Revisiting the use of Iprodione and Trichoderma in the integrated management of onion white rot

The biocontrol properties of Trichoderma species are well documented, but their effectiveness in antagonism of the problematic Sclerotium cepivorum, the causal agent of white rot in Allium species, appears limited with reports of significant control only relating to deliberately-mutated strains of Trichoderma. Our previous studies have indicated the possibility of using selected naturally-occurring strains of the antagonist in the suppression of other diseases; now in vitro and controlled environment in vivo studies have indicated that a degree of control of Onion White Rot is possible, and that the selected antagonist strains can be used in integrated treatments with Iprodione to good effect. The possible value of such treatments is considered in light of other approaches to the suppression of this continuing problem.     

Molybdate inhibits hsp90, induces structural changes in its C-terminal domain, and alters its interactions with substrates

To examine the biochemical mechanism by which hsp90 exerts its essential positive function on certain signal transduction proteins, we characterized the effects of molybdate and geldanamycin on hsp90 function and structure. Molybdate inhibited hsp90-mediated p56lck biogenesis and luciferase renaturation while enforcing salt-stable interactions with these substrates. Molybdate also reduced the amount of free hsp90 present in cell lysates, inhibited hsp90's ability to bind geldanamycin, and induced resistance to proteolysis at a specific region within the C-terminal domain of hsp90. In contrast, the hsp90 inhibitor geldanamycin prevented hsp90 from assuming natural or molybdate-induced conformations that allow salt-stable interactions with substrates. When these compounds were applied sequentially, the order of addition determined the effects observed, indicating that these agents had opposing effects on hsp90. We conclude that a specific region within the C-terminal domain of hsp90 (near residue 600) determines the mode by which hsp90 interacts with substrates and that the ability of hsp90 to cycle between alternative modes of interaction is obligatory for hsp90 function.     

CD40, but not CD154, expression on B cells is necessary for optimal primary B cell responses

CD40 is an important costimulatory molecule for B cells as well as dendritic cells, monocytes, and other APCs. The ligand for CD40, CD154, is expressed on activated T cells, NK cells, mast cells, basophils, and even activated B cells. Although both CD40(-/-) and CD154(-/-) mice have impaired ability to isotype switch, form germinal centers, make memory B cells, and produce Ab, it is not entirely clear whether these defects are intrinsic to B cells, to other APCs, or to T cells. Using bone marrow chimeric mice, we investigated whether CD40 or CD154 must be expressed on B cells for optimal B cell responses in vivo. We demonstrate that CD40 expression on B cells is required for the generation of germinal centers, isotype switching, and sustained Ab production, even when other APCs express CD40. In contrast, the expression of CD154 on B cells is not required for the generation of germinal centers, isotype switching, or sustained Ab production. In fact, B cell responses are completely normal when CD154 expression is limited exclusively to Ag-specific T cells. These results suggest that the interaction of CD154 expressed by activated CD4 T cells with CD40 expressed by B cells is the primary pathway necessary to achieve B cell activation and differentiation and that CD154 expression on B cells does not noticeably facilitate B cell activation and differentiation.     

Glycosylation of Skp1 Promotes Formation of Skp1–Cullin-1–F-box Protein Complexes in Dictyostelium

O₂ sensing in diverse protozoa depends on the prolyl 4 hydroxylation of Skp1 and modification of the resulting hydroxyproline with a series of five sugars. In yeast, plants, and animals, Skp1 is associated with F-box proteins. The Skp1–F-box protein heterodimer can, for many F-box proteins, dock onto cullin-1 en route to assembly of the Skp1–cullin-1–F-box protein–Rbx1 subcomplex of E3^SCFUb ligases. E3^SCFUb ligases conjugate Lys48-polyubiquitin chains onto targets bound to the substrate receptor domains of F-box proteins, preparing them for recognition by the 26S proteasome. In the social amoeba Dictyostelium, we found that O₂ availability was rate-limiting for the hydroxylation of newly synthesized Skp1. To investigate the effect of reduced hydroxylation, we analyzed knockout mutants of the Skp1 prolyl hydroxylase and each of the Skp1 glycosyltransferases. Proteomic analysis of co-immunoprecipitates showed that wild-type cells able to fully glycosylate Skp1 had a greater abundance of an SCF complex containing the cullin-1 homolog CulE and FbxD, a newly described WD40-type F-box protein, than the complexes that predominate in cells defective in Skp1 hydroxylation or glycosylation. Similarly, the previously described FbxA–Skp1CulA complex was also more abundant in glycosylation-competent cells. The CulE interactome also included higher levels of proteasomal regulatory particles when Skp1 was glycosylated, suggesting increased activity consistent with greater association with F-box proteins. Finally, the interactome of FLAG-FbxD was modified when it harbored an F-box mutation that compromised Skp1 binding, consistent with an effect on the abundance of potential substrate proteins. We propose that O₂-dependent posttranslational glycosylation of Skp1 promotes association with F-box proteins and their engagement in functional E3^SCFUb ligases that regulate O₂-dependent developmental progression.Timely protein degradation is a cornerstone of cell cycling and the regulation of numerous physiological and developmental processes. Eukaryotes have evolved an extensive array of polyubiquitination enzymes to tag proteins on a protein-by-protein basis as a recognition marker for degradation in the 26S proteasome. The cullin-RING ubiquitin ligases (CRLs)¹ are a prominent subgroup of these enzymes (1) and consist of an E3 architecture that includes a substrate receptor, an adaptor (in most cases), the cullin scaffold, the RING protein, and an exchangeable E2 ubiquitin donor that has been charged with ubiquitin (Ub) by an E1 enzyme. The first discovered and still prototypic example is the CRL1 class (2), also referred to as SCF on account of the names of its founding subunits, Skp1, cullin-1, and F-box proteins (FBPs). The CRL1 (or SCF) complexes utilize FBPs as substrate receptors, Skp1 as the adaptor linking the FBP to the N-terminal region of cullin-1 (Cul1), and Rbx1 as the RING protein that tethers the E2 Ub donor to the Cul1 C-terminal region (see Fig. 2B). CRL1s can be activated by neddylation of Cul1 by a Nedd8-specific E2, which mobilizes Rbx1 to afford rotational flexibility of the E2 and displaces the inhibitor Cand1, permitting docking of the Skp1–FBP heterodimer (3–5). Deneddylation mediated by the eight-subunit COP9 signalosome is required for in vivo activity, suggesting that Cand1 serves as a substrate exchange factor to allow for re-equilibration of SCF complexes from preexisting subunits. Each reaction cycle requires the exchange of a new E2-Ub and typically assembles a K48-linked polyUb chain that is recognized by the proteasome. Substrate specificity is conferred by FBPs, a gene family that numbers 69 in humans, 20 in budding yeast, 300 in Caenorhabditis elegans, and ∼800 in Arabidopsis. Some characterized FBPs can recognize perhaps a dozen or more substrates, and the coding of recognition and the meaning of their control by the same FBP is under intense investigation (6). Recognition is often activated by posttranslational modification of the substrate (often phosphorylation). Regulation of SCF Ub ligases has centered on the neddylation cycle, which potentially influences all seven known CRLs. Regulation of Skp1, investigated in this paper, would be specific to CRLs possessing Skp1, which include CRL1 and possibly the minor class CRL7 (7).Open in a separate windowFig. 2.Skp1 modification pathway and global analysis of Skp1 interactions. A, Skp1 is sequentially modified by the indicated enzymes (in blue), resulting in the formation of a pentasaccharide at Pro143. B, model of the SCF complex in the context of the overall E3 Ub ligase, from studies in yeast, plants, and animals. Catalysis involves transfer of Ub from an exchangeable Ub-E2 conjugate to the substrate. Removal of Nedd8 by the COP9 signalosome facilitates binding of Cand1 to Cul1, which inhibits binding of Skp1 to Cul1. C, D, vegetative (growth stage) cells were filter-lysed, and a cytosolic fraction prepared via ultracentrifugation was chromatographed on a Superose 12 gel filtration column. Fractions were analyzed via Western blotting (representative examples are shown in C) followed by densitometry (D). The elution position of free Skp1 from a separate trial is indicated.The basic SCF model is thought to be widespread among eukaryotes but has been extensively studied only in fungi/yeasts, plants, and animals. The broad phylogeny represented by protists includes many benign and pathogenic unicellular organisms of great economic, health, and environmental impact. Emerging evidence reveals that Skp1 in some of these groups is subject to a novel form of prolyl 4(trans)-hydroxylation and complex glycosylation (8). The roles of these Skp1 modifications have been most studied in the social amoeba Dictyostelium, which undergoes a starvation-induced developmental program during which individual amoebae chemotactically aggregate into an initial mound that then elongates into a migratory slug. Under appropriate conditions, the slug reorganizes to form a fruiting body consisting of a ball of spores supported by a vertical cellular stalk. The slug-to-fruit switch, referred to as culmination, and sporulation are regulated by checkpoints that are sensitive to multiple factors, including O₂ (9–11). Functional studies of Dictyostelium Skp1 hydroxylation and glycosylation reveal roles in regulating the O₂ dependence of culmination and sporulation (12–14). For example, wild-type (wt) cells require 7% to 10% O₂ and phyA^– requires 18% to 21% O₂ in order to achieve 50% spore formation (a quantitative measure of fruiting body formation), whereas glycosylation mutants exhibit a complex pattern of intermediate requirements (13). In addition, at 21% O₂, phyA^– cells require an additional 3 to 4 h to complete development relative to their wt counterparts (14). In the apicomplexan Toxoplasma gondii, PhyA is also required for Skp1 glycosylation, and phyA^– parasites are deficient in proliferation, especially at low O₂ (15).The idea that O₂ availability is rate limiting for Skp1 modification was originally based on the observation that the Dictyostelium phyA^– phenotype mimics that of wt cells in low O₂ (9). However, the majority of Skp1 is hydroxylated and glycosylated in wt cells even at low O₂ levels where culmination is blocked or delayed. Further analysis of a submerged development model, in which terminal development depended on an atmosphere of 70% to 100% O₂ in order to overcome the diffusion barrier posed by the water layer, showed that at atmospheric O₂ levels of 5% to 21% where sporulation was blocked, unmodified Skp1 accumulated to a higher level than at permissive O₂ levels (10). As Skp1 modifications are thought to be irreversible, this likely resulted from slow hydroxylation of newly synthesized Skp1. To address this in a more physiological setting, we investigated nascent Skp1 directly using metabolic labeling with [³⁵S]Met/Cys and verified that the rate of hydroxylation of newly synthesized Skp1 polypeptide was indeed inversely proportional to O₂ levels, which makes PhyA-mediated hydroxylation of Skp1 an excellent candidate for the primary O₂ sensor for culmination.These modifications of Skp1 are of interest as a novel mechanism regulating the SCF ligase. Previously, we showed that hydroxylation and glycosylation of Dictyostelium Skp1 affect its conformation and promote binding to a soluble FBP, guinea pig Fbs1, in studies of purified proteins (16). Here we show that Dictyostelium Skp1 is indeed a subunit of a canonical SCF complex, as expected. The significance of undermodified Skp1 was examined via interactome analysis of Skp1 isoforms that accumulate in modification pathway mutants. Our findings revealed a lower abundance of SCF complexes than in wt cells, suggesting that Skp1 modification may promote SCF assembly and E3^SCFUb ligase activities that control timely turnover of select proteins involved in developmental progression.     

A spatiotemporal characterization of the effect of p53 phosphorylation on its interaction with MDM2

The interaction of p53 and MDM2 is modulated by the phosphorylation of p53. This mechanism is key to activating p53, yet its molecular determinants are not fully understood. To study the spatiotemporal characteristics of this molecular process we carried out Brownian dynamics simulations of the interactions of the MDM2 protein with a p53 peptide in its wild type state and when phosphorylated at Thr18 (pThr18) and Ser20 (pSer20). We found that p53 phosphorylation results in concerted changes in the topology of the interaction landscape in the diffusively bound encounter complex domain. These changes hinder phosphorylated p53 peptides from binding to MDM2 well before reaching the binding site. The underlying mechanism appears to involve shift of the peptide away from the vicinity of the MDM2 protein, peptide reorientation, and reduction in peptide residence time relative to wild-type p53 peptide. pThr18 and pSr20 p53 peptides experience reduction in residence times by factors of 13.6 and 37.5 respectively relative to the wild-type p53 peptide, indicating a greater role for Ser20 phosphorylation in abrogating p53 MDM2 interactions. These detailed insights into the effect of phosphorylation on molecular interactions are not available from conventional experimental and theoretical approaches and open up new avenues that incorporate molecular interaction dynamics, for stabilizing p53 against MDM2, which is a major focus of anticancer drug lead development.     

A pattern recognition serine proteinase triggers the prophenoloxidase activation cascade in the tobacco hornworm, Manduca sexta

A serine proteinase cascade in insect hemolymph mediates prophenoloxidase activation, a defense mechanism against pathogen or parasite infection. Little is known regarding its initiating proteinase or how this enzyme is activated in response to invading microorganisms. We have isolated from the tobacco hornworm, Manduca sexta, a cDNA encoding a modular protein designated hemolymph proteinase 14 (HP14). It contains five low density lipoprotein receptor class A repeats, a Sushi domain, a unique Cys-rich region, and a proteinase-catalytic domain. The HP14 mRNA exists in fat body and hemocytes of the naive larvae, and its level increases significantly at 24 h after a bacterial challenge. We expressed proHP14 with a carboxyl-terminal hexahistidine tag in a baculovirus/insect cell system and detected the recombinant protein in two forms. The 87-kDa protein was primarily intracellular, whereas the 75-kDa form was present in the medium. Interaction with peptidoglycan resulted in proteolytic processing of the purified zymogen and generation of an amidase activity. Supplementation of hemolymph with proHP14 greatly enhanced prophenoloxidase activation in response to Micrococcus luteus. These data suggest that proHP14 is a pattern recognition protein that binds to bacteria and autoactivates and triggers the prophenoloxidase activation system in the hemolymph of M. sexta.     

A novel role for non-neutralizing antibodies against nucleoprotein in facilitating resistance to influenza virus

Current influenza vaccines elicit Abs to the hemagglutinin and neuraminidase envelope proteins. Due to antigenic drift, these vaccines must be reformulated annually to include the envelope proteins predicted to dominate in the following season. By contrast, vaccination with the conserved nucleoprotein (NP) elicits immunity against multiple serotypes (heterosubtypic immunity). NP vaccination is generally thought to convey protection primarily via CD8 effector mechanisms. However, significant titers of anti-NP Abs are also induced, yet the involvement of Abs in protection has largely been disregarded. To investigate how Ab responses might contribute to heterosubtypic immunity, we vaccinated C57BL/6 mice with soluble rNP. This approach induced high titers of NP-specific serum Ab, but only poorly detectable NP-specific T cell responses. Nevertheless, rNP immunization significantly reduced morbidity and viral titers after influenza challenge. Importantly, Ab-deficient mice were not protected by this vaccination strategy. Furthermore, rNP-immune serum could transfer protection to naive hosts in an Ab-dependent manner. Therefore, Ab to conserved, internal viral proteins, such as NP, provides an unexpected, yet important mechanism of protection against influenza. These results suggest that vaccines designed to elicit optimal heterosubtypic immunity to influenza should promote both Ab and T cell responses to conserved internal proteins.     

Function and structure of lipid storage droplet protein 1 studied in lipoprotein complexes

Triglycerides (TG) stored in lipid droplets (LDs) are the main energy reserve in all animals. The mechanism by which animals mobilize TG is complex and not fully understood. Several proteins surrounding the LDs have been implicated in TG homeostasis such as mammalian perilipin A and insect lipid storage proteins (Lsd). Most of the knowledge on LD-associated proteins comes from studies using cells or LDs leaving biochemical properties of these proteins uncharacterized. Here we describe the purification of recombinant Lsd1 and its reconstitution with lipids to form lipoprotein complexes suitable for functional and structural studies. Lsd1 in the lipid bound state is a predominately α-helical protein. Using lipoprotein complexes containing triolein it is shown that PKA mediated phosphorylation of Lsd1 promoted a 1.7-fold activation of the main fat body lipase demonstrating the direct link between Lsd1 phosphorylation and activation of lipolysis. Serine 20 was identified as the Lsd1-phosphorylation site triggering this effect.     

Cutting edge: organogenesis of nasal-associated lymphoid tissue (NALT) occurs independently of lymphotoxin-alpha (LT alpha) and retinoic acid receptor-related orphan receptor-gamma, but the organization of NALT is LT alpha dependent.

Peyer's patch and nasal-associated lymphoid tissue (NALT) are mucosal lymphoid tissues that appear similar in structure and function. Surprisingly, we found that NALT, unlike Peyer's patch, was formed independently of lymphotoxin (LT)alpha. Furthermore, using mice deficient in the retinoic acid receptor-related orphan receptor-gamma, we found that NALT was formed in the absence of CD4+CD3- cells, which are thought to be the embryonic source of LTalpha. However, we also found that NALT of LTalpha-/- animals was disorganized and lymphopenic, suggesting that the organization and recruitment of lymphocytes within NALT remained dependent on LTalpha. Finally, we demonstrated that both the structure and function of NALT were restored in LTalpha-/- animals upon reconstitution with normal bone marrow. These results demonstrate that the organogenesis of NALT occurs through unique mechanisms.     

Ground wētā in vines of the Awatere Valley,Marlborough: biology,density and distribution

The endemic New Zealand ground wētā (Hemiandrus sp. ‘promontorius’) has a Naturally Uncommon conservation status. This is because of the paucity of information on its density and distribution. Here, the biology, density and distribution of a population of this wētā found in and around vineyards in the Awatere Valley, Marlborough was studied. Wētā density was assessed in vineyards, paddocks and shrublands in this valley. Soil moisture, penetration resistance, pH and organic matter were recorded at locations with and without wētā. Wētā density in vineyards was significantly higher than in either paddocks or shrub habitats. In vineyards, the density of this insect was significantly higher under-vines than in the inter-rows. Higher numbers of this wētā were found in moist soils that required lower force to burrow. Females laid an average of 55 eggs between March and April, which hatched in September. These findings highlight the intersection between agriculture and conservation.