Voltage-dependent anion channels: their roles in plant defense and cell death

The voltage-dependent anion channels (VDACs), mitochondrial outer membrane components, are present in organisms from fungi to animals and plants. They are thought to function in the regulation of metabolite transport between mitochondria and the cytoplasm. Sufficient knowledge on plant VDACs has been accumulated, so that we can here summarize the current information. Then, the involvement of mitochondria in plant defense and cell death is overviewed. While, in mammals, it is suggested that VDAC, also known as a component of the permeability transition pore (PTP) complex formed in the junction site of mitochondrial outer and inner membrane, is a key player in mitochondria-mediated cell death, little is known about the role of plant VDACs in this process. We have shown that plant VDACs are involved in mitochondria-mediated cell death and in defense against a non-host pathogen. In light of the current findings, we discuss the role of the PTP complex and VDAC as its component in plant pathogen defense and cell death.     

Comparative analysis of induction pattern of programmed cell death and defense-related responses during hypersensitive cell death and development of bacterial necrotic leaf spots in eggplant

Pseudomonas cichorii causes necrotic leaf spots (NLS), while Pseudomonas syringae pv. tabaci induces a hypersensitive response (HR) in eggplant. P. cichorii induced cell death at 9 h after inoculation (HAI), reaching a maximum of around 24–30 HAI. On the other hand, cell death was induced 6 HAI with P. syringae pv. tabaci, reaching a maximum of around 12–18 HAI. Superoxide generation was observed in eggplant inoculated with both bacteria. DNA fragmentation, cytochrome c release into the cytosol and expression of defense-related genes such as PR-1 and hsr203J was also induced by inoculation with both bacteria, but these plant reactions were more rapidly induced in eggplant inoculated with P. syringae pv. tabaci rather than those with P. cichorii. Lipid peroxidation and induction of lipoxygenase (LOX) was drastically induced in eggplant inoculated with P. syringae pv. tabaci compared to P. cichorii-inoculated eggplant. Pharmacological studies showed that induction of the cell death, and the NLS or the HR in response to both bacteria was commonly associated with de novo protein synthesis, reactive oxygen species and caspase III-like protease. Interestingly, involvement of lipid peroxidation, LOX, serine protease, and DNase differed between induction of NLS and HR. These results suggest that programmed cell death might be closely associated not only with the HR but also NLS. However, there may be differences not only in the induction kinetics and level of plant responses but also in the infection-related responses between HR and NLS.     

Uncovering the role of VDAC in the regulation of cell life and death

Proper cell activity requires an efficient exchange of molecules between mitochondria and cytoplasm. Lying in the outer mitochondrial membrane, VDAC assumes a crucial position in the cell, forming the main interface between the mitochondrial and the cellular metabolisms. As such, it has been recognized that VDAC plays a crucial role in regulating the metabolic and energetic functions of mitochondria. Indeed, down-regulation of VDAC1 expression by shRNA leads to a decrease in energy production and cell growth. VDAC has also been recognized as a key protein in mitochondria-mediated apoptosis through its involvement in the release of apoptotic proteins located in the inter-membranal space and as the proposed target of pro- and anti-apoptotic members of the Bcl2-family and of hexokinase. Questions, however, remain as to if and how VDAC mediates the transfer of apoptotic proteins from the inter-membranal space to the cytosol. The diameter of the VDAC pore is only about 2.5–3 nm, insufficient for the passage of a folded protein like cytochrome c. New work, however, suggests that pore formation involves the assembly of homo-oligomers of VDAC or hetero-oligomers composed of VDAC and pro-apoptotic proteins, such as Bax. Thus, VDAC appears to represent a convergence point for a variety of cell survival and cell death signals. This review provides insight into the central role of VDAC in mammalian cell life and death, emphasizing VDAC function in the regulation of mitochondria-mediated apoptosis and, as such, its potential as a rational target for new therapeutics.     

Expression of the Hypersensitive Response-assisting Protein in <Emphasis Type="Italic">Arabidopsis</Emphasis> Results in Harpin-dependent Hypersensitive Cell Death in Response to <Emphasis Type="Italic">Erwinia carotovora</Emphasis>

Active defense mechanisms of plants against pathogens often include a rapid plant cell death known as the hypersensitive cell death (HCD). Hypersensitive response-assisting protein (HRAP) isolated from sweet pepper intensifies the harpin_Pss-mediated HCD. Here we demonstrate that constitutive expression of the hrap gene in Arabidopsis results in an enhanced disease resistance towards soft rot pathogen, E. carotovora subsp. carotovora. This resistance was due to the induction of HCD since different HCD markers viz. Athsr3, Athsr4, ion leakage, H₂O₂ and protein kinase were induced. One of the elicitor harpin proteins, HrpN, from Erwinia carotovora subsp. carotovora was able to induce a stronger HCD in hrap-Arabidopsis than non-transgenic controls. To elucidate the role of HrpN, we used E. carotovora subsp. carotovora defective in HrpN production. The hrpN⁻ mutant did not induce disease resistance or HCD markers in hrap-Arabidopsis. These results imply that the disease resistance of hrap-Arabidopsis against a virulent pathogen is harpin dependent.     

Protective Effects of Asiatic Acid on Rotenone- or H<Subscript>2</Subscript>O<Subscript>2</Subscript>-Induced Injury in SH-SY5Y Cells

Parkinson’s disease (PD) is a progressive neurodegenerative disorder with a prevalence of 1–2% in people over the age of 50. Mitochondrial dysfunction occurred in PD patients showing a 15–30% loss of activity in complex I. Asiatic acid (AA), a triterpenoid, is an antioxidant and used for depression treatment, but the effect of AA against PD-like damage has never been reported. In the present study, we investigated the protective effects of AA against H₂O₂ or rotenone-induced cellular injury and mitochondrial dysfunction in SH-SY5Y cells. Mitochondrial membrane potential (MMP) and the expression of voltage-dependent anion channel (VDAC) were detected with or without AA pretreatment following cellular injury to address the possible mechanisms of AA neuroprotection. The results showed that pre-treatment of AA (0.01–100 nM) protected cells against the toxicity induced by rotenone or H₂O₂. In addition, MMP dissipation occurred following the exposure of rotenone, which could be prevented by AA treatment. More interestingly, pre-administration of AA inhibited the elevation of VDAC mRNA and protein levels induced by rotenone(100 nM) or H₂O₂ (300 μM).These data indicate that AA could protect neuronal cells against mitochondrial dysfunctional injury and suggest that AA might be developed as an agent for PD prevention or therapy. Special issue article in honor of Dr. Akitane Mori.     

VDAC regulation: role of cytosolic proteins and mitochondrial lipids

It was recently asserted that the voltage-dependent anion channel (VDAC) serves as a global regulator, or governor, of mitochondrial function (Lemasters and Holmuhamedov, Biochim Biophys Acta 1762:181–190, 2006). Indeed, VDAC, positioned on the interface between mitochondria and the cytosol (Colombini, Mol Cell Biochem 256:107–115, 2004), is at the control point of mitochondria life and death. This large channel plays the role of a “switch” that defines in which direction mitochondria will go: to normal respiration or to suppression of mitochondria metabolism that leads to apoptosis and cell death. As the most abundant protein in the mitochondrial outer membrane (MOM), VDAC is known to be responsible for ATP/ADP exchange and for the fluxes of other metabolites across MOM. It controls them by switching between the open and “closed” states that are virtually impermeable to ATP and ADP. This control has dual importance: in maintaining normal mitochondria respiration and in triggering apoptosis when cytochrome c and other apoptogenic factors are released from the intermembrane space into the cytosol. Emerging evidence indicates that VDAC closure promotes apoptotic signals without direct involvement of VDAC in the permeability transition pore or hypothetical Bax-containing cytochrome c permeable pores. VDAC gating has been studied extensively for the last 30 years on reconstituted VDAC channels. In this review we focus exclusively on physiologically relevant regulators of VDAC gating such as endogenous cytosolic proteins and mitochondrial lipids. Closure of VDAC induced by such dissimilar cytosolic proteins as pro-apoptotic tBid and dimeric tubulin is compared to show that the involved mechanisms are rather distinct. While tBid mostly modulates VDAC voltage gating, tubulin blocks the channel with the efficiency of blockage controlled by voltage. We also discuss how characteristic mitochondrial lipids, phospatidylethanolamine and cardiolipin, could regulate VDAC gating. Overall, we demonstrate that VDAC gating is not just an observation made under artificial conditions of channel reconstitution but is a major mechanism of MOM permeability control.     

Arabidopsis non-host resistance PSS30 gene enhances broad-spectrum disease resistance in the soybean cultivar Williams 82

Non-host resistance (NHR), which protects all members of a plant species from non-adapted or non-host plant pathogens, is the most common form of plant immunity. NHR provides the most durable and robust form of broad-spectrum immunity against non-adaptive pathogens pathogenic to other crop species. In a mutant screen for loss of Arabidopsis (Arabidopsis thaliana) NHR against the soybean (Glycine max (L.) Merr.) pathogen Phytophthora sojae, the Phytophthora sojae-susceptible 30 (pss30) mutant was identified. The pss30 mutant is also susceptible to the soybean pathogen Fusarium virguliforme. PSS30 encodes a folate transporter, AtFOLT1, which was previously localized to chloroplasts and implicated in the transport of folate from the cytosol to plastids. We show that two Arabidopsis folate biosynthesis mutants with reduced folate levels exhibit a loss of non-host immunity against P. sojae. As compared to the wild-type Col-0 ecotype, the steady-state folate levels are reduced in the pss1, atfolt1 and two folate biosynthesis mutants, suggesting that folate is required for non-host immunity. Overexpression of AtFOLT1 enhances immunity of transgenic soybean lines against two serious soybean pathogens, the fungal pathogen F. virguliforme and the soybean cyst nematode (SCN) Heterodera glycines. Transgenic lines showing enhanced SCN resistance also showed increased levels of folate accumulation. This study thus suggests that folate contributes to non-host plant immunity and that overexpression of a non-host resistance gene could be a suitable strategy for generating broad-spectrum disease resistance in crop plants.     

Pea genes associated with non-host disease resistance to Fusarium are also active in race-specific disease resistance to Pseudomonas

A given plant species is able to resist most of the potentially pathogenic microorganisms with which it comes in contact. This phenomenon, known as non-host resistance, can be overcome only by a very small number of true pathogens which can use that plant as a host. In some cases, plants have developed mechanisms for overcoming infection by specific races or strains of a true pathogen. This race-specific resistance can be easily manipulated into agronomically important cultivars by plant breeders. We have previously described nine cDNA clones which represent pea genes active during non-host resistance against the fungus Fusarium solani f. sp. phaseoli. In the present work, we have used these cDNAs as probes to compare non-host resistance with race-specific responses of peas against three races of Pseudomonas syringae pv. pisi. Five of the genes most active during non-host resistance were also active in direct correlation with the phenotypic expression of resistance in race-specific reactions of five differential pea cultivars against three races of Pseudomonas syringae pv. pisi.     

<Emphasis Type="Italic">In vitro</Emphasis> selection and regeneration of chrysanthemum (<Emphasis Type="Italic">Dendranthema grandiflorum</Emphasis> Tzelev) plants resistant to culture filtrate of <Emphasis Type="Italic">Septoria obesa</Emphasis> Syd

Callus cultures derived from leaf segments of chrysanthemum cultivar ‘Snow Ball’ which was susceptible to Septoria obesa were successfully used for in vitro selection for resistance to this pathogenic fungus. Resistant cell lines were selected by culturing callus on growth medium containing various concentrations of S. obesa filtrate. Resistant calluses obtained after two cycles (30 d each cycle) of selection were used for plant regeneration. About 30% of the plants regenerated from the resistant calluses and 70–80% of the plants raised from cuttings had acquired considerable resistance against the pathogen in the field. No phenotypic variation was observed in the selected regenerates.     

Genetic strategies for dissecting mammalian and Drosophila voltage-dependent anion channel functions

Voltage-dependent anion channels (VDACs), also known as mitochondrial porins, are a family of small pore-forming proteins of the mitochondrial outer membrane that are found in all eukaryotes. VDACs are thought to play important roles in the regulated flux of metabolites between the cytosolic and mitochondrial compartments, in overall energy metabolism via interactions with cytosolic kinases, and a debated role in programmed cell death (apoptosis). The mammalian genome contains three VDAC loci termed Vdac1, Vdac2, and Vdac3, raising the question as to what function each isoform may be performing. Based upon expression studies of the mouse VDACs in yeast, biophysical differences can be identified but the physiologic significance of these differences remains unclear. Creation of “knockout” cell lines and mice that lack one or more VDAC isoforms has led to the characterization of distinct phenotypes that provide a different set of insights into function which must be interpreted in the context of complex physiologic systems. Functions in male reproduction, the central nervous system and glucose homeostasis have been identified and require a deeper and more mechanistic examination. Annotation of the genome sequence of Drosophila melanogaster has recently revealed three additional genes (CG17137, CG17139, CG17140) with homology to porin, the previously described gene that encodes the VDAC of D. melanogaster. Molecular analysis of these novel VDACs has revealed a complex pattern of gene organization and expression. Sequence comparisons with other insect VDAC homologs suggest that this gene family evolved through a mechanism of duplication and divergence from an ancestral VDAC gene during the radiation of the genus Drosophila. Striking similarities to mouse VDAC mutants can be found that emphasize the conservation of function over a long evolutionary time frame.     

Molecular cloning and differential expression of an γ-aminobutyrate transaminase gene, OsGABA-T, in rice (Oryza sativa) leaves infected with blast fungus

γ-Aminobutyrate transaminase (GABA-T) catalyzes the conversion of GABA to succinic semialdehyde. Using differential display PCR and cDNA library screening, a full-length GABA-T cDNA (OsGABA-T) was isolated from rice (Oryza sativa) leaves infected with an incompatible race of Magnaporthe grisea. The deduced amino acid sequence comprises 483 amino acid residues and shares 85–69% identity with GABA-T sequences from other plants. OsGABA-T expression is induced by blast fungus infection, mechanical wounding and ultraviolet radiation in rice leaves and is not detected in normal rice organs. This gene is also induced by defense signal molecules such as salicylic acid and abscisic acid, but not by jasmonic acid. Our data suggest that OsGABA-T (GABA shunt) may play a role in restricting the levels of cell death during the host–pathogen interaction.     

Antioxidant and antimicrobial actions of the clubmoss <Emphasis Type="Italic">Lycopodium clavatum</Emphasis> L.

Purpose of the present study was to evaluate antioxidant, antibacterial, antifungal, and antiviral activities of the petroleum ether, chloroform, ethyl acetate and methanol extracts as well as the alkaloid fraction of Lycopodium clavatum L. (LC) from Lycopodiaceae growing in Turkey. Antioxidant activity of the LC extracts was evaluated by 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging method at 0.2 mg/ml using microplate-reader assay. Antiviral assessment of LC extracts was evaluated towards the DNA virus Herpes simplex (HSV) and the RNA virus Parainfluenza (PI-3) using Madin-Darby Bovine Kidney (MDBK) and Vero cell lines. Antibacterial and antifungal activities of the extracts were tested against standard and isolated strains of the following bacteria; Escherichia coli, Pseudomonas aeruginosa, Proteus mirabilis, Acinobacter baumannii, Klebsiella pneumoniae, Staphylococcus aureus, Bacillus subtilis as well as the fungi; Candida albicans and C. parapsilosis. All of the extracts possessed noteworthy activity against ATCC strain of S. aureus (4 μg/ml), while the LC extracts showed reasonable antifungal effect. On the other hand, we found that only the chloroform extract was active against HSV (16–8 μg/ml), while petroleum ether and alkaloid extracts inhibited potently PI-3 (16–4 μg/ml and 32–4 μg/ml, respectively). However, all of the extracts had insignificant antiradical effect on DPPH. In addition, we also analyzed the content of the alkaloid fraction of the plant by capillary gas chromatography-mass spectrometry (GC-MS) and identified lycopodine as the major alkaloid.     

Tomato resistance to Alternaria stem canker: localization in host genotypes and functional expression compared to non-host resistance

Summary The Alternaria stem canker resistance locus (Asc-locus), involved in resistance to the fungal pathogen Alternaria alternata f. sp. lycopersici and in insensitivity to host-specific toxins (AAL-toxins) produced by the pathogen, was genetically mapped on the tomato genome. Susceptibility and resistance were assayed by testing a segregating F₂ population for sensitivity to AAL-toxins in leaf bioassays. Linkage was observed to phenotypic markers solanifolium and sunny, both on chromosome 3. For the Asc-locus, a distance of 18 centiMorgan to solanifolium was calculated, corresponding to position 93 on chromosome 3. This map position of the resistance locus turned out to be the same in three different resistant tomato accessions, one Dutch and two American, that are at least 40 years apart. AAL-toxin sensitivity in susceptible and resistant tomato genotypes was compared with AAL-toxin sensitivity in a non-host Nicotiana tabacum during different levels of plant cell development. In susceptible and resistant tomato genotypes, inhibitory effects were demonstrated at all levels, except for leaves of resistant genotypes. However, during pollen and root development, inhibitory effects on susceptible genotypes were larger than on resistant genotypes. In the non-host Nicotiana tabacum, hardly any effects of AAL-toxins were demonstrated. Apparently, a cellular target site is present in tomato, but not in Nicotiana tabacum. It was concluded that three levels of AAL-toxin sensitivity exist: (1) a susceptible host sensitivity, (2) a resistant host sensitivity, (3) a non-host sensitivity, and that the resistance mechanism operating in tomato is different from that operating in Nicotiana tabacum.     

UDP-Gal: <Emphasis Type="Italic">N</Emphasis>-acetylglucosamine β 1–4 galactosyltransferase expressing live attenuated parasites as vaccine for visceral leishmaniasis

As compared to cutaneous leishmaniasis, vaccination against visceral leishmaniasis (VL) has received limited attention. In this study, we demonstrate for the first time that an UDP-Galactose: N-acetylglucosamine β 1–4 galactosyltransferase (GenBank Accession No. EF159943) expressing attenuated LD clonal population (A-LD) is able to confer protection against the experimental challenge with the virulent LD AG83 parasite. A-LD was also effective in established leishmania infection. The vaccinated animals showed both cell mediated (in vitro T-cell proliferation, and DTH response) and humoral responses (Th1 type). These results demonstrate the potential of the attenuated clones as an immunotherapeutic and immunoprophylactic agent against visceral leishmaniasis.     

Population densities of herbivorous lepidopterans in diverse cruciferous cropping habitats: Effects of mixed cropping and using a living mulch

Two field experiments were conducted in Hawaii to determine the responses of lepidopteran pests and their associated natural enemies to broccoli (Brassica oleracea), grown in monoculture or interplanted with chili pepper (Capsicum annuum), or yellow sweetclover (YSC) (Melilotus officinalis). The population responses of Artogeia rapae and Trichoplusia ni to habitat types were not consistent during the field trials. Additionally, their reactions to habitat diversification differed according to non-host plant species. For example, Trichoplusia ni densities were significantly lower in the broccoli–YSC habitat contrasted with the broccoli–pepper habitat on most sampling dates. Similarly, the proportion of plants damaged by Hellula undalis was significantly lower in the broccoli–YSC habitat contrasted with broccoli–pepper habitat. Findings from the study, suggest that biological control agents (e.g., spiders, parasitoids, and␣entomopathogens) are the most significant factors influencing the abundance of A.␣rapae in habitat type and that these agents inflict greater mortality on A. rapae in diculture than monoculture habitats.     

Characterization of Virulence Function of Pseudomonas cichorii Avirulence Protein E1 (AvrE1) during Host Plant Infection

Pseudomonas cichorii secretes effectors that suppress defense mechanisms in host plants. However, the function of these effectors, including avirulence protein E1 (AvrE1), in the pathogenicity of P. cichorii, remains unexplored. In this study, to investigate the function of avrE1 in P. cichorii JBC1 (PcJBC1), we created an avrE1-deficient mutant (JBC1^ΔavrE1) using CRISPR/Cas9. The disease severity caused by JBC1^ΔavrE1 in tomato plants significantly decreased by reducing water soaking during early infection stage, as evidenced by the electrolyte leakage in infected leaves. The disease symptoms caused by JBC1^ΔavrE1 in the cabbage midrib were light-brown spots compared to the dark-colored ones caused by PcJBC1, which indicates the role of AvrE1 in cell lysis. The avrE1-deficient mutant failed to elicit cell death in non-host tobacco plants. Disease severity and cell death caused by JBC1^ΔavrE1 in host and non-host plants were restored through heterologous complementation with avrE1 from Pseudomonas syringae pv. tomato DC3000 (PstDC3000). Overall, our results indicate that avrE1 contributes to cell death during early infection, which consequently increases disease development in host plants. The roles of PcJBC1 AvrE1 in host cells remain to be elucidated.