Roles and Programming of Arabidopsis ARGONAUTE Proteins during Turnip Mosaic Virus Infection

In eukaryotes, ARGONAUTE proteins (AGOs) associate with microRNAs (miRNAs), short interfering RNAs (siRNAs), and other classes of small RNAs to regulate target RNA or target loci. Viral infection in plants induces a potent and highly specific antiviral RNA silencing response characterized by the formation of virus-derived siRNAs. Arabidopsis thaliana has ten AGO genes of which AGO1, AGO2, and AGO7 have been shown to play roles in antiviral defense. A genetic analysis was used to identify and characterize the roles of AGO proteins in antiviral defense against Turnip mosaic virus (TuMV) in Arabidopsis. AGO1, AGO2 and AGO10 promoted anti-TuMV defense in a modular way in various organs, with AGO2 providing a prominent antiviral role in leaves. AGO5, AGO7 and AGO10 had minor effects in leaves. AGO1 and AGO10 had overlapping antiviral functions in inflorescence tissues after systemic movement of the virus, although the roles of AGO1 and AGO10 accounted for only a minor amount of the overall antiviral activity. By combining AGO protein immunoprecipitation with high-throughput sequencing of associated small RNAs, AGO2, AGO10, and to a lesser extent AGO1 were shown to associate with siRNAs derived from silencing suppressor (HC-Pro)-deficient TuMV-AS9, but not with siRNAs derived from wild-type TuMV. Co-immunoprecipitation and small RNA sequencing revealed that viral siRNAs broadly associated with wild-type HC-Pro during TuMV infection. These results support the hypothesis that suppression of antiviral silencing during TuMV infection, at least in part, occurs through sequestration of virus-derived siRNAs away from antiviral AGO proteins by HC-Pro. These findings indicate that distinct AGO proteins function as antiviral modules, and provide a molecular explanation for the silencing suppressor activity of HC-Pro.     

Ubiquitous urease affects soybean susceptibility to fungi

The soybean ubiquitous urease (encoded by GmEu4) is responsible for recycling metabolically derived urea. Additional biological roles have been demonstrated for plant ureases, notably in toxicity to other organisms. However, urease enzymatic activity is not related to its toxicity. The role of GmEu4 in soybean susceptibility to fungi was investigated in this study. A differential expression pattern of GmEu4 was observed in susceptible and resistant genotypes of soybeans over the course of a Phakopsora pachyrhizi infection, especially 24 h after infection. Twenty-nine adult, transgenic soybean plants, representing six independently transformed lines, were obtained. Although the initial aim of this study was to overexpress GmEu4, the transgenic plants exhibited GmEu4 co-suppression and decreased ureolytic activity. The growth of Rhizoctonia solani, Phomopsis sp., and Penicillium herguei in media containing a crude protein extract from either transgenic or non-transgenic leaves was evaluated. The fungal growth was higher in the protein extracts from transgenic urease-deprived plants than in extracts from non-transgenic controls. When infected by P. pachyrhizi uredospores, detached leaves of urease-deprived plants developed a significantly higher number of lesions, pustules and erupted pustules than leaves of non-transgenic plants containing normal levels of the enzyme. The results of the present work show that the soybean plants were more susceptible to fungi in the absence of urease. It was not possible to overexpress active GmEu4. For future work, overexpression of urease fungitoxic peptides could be attempted as an alternative approach.     

Model peptides mimic the structure and function of the N-terminus of the pore-forming toxin sticholysin II

To investigate the role of the N-terminal region in the lytic mechanism of the pore-forming toxin sticholysin II (St II), we studied the conformational and functional properties of peptides encompassing the first 30 residues of the protein. Peptides containing residues 1-30 (P1-30) and 11-30 (P11-30) were synthesized and their conformational properties were examined in aqueous solution as a function of peptide concentration, pH, ionic strength, and addition of the secondary structure-inducing solvent trifluoroethanol (TFE). CD spectra showed that increasing concentration, pH, and ionic strength led to aggregation of P1-30; as a consequence, the peptide acquired beta-sheet conformation. In contrast, P11-30 exhibited practically no conformational changes under the same conditions, remaining essentially structureless. Moreover, this peptide did not undergo aggregation. These differences clearly point to the modulating effect of the first 10 hydrophobic residues on the peptides aggregation and conformational properties. In TFE both the first ten hydrophobic peptides acquired alpha-helical conformation, albeit to a different extent, P11-30 displayed lower alpha-helical content. P1-30 presented a larger fraction of residues in alpha-helical conformation in TFE than that found in St II's crystal structure for that portion of the protein. Since TFE mimics the membrane environment, such increase in helical content could also occur upon toxin binding to membranes and represent a step in the mechanism of pore formation. The peptides conformational properties correlated well with their functional behavior. Thus, P1-30 exhibited much higher hemolytic activity than P11-30. In addition, P11-30 was able to block the toxin's hemolytic activity. The size of pores formed in red blood cells by P1-30 was estimated by measuring the permeability to PEGs of different molecular mass. The pore radius (0.95 +/- 0.01 nm) was very similar to that of the pore formed by the toxin. The results demonstrate that the synthetic peptide P1-30 is a good model of St II conformation and function and emphasize the contribution of the toxin's N-terminal region, and, in particular, the hydrophobic residues 1-10 to pore formation.     

Chemical Deterrence of a Marine Cyanobacterium against Sympatric and Non-sympatric Consumers

This study investigates the influence of mesograzer prior exposure to toxic metabolites on palatability of the marine cyanobacterium, Lyngbya majuscula. We examined the palatability of L. majuscula crude extract obtained from a bloom in Moreton Bay, South East Queensland, Australia, containing lyngbyatoxin-a (LTA) and debromoaplysiatoxin (DAT), to two groups: (1) mesograzers of L. majuscula from Guam where LTA and DAT production is rare; and (2) macro- and mesograzers found feeding on L. majuscula blooms in Moreton Bay where LTA and DAT are often prevalent secondary metabolites. Pair-wise feeding assays using artificial diets consisting of Ulva clathrata suspended in agar (control) or coated with Moreton Bay L. majuscula crude extracts (treatment) were used to determine palatability to a variety of consumers. In Guam, the amphipods, Parhyale hawaiensis and Cymadusa imbroglio; the majid crab Menaethius monoceros; and the urchin Echinometra mathaei were significantly deterred by the Moreton Bay crude extract. The sea hares, Stylocheilus striatus, from Guam were stimulated to feed by treatment food whereas S. striatus collected from Moreton Bay showed no discrimination between food types. In Moreton Bay, the cephalaspidean Diniatys dentifer and wild caught rabbitfish Siganus fuscescens were significantly deterred by the crude extract. However, captive-bred S. fuscescens with no known experience with L. majuscula did not clearly discriminate between food choices. Lyngbya majuscula crude extract deters feeding by most mesograzers regardless of prior contact or association with blooms.     

Short-term consequences of slash-and-burn practices on the arbuscular mycorrhizal fungi of a tropical dry forest

Rates of land conversion from forest to cultivated land by slash-and-burn practices are higher in tropical dry forest (TDF) than any other Neotropical forest type. This study examined the short-term consequences of the slash-and-burn process on arbuscular mycorrhizal fungi (AMF). We expected that slash-and-burn would reduce mycorrhizal colonization and propagules and change species richness and composition. Soil and root samples were taken from TDF control and pasture plots originated after slash-and-burn at four dates during the year of conversion to examine species composition, spore abundance, and infective propagules. Additionally, spore abundance and viability and viable intraradical colonization were measured twice during the second year after conversion. Forest and pasture plots maintained similar species richness and an overall 84% similarity during the first year after conversion. Infective propagules were reduced in pasture plots during the first year after slash-and-burn, whereas spore abundance and intraradical colonization remained similar in TDF and pasture plots both years of the study. Our results suggest, contrary to the expected, that forest conversion by means of slash-and-burn followed by cultivation resulted in few immediate changes in the AMF communities, likely because of the low heat conductivity of the soil and rapid combustion of plant residues.     

Effects of gonadotropin-exposed medium with high concentrations of progesterone and estradiol-17β on in vitro maturation of canine oocytes

During the process of maturation in the oviduct, canine oocytes in the germinal vesicle stage are exposed to decreasing levels of estradiol-17β and increasing levels of progesterone. However, hormone concentrations in the microenvironments in which they act are higher than serum concentrations. Therefore, the aim of the present study was to compare the meiotic competence of canine oocytes harvested from anestrous bitches in culture medium containing high concentrations (20 μg ml⁻¹) of estradiol-17β and/or progesterone in association to gonadotropins (luteinizing hormone and follicle-stimulating hormone) using three different maturation periods (48, 72, and 96 h). Oocytes were cultured in tissue culture medium (TCM-199) and arranged in four experimental groups: group control, group E2 (estradiol-17β), group P4 (progesterone), and group E2 + P4. Regardless of the maturation period, groups P4 and E2 + P4 presented statistically higher rate of germinal vesicle breakdown oocytes compared to the group control and group E2. There were no significant differences among groups on germinal vesicle, metaphase I, metaphase II, and degenerated or unidentifiable oocytes rates. The mean percentage of metaphase II oocytes was higher at 96 h when compared to 72 h. Results of the present research indicate no influence of estradiol-17β supplementation, unless in association with progesterone. There is an evidence of the positive effect of progesterone on germinal vesicle breakdown. Results also showed that extended periods of in vitro maturation affect positively maturation rates to metaphase II of low competent oocytes harvested from anestrous bitches, independent of the maturation media. In conclusion, high concentrations of steroids, especially progesterone, have positive effect on in vitro oocyte maturation when the oocytes are derived from the anestrous status.     

The E3 Ubiquitin Ligase Triad3A Negatively Regulates the RIG-I/MAVS Signaling Pathway by Targeting TRAF3 for Degradation

The primary role of the innate immune response is to limit the spread of infectious pathogens, with activation of Toll-like receptor (TLR) and RIG-like receptor (RLR) pathways resulting in a pro-inflammatory response required to combat infection. Limiting the activation of these signaling pathways is likewise essential to prevent tissue injury in the host. Triad3A is an E3 ubiquitin ligase that interacts with several components of TLR signaling and modulates TLR activity. In the present study, we demonstrate that Triad3A negatively regulates the RIG-I RNA sensing pathway through Lys⁴⁸-linked, ubiquitin-mediated degradation of the tumor necrosis factor receptor-associated factor 3 (TRAF3) adapter. Triad3A was induced following dsRNA exposure or virus infection and decreased TRAF3 levels in a dose-dependent manner; moreover, Triad3A expression blocked IRF-3 activation by Ser-396 phosphorylation and inhibited the expression of type 1 interferon and antiviral genes. Lys⁴⁸-linked ubiquitination of TRAF3 by Triad3A increased TRAF3 turnover, whereas reduction of Triad3A expression by stable shRNA expression correlated with an increase in TRAF3 protein expression and enhancement of the antiviral response following VSV or Sendai virus infection. Triad3A and TRAF3 physically interacted together, and TRAF3 residues Y440 and Q442—previously shown to be important for association with the MAVS adapter—were also critical for Triad3A. Point mutation of the TRAF-Interacting-Motif (TIM) of Triad3A abrogated its ability to interact with TRAF3 and modulate RIG-I signaling. TRAF3 appears to undergo sequential ubiquitin “immuno-editing” following virus infection that is crucial for regulation of RIG-I-dependent signaling to the antiviral response. Thus, Triad3A represents a versatile E3 ubiquitin ligase that negatively regulates RIG-like receptor signaling by targeting TRAF3 for degradation following RNA virus infection.     

Evolution of culture broth rheological properties during propagation of the entomopathogenic nematode Steinernema carpocapsae,in submerged monoxenic culture

This article presents the evolution of culture broth rheological properties during monoxenic cultures of Steinernema carpocapsae in cylindrical bottles agitated orbitally. Rheological properties were evaluated in simple-shear flow conditions and were well-modeled by the Ostwald-de Waele model. Rheological properties varied from slightly dilatant, n = 1.2 (-), to moderately pseudoplastic flow behavior, n = 0.6 (-). Nematode concentrations increased from 750 +/- 190 to 130 900 +/- 6900 nematodes/mL, and the apparent viscosity (eta(a)) evolved from 4.5 +/- 0.7 to 46.6 +/- 3.2 mPa.s during the fermentations. Rheological behavior did not appear to be strongly influenced by nematode number and/or its stage of development; however, the release of substances from the decomposition of nematode cadavers appeared to be of great importance. Among the different developmental stages of the nematodes, only juveniles of the first stage (J1) were highly susceptible to the shearing conditions tested (shear stress, tau(r)()(theta), from 0.9 to 3.5 Pa during periods of 80-100 min), resulting in the viability loss of 85% of J1 nematodes.