Involvement of TatD nuclease during programmed cell death in the protozoan parasite Trypanosoma brucei

In this report, we describe the involvement of TatD nuclease during programmed cell death (PCD) in the human protozoan parasite Trypanosoma brucei. T. brucei TatD nuclease showed intrinsic DNase activity, was localized in the cytoplasm and translocated to the nucleus when cells were treated with inducers previously demonstrated to cause PCD in T. brucei. Overexpression of TatD nuclease resulted in elevated PCD and conversely, loss of TatD expression by RNAi conferred significant resistance to the induction of PCD in T. brucei. Co‐immunoprecipitation studies revealed that TatD nuclease interacts with endonucleaseG suggesting that these two nucleases could form a DNA degradation complex in the nucleus. Together, biochemical activity, RNAi and subcellular localization results demonstrate the role of TatD nuclease activity in DNA degradation during PCD in these evolutionarily ancient eukaryotic organisms. Further, in conjunction with endonucleaseG, TatD may represent a critical nuclease in a caspase‐independent PCD pathway in trypanosomatid parasites since caspases have not been identified in these organisms.     

Interferon‐inducible protein,P56, inhibits HPV DNA replication by binding to the viral protein E1

Type I interferon (IFN) inhibits, by an unknown mechanism, the replication of human papillomaviruses (HPV), which are major human pathogens, Here, we present evidence that P56 (a protein), the expression of which is strongly induced by IFN, double‐stranded RNA and viruses, mediates the anti‐HPV effect of IFN. Ectopic expression of P56 inhibited HPV DNA replication and its ablation in IFN‐treated cells alleviated the inhibitory effect of IFN on HPV DNA replication. Protein–protein interaction and mutational analyses established that the antiviral effect of P56 was mediated by its direct interaction with the DNA replication origin‐binding protein E1 of several strains of HPV, through the tetratricopeptide repeat 2 in the N‐terminal region of P56 and the C‐terminal region of E1. In vivo, the interaction with P56, a cytoplasmic protein, caused translocation of E1 from the nucleus to the cytoplasm. In vitro, recombinant P56, or a small fragment derived from it, inhibited the DNA helicase activity of E1 and E1‐mediated HPV DNA replication. These observations delineate the molecular mechanism of IFN's antiviral action against HPV.     

Set‐point control of RD21 protease activity by AtSerpin1 controls cell death in Arabidopsis

Programmed cell death (PCD) in plants plays a key role in defense response and is promoted by the release of compartmentalized proteases to the cytoplasm. Yet the exact identity and control of these proteases is poorly understood. Serpins are an important group of proteins that uniquely curb the activity of proteases by irreversible inhibition; however, their role in plants remains obscure. Here we show that during cell death the Arabidopsis serpin protease inhibitor, AtSerpin1, exhibits a pro‐survival function by inhibiting its target pro‐death protease, RD21. AtSerpin1 accumulates in the cytoplasm and RD21 accumulates in the vacuole and in endoplasmic reticulum bodies. Elicitors of cell death, including the salicylic acid agonist benzothiadiazole and the fungal toxin oxalic acid, stimulated changes in vacuole permeability as measured by the changes in the distribution of marker dye. Concomitantly, a covalent AtSerpin1–RD21 complex was detected indicative of a change in protease compartmentalization. Furthermore, mutant plants lacking RD21 or plants with AtSerpin1 over‐expression exhibited significantly less elicitor‐stimulated PCD than plants lacking AtSerpin1. The necrotrophic fungi Botrytis cinerea and Sclerotina sclerotiorum secrete oxalic acid as a toxin that stimulates cell death. Consistent with a pro‐death function for RD21 protease, the growth of these necrotrophs was compromised in plants lacking RD21 but accelerated in plants lacking AtSerpin1. The results indicate that AtSerpin1 controls the pro‐death function of compartmentalized protease RD21 by determining a set‐point for its activity and limiting the damage induced during cell death.     

Apoptosis-like programmed cell death in the grey mould fungus Botrytis cinerea: genes and their role in pathogenicity

A considerable number of fungal homologues of human apoptotic genes have been identified in recent years. Nevertheless, we are far from being able to connect the different pieces and construct a primary structure of the fungal apoptotic regulatory network. To get a better picture of the available fungal components, we generated an automatic search protocol that is based on protein sequences together with a domain-centred approach. We used this protocol to search all the available fungal databases for domains and homologues of human apoptotic proteins. Among all known apoptotic domains, only the BIR [baculovirus IAP (inhibitor of apoptosis protein) repeat] domain was found in fungi. A single protein with one or two BIR domains is present in most (but not all) fungal species. We isolated the BIR-containing protein from the grey mould fungus Botrytis cinerea and determined its role in apoptosis and pathogenicity. We also isolated and analysed BcNMA, a homologue of the yeast NMA11 gene. Partial knockout or overexpression strains of BcBIR1 confirmed that BcBir1 is anti-apoptotic and this activity was assigned to the N'-terminal part of the protein. Plant infection assays showed that the fungus undergoes massive PCD (programmed cell death) during early stages of infection. Further studies showed that fungal virulence was fully correlated with the ability of the fungus to cope with plant-induced PCD. Together, our result show that BcBir1 is a major regulator of PCD in B. cinerea and that proper regulation of the host-induced PCD is essential for pathogenesis in this and other similar fungal pathogens.     

New insights in phytoplasma‐vector interaction: acquisition and inoculation of flavescence dorée phytoplasma by Scaphoideus titanus adults in a short window of time

The leafhopper Scaphoideus titanus is able to transmit 16SrV phytoplasmas agents of grapevine's flavescence dorée (FD) within 30–45 days, following an acquisition access period (AAP) of a few days feeding on infected plants as a nymph, a latency period (LP) of 3–5 weeks becoming meanwhile an adult, and an inoculation access period (IAP) of a few days on healthy plants. However, several aspects of FD epidemiology suggest how the whole transmission process may take less time, and may start directly with adults of the insect vector. Transmission experiments have been set up under lab condition. Phytoplasma‐free S. titanus adults were placed on broad bean (BB) plants (Vicia faba) infected by FD‐C (16SrV‐C) phytoplasmas for an AAP = 7 days. Afterwards, they were immediately moved onto healthy BB for IAP, which were changed every 7 days, obtaining three timings of inoculation: IAP 1, IAP 2 and IAP 3, lasting 7, 14 and 21 days from the end of AAP, respectively. DNA was extracted from plants and insects, and PCR tests were performed to identify FD phytoplasmas. Insects were dissected and fluorescence in situ hybridisation was made to detect the presence of phytoplasmas in midguts and salivary glands. The rate of infection in insects ranged 46–68% without significant differences among IAPs. Inoculation in plants succeeded in all IAPs, at a rate of 16–23% (no significant differences). Phytoplasma load was significantly higher in IAP 3 than IAP 1–2 for both plants and insects. Phytoplasmas were identified both in midgut and salivary glands of S. titanus at all IAP times. The possible implications of these results in the epidemiology of flavescence dorée are discussed.     

Expression of the Theobroma cacao Bax‐inhibitor‐1 gene in tomato reduces infection by the hemibiotrophic pathogen Moniliophthora perniciosa

Programmed cell death (PCD) plays a key role in plant responses to pathogens, determining the success of infection depending on the pathogen lifestyle and on which participant of the interaction triggers cell death. The hemibiotrophic basidiomycete Moniliophthora perniciosa is the causal agent of witches' broom disease of Theobroma cacao L. (cacao), a serious constraint for production in South America and the Caribbean. It has been hypothesized that M. perniciosa pathogenesis involves PCD, initially as a plant defence mechanism, which is diverted by the fungus to induce necrosis during the dikaryotic phase of the mycelia. Here, we evaluated whether the expression of a cacao anti‐apoptotic gene would affect the incidence and severity of M. perniciosa infection using the ‘Micro‐Tom’ (MT) tomato as a model. The cacao Bax‐inhibitor‐1 (TcBI‐1) gene, encoding a putative basal attenuator of PCD, was constitutively expressed in MT to evaluate function. Transformants expressing TcBI‐1, when treated with tunicamycin, an inducer of endoplasmic reticulum stress, showed a decrease in cell peroxidation. When the same transformants were inoculated with the necrotrophic fungal pathogens Sclerotinia sclerotiorum, Sclerotium rolfsii and Botrytis cinerea, a significant reduction in infection severity was observed, confirming TcBI‐1 function. After inoculation with M. perniciosa, TcBI‐1 transformant lines showed a significant reduction in disease incidence compared with MT. The overexpression of TcBI‐1 appears to affect the ability of germinating spores to penetrate susceptible tissues, restoring part of the non‐host resistance in MT against the S‐biotype of M. perniciosa.     

Re‐targeting of a plant defense protease by a cyst nematode effector

Plants mount defense responses during pathogen attacks, and robust host defense suppression by pathogen effector proteins is essential for infection success. 4E02 is an effector of the sugar beet cyst nematode Heterodera schachtii. Arabidopsis thaliana lines expressing the effector‐coding sequence showed altered expression levels of defense response genes, as well as higher susceptibility to both the biotroph H. schachtii and the necrotroph Botrytis cinerea, indicating a potential suppression of defenses by 4E02. Yeast two‐hybrid analyses showed that 4E02 targets A. thaliana vacuolar papain‐like cysteine protease (PLCP) ‘Responsive to Dehydration 21A’ (RD21A), which has been shown to function in the plant defense response. Activity‐based protein profiling analyses documented that the in planta presence of 4E02 does not impede enzymatic activity of RD21A. Instead, 4E02 mediates a re‐localization of this protease from the vacuole to the nucleus and cytoplasm, which is likely to prevent the protease from performing its defense function and at the same time, brings it in contact with novel substrates. Yeast two‐hybrid analyses showed that RD21A interacts with multiple host proteins including enzymes involved in defense responses as well as carbohydrate metabolism. In support of a role in carbohydrate metabolism of RD21A after its effector‐mediated re‐localization, we observed cell wall compositional changes in 4E02 expressing A. thaliana lines. Collectively, our study shows that 4E02 removes RD21A from its defense‐inducing pathway and repurposes this enzyme by targeting the active protease to different cell compartments.     

Doom, a product of the Drosophila mod(mdg4) gene, induces apoptosis and binds to baculovirus inhibitor-of-apoptosis proteins.

A family of baculovirus inhibitor-of-apoptosis (IAP) genes is present in mammals, insects, and baculoviruses, but the mechanism by which they block apoptosis is unknown. We have identified a protein encoded by the Drosophila mod(mdg4) gene which bound to the baculovirus IAPs. This protein induced rapid apoptosis in insect cells, and consequently we have named it Doom. Baculovirus IAPs and P35, an inhibitor of aspartate-specific cysteine proteases, blocked Doom-induced apoptosis. The carboxyl terminus encoded by the 3' exon of the doom cDNA, which distinguishes it from other mod(mdg4) cDNAs, was responsible for induction of apoptosis and engagement of the IAPs. Doom localized to the nucleus, while the IAPs localized to the cytoplasm, but when expressed together, Doom and the IAPs both localized in the nucleus. Thus, IAPs might block apoptosis by interacting with and modifying the behavior of Doom-like proteins that reside in cellular apoptotic pathways.     

Induction of nucleolin translocation by acharan sulfate in A549 human lung adenocarcinoma

Acharan sulfate (AS), isolated from the giant African snail Achatina fulica, is a novel glycosaminoglycan, consisting primarily of the repeating disaccharide structure α‐D ‐N‐acetylglucosaminyl (1 → 4) 2‐sulfoiduronic acid. AS shows anti‐tumor activity in vitro and in vivo. Despite this activity, AS is only weakly cytotoxic towards cancer cells. We examine the interactions between AS and cell‐surface proteins in an effort to explain this anti‐tumor activity. Using flow cytometry and affinity column chromatography, we confirm that AS has strong affinity to specific cell‐surface proteins including nucleolin (NL) in A549 human lung adenocarcinomas. Surprisingly, we found the translocation of NL from nucleus to cytoplasm under the stimulation of AS (100 µg/ml) in vitro. Also, as NL exits the nucleus, the levels of growth factors such as bFGF and signaling cascade proteins, such as p38, p53, and pERK, are altered. These results suggest that the communication between AS and NL plays a critical role on signal transduction in tumor inhibition. J. Cell. Biochem. 110: 1272–1278, 2010. Published 2010 Wiley‐Liss, Inc.     

Localization, regulation and function of Type II phosphatidylinositol 5-phosphate 4-kinases

Our recent studies of the Type II PtdIns5P 4-kinases have revealed that the Type IIα isoform is very much more active than the IIβ or IIγ isoforms, and that it can (and does physiologically) heterodimerize with them. This suggests the idea that the Type IIα enzyme is targeted to the nucleus (by dimerization with Type IIβ), to secretory/transport vesicles (by dimerization with Type IIγ), or to the cytoplasm (as a homodimer), with the relative proportions of PtdIns5P 4-kinase activity at these localizations being regulated by the relative amounts of the three Type II isoforms expressed in any cell. The targeting to vesicles by PtdIns5P 4-kinase IIγ is likely to be of particular significance in epithelial cells in specific regions of the kidney tubules and in a sub-population of neurons in the brain and the spinal cord. The relationship between this dimerization between Type II PtdIns5P 4-kinase isoforms and the known ability of Type IIα PtdIns5P 4-kinase to associate with Type I PtdIns4P 5-kinases remains to be explored.     

Heterologous expression of the immunomodulatory protein gene from Ganoderma sinense in the basidiomycete Coprinopsis cinerea

Aims: FIP‐gsi, a fungal immunomodulatory protein found in Ganoderma sinense, has antitumour, anti‐allergy and immunomodulatory activities and is regulated by the fip‐gsi gene. In this study, we aimed to express the fip‐gsi gene from G. sinense in Coprinopsis cinerea to increase yield of FIPs‐gsi. Methods and Results: A fungal expression vector pBfip‐gsi containing the gpd promoter from Agaricus bisporus and the fip‐gsi gene from the G. sinense was constructed and transformed into C. cinerea. PCR and Southern blotting analysis verified the successful integration of the exogenous gene fip‐gsi into the genome of C. cinerea. RT‐PCR and Northern blotting analysis confirmed that the fip‐gsi gene was transcribed in C. cinerea. The yield of the FIP‐gsi protein reached 314 mg kg^?1 fresh mycelia. The molecular weight of the FIP‐gsi was 13 kDa, and the FIP‐gsi was capable of hemagglutinating mouse red blood cells, but no such activity was observed towards human red blood cells in vitro. Conclusions: The fip‐gsi from G. sinense has been successfully translated in C. cinerea, and the yield of bioactive FIP‐gsi protein was high. Significance and Impact of the Study: This is the first report using the C. cinerea for the heterologous expression of FIP‐gsi protein and it might supply a basis for large‐scale production of the protein.     

Acoustic modulation of neural activity in the preoptic area and ventral hypothalamus of the green treefrog (Hyla cinerea)

Summary Responses of neurons in the preoptic area and ventral hypothalamus to conspecific mating calls or white noise bursts were examined in male green treefrogs (Hyla cinerea) during different seasons. In the winter, 34.3% of preoptic neurons and 46.7% of ventral hypothalamic cells demonstrated significant changes in activity level during presentation of a conspecific mating call. In contrast, only 13.3% of preoptic units and 16.7% of ventral hypothalamic cells responded to the white noise. The percentage of preoptic and hypothalamic units responding to the advertisement call did not differ significantly during the summer breeding season. Type I units exhibited a dramatic increase in activity during acoustic stimulation followed by a rapid return to baseline activity levels after stimulus offset. Type II cells showed a robust activity increase during stimulation, but maintained an intermediate activity level after stimulus offset. In the preoptic area, a third response type exhibited suppressed activity during acoustic stimulation. Although seasonal condition did not alter the percentage of acoustically responsive units within either nucleus, the proportion of Type I units in the ventral hypothalamus was greatest during the summer.Abbreviations MC mating call - NS no stimulus - POA preoptic area - VH ventral hypothalamus - WN white noise     

Nxf1 Natural Variant E610G Is a Semi-dominant Suppressor of IAP-Induced RNA Processing Defects

Endogenous retroviruses and retrotransposons contribute functional genetic variation in animal genomes. In mice, Intracisternal A Particles (IAPs) are a frequent source of both new mutations and polymorphism across laboratory strains. Intronic IAPs can induce alternative RNA processing choices, including alternative splicing. We previously showed IAP I∆1 subfamily insertional mutations are suppressed by a wild-derived allele of the major mRNA export factor, Nxf1. Here we show that a wider diversity of IAP insertions present in the mouse reference sequence induce insertion-dependent alternative processing that is suppressed by Nxf1^CAST alleles. These insertions typically show more modest gene expression changes than de novo mutations, suggesting selection or attenuation. Genome-wide splicing-sensitive microarrays and gene-focused assays confirm specificity of Nxf1 genetic modifier activity for IAP insertion alleles. Strikingly, CRISPR/Cas9-mediated genome editing demonstrates that a single amino acid substitution in Nxf1, E610G, is sufficient to recreate a quantitative genetic modifier in a co-isogenic background.     

Brief in vitro study on Botrytis cinerea and Aspergillus carbonarius regarding growth and ochratoxin A

Aims: To evaluate the effect of Botrytis cinerea growth on ochratoxin A (OTA) production by Aspergillus carbonarius and degradation. Methods and Results: OTA‐producing A. carbonarius and B. cinerea were grown on grape‐like medium at 20°C for 7 days. Radii of colonies were daily recorded and OTA was analysed. In addition, each B. cinerea isolate was inoculated on grape‐like synthetic nutrient medium (SNM) paired with each A. carbonarius isolate at a distance of 45 mm. Botrytis cinerea isolates were also grown in OTA‐spiked SNM. Growth rates of B. cinerea and A. carbonarius were 20 and 7·5 mm day^?1, respectively. The growth of the colonies of each species stopped when they contacted each other in paired cultures. OTA production by A. carbonarius in the contact area was affected by B. cinerea, but no clear trend was observed. All B. cinerea isolates showed to degrade between 24·2% and 26·7% of OTA from spiked SNM. Conclusions: The ecological advantage of B. cinerea, in terms of growth rate, vs. OTA‐producing Aspergillus in some wine‐growing regions and its ability to degrade OTA may explain the low levels of this toxin in noble wines. Significance and Impact of the Study: At determinate conditions, the presence of B. cinerea in grapes with A. carbonarius may help in reducing OTA accumulation.     

Macromolecular Toxins Secreted by <Emphasis Type="Italic">Botrytis cinerea</Emphasis> Induce Programmed Cell Death in Arabidopsis Leaves

Botrytis cinerea causes grey mold disease in crops and horticultural plants. It is suspected to kill plant cells via secreted toxins and to derive nutrients from dead or dying cells. However, whether macromolecular phytotoxins (MPs) secreted by B. cinerea induce necrosis or also trigger a programmed cell death (PCD) remains to be determined. We have previously partially characterized MPs secreted by B. cinerea. Here we isolated MPs from B. cinerea culture and applied them to leaf cells, assessing PCD over the following 120 h. Cell death was assessed by propidium iodide (PI) and 4′,6-diamidino-2-phenylindole (DAPI) staining. Catalase (CAT), peroxidase (POD) activity and the cytochrome c/a ratio were assessed by spectrophotometer. POD isomers were measured using the benzidine acetate method. In Arabidopsis thaliana (L.) Heynh. exposed to B. cinerea MPs, we observed chromatin condensation and marginalization, nuclear substance leakage and accumulation of autofluorescent materials in the cell wall. Furthermore, B. cinerea MPs induced release of cytochrome c from the mitochondria into the cytosol. Moreover, CAT and POD activity was upregulated and the POD isoenzyme pattern was altered. In conclusion, A. thaliana exposed to B. cinerea MPs exhibits multiple hallmarks of PCD, suggesting that B. cinerea induces PCD in host cells through secreted macromolecules.     

Inhibitor of Apoptosis Proteins Physically Interact with and Block Apoptosis Induced by Drosophila Proteins HID and GRIM

Reaper (RPR), HID, and GRIM activate apoptosis in cells programmed to die during Drosophila development. We have previously shown that transient overexpression of RPR in the lepidopteran SF-21 cell line induces apoptosis and that members of the inhibitor of apoptosis (IAP) family of antiapoptotic proteins can inhibit RPR-induced apoptosis and physically interact with RPR through their BIR motifs (D. Vucic, W. J. Kaiser, A. J. Harvey, and L. K. Miller, Proc. Natl. Acad. Sci. USA 94:10183–10188, 1997). In this study, we found that transient overexpression of HID and GRIM also induced apoptosis in the SF-21 cell line. Baculovirus and Drosophila IAPs blocked HID- and GRIM-induced apoptosis and also physically interacted with them through the BIR motifs of the IAPs. The region of sequence similarity shared by RPR, HID, and GRIM, the N-terminal 14 amino acids of each protein, was required for the induction of apoptosis by HID and its binding to IAPs. When stably overexpressed by fusion to an unrelated, nonapoptotic polypeptide, the N-terminal 37 amino acids of HID and GRIM were sufficient to induce apoptosis and confer IAP binding activity. However, GRIM was more complex than HID since the C-terminal 124 amino acids of GRIM retained apoptosis-inducing and IAP binding activity, suggesting the presence of two independent apoptotic motifs within GRIM. Coexpression of IAPs with HID stabilized HID levels and resulted in the accumulation of HID in punctate perinuclear locations which coincided with IAP localization. The physical interaction of IAPs with RPR, HID, and GRIM provides a common molecular mechanism for IAP inhibition of these Drosophila proapoptotic proteins.