BON1 interacts with the protein kinases BIR1 and BAK1 in modulation of temperature-dependent plant growth and cell death in Arabidopsis

The Arabidopsis copine gene BON1 encodes a calcium-dependent phospholipid-binding protein involved in plant growth homeostasis and disease resistance. However, the biochemical and molecular mechanisms by which BON1 modulates plant growth and defense responses are not well understood. Here, we show that BON1 interacts physically with the leucine-rich-repeat receptor-like kinases BIR1 (BAK1-interacting receptor-like kinase 1) and pathogen-associated molecular pattern (PAMP) receptor regulator BAK1 in vitro and in vivo. Additionally, bon1 and bir1 mutants exhibit synergistic interaction. While a bir1 null mutant has similar growth and cell-death defects compared with bon1, a bir1 bon1 double mutant displays more severe phenotypes than does the single mutants. The bon1-1 and bir1-1 phenotypes are partially suppressed by overexpression of BIR1 and BON1, respectively. Furthermore, the bir1 phenotype is attenuated by a loss-of-function mutation in the resistance (R) gene SNC1 (Suppressor of npr1-1, constitutive 1), which mediates defense responses in bon1. Intriguingly, BON1 and BIR1 can be phosphorylated by BAK1 in vitro. Our findings suggest that BIR1 functions as a negative regulator of plant resistance and that BON1 and BIR1 might modulate both PAMP- and R protein-triggered immune responses.     

The crystal structure of Arabidopsis BON1 provides insights into the copine protein family

The Arabidopsis thaliana BON1 gene product is a member of the evolutionary conserved eukaryotic calcium‐dependent membrane‐binding protein family. The copine protein is composed of two C2 domains (C2A and C2B) followed by a vWA domain. The BON1 protein is localized on the plasma membrane, and is known to suppress the expression of immune receptor genes and to positively regulate stomatal closure. The first structure of this protein family has been determined to 2.5‐Å resolution and shows the structural features of the three conserved domains C2A, C2B and vWA. The structure reveals the third Ca²⁺‐binding region in C2A domain is longer than classical C2 domains and a novel Ca²⁺ binding site in the vWA domain. The structure of BON1 bound to Mn²⁺ is also presented. The binding of the C2 domains to phospholipid (PSF) has been modeled and provides an insight into the lipid‐binding mechanism of the copine proteins. Furthermore, the selectivity of the separate C2A and C2B domains and intact BON1 to bind to different phospholipids has been investigated, and we demonstrated that BON1 could mediate aggregation of liposomes in response to Ca²⁺. These studies have formed the basis of further investigations into the important role that the copine proteins play in vivo.     

Autophagy promotes programmed cell death and corpse clearance in specific cell types of the Arabidopsis root cap

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The Arabidopsis gain-of-function mutant dll1 spontaneously develops lesions mimicking cell death associated with disease

We describe the characterization of a novel gain-of-function Arabidopsis mutant, dll1 (disease-like lesions1), which spontaneously develops lesions mimicking bacterial speck disease and constitutively expresses biochemical and molecular markers associated with pathogen infection. Despite the constitutive expression of defense-related responses, dll1 is unable to suppress the growth of virulent pathogens. However, dll1 elicits normal hypersensitive response in response to avirulent pathogens, thus indicating that dll1 is not defective in the induction of normal resistance responses. The lesion+ leaves of dll1 support the growth of hrcC mutant of Pseudomonas syringae, which is defective in the transfer of virulence factors into the plant cells, and therefore non-pathogenic to wild-type Col-0 plants. This suggests that dll1 intrinsically expresses many of the cellular processes that are required for pathogen growth during disease. Epistasis analyses reveal that salicylic acid and NPR1 are required for lesion formation, while ethylene modulates lesion development in dll1, suggesting that significant overlap exist between the signalling pathways leading to resistance- and disease-associated cell death. Our results suggest that host cell death during compatible interactions, at least in part, is genetically controlled by the plant and DLL1 may positively regulate this process.     

Fine-mapping of an Arabidopsis cell death mutation locus

An Arabidopsis cell death mutation locus was mapped to chromosome 2 between /GS1 and mi421. The YAC clone ends, CIC9A3R, CIC11C7L, CIC2G5R and RFLP marker CDs3 within this interval, were used to probe TAMU BAC library and 31 BAC clones were obtained. A BAC contig encompassing the mutation locus, which consists of T6P5, T7M23, T12A21, T8L6 and T18A18, was identified by Southern hybridization with the BAC ends as probes. 11 CAPS and 12 STS markers were developed in this region. These results will facilitate map-based cloning of the genes and sequencing of the genomic DNA in this region.     

Fine-mapping of an Arabidopsis cell death mutation locus

An Arabidopsis cell death mutation locus was mapped to chromosome 2 between lGS1 and mi421. The YAC clone ends, CIC9A3R, CIC11C7L, CIC2G5R and RFLP marker CDs3 within this interval, were used to probe TAMU BAC library and 31 BAC clones were obtained. A BAC contig encompassing the mutation locus, which consists of T6P5, T7M23, T12A21, T8L6 and T18A18, was identified by Southern hybridization with the BAC ends as probes. 11 CAPS and 12 STS markers were developed in this region. These results will facilitate map-based cloning of the genes and sequencing of the genomic DNA in this region.     

The Arabidopsis aberrant growth and death2 mutant shows resistance to Pseudomonas syringae and reveals a role for NPR1 in suppressing hypersensitive cell death

A novel Arabidopsis mutant has been identified with constitutive expression of GST1-GUS using plants with a pathogen-responsive reporter transgene containing the beta-glucuronidase (GUS) coding region driven by the GST1 promoter. The recessive mutant, called agd2 (aberrant growth and death2), has salicylic acid (SA)-dependent increased resistance to virulent and avirulent strains of the bacterial pathogen Pseudomonas syringae, elevated SA levels, a low level of spontaneous cell death, callose deposition, and enlarged cells in leaves. The enhanced resistance of agd2 to virulent P. syringae requires the SA signaling component NONEXPRESSOR OF PR1 (NPR1). However, agd2 renders the resistance response to P. syringae carrying avrRpt2 NPR1-independent. Thus agd2 affects both an SA- and NPR1-dependent general defense pathway and an SA-dependent, NPR1-independent pathway that is active during the recognition of avirulent P. syringae. agd2 plants also fail to show a hypersensitive cell death response (HR) unless NPR1 is removed. This novel function for NPR1 is also apparent in otherwise wild-type plants: npr1 mutants show a stronger HR, while NPR1-overproducing plants show a weaker HR when infected with P. syringae carrying the avrRpm1 gene. Spontaneous cell death in agd2 is partially suppressed by npr1, indicating that NPR1 can suppress or enhance cell death depending on the cellular context. agd2 plants depleted of SA show a dramatic exacerbation of the cell-growth phenotype and increased callose deposition, suggesting a role for SA in regulating growth and this cell-wall modification. AGD2 may function in cell death and/or growth control as well as the defense response, similarly to what has been described in animals for the functions of NFkappaB.     

A role for salicylic acid and NPR1 in regulating cell growth in Arabidopsis

Salicylic acid (SA) plays a key role in activating defenses and cell death during plant-pathogen interactions. In response to some pathogens, SA also limits the extent of cell death, indicating that it acts positively or negatively depending on the host-pathogen interaction. In addition, we previously showed that SA affects cell growth in the Arabidopsis defense-related mutants accelerated cell death 6-1 (acd6-1) and aberrant growth and death 2 (agd2). Using acd6-1, agd2 and two other defense-related mutants, lesion simulating disease 6 (lsd6), suppressor of SA-insensitivity (ssi1), we show here in detail that SA regulates cell growth by specifically affecting cell enlargement, endoreduplication and/or cell division. We find that SA can act either positively or negatively to regulate cell growth depending on the context in which signaling occurs. Additionally, Nonexpressor of PR 1 (NPR1), a key SA signaling protein important for regulating defenses and cell death, also acts to promote cell division and/or suppress endoreduplication during leaf development. We propose that SA interacts with multiple receptors or signaling pathways to control cellular alterations during normal development, pathogen attack and/or stress situations. We suggest that SA and NPR1 play broader roles in cell fate control than has previously been understood.     

Accelerated cell death 2 suppresses mitochondrial oxidative bursts and modulates cell death in Arabidopsis

The Arabidopsis ACCELERATED CELL DEATH 2 (ACD2) protein protects cells from programmed cell death (PCD) caused by endogenous porphyrin‐related molecules like red chlorophyll catabolite or exogenous protoporphyrin IX. We previously found that during bacterial infection, ACD2, a chlorophyll breakdown enzyme, localizes to both chloroplasts and mitochondria in leaves. Additionally, acd2 cells show mitochondrial dysfunction. In plants with acd2 and ACD2 ⁺ sectors, ACD2 functions cell autonomously, implicating a pro‐death ACD2 substrate as being cell non‐autonomous in promoting the spread of PCD. ACD2 targeted solely to mitochondria can reduce the accumulation of an ACD2 substrate that originates in chloroplasts, indicating that ACD2 substrate molecules are likely to be mobile within cells. Two different light‐dependent reactive oxygen bursts in mitochondria play prominent and causal roles in the acd2 PCD phenotype. Finally, ACD2 can complement acd2 when targeted to mitochondria or chloroplasts, respectively, as long as it is catalytically active: the ability to bind substrate is not sufficient for ACD2 to function in vitro or in vivo. Together, the data suggest that ACD2 localizes dynamically during infection to protect cells from pro‐death mobile substrate molecules, some of which may originate in chloroplasts, but have major effects on mitochondria.     

Mechanism of basic fibroblast growth factor-induced cell death

Basic fibroblast growth factor (bFGF) is a potent mitogen for a number of different cell types. Its over-expression has been implicated in transformation and malignant progression. The use of bFGF to treat malignancy is therefore counterintuitive. However, recent studies have shown bFGF-induces cell death in some tumour types. This mini review will summarise the most recent findings on bFGF-induced cell death and discuss its potential mechanism of action.     

Epidermal growth factor and insulin-like growth factor-1 protect MDA-231 cells from death induced by actinomycin D: The involvement of growth factors in drug resistance

Summary In the present study, we investigated the ability of epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), and insulin to protect the human breast cancer cell line MDA-231 from death induced by the antitumor drug actinomycin D (ACT-D). ACT-D is an inhibitor of RNA and protein synthesis, and its cytotoxicity may result due to continuous depletion in some vital protein molecules. Cell death was induced in the MDA-231 cells by either continuous exposure to a low dose of ACT-D (0.2μg/ml), or by a short-time exposure to a high dose of ACT-D (2μg/ml) and further culturing in the absence of the drug. Cell death was evaluated by the trypan blue dye exclusion test, the release of lactic dehydrogenase into the culture medium, and the depletion in the cellular ATP content. EGF and IGF-1, each at an optimal concentration of 20 ng/ml, enhanced substantially survival of cells exposed either to a low or a high dose of ACT-D. The combination of EGF (10 ng/ml) and IGF-1 (10 ng/ml) had an additive survival effect, which proposes that each of the growth factors enhanced survival by a distinct pathway. Insulin up to 40 ng/ml had no effect on cell survival. Pretreatment of the cells for 1 to 5 h with EGF and IGF-1 protected cells from the cytotoxic effect of ACT-D. Exposure of the cells to 2μg/ml of ACT-D for 1 h resulted in a drastic inhibition in uridine incorporation and only in a slight inhibition in leucine incorporation. Further incubation in the absence of ACT-D resulted in a continuous decrease in uridine and in leucine incorporation, either in the absence or presence of the growth factors. However, EGF and IGF-1, but not insulin, attenuated significantly this continuous decrease. We assume that EGF and IGF-1 protect cell viability by a mechanism that maintains a critical level of some vital protein molecule above the critical level at which cells die. Our finding that EGF and IGF-1 induced resistance to ACT-D suggests that growth factors may be involved in the mechanism of drug resistance.     

Molecular mechanisms of programmed cell death

Programmed cell death is currently under active investigation. A recent meeting focused on the molecular machinery of programmed cell death and on its role in the pathogenesis of human diseases.     

RPN1a, a 26S proteasome subunit, is required for innate immunity in Arabidopsis

Accumulating evidence shows that proper degradation of proteins that affect defense responses in a positive or negative manner is critical in plant immunity. However, the role of plant degradation systems such as the 26S proteasome in plant immunity is not well understood. Loss‐of‐function mutations in EDR2 (ENHANCED DISEASE RESISTANCE 2) lead to increased resistance to the adapted biotrophic powdery mildew pathogen Golovinomyces cichoracearum. To study the molecular interactions between powdery mildew pathogen and Arabidopsis, we performed a screen for suppressors of edr2 and found that mutation in the gene that encodes RPN1a, a subunit of the 26S proteasome, suppressed edr2‐associated disease resistance phenotypes. In addition, RPN1a is required for edr1‐ and pmr4‐mediated powdery mildew resistance and mildew‐induced cell death. Furthermore, we show that rpn1a displayed enhanced susceptibility to the fungal pathogen G. cichoracearum and to virulent and avirulent bacterial Pto DC3000 strains, which indicated that rpn1a has defects in basal defense and resistance (R) protein‐mediated defense. RPN1a–GFP localizes to both the nucleus and cytoplasm. Accumulation of RPN1a is affected by salicylic acid (SA) and the rpn1a mutant has defects in SA accumulation upon Pto DC3000 infection. Further analysis revealed that two other subunits of the 26S proteasome, RPT2a and RPN8a are also involved in edr2‐mediated disease resistance. Based on these results, we conclude that RPN1a is required for basal defense and R protein‐mediated defense. Our data provide evidence that some subunits of the 26S proteasome are involved in innate immunity in Arabidopsis.     

A mutation in the GTP hydrolysis site of Arabidopsis dynamin-related protein 1E confers enhanced cell death in response to powdery mildew infection

We screened for mutants of Arabidopsis thaliana that displayed enhanced disease resistance to the powdery mildew pathogen Erysiphe cichoracearum and identified the edr3 mutant, which formed large gray lesions upon infection with E. cichoracearum and supported very little sporulation. The edr3-mediated disease resistance and cell death phenotypes were dependent on salicylic acid signaling, but independent of ethylene and jasmonic acid signaling. In addition, edr3 plants displayed enhanced susceptibility to the necrotrophic fungal pathogen Botrytis cinerea, but showed normal responses to virulent and avirulent strains of Pseudomonas syringae pv. tomato. The EDR3 gene was isolated by positional cloning and found to encode Arabidopsis dynamin-related protein 1E (DRP1E). The edr3 mutation caused an amino acid substitution in the GTPase domain of DRP1E (proline 77 to leucine) that is predicted to block GTP hydrolysis, but not GTP binding. A T-DNA insertion allele in DRP1E did not cause powdery mildew-induced lesions, suggesting that this phenotype is caused by DRP1E being locked in the GTP-bound state, rather than by a loss of DRP1E activity. Analysis of DRP1E-green fluorescent protein fusion proteins revealed that DRP1E is at least partially localized to mitochondria. These observations suggest a mechanistic link between salicylic acid signaling, mitochondria and programmed cell death in plants.     

Thermographic visualization of cell death in tobacco and Arabidopsis

Pending cell death was visualized by thermographic imaging in bacterio‐opsin transgenic tobacco plants. Cell death in these plants was characterized by a complex lesion phenotype. Isolated cell death lesions were preceded by a colocalized thermal effect, as previously observed at sites infected by tobacco mosaic virus (TMV) ( Chaerle et al. 1999 Nature Biotechnology 17, 813–816). However, in most cases, a coherent front of higher temperature, trailed by cell death, initiated at the leaf base and expanded over the leaf lamina. In contrast to the homogenous thermal front, cell death was first visible close to the veins, and subsequently appeared as discrete spots on the interveinal tissue, as cell death spread along the veins. Regions with visible cell death had a lower temperature because of water evaporation from damaged cells. In analogy with previous observations on the localized tobacco–TMV interaction ( Chaerle et al. 1999 ), the kinetics of thermographic and continuous gas exchange measurements indicated that stomatal closure preceded tissue collapse. Localized spontaneous cell death could also be presymptomatically visualized in the Arabidopsis lsd2 mutant.     

Pharmacological eEF2K activation promotes cell death and inhibits cancer progression

Activation of the elongation factor 2 kinase (eEF2K) leads to the phosphorylation and inhibition of the elongation factor eEF2, reducing mRNA translation rates. Emerging evidence indicates that the regulation of factors involved in protein synthesis may be critical for controlling diverse biological processes including cancer progression. Here we show that inhibitors of the HIV aspartyl protease (HIV‐PIs), nelfinavir in particular, trigger a robust activation of eEF2K leading to the phosphorylation of eEF2. Beyond its anti‐viral effects, nelfinavir has antitumoral activity and promotes cell death. We show that nelfinavir‐resistant cells specifically evade eEF2 inhibition. Decreased cell viability induced by nelfinavir is impaired in cells lacking eEF2K. Moreover, nelfinavir‐mediated anti‐tumoral activity is severely compromised in eEF2K‐deficient engrafted tumors in vivo. Our findings imply that exacerbated activation of eEF2K is detrimental for tumor survival and describe a mechanism explaining the anti‐tumoral properties of HIV‐PIs.     

The exocyst subunit Exo70B1 is involved in the immune response of Arabidopsis thaliana to different pathogens and cell death

Components of the vesicle trafficking machinery are central to the immune response in plants. The role of vesicle trafficking during pre-invasive penetration resistance has been well documented. However, emerging evidence also implicates vesicle trafficking in early immune signaling. Here we report that Exo70B1, a subunit of the exocyst complex which mediates early tethering during exocytosis is involved in resistance. We show that exo70B1 mutants display pathogen-specific immuno-compromised phenotypes. We also show that exo70B1 mutants display lesion-mimic cell death, which in combination with the reduced responsiveness to pathogen-associated molecular patterns (PAMPs) results in complex immunity-related phenotypes.     

自噬在细胞存活和死亡中的作用

自噬是亚细胞膜结构发生动态变化并经溶酶体介导对细胞内蛋白质和细胞器降解的过程.通过平衡细胞合成和分解代谢,自噬稳定细胞内环境,维持细胞的存活.然而,过度自噬可导致细胞发生Ⅱ型程序性细胞死亡.自噬与凋亡在细胞死亡过程中的关系十分密切.本文对自噬的过程及其在细胞存活和死亡中的作用作一综述.