RESUMEN
Iron- and reactive oxygen species (ROS)-dependent ferroptosis occurs in plant cells. Ca2+ acts as a conserved key mediator to control plant immune responses. Here, we report a novel role of cytoplasmic Ca2+ influx regulating ferroptotic cell death in rice immunity using pharmacological approaches. High Ca2+ influx triggered iron-dependent ROS accumulation, lipid peroxidation, and subsequent hypersensitive response (HR) cell death in rice (Oryza sativa). During Magnaporthe oryzae infection, 14 different Ca2+ influx regulators altered Ca2+, ROS and Fe2+ accumulation, glutathione reductase (GR) expression, glutathione (GSH) depletion and lipid peroxidation, leading to ferroptotic cell death in rice. High Ca2+ levels inhibited the reduction of glutathione isulphide (GSSG) to GSH in vitro. Ca2+ chelation by ethylene glycol-bis (2-aminoethylether)-N, N, N', N'-tetra-acetic acid (EGTA) suppressed apoplastic Ca2+ influx in rice leaf sheaths during infection. Blocking apoplastic Ca2+ influx into the cytoplasm by Ca2+ chelation effectively suppressed Ca2+-mediated iron-dependent ROS accumulation and ferroptotic cell death. By contrast, acibenzolar-S-methyl (ASM), a plant defense activator, significantly enhanced Ca2+ influx, as well as ROS and iron accumulation to trigger ferroptotic cell death in rice. The cytoplasmic Ca2+ influx through calcium-permeable cation channels, including the putative resistosomes, could mediate iron- and ROS-dependent ferroptotic cell death under reduced GR expression levels in rice immune responses.
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Ferritin is a ubiquitous iron storage protein that regulates iron homeostasis and oxidative stress in plants. Iron plays an important role in ferroptotic cell death response of rice (Oryza sativa) to Magnaporthe oryzae infection. Here, we report that rice ferritin 2, OsFER2, is required for iron- and reactive oxygen species (ROS)-dependent ferroptotic cell death and defense response against the avirulent M. oryzae INA168. The full-length ferritin OsFER2 and its transit peptide were localized to the chloroplast, the most Fe-rich organelle for photosynthesis. This suggests that the transit peptide acts as a signal peptide for the rice ferritin OsFER2 to move into chloroplasts. OsFER2 expression is involved in rice resistance to M. oryzae infection. OsFER2 knock-out in wild-type rice HY did not induce ROS and ferric ion (Fe3+) accumulation, lipid peroxidation and hypersensitive response (HR) cell death, and also downregulated the defense-related genes OsPAL1, OsPR1-b, OsRbohB, OsNADP-ME2-3, OsMEK2 and OsMPK1, and vacuolar membrane transporter OsVIT2 expression. OsFER2 complementation in ΔOsfer2 knock-out mutants restored ROS and iron accumulation and HR cell death phenotypes during infection. The iron chelator deferoxamine, the lipid-ROS scavenger ferrostatin-1, the actin microfilament polymerization inhibitor cytochalasin E and the redox inhibitor diphenyleneiodonium suppressed ROS and iron accumulation and HR cell death in rice leaf sheaths. However, the small-molecule inducer erastin did not trigger iron-dependent ROS accumulation and HR cell death induction in ΔOsfer2 mutants. These combined results suggest that OsFER2 expression positively regulates iron- and ROS-dependent ferroptotic cell death and defense response in rice-M. oryzae interactions.
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Nodule inception (NIN)-like proteins (NLPs) have a central role in nitrate signaling to mediate plant growth and development. Here, we report that OsNLP2 negatively regulates ferroptotic cell death and immune responses in rice during Magnaporthe oryzae infection. OsNLP2 was localized to the plant cell nucleus, suggesting that it acts as a transcription factor. OsNLP2 expression was involved in susceptible disease development. ΔOsnlp2 knockout mutants exhibited reactive oxygen species (ROS) and iron-dependent ferroptotic hypersensitive response (HR) cell death in response to M. oryzae. Treatments with the iron chelator deferoxamine, lipid-ROS scavenger ferrostatin-1, actin polymerization inhibitor cytochalasin A, and NADPH oxidase inhibitor diphenyleneiodonium suppressed the accumulation of ROS and ferric ions, lipid peroxidation, and HR cell death, which ultimately led to successful M. oryzae colonization in ΔOsnlp2 mutants. The loss-of-function of OsNLP2 triggered the expression of defense-related genes including OsPBZ1, OsPIP-3A, OsWRKY104, and OsRbohB in ΔOsnlp2 mutants. ΔOsnlp2 mutants exhibited broad-spectrum, nonspecific resistance to diverse M. oryzae strains. These combined results suggest that OsNLP2 acts as a negative regulator of ferroptotic HR cell death and defense responses in rice, and may be a valuable gene source for molecular breeding of rice with broad-spectrum resistance to blast disease.
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Mitogen-activated protein kinase (MAPK) signaling is required for plant cell death responses to invading microbial pathogens. Iron- and reactive oxygen species (ROS)-dependent ferroptotic cell death occurs in rice (Oryza sativa) during an incompatible rice-Magnaporthe oryzae interaction. Here, we show that rice MAP kinase (OsMEK2 and OsMPK1) signaling cascades are involved in iron- and ROS-dependent ferroptotic cell death responses of rice to M. oryzae infection using OsMEK2 knock-out mutant and OsMEK2 and OsMPK1 overexpression rice plants. The OsMPK1:GFP and OsWRKY90:GFP transcription factor were localized to the nuclei, suggesting that OsMPK1 in the cytoplasm moves into the nuclei to interact with the WRKY90. M. oryzae infection in ΔOsmek2 knock-out plants did not trigger iron and ROS accumulation and lipid peroxidation, and also downregulated OsMPK1, OsWRKY90, OsRbohB, and OsPR-1b expression. However, 35S:OsMEK2 overexpression induced ROS- and iron-dependent cell death in rice. The downstream MAP kinase (OsMPK1) overexpression induced ROS- and iron-dependent ferroptotic cell death response to virulent M. oryzae infection. The small-molecule ferroptosis inhibitor ferrostatin-1 suppressed iron- and ROS-dependent ferroptotic cell death in 35S:OsMPK1 overexpression plants. However, the small-molecule inducer erastin triggered iron- and lipid ROS-dependent, but OsMEK2-independent, ferroptotic cell death during M. oryzae infection. Disease (susceptibility)-related cell death was lipid ROS-dependent, but iron-independent in the ΔOsmek2 knock-out mutant during the late M. oryzae infection stage. These combined results suggest that OsMEK2 and OsMPK1 expression positively regulates iron- and ROS-dependent ferroptotic cell death, and blast disease (susceptibility)-related cell death was ROS-dependent but iron-independent in rice-M. oryzae interactions.
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Hypersensitive response (HR) cell death is the most effective plant immune response restricting fungal pathogen invasion. Here, we report that incompatible rice (Oryza sativa) Magnaporthe oryzae interactions induce iron- and reactive oxygen species (ROS)-dependent ferroptotic cell death in rice cells. Ferric ions and ROS (i.e., H2O2) accumulated in tissues undergoing HR cell death of rice leaf sheath tissues during avirulent M. oryzae infection. By contrast, iron did not accumulate in rice cells during virulent M. oryzae infection or treatment with the fungal elicitor chitin. Avirulent M. oryzae infection in ΔOs-nadp-me2-3 mutant rice did not trigger iron and ROS accumulation and suppressed HR cell death, suggesting that NADP-malic enzyme2 is required for ferroptotic cell death in rice. The small-molecule ferroptosis inhibitors deferoxamine, ferrostatin-1, and cytochalasin E and the NADPH oxidase inhibitor diphenyleneiodonium suppressed iron-dependent ROS accumulation and lipid peroxidation to completely attenuate HR cell death in rice sheaths during avirulent M. oryzae infection. By contrast, the small-molecule inducer erastin triggered iron-dependent ROS accumulation and glutathione depletion, which ultimately led to HR cell death in rice in response to virulent M. oryzae These combined results demonstrate that iron- and ROS-dependent signaling cascades are involved in the ferroptotic cell death pathway in rice to disrupt M. oryzae infection.
Asunto(s)
Hierro/metabolismo , Magnaporthe/patogenicidad , Oryza/metabolismo , Oryza/microbiología , Especies Reactivas de Oxígeno/metabolismo , Ciclohexilaminas/farmacología , Citocalasinas/farmacología , Deferoxamina/farmacología , Peroxidación de Lípido/efectos de los fármacos , Fenilendiaminas/farmacologíaRESUMEN
Microbial pathogens have evolved protein effectors to promote virulence and cause disease in host plants. Pathogen effectors delivered into plant cells suppress plant immune responses and modulate host metabolism to support the infection processes of pathogens. Reactive oxygen species (ROS) act as cellular signaling molecules to trigger plant immune responses, such as pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) and effector-triggered immunity. In this review, we discuss recent insights into the molecular functions of pathogen effectors that target multiple steps in the ROS signaling pathway in plants. The perception of PAMPs by pattern recognition receptors leads to the rapid and strong production of ROS through activation of NADPH oxidase Respiratory Burst Oxidase Homologs (RBOHs) as well as peroxidases. Specific pathogen effectors directly or indirectly interact with plant nucleotide-binding leucine-rich repeat receptors to induce ROS production and the hypersensitive response in plant cells. By contrast, virulent pathogens possess effectors capable of suppressing plant ROS bursts in different ways during infection. PAMP-triggered ROS bursts are suppressed by pathogen effectors that target mitogen-activated protein kinase cascades. Moreover, pathogen effectors target vesicle trafficking or metabolic priming, leading to the suppression of ROS production. Secreted pathogen effectors block the metabolic coenzyme NADP-malic enzyme, inhibiting the transfer of electrons to the NADPH oxidases (RBOHs) responsible for ROS generation. Collectively, pathogen effectors may have evolved to converge on a common host protein network to suppress the common plant immune system, including the ROS burst and cell death response in plants.
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MAIN CONCLUSION: Pepper leucine-rich repeat protein (CaLRR1) interacts with defense response proteins to regulate plant cell death and immunity. This review highlights the current understanding of the molecular functions of CaLRR1 and its interactor proteins. Plant cell death and immune responses to microbial pathogens are controlled by complex and tightly regulated molecular signaling networks. Xanthomonas campestris pv. vesicatoria (Xcv)-inducible pepper (Capsicum annuum) leucine-rich repeat protein 1 (CaLRR1) serves as a molecular marker for plant cell death and immunity signaling. In this review, we discuss recent advances in elucidating the functional roles of CaLRR1 and its interacting plant proteins, and understanding how they are involved in the cell death and defense responses. CaLRR1 physically interacts with pepper pathogenesis-related proteins (CaPR10 and CaPR4b) and hypersensitive-induced reaction protein (CaHIR1) to regulate plant cell death and defense responses. CaLRR1 is produced in the cytoplasm and trafficked to the extracellular matrix. CaLRR1 binds to CaPR10 in the cytoplasm and CaPR4b and CaHIR1 at the plasma membrane. CaLRR1 synergistically accelerates CaPR10-triggered hypersensitive cell death, but negatively regulates CaPR4b- and CaHIR1-triggered cell death. CaHIR1 interacts with Xcv filamentous hemagglutinin (Fha1) to trigger disease-associated cell death. The subcellular localization and cellular function of these CaLRR1 interactors during plant cell death and defense responses were elucidated by Agrobacterium-mediated transient expression, virus-induced gene silencing, and transgenic overexpression studies. CaPR10, CaPR4b, and CaHIR1 positively regulate defense signaling mediated by salicylic acid and reactive oxygen species, thereby activating hypersensitive cell death and disease resistance. A comprehensive understanding of the molecular functions of CaLRR1 and its interacting protein partners in cell death and defense responses will provide valuable information for the molecular genetics of plant disease resistance, which could be exploited as a sustainable disease management strategy.
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Capsicum/genética , Muerte Celular/genética , Inmunidad de la Planta/genética , Proteínas de Plantas/genética , Proteínas/genética , Capsicum/metabolismo , Proteínas Repetidas Ricas en Leucina , Enfermedades de las Plantas , Proteínas de Plantas/fisiología , Proteínas/fisiologíaRESUMEN
MAIN CONCLUSION: Xanthomonas effector AvrBsT interacts with plant defense proteins and triggers cell death and defense response. This review highlights our current understanding of the molecular functions of AvrBsT and its host interactor proteins. The AvrBsT protein is a member of a growing family of effector proteins in both plant and animal pathogens. Xanthomonas type III effector AvrBsT, a member of the YopJ/AvrRxv family, suppresses plant defense responses in susceptible hosts, but triggers cell death signaling leading to hypersensitive response (HR) and defense responses in resistant plants. AvrBsT interacts with host defense-related proteins to trigger the HR cell death and defense responses in plants. Here, we review and discuss recent progress in understanding the molecular functions of AvrBsT and its host interactor proteins in pepper (Capsicum annuum). Pepper arginine decarboxylase1 (CaADC1), pepper aldehyde dehydrogenase1 (CaALDH1), pepper heat shock protein 70a (CaHSP70a), pepper suppressor of the G2 allele of skp1 (CaSGT1), pepper SNF1-related kinase1 (SnRK1), and Arabidopsis acetylated interacting protein1 (ACIP1) have been identified as AvrBsT interactors in pepper and Arabidopsis. Gene expression profiling, virus-induced gene silencing, and transient transgenic overexpression approaches have advanced the functional characterization of AvrBsT-interacting proteins in plants. AvrBsT is localized in the cytoplasm and forms protein-protein complexes with host interactors. All identified AvrBsT interactors regulate HR cell death and defense responses in plants. Notably, CaSGT1 physically binds to both AvrBsT and pepper receptor-like cytoplasmic kinase1 (CaPIK1) in the cytoplasm. During infection with Xanthomonas campestris pv. vesicatoria strain Ds1 (avrBsT), AvrBsT is phosphorylated by CaPIK1 and forms the active AvrBsT-CaSGT1-CaPIK1 complex, which ultimately triggers HR cell death and defense responses. Collectively, the AvrBsT interactor proteins are involved in plant cell death and immunity signaling.
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Proteínas Bacterianas/metabolismo , Capsicum/microbiología , Interacciones Huésped-Patógeno , Células Vegetales/microbiología , Xanthomonas campestris/patogenicidad , Aldehído Deshidrogenasa/genética , Aldehído Deshidrogenasa/metabolismo , Proteínas Bacterianas/genética , Capsicum/metabolismo , Carboxiliasas/genética , Carboxiliasas/metabolismo , Muerte Celular , Proteínas HSP70 de Choque Térmico/metabolismo , Células Vegetales/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Transducción de Señal , Xanthomonas campestris/metabolismo , Xanthomonas campestris/fisiologíaRESUMEN
MAIN CONCLUSION: Proteomics and functional analyses of the Arabidopsis - Pseudomonas syringae pv. tomato interactions reveal that Arabidopsis nitrilases are required for plant defense and R gene-mediated resistant responses to microbial pathogens. A high-throughput in planta proteome screen has identified Arabidopsis nitrilase 2 (AtNIT2), which was de novo-induced by Pseudomonas syringae pv. tomato (Pst) infection. The AtNIT2, AtNIT3, and AtNIT4 genes, but not AtNIT1, were distinctly induced in Arabidopsis leaves by Pst infection. Notably, avirulent Pst DC3000 (avrRpt2) infection led to significant induction of AtNIT2 and AtNIT4 in leaves. Pst DC3000 and Pst DC3000 (avrRpt2) significantly grew well in leaves of nitrilase transgenic (nit2i-2) and mutant (nit1-1 and nit3-1) lines compared to the wild-type leaves. In contrast, NIT2 overexpression in nit2 mutants led to significantly high growth of the two Pst strains in leaves. The nitrilase transgenic and mutant lines exhibited enhanced susceptibility to Hyaloperonospora arabidopsidis infection. The nit2 mutation enhanced Pst DC3000 (avrRpt2) growth in salicylic acid (SA)-deficient NahG transgenic and sid2 and npr1 mutant lines. Infection with Pst DC3000 or Pst DC3000 (avrRpt2) induced lower levels of indole-3-acetic acid (IAA) in nit2i and nit2i NahG plants than in wild-type plants, but did not alter the IAA level in NahG transgenic plants. This suggests that Arabidopsis nitrilase 2 is involved in IAA signaling of defense and R gene-mediated resistance responses to Pst infection. Quantification of SA in these transgenic and mutant plants demonstrates that Arabidopsis nitrilase 2 is not required for SA-mediated defense response to the virulent Pst DC3000 but regulates SA-mediated resistance to the avirulent Pst DC3000 (avrRpt2). These results collectively suggest that Arabidopsis nitrilase genes are involved in plant defense and R gene-mediated resistant responses to microbial pathogens.
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Aminohidrolasas/fisiología , Proteínas de Arabidopsis/fisiología , Arabidopsis/metabolismo , Resistencia a la Enfermedad/genética , Aminohidrolasas/química , Aminohidrolasas/genética , Aminohidrolasas/metabolismo , Arabidopsis/genética , Arabidopsis/microbiología , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Mutagénesis Insercional , Filogenia , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Hojas de la Planta/microbiología , Plantas Modificadas Genéticamente/metabolismo , Plantas Modificadas Genéticamente/microbiología , Proteómica , Pseudomonas syringae/fisiología , Análisis de Secuencia de Proteína , Transducción de Señal/genéticaRESUMEN
Carbohydrate-binding proteins, commonly referred to as lectins or agglutinins, function in defense responses to microbial pathogens. Pepper (Capsicum annuum) GNA-related lectin and PAN-domain protein gene CaGLP1 was isolated and functionally characterized from pepper leaves infected with Xanthomonas campestris pv. vesicatoria (Xcv). CaGLP1 contained an amine-terminus prokaryotic membrane lipoprotein lipid attachment site, a Galanthus nivalis agglutinin (GNA)-related lectin domain responsible for the recognition of high-mannose N-glycans, and a carboxyl-terminus PAN/apple domain. RNA gel blot and immunoblot analyses determined that CaGLP1 was strongly induced in pepper by compatible and incompatible Xcv infection. CaGLP1 protein localized primarily to the plasma membrane and exhibited mannose-binding specificity. CaGLP1-silenced pepper plants were more susceptible to compatible or incompatible Xcv infection compared with that of non-silenced control plants. CaGLP1 silencing in pepper leaves did not accumulate H2O2 and induce cell death during incompatible Xcv infection. Defense-related CaDEF1 (defensin) gene expression was significantly reduced in CaGLP1-silenced pepper plants. CaGLP1-overexpression in Arabidopsis thaliana enhanced resistance to Pseudomonas syringae pv. tomato. Defense-related AtPDF1.2 expression was elevated in CaGLP1-overexpression lines. Together, these results suggest that CaGLP1 is required for plant cell death and defense responses through the reactive oxygen species burst and downstream defense-related gene expression in response to bacterial pathogen challenge.
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Capsicum/genética , Muerte Celular , Enfermedades de las Plantas/inmunología , Inmunidad de la Planta , Proteínas de Plantas/genética , Transducción de Señal , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/microbiología , Capsicum/metabolismo , Capsicum/microbiología , Enfermedades de las Plantas/microbiología , Hojas de la Planta/microbiología , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Plantas Modificadas Genéticamente/microbiología , Pseudomonas syringae/fisiología , Xanthomonas campestris/fisiologíaRESUMEN
Plants are constantly exposed to a variety of biotic and abiotic stresses, which include pathogens and conditions of high salinity, low temperature, and drought. Abscisic acid (ABA) is a major plant hormone involved in signal transduction pathways that mediate the defense response of plants to abiotic stress. Previously, we isolated Ring finger protein gene (CaRING1) from pepper (Capsicum annuum), which is associated with resistance to bacterial pathogens, accompanied by hypersensitive cell death. Here, we report a new function of the CaRING1 gene product in the ABA-mediated defense responses of plants to dehydration stress. The expression of the CaRING1 gene was induced in pepper leaves treated with ABA or exposed to dehydration or NaCl. Virus-induced gene silencing of CaRING1 in pepper plants exhibited low degree of ABA-induced stomatal closure and high levels of transpirational water loss in dehydrated leaves. These led to be more vulnerable to dehydration stress in CaRING1-silenced pepper than in the control pepper, accompanied by reduction of ABA-regulated gene expression and low accumulation of ABA and H2O2. In contrast, CaRING1-overexpressing transgenic plants showed enhanced sensitivity to ABA during the seedling growth and establishment. These plants were also more tolerant to dehydration stress than the wild-type plants because of high ABA accumulation, enhanced stomatal closure and increased expression of stress-responsive genes. Together, these results suggest that the CaRING1 acts as positive factor for dehydration tolerance in Arabidopsis by modulating ABA biosynthesis and ABA-mediated stomatal closing and gene expression.
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Ácido Abscísico/fisiología , Capsicum/fisiología , Deshidratación/fisiopatología , Reguladores del Crecimiento de las Plantas/fisiología , Proteínas de Plantas/fisiología , Arabidopsis/genética , Arabidopsis/fisiología , Capsicum/genética , Deshidratación/genética , Silenciador del Gen/fisiología , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Phosphoenolpyruvate carboxykinase, a member of the lyase family, is involved in the metabolic pathway of gluconeogenesis in organisms. Although the major function of PEPCK in gluconeogenesis is well established, it is unclear whether this enzyme is involved in plant immunity. Here, we isolated and identified the pepper (Capsicum annuum) PEPCK (CaPEPCK1) gene from pepper leaves infected with Xanthomonas campestris pv. vesicatoria (Xcv). CaPEPCK1 was strongly expressed in pepper leaves during the incompatible interaction with avirulent Xcv and in response to environmental stresses, especially salicylic acid (SA) treatment. PEPCK activity was low in healthy leaves but dramatically increased in avirulent Xcv-infected leaves. Knock-down expression of CaPEPCK1 by virus-induced gene silencing resulted in high levels of susceptibility to both virulent and avirulent Xcv infection. CaPEPCK1 silencing in pepper compromised induction of the basal defense-marker genes CaPR1 (pathogenesis-related 1 protein), CaPR10 (pathogenesis-related 10 protein) and CaDEF1 (defensin) during Xcv infection. SA accumulation was also significantly suppressed in the CaPEPCK1-silenced pepper leaves infected with Xcv. CaPEPCK1 in an Arabidopsis overexpression (OX) line inhibited the proliferation of Pseudomonas syringae pv. tomato (Pst) and Hyaloperonospora arabidopsidis (Hpa). CaPEPCK1-OX plants developed more rapidly, with enlarged leaves, compared to wild-type plants. The T-DNA insertion Arabidopsis orthologous mutants pck1-3 and pck1-4 were more susceptible to the bacterial Pst and oomycete Hpa pathogens than the wild type. Taken together, these results suggest that CaPEPCK positively contributes to plant innate immunity against hemibiotrophic bacterial and obligate biotrophic oomycete pathogens.
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Capsicum/inmunología , Oomicetos , Fosfoenolpiruvato Carboxiquinasa (ATP)/fisiología , Enfermedades de las Plantas/inmunología , Inmunidad de la Planta/fisiología , Xanthomonas campestris , Capsicum/enzimología , Capsicum/genética , Capsicum/fisiología , Fosfoenolpiruvato Carboxiquinasa (ATP)/genética , Fosfoenolpiruvato Carboxiquinasa (ATP)/aislamiento & purificación , Fosfoenolpiruvato Carboxiquinasa (ATP)/metabolismo , Enfermedades de las Plantas/microbiología , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Xanthomonas type III effector AvrBsT induces hypersensitive cell death and defence responses in pepper (Capsicum annuum) and Nicotiana benthamiana. Little is known about the host factors that interact with AvrBsT. Here, we identified pepper aldehyde dehydrogenase 1 (CaALDH1) as an AvrBsT-interacting protein. Bimolecular fluorescence complementation and co-immunoprecipitation assays confirmed the interaction between CaALDH1 and AvrBsT in planta. CaALDH1:smGFP fluorescence was detected in the cytoplasm. CaALDH1 expression in pepper was rapidly and strongly induced by avirulent Xanthomonas campestris pv. vesicatoria (Xcv) Ds1 (avrBsT) infection. Transient co-expression of CaALDH1 with avrBsT significantly enhanced avrBsT-triggered cell death in N. benthamiana leaves. Aldehyde dehydrogenase activity was higher in leaves transiently expressing CaALDH1, suggesting that CaALDH1 acts as a cell death enhancer, independently of AvrBsT. CaALDH1 silencing disrupted phenolic compound accumulation, H2O2 production, defence response gene expression, and cell death during avirulent Xcv Ds1 (avrBsT) infection. Transgenic Arabidopsis thaliana overexpressing CaALDH1 exhibited enhanced defence response to Pseudomonas syringae pv. tomato and Hyaloperonospora arabidopsidis infection. These results indicate that cytoplasmic CaALDH1 interacts with AvrBsT and promotes plant cell death and defence responses.
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Aldehído Deshidrogenasa/metabolismo , Proteínas Bacterianas/metabolismo , Capsicum/enzimología , Regulación de la Expresión Génica de las Plantas , Enfermedades de las Plantas/inmunología , Xanthomonas campestris/fisiología , Aldehído Deshidrogenasa/genética , Arabidopsis/enzimología , Arabidopsis/genética , Arabidopsis/inmunología , Arabidopsis/fisiología , Proteínas Bacterianas/genética , Capsicum/genética , Capsicum/inmunología , Capsicum/fisiología , Muerte Celular , Peróxido de Hidrógeno/metabolismo , Oomicetos/fisiología , Inmunidad de la Planta , Hojas de la Planta/enzimología , Hojas de la Planta/genética , Hojas de la Planta/inmunología , Hojas de la Planta/fisiología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Pseudomonas syringae/fisiología , Proteínas Recombinantes de Fusión , Transducción de Señal , Nicotiana/enzimología , Nicotiana/genética , Nicotiana/inmunología , Nicotiana/fisiologíaRESUMEN
The pepper receptor-like cytoplasmic protein kinase, CaPIK1, which mediates signalling of plant cell death and defence responses was previously identified. Here, the identification of a class IV chitinase, CaChitIV, from pepper plants (Capsicum annuum), which interacts with CaPIK1 and promotes CaPIK1-triggered cell death and defence responses, is reported. CaChitIV contains a signal peptide, chitin-binding domain, and glycol hydrolase domain. CaChitIV expression was up-regulated by Xanthomonas campestris pv. vesicatoria (Xcv) infection. Notably, avirulent Xcv infection rapidly induced CaChitIV expression in pepper leaves. Bimolecular fluorescence complementation and co-immunoprecipitation revealed that CaPIK1 interacts with CaChitIV in planta, and that the CaPIK1-CaChitIV complex is localized mainly in the cytoplasm and plasma membrane. CaChitIV is also localized in the endoplasmic reticulum. Transient co-expression of CaChitIV with CaPIK1 enhanced CaPIK1-triggered cell death response and reactive oxygen species (ROS) and nitric oxide (NO) bursts. Co-silencing of both CaChitIV and CaPIK1 in pepper plants conferred enhanced susceptibility to Xcv infection, which was accompanied by a reduced induction of cell death response, ROS and NO bursts, and defence response genes. Ectopic expression of CaPIK1 in Arabidopsis enhanced basal resistance to Hyaloperonospora arabidopsidis infection. Together, the results suggest that CaChitIV positively regulates CaPIK1-triggered cell death and defence responses through its interaction with CaPIK1.
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Capsicum/enzimología , Interacciones Huésped-Patógeno , Oomicetos/patogenicidad , Enfermedades de las Plantas/inmunología , Inmunidad de la Planta , Proteínas de Plantas/metabolismo , Xanthomonas campestris/patogenicidad , Arabidopsis/enzimología , Arabidopsis/genética , Arabidopsis/inmunología , Capsicum/genética , Capsicum/inmunología , Muerte Celular , Quitinasas/genética , Quitinasas/metabolismo , Genes Reporteros , Óxido Nítrico/metabolismo , Enfermedades de las Plantas/microbiología , Hojas de la Planta/enzimología , Hojas de la Planta/genética , Hojas de la Planta/inmunología , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Ácido Salicílico/metabolismo , Transducción de SeñalRESUMEN
In plants, lipoxygenases (LOXs) are involved in various physiological processes, including defense responses to biotic and abiotic stresses. Our previous study had shown that the pepper 9-LOX gene, CaLOX1, plays a crucial role in cell death due to pathogen infection. Here, the function of CaLOX1 in response to osmotic, drought and high salinity stress was examined using CaLOX1-overexpressing (CaLOX1-OX) Arabidopsis plants. Changes in the temporal expression pattern of the CaLOX1 gene were observed when pepper leaves were treated with drought and high salinity, but not when treated with ABA, the primary hormone in response to drought stress. During seed germination and seedling development, CaLOX1-OX plants were more tolerant to ABA, mannitol and high salinity than wild-type plants. In contrast, expression of the ABA-responsive marker genes RAB18 and RD29B was higher in CaLOX1-OX Arabidopsis plants than in wild-type plants. In response to high salinity, CaLOX1-OX plants exhibited enhanced tolerance, compared with the wild type, which was accompanied by decreased accumulation of H2O2 and high levels of RD20, RD29A, RD29B and P5CS gene expression. Similarly, CaLOX1-OX plants were also more tolerant than wild-type plants to severe drought stress. H2O2 production and the relative increase in lipid peroxidation were lower, and the expression of COR15A, DREB2A, RD20, RD29A and RD29B was higher in CaLOX1-OX plants, relative to wild-type plants. Taken together, our results indicate that CaLOX1 plays a crucial role in plant stress responses by modulating the expression of ABA- and stress-responsive marker genes, lipid peroxidation and H2O2 production.
Asunto(s)
Capsicum/enzimología , Capsicum/fisiología , Sequías , Lipooxigenasa/metabolismo , Presión Osmótica , Salinidad , Estrés Fisiológico , Ácido Abscísico/farmacología , Adaptación Fisiológica/efectos de los fármacos , Adaptación Fisiológica/genética , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Arabidopsis/fisiología , Capsicum/efectos de los fármacos , Capsicum/genética , Regulación de la Expresión Génica de las Plantas , Germinación/efectos de los fármacos , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Tolerancia a la Sal/efectos de los fármacos , Tolerancia a la Sal/genética , Semillas/efectos de los fármacos , Semillas/crecimiento & desarrollo , Cloruro de Sodio/farmacología , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/genéticaRESUMEN
Xanthomonas campestris pv. vesicatoria (Xcv) type III effector AvrBsT triggers programmed cell death (PCD) and activates the hypersensitive response (HR) in plants. Here, we isolated and identified the plasma membrane localized pathogenesis-related (PR) protein 4c gene (CaPR4c) from pepper (Capsicum annuum) leaves undergoing AvrBsT-triggered HR cell death. CaPR4c encodes a protein with a signal peptide and a Barwin domain. Recombinant CaPR4c protein expressed in Escherichia coli exhibited cysteine protease-inhibitor activity and ribonuclease (RNase) activity. Subcellular localization analyses revealed that CaPR4c localized to the plasma membrane in plant cells. CaPR4c expression was rapidly and specifically induced by avirulent Xcv (avrBsT) infection. Transient expression of CaPR4c caused HR cell death in pepper leaves, which was accompanied by enhanced accumulation of H2 O2 and significant induction of some defense-response genes. Deletion of the signal peptide from CaPR4c abolished the induction of HR cell death, indicating a requirement for plasma membrane localization of CaPR4c for HR cell death. CaPR4c silencing in pepper disrupted both basal and AvrBsT-triggered resistance responses, and enabled Xcv proliferation in infected leaves. H2 O2 accumulation, cell-death induction, and defense-response gene expression were distinctly reduced in CaPR4c-silenced pepper. CaPR4c overexpression in transgenic Arabidopsis plants conferred greater resistance against infection by Pseudomonas syringae pv. tomato and Hyaloperonospora arabidopsidis. These results collectively suggest that CaPR4c plays an important role in plant cell death and defense signaling.
Asunto(s)
Capsicum/metabolismo , Muerte Celular , Inhibidores de Cisteína Proteinasa/metabolismo , Proteínas de la Membrana/fisiología , Células Vegetales/fisiología , Proteínas de Plantas/fisiología , Transducción de Señal , Arabidopsis/genética , Capsicum/citología , Capsicum/inmunología , Membrana Celular , Inhibidores de Cisteína Proteinasa/análisis , Resistencia a la Enfermedad/fisiología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Hojas de la Planta/citología , Hojas de la Planta/inmunología , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/inmunología , Plantas Modificadas Genéticamente/metabolismo , Xanthomonas campestris/fisiologíaRESUMEN
Plants use a variety of innate immune regulators to trigger cell death and defense responses against pathogen attack. We identified pepper (Capsicum annuum) GLYCINE-RICH RNA-BINDING PROTEIN1 (CaGRP1) as a RECEPTOR-LIKE CYTOPLASMIC PROTEIN KINASE1 (CaPIK1)-interacting partner, based on bimolecular fluorescence complementation and coimmunoprecipitation analyses as well as gene silencing and transient expression analysis. CaGRP1 contains an N-terminal RNA recognition motif and a glycine-rich region at the C-terminus. The CaGRP1 protein had DNA- and RNA-binding activity in vitro. CaGRP1 interacted with CaPIK1 in planta. CaGRP1 and CaGRP1-CaPIK1 complexes were localized to the nucleus in plant cells. CaPIK1 phosphorylated CaGRP1 in vitro and in planta. Transient coexpression of CaGRP1 with CaPIK1 suppressed the CaPIK1-triggered cell death response, accompanied by a reduced CaPIK1-triggered reactive oxygen species (ROS) burst. The RNA recognition motif region of CaGRP1 was responsible for the nuclear localization of CaGRP1 as well as the suppression of the CaPIK1-triggered cell death response. CaGRP1 silencing in pepper conferred enhanced resistance to Xanthomonas campestris pv vesicatoria (Xcv) infection; however, CaPIK1-silenced plants were more susceptible to Xcv. CaGRP1 interacts with CaPIK1 and negatively regulates CaPIK1-triggered cell death and defense responses by suppressing ROS accumulation.
Asunto(s)
Capsicum/metabolismo , Interacciones Huésped-Patógeno , Proteínas de Plantas/metabolismo , Capsicum/citología , Capsicum/microbiología , Muerte Celular/genética , Regulación de la Expresión Génica de las Plantas , Silenciador del Gen , Fosforilación , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Xanthomonas campestris/patogenicidadRESUMEN
Pepper (Capsicum annuum L.) provides a good experimental system for studying the molecular and functional genomics underlying the ability of plants to defend themselves against microbial pathogens. Cell death is a genetically programmed response that requires specific host cellular factors. Hypersensitive response (HR) is defined as rapid cell death in response to a pathogen attack. Pepper plants respond to pathogen attacks by activating genetically controlled HR- or disease-associated cell death. HR cell death, specifically in incompatible interactions between pepper and Xanthomonas campestris pv. vesicatoria, is mediated by the molecular genetics and biochemical machinery that underlie pathogen-induced cell death in plants. Gene expression profiles during the HR-like cell death response, virus-induced gene silencing and transient and transgenic overexpression approaches are used to isolate and identify HR- or disease-associated cell death genes in pepper plants. Reactive oxygen species, nitric oxide, cytosolic calcium ion and defense-related hormones such as salicylic acid, jasmonic acid, ethylene and abscisic acid are involved in the execution of pathogen-induced cell death in plants. In this review, we summarize recent molecular and cellular studies of the pepper cell death-mediated defense response, highlighting the signaling events of cell death in disease-resistant pepper plants. Comprehensive knowledge and understanding of the cellular functions of pepper cell death response genes will aid the development of novel practical approaches to enhance disease resistance in pepper, thereby helping to secure the future supply of safe and nutritious pepper plants worldwide.
Asunto(s)
Capsicum/genética , Resistencia a la Enfermedad/genética , Enfermedades de las Plantas/genética , Transducción de Señal/genética , Capsicum/metabolismo , Capsicum/microbiología , Muerte Celular/genética , Perfilación de la Expresión Génica , Interacciones Huésped-Patógeno , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteómica , Xanthomonas campestris/fisiologíaRESUMEN
Heat shock proteins (HSPs) function as molecular chaperones and are essential for the maintenance and/or restoration of protein homeostasis. The genus Xanthomonas type III effector protein AvrBsT induces hypersensitive cell death in pepper (Capsicum annuum). Here, we report the identification of the pepper CaHSP70a as an AvrBsT-interacting protein. Bimolecular fluorescence complementation and coimmunoprecipitation assays confirm the specific interaction between CaHSP70a and AvrBsT in planta. The CaHSP70a peptide-binding domain is essential for its interaction with AvrBsT. Heat stress (37°C) and Xanthomonas campestris pv vesicatoria (Xcv) infection distinctly induce CaHSP70a in pepper leaves. Cytoplasmic CaHSP70a proteins significantly accumulate in pepper leaves to induce the hypersensitive cell death response by Xcv (avrBsT) infection. Transient CaHSP70a overexpression induces hypersensitive cell death under heat stress, which is accompanied by strong induction of defense- and cell death-related genes. The CaHSP70a peptide-binding domain and ATPase-binding domain are required to trigger cell death under heat stress. Transient coexpression of CaHSP70a and avrBsT leads to cytoplasmic localization of the CaHSP70a-AvrBsT complex and significantly enhances avrBsT-triggered cell death in Nicotiana benthamiana. CaHSP70a silencing in pepper enhances Xcv growth but disrupts the reactive oxygen species burst and cell death response during Xcv infection. Expression of some defense marker genes is significantly reduced in CaHSP70a-silenced leaves, with lower levels of the defense hormones salicylic acid and jasmonic acid. Together, these results suggest that CaHSP70a interacts with the type III effector AvrBsT and is required for cell death and immunity in plants.
Asunto(s)
Proteínas Bacterianas/metabolismo , Capsicum/citología , Capsicum/inmunología , Proteínas HSP70 de Choque Térmico/metabolismo , Células Vegetales/metabolismo , Inmunidad de la Planta , Proteínas de Plantas/metabolismo , Sistemas de Secreción Bacterianos , Capsicum/genética , Capsicum/microbiología , Muerte Celular , Ciclopentanos/metabolismo , Resistencia a la Enfermedad/inmunología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Silenciador del Gen , Genes de Plantas , Proteínas HSP70 de Choque Térmico/química , Respuesta al Choque Térmico , Oxilipinas/metabolismo , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Hojas de la Planta/citología , Hojas de la Planta/metabolismo , Hojas de la Planta/microbiología , Proteínas de Plantas/química , Plantas Modificadas Genéticamente , Unión Proteica , Estructura Terciaria de Proteína , Especies Reactivas de Oxígeno/metabolismo , Ácido Salicílico/metabolismo , Eliminación de Secuencia , Fracciones Subcelulares/metabolismo , Técnicas del Sistema de Dos Híbridos , Xanthomonas campestris/fisiologíaRESUMEN
Formate dehydrogenase (FDH; EC 1.2.1.2) is an NAD-dependent enzyme that catalyzes the oxidation of formate to carbon dioxide. Here, we report the identification and characterization of pepper (Capsicum annuum) mitochondrial FDH1 as a positive regulator of cell death and defense responses. Transient expression of FDH1 caused hypersensitive response (HR)-like cell death in pepper and Nicotiana benthamiana leaves. The D-isomer -: specific 2-hydroxyacid dehydrogenase signatures of FDH1 were required for the induction of HR-like cell death and FDH activity. FDH1 contained a mitochondrial targeting sequence at the N-terminal region; however, mitochondrial localization of FDH1 was not essential for the induction of HR-like cell death and FDH activity. FDH1 silencing in pepper significantly attenuated the cell death response and salicylic acid levels but stimulated growth of Xanthomonas campestris pv vesicatoria. By contrast, transgenic Arabidopsis (Arabidopsis thaliana) overexpressing FDH1 exhibited greater resistance to Pseudomonas syringae pv tomato in a salicylic acid-dependent manner. Arabidopsis transfer DNA insertion mutant analysis indicated that AtFDH1 expression is required for basal defense and resistance gene-mediated resistance to P. syringae pv tomato infection. Taken together, these data suggest that FDH1 has an important role in HR-like cell death and defense responses to bacterial pathogens.