RESUMEN
Membrane trafficking is required during plant immune responses, but its contribution to the hypersensitive response (HR), a form of programmed cell death (PCD) associated with effector-triggered immunity, is not well understood. HR is induced by nucleotide binding-leucine-rich repeat (NB-LRR) immune receptors and can involve vacuole-mediated processes, including autophagy. We previously isolated lazarus (laz) suppressors of autoimmunity-triggered PCD in the Arabidopsis thaliana mutant accelerated cell death11 (acd11) and demonstrated that the cell death phenotype is due to ectopic activation of the LAZ5 NB-LRR. We report here that laz4 is mutated in one of three VACUOLAR PROTEIN SORTING35 (VPS35) genes. We verify that LAZ4/VPS35B is part of the retromer complex, which functions in endosomal protein sorting and vacuolar trafficking. We show that VPS35B acts in an endosomal trafficking pathway and plays a role in LAZ5-dependent acd11 cell death. Furthermore, we find that VPS35 homologs contribute to certain forms of NB-LRR protein-mediated autoimmunity as well as pathogen-triggered HR. Finally, we demonstrate that retromer deficiency causes defects in late endocytic/lytic compartments and impairs autophagy-associated vacuolar processes. Our findings indicate important roles of retromer-mediated trafficking during the HR; these may include endosomal sorting of immune components and targeting of vacuolar cargo.
Asunto(s)
Apoptosis , Proteínas de Arabidopsis/metabolismo , Arabidopsis/citología , Arabidopsis/inmunología , Complejos Multiproteicos/metabolismo , Inmunidad de la Planta , Arabidopsis/genética , Autofagia , Resistencia a la Enfermedad/inmunología , Endocitosis , Genes de Plantas , Proteínas Fluorescentes Verdes/metabolismo , Cuerpos Multivesiculares/metabolismo , Mutación , Enfermedades de las Plantas/inmunología , Unión Proteica , Subunidades de Proteína/metabolismo , Transporte de Proteínas , Homología de Secuencia de AminoácidoRESUMEN
Plants need to finely balance resources allocated to growth and immunity to achieve optimal fitness. A tradeoff between pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and brassinosteroid (BR)-mediated growth was recently reported, but more information about the underlying mechanisms is needed. Here, we identify the basic helix-loop-helix (bHLH) transcription factor homolog of brassinosteroid enhanced expression2 interacting with IBH1 (HBI1) as a negative regulator of PTI signaling in Arabidopsis (Arabidopsis thaliana). HBI1 expression is down-regulated in response to different PAMPs. HBI1 overexpression leads to reduced PAMP-triggered responses. This inhibition correlates with reduced steady-state expression of immune marker genes, leading to increased susceptibility to the bacterium Pseudomonas syringae. Overexpression of the HBI1-related bHLHs brassinosteroid enhanced expression2 (BEE2) and cryptochrome-interacting bHLH (CIB1) partially inhibits immunity, indicating that BEE2 and CIB1 may act redundantly with HBI1. In contrast to its expression pattern upon PAMP treatment, HBI1 expression is enhanced by BR treatment. Also, HBI1-overexpressing plants are hyperresponsive to BR and more resistant to the BR biosynthetic inhibitor brassinazole. HBI1 is nucleus localized, and a mutation in a conserved leucine residue within the first helix of the protein interaction domain impairs its function in BR signaling. Interestingly, HBI1 interacts with several inhibitory atypical bHLHs, which likely keep HBI1 under negative control. Hence, HBI1 is a positive regulator of BR-triggered responses, and the negative effect of PTI is likely due to the antagonism between BR and PTI signaling. This study identifies a novel component involved in the complex tradeoff between innate immunity and BR-regulated growth.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/inmunología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Inmunidad de la Planta , Secuencia de Aminoácidos , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/química , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/química , Brasinoesteroides/biosíntesis , Brasinoesteroides/farmacología , Secuencia Conservada/genética , Regulación hacia Abajo , Regulación de la Expresión Génica de las Plantas , Genes Dominantes , Inmunidad Innata , Leucina/genética , Datos de Secuencia Molecular , Mutación/genética , Unión Proteica , Receptores de Reconocimiento de Patrones/metabolismo , Homología de Secuencia de AminoácidoRESUMEN
Asparagine-linked glycosylation of proteins is an essential cotranslational and posttranslational protein modification in plants. The central step in this process is the transfer of a preassembled oligosaccharide to nascent proteins in the endoplasmic reticulum by the oligosaccharyltransferase (OST) complex. Despite the importance of the catalyzed reaction, the composition and the function of individual OST subunits are still ill defined in plants. Here, we report the function of the highly conserved OST subunit OST3/6. We have identified a mutant in the OST3/6 gene that causes overall underglycosylation of proteins and affects the biogenesis of the receptor kinase EF-TU RECEPTOR involved in innate immunity and the endo-ß-1,4-glucanase KORRIGAN1 required for cellulose biosynthesis. Notably, the ost3/6 mutation does not affect mutant variants of the receptor kinase BRASSINOSTEROID-INSENSITIVE1. OST3/6 deficiency results in activation of the unfolded protein response and causes hypersensitivity to salt/osmotic stress and to the glycosylation inhibitor tunicamycin. Consistent with its role in protein glycosylation, OST3/6 resides in the endoplasmic reticulum and interacts with other subunits of the OST complex. Together, our findings reveal the importance of Arabidopsis (Arabidopsis thaliana) OST3/6 for the efficient glycosylation of specific glycoproteins involved in different physiological processes and shed light on the composition and function of the plant OST complex.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Hexosiltransferasas/metabolismo , Proteínas de la Membrana/metabolismo , Inmunidad de la Planta , Estrés Fisiológico , Secuencia de Aminoácidos , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Arabidopsis/fisiología , Proteínas de Arabidopsis/genética , Celulasa/genética , Celulasa/metabolismo , Celulosa/metabolismo , Retículo Endoplásmico/metabolismo , Glicoproteínas , Glicosilación , Hexosiltransferasas/genética , Manitol/farmacología , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Mutación , Mapeo de Interacción de Proteínas , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Receptores de Reconocimiento de Patrones/genética , Receptores de Reconocimiento de Patrones/metabolismo , Proteínas Recombinantes de Fusión , Plantones/efectos de los fármacos , Plantones/enzimología , Plantones/genética , Plantones/fisiología , Alineación de Secuencia , Cloruro de Sodio/farmacología , Tunicamicina/farmacologíaRESUMEN
Recognition of pathogen-associated molecular patterns (PAMPs) by surface-localized pattern recognition receptors (PRRs) constitutes an important layer of innate immunity in plants. The leucine-rich repeat (LRR) receptor kinases EF-TU RECEPTOR (EFR) and FLAGELLIN SENSING2 (FLS2) are the PRRs for the peptide PAMPs elf18 and flg22, which are derived from bacterial EF-Tu and flagellin, respectively. Using coimmunoprecipitation and mass spectrometry analyses, we demonstrated that EFR and FLS2 undergo ligand-induced heteromerization in planta with several LRR receptor-like kinases that belong to the SOMATIC-EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) family, including BRASSINOSTEROID INSENSITIVE1-ASSOCIATED KINASE1/SERK3 (BAK1/SERK3) and BAK1-LIKE1/SERK4 (BKK1/SERK4). Using a novel bak1 allele that does not exhibit pleiotropic defects in brassinosteroid and cell death responses, we determined that BAK1 and BKK1 cooperate genetically to achieve full signaling capability in response to elf18 and flg22 and to the damage-associated molecular pattern AtPep1. Furthermore, we demonstrated that BAK1 and BKK1 contribute to disease resistance against the hemibiotrophic bacterium Pseudomonas syringae and the obligate biotrophic oomycete Hyaloperonospora arabidopsidis. Our work reveals that the establishment of PAMP-triggered immunity (PTI) relies on the rapid ligand-induced recruitment of multiple SERKs within PRR complexes and provides insight into the early PTI signaling events underlying this important layer of plant innate immunity.
Asunto(s)
Proteínas de Arabidopsis/inmunología , Arabidopsis/enzimología , Arabidopsis/inmunología , Inmunidad Innata , Oomicetos/patogenicidad , Enfermedades de las Plantas/inmunología , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/inmunología , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/microbiología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ligandos , Datos de Secuencia Molecular , Complejos Multienzimáticos/genética , Complejos Multienzimáticos/metabolismo , Oomicetos/inmunología , Péptidos/genética , Péptidos/metabolismo , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/genética , Pseudomonas syringae/inmunología , Pseudomonas syringae/patogenicidad , Proteínas Recombinantes de Fusión/metabolismo , Transducción de SeñalRESUMEN
Certain pathogens deliver effectors into plant cells to modify host protein targets and thereby suppress immunity. These target modifications can be detected by intracellular immune receptors, or Resistance (R) proteins, that trigger strong immune responses including localized host cell death. The accelerated cell death 11 (acd11) "lesion mimic" mutant of Arabidopsis thaliana exhibits autoimmune phenotypes such as constitutive defense responses and cell death without pathogen perception. ACD11 encodes a putative sphingosine transfer protein, but its precise role during these processes is unknown. In a screen for lazarus (laz) mutants that suppress acd11 death we identified two genes, LAZ2 and LAZ5. LAZ2 encodes the histone lysine methyltransferase SDG8, previously shown to epigenetically regulate flowering time via modification of histone 3 (H3). LAZ5 encodes an RPS4-like R-protein, defined by several dominant negative alleles. Microarray and chromatin immunoprecipitation analyses showed that LAZ2/SDG8 is required for LAZ5 expression and H3 lysine 36 trimethylation at LAZ5 chromatin to maintain a transcriptionally active state. We hypothesize that LAZ5 triggers cell death in the absence of ACD11, and that cell death in other lesion mimic mutants may also be caused by inappropriate activation of R genes. Moreover, SDG8 is required for basal and R protein-mediated pathogen resistance in Arabidopsis, revealing the importance of chromatin remodeling as a key process in plant innate immunity.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/genética , Proteínas de Arabidopsis/genética , Arabidopsis/inmunología , Epigénesis Genética/fisiología , Proteínas de Transporte de Membrana/genética , Receptores Inmunológicos/genética , Proteínas Reguladoras de la Apoptosis/fisiología , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/fisiología , Proteínas de Arabidopsis/fisiología , Autoinmunidad/fisiología , Muerte Celular/genética , Muerte Celular/inmunología , Ensamble y Desensamble de Cromatina/genética , Ensamble y Desensamble de Cromatina/inmunología , Ensamble y Desensamble de Cromatina/fisiología , Epigénesis Genética/inmunología , Regulación de la Expresión Génica de las Plantas/fisiología , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , N-Metiltransferasa de Histona-Lisina/fisiología , Inmunidad Innata/genética , Proteínas de Transporte de Membrana/fisiología , Receptores Inmunológicos/fisiologíaRESUMEN
To optimize fitness a plant should monitor its metabolism to appropriately control growth and defense. Primary metabolism can be measured by the universally conserved TOR (Target of Rapamycin) pathway to balance growth and development with the available energy and nutrients. Recent work suggests that plants may measure defense metabolites to potentially provide a strategy ensuring fast reallocation of resources to coordinate plant growth and defense. There is little understanding of mechanisms enabling defense metabolite signaling. To identify mechanisms of defense metabolite signaling, we used glucosinolates, an important class of plant defense metabolites. We report novel signaling properties specific to one distinct glucosinolate, 3-hydroxypropylglucosinolate across plants and fungi. This defense metabolite, or derived compounds, reversibly inhibits root growth and development. 3-hydroxypropylglucosinolate signaling functions via genes in the ancient TOR pathway. If this event is not unique, this raises the possibility that other evolutionarily new plant metabolites may link to ancient signaling pathways.
Asunto(s)
Glucosinolatos/metabolismo , Reguladores del Crecimiento de las Plantas/metabolismo , Raíces de Plantas/crecimiento & desarrollo , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Hongos/metabolismo , Plantas/metabolismoRESUMEN
Plant perception of pathogen-associated molecular patterns (PAMPs) triggers a phosphorylation relay leading to PAMP-triggered immunity (PTI). Despite increasing knowledge of PTI signaling, how immune homeostasis is maintained remains largely unknown. Here we describe a forward-genetic screen to identify loci involved in PTI and characterize the Arabidopsis calcium-dependent protein kinase CPK28 as a negative regulator of immune signaling. Genetic analyses demonstrate that CPK28 attenuates PAMP-triggered immune responses and antibacterial immunity. CPK28 interacts with and phosphorylates the plasma-membrane-associated cytoplasmic kinase BIK1, an important convergent substrate of multiple pattern recognition receptor (PRR) complexes. We find that BIK1 is rate limiting in PTI signaling and that it is continuously turned over to maintain cellular homeostasis. We further show that CPK28 contributes to BIK1 turnover. Our results suggest a negative regulatory mechanism that continually buffers immune signaling by controlling the turnover of this key signaling kinase.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Inmunidad de la Planta/genética , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Secuencia de Aminoácidos , Arabidopsis/genética , Arabidopsis/inmunología , Proteínas de Arabidopsis/genética , Sitios Genéticos , Datos de Secuencia Molecular , Fosforilación , Enfermedades de las Plantas/inmunología , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
Innate immunity relies on the perception of pathogen-associated molecular patterns (PAMPs) by pattern-recognition receptors (PRRs) located on the host cell's surface. Many plant PRRs are kinases. Here, we report that the Arabidopsis receptor kinase EF-TU RECEPTOR (EFR), which perceives the elf18 peptide derived from bacterial elongation factor Tu, is activated upon ligand binding by phosphorylation on its tyrosine residues. Phosphorylation of a single tyrosine residue, Y836, is required for activation of EFR and downstream immunity to the phytopathogenic bacterium Pseudomonas syringae. A tyrosine phosphatase, HopAO1, secreted by P. syringae, reduces EFR phosphorylation and prevents subsequent immune responses. Thus, host and pathogen compete to take control of PRR tyrosine phosphorylation used to initiate antibacterial immunity.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/inmunología , Arabidopsis/microbiología , Proteínas Bacterianas/metabolismo , Factor Tu de Elongación Peptídica/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Pseudomonas syringae/patogenicidad , Receptores de Reconocimiento de Patrones/metabolismo , Proteínas de Arabidopsis/agonistas , Péptidos/metabolismo , Péptidos/farmacología , Fosforilación , Pseudomonas syringae/enzimología , Receptores de Reconocimiento de Patrones/agonistas , Tirosina/metabolismoRESUMEN
Salicylic acid (SA) is implicated in the induction of programmed cell death (PCD) associated with pathogen defense responses because SA levels increase in response to PCD-inducing infections, and PCD development can be inhibited by expression of salicylate hydroxylase encoded by the bacterial nahG gene. The acd11 mutant of Arabidopsis (Arabidopsis thaliana L. Heynh.) activates PCD and defense responses that are fully suppressed by nahG. To further study the role of SA in PCD induction, we compared phenotypes of acd11/nahG with those of acd11/eds5-1 and acd11/sid2-2 mutants deficient in a putative transporter and isochorismate synthase required for SA biosynthesis. We show that sid2-2 fully suppresses SA accumulation and cell death in acd11, although growth inhibition and premature leaf chlorosis still occur. In addition, application of exogenous SA to acd11/sid2-2 is insufficient to restore cell death. This indicates that isochorismate-derived compounds other than SA are required for induction of PCD in acd11 and that some acd11 phenotypes require NahG-degradable compounds not synthesized via isochorismate.