cells to various cell types. In flowering plants, the shoot apical meristem (SAM) contains a pluripotent stem cell population which generates the aerial part of plants including the germ cells. Under appropriate conditions, the SAM undergoes a developmental transition from a leaf-forming vegetative SAM to an inflorescence- and flower-forming reproductive SAM. While SAM characteristics are largely altered in this transition, the complete picture of DNA methylation remains elusive. Here, by analyzing whole-genome DNA methylation of isolated rice SAMs in the vegetative and reproductive stages, we show that methylation at CHH sites is kept high, particularly at transposable elements (TEs), in the vegetative SAM relative to the differentiated leaf, and increases in the reproductive SAM via the RNA-dependent DNA methylation pathway. We also show that half of the TEs that were highly methylated in gametes had already undergone CHH hypermethylation in the SAM. Our results indicate that changes in DNA methylation begin in the SAM long before germ cell differentiation to protect the genome from harmful TEs.
Assuntos
Metilação de DNA , Meristema/crescimento & desenvolvimento , Meristema/genética , Oryza/genética , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/genética , Elementos de DNA Transponíveis , Biologia do Desenvolvimento , Epigenômica , Flores , Regulação da Expressão Gênica de Plantas , Inflorescência , Folhas de Planta/metabolismo , Proteínas de Plantas/genéticaRESUMO
Resistance (R) genes encode intracellular nucleotide-binding/leucine-rich repeat-containing (NLR) family proteins that serve as critical plant immune receptors to induce effector-triggered immunity (ETI). NLR proteins possess a tripartite domain architecture consisting of an N-terminal variable region, a central nucleotide-binding domain, and a C-terminal leucine-rich repeat. N-terminal coiled-coil (CC) or Toll-interleukin 1 receptor (TIR) domains of R proteins appear to serve as platforms to trigger immune responses, because overexpression of the CC or TIR domain of some R proteins is sufficient to induce an immune response. Because direct downstream signaling molecules of R proteins remain obscure, the molecular mechanisms by which R proteins regulate downstream signaling are largely unknown. We reported previously that a rice R protein named Pit triggers ETI through a small GTPase, OsRac1, although how Pit activates OsRac1 is unclear. Here, we identified OsSPK1, a DOCK family guanine nucleotide exchange factor, as an interactor of Pit and activator for OsRac1. OsSPK1 contributes to signaling by two disease-resistance genes, Pit and Pia, against the rice blast fungus Magnaporthe oryzae and facilitates OsRac1 activation in vitro and in vivo. The CC domain of Pit is required for its binding to OsSPK1, OsRac1 activation, and the induction of cell death. Overall, we conclude that OsSPK1 is a direct and key signaling target of Pit-mediated immunity. Our results shed light on how R proteins trigger ETI through direct downstream molecules.
Assuntos
Oryza/genética , Oryza/imunologia , Doenças das Plantas/imunologia , Proteínas de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas , Magnaporthe , Doenças das Plantas/microbiologia , Proteínas de Plantas/genéticaRESUMO
BACKGROUND: Small GTPases act as molecular switches that regulate various plant responses such as disease resistance, pollen tube growth, root hair development, cell wall patterning and hormone responses. Thus, to monitor their activation status within plant cells is believed to be the key step in understanding their roles. RESULTS: We have established a plant version of a Förster resonance energy transfer (FRET) probe called Ras and interacting protein chimeric unit (Raichu) that can successfully monitor activation of the rice small GTPase OsRac1 during various defence responses in cells. Here, we describe the protocol for visualizing spatiotemporal activity of plant Rac/ROP GTPase in living plant cells, transfection of rice protoplasts with Raichu-OsRac1 and acquisition of FRET images. CONCLUSIONS: Our protocol should be adaptable for monitoring activation for other plant small GTPases and protein-protein interactions for other FRET sensors in various plant cells.
RESUMO
Hd3a, a rice homolog of FLOWERING LOCUS T (FT), is a florigen that induces flowering. Hd3a forms a ternary 'florigen activation complex' (FAC) with 14-3-3 protein and OsFD1 transcription factor, a rice homolog of FD that induces transcription of OsMADS15, a rice homolog of APETALA1 (AP1), which leads to flowering. TERMINAL FLOWER 1 (TFL1) represses flowering and controls inflorescence architecture. However, the molecular basis for floral repression by TFL1 remains poorly understood. Here we show that RICE CENTRORADIALIS (RCN), rice TFL1-like proteins, compete with Hd3a for 14-3-3 binding. All four RCN genes are predominantly expressed in the vasculature, and RCN proteins are transported to the shoot apex to antagonize florigen activity and regulate inflorescence development. The antagonistic function of RCN to Hd3a is dependent on its 14-3-3 binding activity. Our results suggest a molecular basis for regulation of the balance between florigen FT and anti-florigen TFL1.
Assuntos
Proteínas 14-3-3/metabolismo , Inflorescência/crescimento & desenvolvimento , Inflorescência/metabolismo , Oryza/crescimento & desenvolvimento , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Florígeno/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas , Inflorescência/efeitos dos fármacos , Meristema/efeitos dos fármacos , Meristema/metabolismo , Modelos Biológicos , Especificidade de Órgãos/genética , Oryza/efeitos dos fármacos , Oryza/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Ligação Proteica/efeitos dos fármacosRESUMO
Photoperiod-regulated flowering and potato tuber formation involve leaf-produced mobile signals, florigen and tuberigen, respectively. The major protein component of florigen has been identified as the FLOWERING LOCUS T (FT) protein. In rice, an FT-like protein, Heading date 3a (Hd3a), induces flowering by making the florigen activation complex (FAC) through interactions with 14-3-3 and OsFD1, a rice FD-like protein. In potato, StSP6A, an FT-like protein, was identified as a major component of tuberigen. However, the molecular mechanism of how StSP6A triggers tuber formation remains elusive. Here we analyzed the significance of the formation of a complex including StSP6A, 14-3-3 and FD-like proteins in tuberization. Yeast two-hybrid, bimolecular fluorescence complementation and in vitro pull-down assays showed that StSP6A and StFDL1, a potato FD-like protein, interact with St14-3-3s. StSP6A overexpression induced early tuberization in a 14-3-3-dependent manner, and suppression of StFDL1 delayed tuberization. These results strongly suggest that an FAC-like complex, the tuberigen activation complex (TAC), comprised of StSP6A, St14-3-3s and StFDL1, regulates potato tuber formation.
Assuntos
Proteínas de Plantas/metabolismo , Tubérculos/metabolismo , Tubérculos/fisiologia , Solanum tuberosum/metabolismo , Solanum tuberosum/fisiologia , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Tubérculos/genética , Ligação Proteica , Solanum tuberosum/genéticaRESUMO
Rice is one of the most important food crops, feeding about half population in the world. Rice pathogens cause enormous damage to rice production worldwide. In plant immunity research, considerable progress has recently been made in our understanding of the molecular mechanisms underlying microbe-associated molecular pattern (MAMP)-triggered immunity. Using genome sequencing and molecular techniques, a number of new MAMPs and their receptors have been identified in the past two decades. Notably, the mechanisms for chitin perception via the lysine motif (LysM) domain-containing receptor OsCERK1, as well as the mechanisms for bacterial MAMP (e.g. flg22, elf18) perception via the leucine-rich repeat (LRR) domain-containing receptors FLS2 and EFR, have been clarified in rice and Arabidopsis, respectively. In chitin signaling in rice, two direct substrates of OsCERK1, Rac/ROP GTPase guanine nucleotide exchange factor OsRacGEF1 and receptor-like cytoplasmic kinase OsRLCK185, have been identified as components of the OsCERK1 complex and are rapidly phosphorylated by OsCERK1 in response to chitin. Interestingly, OsCERK1 also participates in symbiosis with arbuscular mycorrhizal fungi (AMF) in rice and plays a role in the recognition of short-chitin molecules (CO4/5), which are symbiotic signatures included in AMF germinated spore exudates and induced by synthetic strigolactone. Thus, OsCERK1 contributes to both immunity and symbiotic responses. In this review, we describe recent studies on pathways involved in rice immunity and symbiotic signaling triggered by interactions with microorganisms. In addition, we describe recent advances in genetic engineering by using plant immune receptors and symbiotic microorganisms to enhance disease resistance of rice.
RESUMO
Numerous plant defense-related proteins are thought to congregate in plasma membrane microdomains, which consist mainly of sphingolipids and sterols. However, the extent to which microdomains contribute to defense responses in plants is unclear. To elucidate the relationship between microdomains and innate immunity in rice (Oryza sativa), we established lines in which the levels of sphingolipids containing 2-hydroxy fatty acids were decreased by knocking down two genes encoding fatty acid 2-hydroxylases (FAH1 and FAH2) and demonstrated that microdomains were less abundant in these lines. By testing these lines in a pathogen infection assay, we revealed that microdomains play an important role in the resistance to rice blast fungus infection. To illuminate the mechanism by which microdomains regulate immunity, we evaluated changes in protein composition, revealing that microdomains are required for the dynamics of the Rac/ROP small GTPase Rac1 and respiratory burst oxidase homologs (Rbohs) in response to chitin elicitor. Furthermore, FAHs are essential for the production of reactive oxygen species (ROS) after chitin treatment. Together with the observation that RbohB, a defense-related NADPH oxidase that interacts with Rac1, is localized in microdomains, our data indicate that microdomains are required for chitin-induced immunity through ROS signaling mediated by the Rac1-RbohB pathway.
Assuntos
Microdomínios da Membrana/genética , Microdomínios da Membrana/metabolismo , Oryza/metabolismo , Imunidade Vegetal/fisiologia , Proteínas de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Oryza/genética , Imunidade Vegetal/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Ligação Proteica , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
Molecular links between receptor-kinases and Rac/ROP family small GTPases mediated by activator guanine nucleotide exchange factors (GEFs) govern diverse biological processes. However, it is unclear how the Rac/ROP GTPases orchestrate such a wide variety of activities. Here, we show that rice OsRacGEF1 forms homodimers, and heterodimers with OsRacGEF2, at the plasma membrane (PM) and the endoplasmic reticulum (ER). OsRacGEF2 does not bind directly to the receptor-like kinase (RLK) OsCERK1, but forms a complex with OsCERK1 through OsRacGEF1 at the ER. This complex is transported from ER to the PM and there associates with OsRac1, resulting in the formation of a stable immune complex. Such RLK-GEF heterodimer complexes may explain the diversity of Rac/ROP family GTPase signalings.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Oryza/metabolismo , Multimerização Proteica , Sequência de Aminoácidos , Fluorescência , Fatores de Troca do Nucleotídeo Guanina/química , Dados de Sequência Molecular , Oryza/citologia , Células Vegetais/metabolismo , Proteínas de Plantas/metabolismo , Ligação ProteicaRESUMO
Accumulating evidence indicates that the FLOWERING LOCUS T (FT) protein is the mobile floral signal known as florigen. A rice FT homolog, Heading date 3a (Hd3a), is transported from the phloem to shoot apical cells, where it interacts with 14-3-3 proteins and transcription factor OsFD1 to form a florigen activation complex (FAC) that activates a rice homolog of the floral identity gene APETALA1. Recent studies showed that florigen has roles in plant development beyond flowering; however, the exact nature of these roles is not well understood. It is not clear whether FT is transported to organs outside the shoot apex, and whether FAC formation is required for processes other than flowering. We show here that the Hd3a protein accumulates in axillary meristems to promote branching, and that FAC formation is required. Analysis of transgenic plants revealed that Hd3a promotes branching through lateral bud outgrowth. Hd3a protein produced in the phloem reached the axillary meristem in the lateral bud, and its transport was required for promotion of branching. Moreover, mutant Hd3a proteins defective in FAC formation but competent with respect to transport did not promote branching. Finally, we show that Hd3a promotes branching independently from strigolactone and FC1, a transcription factor that inhibits branching in rice. Together, these results suggest that Hd3a functions as a mobile signal for branching in rice.
Assuntos
Oryza/crescimento & desenvolvimento , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Florígeno/metabolismo , Meristema/genética , Meristema/crescimento & desenvolvimento , Meristema/metabolismo , Oryza/genética , Proteínas de Plantas/genéticaRESUMO
Floral induction is a crucial developmental step in higher plants. Florigen, a mobile floral activator that is synthesized in the leaf and transported to the shoot apex, was recently identified as a protein encoded by FLOWERING LOCUS T (FT) and its orthologs; the rice florigen is Heading date 3a (Hd3a) protein. The 14-3-3 proteins mediate the interaction of Hd3a with the transcription factor OsFD1 to form a ternary structure called the florigen activation complex on the promoter of OsMADS15, a rice APETALA1 ortholog. However, crucial information, including the spatiotemporal overlap among FT-like proteins and the components of florigen activation complex and downstream genes, remains unclear. Here, we confirm that Hd3a coexists, in the same regions of the rice shoot apex, with the other components of the florigen activation complex and its transcriptional targets. Unexpectedly, however, RNA-sequencing analysis of shoot apex from wild-type and RNA-interference plants depleted of florigen activity revealed that 4,379 transposable elements (TEs; 58% of all classifiable rice TEs) were expressed collectively in the vegetative and reproductive shoot apex. Furthermore, in the reproductive shoot apex, 214 TEs were silenced by florigen. Our results suggest a link between floral induction and regulation of TEs.
Assuntos
Elementos de DNA Transponíveis/genética , Flores/fisiologia , Inativação Gênica , Meristema/fisiologia , Oryza/genética , Oryza/fisiologia , Proteínas de Plantas/metabolismo , Sequência de Bases , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Florígeno/farmacologia , Flores/efeitos dos fármacos , Flores/genética , Flores/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Inativação Gênica/efeitos dos fármacos , Genes de Plantas , Inflorescência/efeitos dos fármacos , Inflorescência/metabolismo , Meristema/efeitos dos fármacos , Meristema/genética , Organogênese/efeitos dos fármacos , Oryza/efeitos dos fármacos , Fenótipo , Proteínas de Plantas/genética , Transporte Proteico/efeitos dos fármacos , Reprodutibilidade dos Testes , Reprodução/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
A complex consisting of evolutionarily conserved FD, flowering locus T (FT) proteins is a regulator of floral transition. Intriguingly, FT orthologs are also implicated in developmental transitions distinct from flowering, such as photoperiodic control of bulbing in onions, potato tuberization, and growth cessation in trees. However, whether an FT-FD complex participates in these transitions and, if so, its mode of action, are unknown. We identified two closely related FD homologs, FD-like 1 (FDL1) and FD-like 2 (FDL2), in the model tree hybrid aspen. Using gain of function and RNAi-suppressed FDL1 and FDL2 transgenic plants, we show that FDL1 and FDL2 have distinct functions and a complex consisting of FT and FDL1 mediates in photoperiodic control of seasonal growth. The downstream target of the FT-FD complex in photoperiodic control of growth is Like AP1 (LAP1), a tree ortholog of the floral meristem identity gene APETALA1. Intriguingly, FDL1 also participates in the transcriptional control of adaptive response and bud maturation pathways, independent of its interaction with FT, presumably via interaction with abscisic acid insensitive 3 (ABI3) transcription factor, a component of abscisic acid (ABA) signaling. Our data reveal that in contrast to its primary role in flowering, FD has dual roles in the photoperiodic control of seasonal growth and stress tolerance in trees. Thus, the functions of FT and FD have diversified during evolution, and FD homologs have acquired roles that are independent of their interaction with FT.
Assuntos
Adaptação Fisiológica , Florígeno/metabolismo , Fotoperíodo , Árvores/fisiologia , Árvores/crescimento & desenvolvimentoRESUMO
In plants, sophisticated forms of immune systems have developed to cope with a variety of pathogens. Accumulating evidence indicates that Rac (also known as Rop), a member of the Rho family of small GTPases, is a key regulator of immunity in plants and animals. Like other small GTPases, Rac/Rop GTPases function as a molecular switch downstream of immune receptors by cycling between GDP-bound inactive and GTP-bound active forms in cells. Rac/Rop GTPases trigger various immune responses, thereby resulting in enhanced disease resistance to pathogens. In this review, we highlight recent studies that have contributed to our current understanding of the Rac/Rop family GTPases and the upstream and downstream proteins involved in plant immunity. We also compare the features of effector-triggered immunity between plants and animals, and discuss the in vivo monitoring of Rac/Rop activation.
RESUMO
Cell-to-cell communication is a fundamental mechanism for coordinating developmental and physiological events in multicellular organisms. Heterotrimeric G proteins are key molecules that transmit extracellular signals; similarly, CLAVATA signaling is a crucial regulator in plant development. Here, we show that Arabidopsis thaliana Gß mutants exhibit an enlarged stem cell region, which is similar to that of clavata mutants. Our genetic and cell biological analyses suggest that the G protein beta-subunit1 AGB1 and RPK2, one of the major CLV3 peptide hormone receptors, work synergistically in stem cell homeostasis through their physical interactions. We propose that AGB1 and RPK2 compose a signaling module to facilitate meristem development.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Câmbio/metabolismo , Proliferação de Células , Subunidades beta da Proteína de Ligação ao GTP/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Arabidopsis/genética , Câmbio/fisiologia , Subunidades beta da Proteína de Ligação ao GTP/genética , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Transdução de SinaisRESUMO
Rac/Rop proteins are Rho-type small GTPases that act as molecular switches in plants. Recent studies have identified these proteins as key components in many major plant signaling pathways, such as innate immunity, pollen tube growth, and root hair formation. In rice, the Rac/Rop protein OsRac1 plays an important role in regulating the production of reactive oxygen species (ROS) by the NADPH oxidase OsRbohB during innate immunity. However, the molecular mechanism by which OsRac1 regulates OsRbohB remains unknown. Here, we report the crystal structure of OsRac1 complexed with the non-hydrolyzable GTP analog guanosine 5'-(ß,γ-imido)triphosphate at 1.9 Å resolution; this represents the first active-form structure of a plant small GTPase. To elucidate the ROS production in rice cells, structural information was used to design OsRac1 mutants that displayed reduced binding to OsRbohB. Only mutations in the OsRac1 Switch I region showed attenuated interactions with OsRbohB in vitro. In particular, Tyr(39) and Asp(45) substitutions suppressed ROS production in rice cells, indicating that these residues are critical for interaction with and activation of OsRbohB. Structural comparison of active-form OsRac1 with AtRop9 in its GDP-bound inactive form showed a large conformational difference in the vicinity of these residues. Our results provide new insights into the molecular mechanism of the immune response through OsRac1 and the various cellular responses associated with plant Rac/Rop proteins.
Assuntos
Guanilil Imidodifosfato/química , NADPH Oxidases/química , Oryza/química , Fosfatos de Fosfatidilinositol/química , Proteínas de Plantas/química , Proteínas rac1 de Ligação ao GTP/química , Sequência de Aminoácidos , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Regulação da Expressão Gênica de Plantas , Guanilil Imidodifosfato/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Mutação , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Oryza/enzimologia , Oryza/genética , Oryza/imunologia , Oxirredução , Fosfatos de Fosfatidilinositol/metabolismo , Imunidade Vegetal , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Espécies Reativas de Oxigênio/química , Espécies Reativas de Oxigênio/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Plant resistance proteins of the class of nucleotide-binding and leucine-rich repeat domain proteins (NB-LRRs) are immune sensors which recognize pathogen-derived molecules termed avirulence (AVR) proteins. We show that RGA4 and RGA5, two NB-LRRs from rice, interact functionally and physically to mediate resistance to the fungal pathogen Magnaporthe oryzae and accomplish different functions in AVR recognition. RGA4 triggers an AVR-independent cell death that is repressed in the presence of RGA5 in both rice protoplasts and Nicotiana benthamiana. Upon recognition of the pathogen effector AVR-Pia by direct binding to RGA5, repression is relieved and cell death occurs. RGA4 and RGA5 form homo- and hetero-complexes and interact through their coiled-coil domains. Localization studies in rice protoplast suggest that RGA4 and RGA5 localize to the cytosol. Upon recognition of AVR-Pia, neither RGA4 nor RGA5 is re-localized to the nucleus. These results establish a model for the interaction of hetero-pairs of NB-LRRs in plants: RGA4 mediates cell death activation, while RGA5 acts as a repressor of RGA4 and as an AVR receptor.
Assuntos
Resistência à Doença , Magnaporthe/crescimento & desenvolvimento , Magnaporthe/imunologia , Oryza/imunologia , Oryza/microbiologia , Proteínas de Plantas/imunologia , Proteínas de Plantas/metabolismo , Morte Celular , Modelos Biológicos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Protoplastos/fisiologia , Nicotiana/imunologia , Nicotiana/microbiologiaRESUMO
Oryza sativa Pto-interacting protein 1a (OsPti1a), an ortholog of tomato (Solanum lycopersicum) SlPti1, functions as a negative regulator of innate immunity in rice (Oryza sativa). In ospti1a mutants, the activation of immune responses, including hypersensitive response-like cell death, is caused by loss of the OsPti1a protein; however, it is as yet unclear how OsPti1a suppresses immune responses. Here, we report that OsPti1a localizes to detergent-resistant membrane fractions of the plasma membrane through lipid modification of the protein's amino terminus, which is highly conserved among Pti1 orthologs in several plant species. Importantly, mislocalization of OsPti1a after deletion of its amino terminus reduced its ability to complement the mutant phenotypes, including hypersensitive response-like cell death. Furthermore, complex formation of OsPti1a depends on its amino terminus-mediated membrane localization. Liquid chromatography-tandem mass spectrometry analysis of OsPti1a complex-interacting proteins identified several defense-related proteins. Collectively, these findings indicate that appropriate complex formation by OsPti1a at the plasma membrane is required for the negative regulation of plant immune responses in rice.
Assuntos
Oryza/enzimologia , Proteínas de Plantas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Sequência de Aminoácidos , Membrana Celular/enzimologia , Sequência Conservada , Lipoilação , Dados de Sequência Molecular , Oryza/imunologia , FenótipoRESUMO
Nucleotide binding domain and leucine-rich repeat (NLR)-containing family proteins function as intracellular immune sensors in both plants and animals. In plants, the downstream components activated by NLR family proteins and the immune response mechanisms induced by these downstream molecules are largely unknown. We have previously found that the small GTPase OsRac1, which acts as a molecular switch in rice immunity, is activated by Pit, an NLR-type resistance (R) protein to rice blast fungus, and this activation plays critical roles in Pit-mediated immunity. However, the sites and mechanisms of activation of Pit in vivo remain unknown. To clarify the mechanisms involved in the localization of Pit, we searched for consensus sequences in Pit that specify membrane localization and found a pair of potential palmitoylation sites in the N-terminal coiled-coil region. Although wild-type Pit was localized mainly to the plasma membrane, this membrane localization was compromised in a palmitoylation-deficient mutant of Pit. The palmitoylation-deficient Pit displayed significantly lower affinity for OsRac1 on the plasma membrane, thereby resulting in failures of the Pit-mediated cell death, the production of reactive oxygen species, and disease resistance to rice blast fungus. These results indicate that palmitoylation-dependent membrane localization of Pit is required for the interaction with and the activation of OsRac1 and that OsRac1 activation by Pit is vital for Pit-mediated disease resistance to rice blast fungus.