Silencing of a BYPASS1 homolog results in root-independent pleiotrophic developmental defects in Nicotiana benthamiana

The Arabidopsis bypass1 mutant (bps1) exhibits defective shoot and root growth that is associated with constitutive production of a root-derived carotenoid-related signal (Van Norman et al., Curr Biol 14:1739-1746, 2004). Since the identity of the signal and the function of BPS1 are still unknown, we investigated effects of BPS1 depletion in Nicotiana benthamiana to elucidate BPS1 function in plant growth and development. The predicted protein of NbBPS1, a BPS1 homolog of N. benthamiana, contains a central transmembrane domain, and a NbBPS1:GFP fusion protein was mainly associated with the endoplasmic reticulum. Virus-induced gene silencing (VIGS) of NbBPS1 resulted in pleiotrophic phenotypes, including growth retardation and abnormal leaf development. At the cellular level, the plants exhibited hyperproliferation of the cambial cells and defective xylem differentiation during stem vascular development. Hyperactivity of the cambium was associated with an elevated auxin and cytokinin response. In contrast, the leaves had reduced numbers of cells with increased cell size and elevated endoreduplication. Cell death in NbBPS1 VIGS leaves started with vacuole collapse, followed by degeneration of the organelles. Interestingly, these phenotypes were mainly caused by silencing of NbBPS1 in the aerial parts of the plants, different from the case of the Arabidopsis bps1 mutant. These results suggest that NbBPS1 plays a role in the control of cell division and differentiation in the cambium of N. benthamiana, and BPS homologs may have a diverse function in different tissues and in different species.     

Cell death triggered by the P25 protein in Potato virus X-associated synergisms results from endoplasmic reticulum stress in Nicotiana benthamiana

The synergistic interaction of Potato virus X (PVX) with a number of potyviruses results in systemic necrosis in Nicotiana spp. Previous investigations have indicated that the viral suppressor of RNA silencing (VSR) protein P25 of PVX triggers systemic necrosis in PVX-associated synergisms in a threshold-dependent manner. However, little is still known about the cellular processes that lead to this necrosis, and whether the VSR activity of P25 is involved in its elicitation. Here, we show that transient expression of P25 in the presence of VSRs from different viruses, including the helper component-proteinase (HC-Pro) of potyviruses, induces endoplasmic reticulum (ER) stress and the unfolded protein response (UPR), which ultimately lead to ER collapse. However, the host RNA silencing pathway was dispensable for the elicitation of cell death by P25. Confocal microscopy studies in leaf patches co-expressing P25 and HC-Pro showed dramatic alterations in ER membrane structures, which correlated with the up-regulation of bZIP60 and several ER-resident chaperones, including the ER luminal binding protein (BiP). Overexpression of BiP alleviated the cell death induced by the potexviral P25 protein when expressed together with VSRs derived from different viruses. Conversely, silencing of the UPR master regulator, bZIP60, led to an increase in cell death elicited by the P25/HC-Pro combination as well as by PVX-associated synergism. In addition to its role as a negative regulator of P25-induced cell death, UPR partially restricted PVX infection. Thus, systemic necrosis caused by PVX-associated synergistic infections is probably the effect of an unmitigated ER stress following the overaccumulation of a viral protein, P25, with ER remodelling activity.     

The plant cell death suppressor Adi3 interacts with the autophagic protein Atg8h

The tomato AGC protein kinase Adi3 is known to function as a suppressor of PCD and silencing of Adi3 leads to spontaneous cell death on leaves and stems. In an effort to isolate Adi3 interacting proteins, a yeast two-hybrid screen was carried out and identified the autophagy protein Atg8h as an Adi3 interactor. This interaction occurred independent of the kinase activity status of Adi3. Silencing of genes involved in autophagy is known to eliminate the restriction of pathogen-induced PCD to a few cells and leads to run away PCD. Cosilencing Adi3 with several autophagy genes lead to the same run away cell death suggesting Adi3 may be involved in autophagic regulation of PCD.     

DspA/E, a type III effector of Erwinia amylovora, is required for early rapid growth in Nicotiana benthamiana and causes NbSGT1-dependent cell death

DspA/E is a pathogenicity factor of Erwinia amylovora that is translocated into the plant cell cytoplasm through an Hrp type III secretion system. Transient expression of dspA/E in Nicotiana benthamiana or yeast induced cell death, as it does in N. tabacum and apple as described previously. DspA/E-induced cell death in N. benthamiana was not inhibited by coexpression of AvrPtoB of Pseudomonas syringae pv. tomato , which inhibits programmed cell death (PCD) induced by several other elicitors in plants. Silencing of NbSGT1 , the expression of which is required for PCD mediated by several resistance proteins of plants, prevented DspA/E-induced cell death in N. benthamiana. However, silencing of NbRAR1 , or two MAP kinase kinase genes, which are required for PCD associated with many resistance genes in plants, did not prevent cell death induced by DspA/E. Silencing of NbSGT1 also compromised non-host resistance against E. amylovora . E. amylovora grew rapidly within the first 24 h after infiltration in N. benthamiana , and DspA/E was required for this early rapid growth. However, bacterial cell numbers decreased after 24 h in TRV-vector-transformed plants, whereas a dspA/E mutant strain grew to high populations in NbSGT1 -silenced plants. Our results indicate that DspA/E enhances virulence of E. amylovora in N. benthamiana, but the bacteria are then recognized by the plant, resulting in PCD and death of bacterial cells or restriction of bacterial cell growth.     

Shiga toxins induce autophagic cell death in intestinal epithelial cells via the endoplasmic reticulum stress pathway

Shiga toxins (Stxs) are a family of cytotoxic proteins that lead to the development of bloody diarrhea, hemolytic-uremic syndrome, and central nervous system complications caused by bacteria such as S. dysenteriae, E. coli O157:H7 and E. coli O104:H4. Increasing evidence indicates that macroautophagy (autophagy) is a key factor in the cell death induced by Stxs. However, the associated mechanisms are not yet clear. This study showed that Stx2 induces autophagic cell death in Caco-2 cells, a cultured line model of human enterocytes. Inhibition of autophagy using pharmacological inhibitors, such as 3-methyladenine and bafilomycin A₁, or silencing of the autophagy genes ATG12 or BECN1 decreased the Stx2-induced death in Caco-2 cells. Furthermore, there were numerous instances of dilated endoplasmic reticulum (ER) in the Stx2-treated Caco-2 cells, and repression of ER stress due to the depletion of viable candidates of DDIT3 and NUPR1. These processes led to Stx2-induced autophagy and cell death. Finally, the data showed that the pseudokinase TRIB3-mediated DDIT3 expression and AKT1 dephosphorylation upon ER stress were triggered by Stx2. Thus, the data indicate that Stx2 causes autophagic cell death via the ER stress pathway in intestinal epithelial cells.     

The fatal effect of tungsten on <Emphasis Type="Italic">Pisum sativum</Emphasis> L. root cells: indications for endoplasmic reticulum stress-induced programmed cell death

Programmed cell death (PCD) is a widespread response of plants against abiotic stress, such as heavy metal toxicity. Tungsten (W) is increasingly considered toxic for plants since it irreversibly affects their growth. Therefore, we investigated whether W could induce some kind of PCD in plants, like other heavy metals do. The morphology of cell and nucleus, the integrity of the cytoskeleton, Evans Blue absorbance and the expression of PCD-related genes were used as indicators of PCD in W-treated roots of Pisum sativum (pea). TEM and fluorescence microscopy revealed mitotic cycle arrest, protoplast shrinkage, disruption of the cytoskeleton and chromatin condensation and peripheral distribution in the nucleus of W-affected cells. Moreover, Evans Blue absorbance in roots increased in relation to the duration of W treatment. These effects were suppressed by inhibitors of the 26S proteasome, caspases and endoplasmic reticulum stress. In addition, silencing of DAD-1 and induction of HSR203J, BiP-D, bZIP28 and bZIP60 genes were also recorded in W-treated pea roots by semi-quantitative RT-PCR. The above observations show that W induces a kind of PCD in pea roots, further substantiating its toxicity for plants. Data imply that endoplasmic reticulum stress-unfolded protein response may be involved in W-induced PCD.     

The mutualistic fungus Piriformospora indica colonizes Arabidopsis roots by inducing an endoplasmic reticulum stress-triggered caspase-dependent cell death

In Arabidopsis thaliana roots, the mutualistic fungus Piriformospora indica initially colonizes living cells, which die as the colonization proceeds. We aimed to clarify the molecular basis of this colonization-associated cell death. Our cytological analyses revealed endoplasmic reticulum (ER) swelling and vacuolar collapse in invaded cells, indicative of ER stress and cell death during root colonization. Consistent with this, P. indica-colonized plants were hypersensitive to the ER stress inducer tunicamycin. By clear contrast, ER stress sensors bZIP60 and bZIP28 as well as canonical markers for the ER stress response pathway, termed the unfolded protein response (UPR), were suppressed at the same time. Arabidopsis mutants compromised in caspase 1-like activity, mediated by cell death-regulating vacuolar processing enzymes (VPEs), showed reduced colonization and decreased cell death incidence. We propose a previously unreported microbial invasion strategy during which P. indica induces ER stress but inhibits the adaptive UPR. This disturbance results in a VPE/caspase 1-like-mediated cell death, which is required for the establishment of the symbiosis. Our results suggest the presence of an at least partially conserved ER stress-induced caspase-dependent cell death pathway in plants as has been reported for metazoans.     

DAP-kinase is a mediator of endoplasmic reticulum stress-induced caspase activation and autophagic cell death

Damage to endoplasmic reticulum (ER) homeostasis that cannot be corrected by the unfolded protein response activates cell death. Here, we identified death-associated protein kinase (DAPk) as an important component in the ER stress-induced cell death pathway. DAPk-/- mice are protected from kidney damage caused by injection of the ER stress-inducer tunicamycin. Likewise, the cell death response to ER stress-inducers is reduced in DAPk-/- primary fibroblasts. Both caspase activation and autophagy induction, events that are activated by ER stress and precede cell death, are significantly attenuated in the DAPk null cells. Notably, in this cellular setting, autophagy serves as a second cell killing mechanism that acts in concert with apoptosis, as the depletion of Atg5 or Beclin1 from fibroblasts significantly protected from ER stress-induced death when combined with caspase-3 depletion. We further show that ER stress promotes the catalytic activity of DAPk by causing dephosphorylation of an inhibitory autophosphorylation on Ser(308) by a PP2A-like phosphatase. Thus, DAPk constitutes a critical integration point in ER stress signaling, transmitting these signals into two distinct directions, caspase activation and autophagy, leading to cell death.     

Identification of three MAPKKKs forming a linear signaling pathway leading to programmed cell death in

ABSTRACT: BACKGROUND: The mitogen-activated protein kinase (MAPK) cascade is an evolutionarily ancient mechanism of signal transduction found in eukaryotic cells. In plants, MAPK cascades are associated with responses to various abiotic and biotic stresses such as plant pathogens. MAPK cascades function through sequential phosphorylation: MAPK kinase kinases (MAPKKKs) phosphorylate MAPK kinases (MAPKKs), and phosphorylated MAPKKs phosphorylate MAPKs. Of these three types of kinase, the MAPKKKs exhibit the most divergence in the plant genome. Their great diversity is assumed to allow MAPKKKs to regulate many specific signaling pathways in plants despite the relatively limited number of MAPKKs and MAPKs. Although some plant MAPKKKs, including the MAPKKKalpha of Nicotiana benthamiana (NbMAPKKKalpha), are known to play crucial roles in plant defense responses, the functional relationship among MAPKKK genes is poorly understood. Here, we performed a comparative functional analysis of MAPKKKs to investigate the signaling pathway leading to the defense response. RESULTS: We cloned three novel MAPKKK genes from N. benthamiana: NbMAPKKKbeta, NbMAPKKKgamma, and NbMAPKKKepsilon2. Transient overexpression of full-length NbMAPKKKbeta or NbMAPKKKgamma or their kinase domains in N. benthamiana leaves induced hypersensitive response (HR)-like cell death associated with hydrogen peroxide production. This activity was dependent on the kinase activity of the overexpressed MAPKKK. In addition, virus-induced silencing of NbMAPKKKbeta or NbMAPKKKgamma expression significantly suppressed the induction of programmed cell death (PCD) by viral infection. Furthermore, in epistasis analysis of the functional relationships among NbMAPKKKbeta, NbMAPKKKgamma, and NbMAPKKKalpha (previously shown to be involved in plant defense responses) conducted by combining transient overexpression analysis and virus-induced gene silencing, silencing of NbMAPKKKalpha suppressed cell death induced by the overexpression of the NbMAPKKKbeta kinase domain or of NbMAPKKKgamma, but silencing of NbMAPKKKbeta failed to suppress cell death induced by the overexpression of NbMAPKKKalpha or NbMAPKKKgamma. Silencing of NbMAPKKKgamma suppressed cell death induced by the NbMAPKKKbeta kinase domain but not that induced by NbMAPKKKalpha. CONCLUSIONS: These results demonstrate that in addition to NbMAPKKKalpha, NbMAPKKKbeta and NbMAPKKKgamma also function as positive regulators of PCD. Furthermore, these three MAPKKKs form a linear signaling pathway leading to PCD; this pathway proceeds from NbMAPKKKbeta to NbMAPKKKgamma to NbMAPKKKalpha.     

The binding protein BiP attenuates stress-induced cell death in soybean via modulation of the N-rich protein-mediated signaling pathway

The molecular chaperone binding protein (BiP) participates in the constitutive function of the endoplasmic reticulum (ER) and protects the cell against stresses. In this study, we investigated the underlying mechanism by which BiP protects plant cells from stress-induced cell death. We found that enhanced expression of BiP in soybean (Glycine max) attenuated ER stress- and osmotic stress-mediated cell death. Ectopic expression of BiP in transgenic lines attenuated the leaf necrotic lesions that are caused by the ER stress inducer tunicamycin and also maintained shoot turgidity upon polyethylene glycol-induced dehydration. BiP-mediated attenuation of stress-induced cell death was confirmed by the decreased percentage of dead cell, the reduced induction of the senescence-associated marker gene GmCystP, and reduced DNA fragmentation in BiP-overexpressing lines. These phenotypes were accompanied by a delay in the induction of the cell death marker genes N-RICH PROTEIN-A (NRP-A), NRP-B, and GmNAC6, which are involved in transducing a cell death signal generated by ER stress and osmotic stress through the NRP-mediated signaling pathway. The prosurvival effect of BiP was associated with modulation of the ER stress- and osmotic stress-induced NRP-mediated cell death signaling, as determined in transgenic tobacco (Nicotiana tabacum) lines with enhanced (sense) and suppressed (antisense) BiP levels. Enhanced expression of BiP prevented NRP- and NAC6-mediated chlorosis and the appearance of senescence-associated markers, whereas silencing of endogenous BiP accelerated the onset of leaf senescence mediated by NRPs and GmNAC6. Collectively, these results implicate BiP as a negative regulator of the stress-induced NRP-mediated cell death response.