Iso-lines and inbred-lines confirmed loci that underlie resistance from cultivar ‘Hartwig’ to three soybean cyst nematode populations

Soybean [Glycine max (L.) Merr.] cultivars varied in their resistance to different populations of the soybean cyst nematode (SCN), Heterodera glycines, called HG Types. The rhg1 locus on linkage group G was necessary for resistance to all HG types. However, the loci for resistance to H. glycines HG Type 1.3- (race 14) and HG Type 1.2.5- (race 2) of the soybean cyst nematode have varied in their reported locations. The aims were to compare the inheritance of resistance to three nematode HG Types in a population segregating for resistance to SCN and to identify the underlying quantitative trait loci (QTL). ‘Hartwig’, a soybean cultivar resistant to most SCN HG Types, was crossed with the susceptible cultivar ‘Flyer’. A total of 92 F5-derived recombinant inbred lines (RILs; or inbred lines) and 144 molecular markers were used for map development. The rhg1 associated QTL found in earlier studies were confirmed and shown to underlie resistance to all three HG Types in RILs (Satt309; HG Type 0, P = 0.0001 R ² = 22%; Satt275; HG Type 1.3, P = 0.001, R ² = 14%) and near isogeneic lines (NILs; or iso-lines; Satt309; HG Type 1.2.5-, P = 0.001 R ² = 24%). A new QTL underlying resistance to HG Type 1.2.5- was detected on LG D2 (Satt574; P = 0.001, R ² = 11%) among 14 RILs resistant to the other HG types. The locus was confirmed in a small NIL population consisting of 60 plants of ten genotypes (P = 0.04). This QTL (cqSCN-005) is located in an interval previously associated with resistance to both SDS leaf scorch from ‘Pyramid’ and ‘Ripley’ (cqSDS-001) and SCN HG Type 1.3- from Hartwig and Pyramid. The QTL detected will allow marker assisted selection for multigenic resistance to complex nematode populations in combination with sudden death syndrome resistance (SDS) and other agronomic traits.     

Conformational Changes in the Capsid of a Calicivirus upon Interaction with Its Functional Receptor

Nonenveloped viral capsids are metastable structures that undergo conformational changes during virus entry that lead to interactions of the capsid or capsid fragments with the cell membrane. For members of the Caliciviridae, neither the nature of these structural changes in the capsid nor the factor(s) responsible for inducing these changes is known. Feline junctional adhesion molecule A (fJAM-A) mediates the attachment and infectious viral entry of feline calicivirus (FCV). Here, we show that the infectivity of some FCV isolates is neutralized following incubation with the soluble receptor at 37°C. We used this property to select mutants resistant to preincubation with the soluble receptor. We isolated and sequenced 24 soluble receptor-resistant (srr) mutants and characterized the growth properties and receptor-binding activities of eight mutants. The location of the mutations within the capsid structure of FCV was mapped using a new 3.6-Å structure of native FCV. The srr mutations mapped to the surface of the P2 domain were buried at the protruding domain dimer interface or were present in inaccessible regions of the capsid protein. Coupled with data showing that both the parental FCV and the srr mutants underwent increases in hydrophobicity upon incubation with the soluble receptor at 37°C, these findings indicate that FCV likely undergoes conformational change upon interaction with its receptor. Changes in FCV capsid conformation following its interaction with fJAM-A may be important for subsequent interactions of the capsid with cellular membranes, membrane penetration, and genome delivery.The interactions between viruses and receptors on the surface of host cells strongly influence viral pathogenesis and regulate morbidity and mortality in the host. Virus-receptor interactions determine the types of cells that can be infected, the pathway of entry into the cell, and the efficiency of productive infection. Interactions between nonenveloped virus capsids and their receptor(s) trigger one or more steps required for infectious entry. These steps can include interaction with other receptors, exposure to low pH or endosomal proteases, or other factors. Ultimately, one or more of these interactions induce changes in capsid conformation that result in the exposure of hydrophobic regions or release of a lipid-seeking factor that can interact with and disrupt the limiting cellular membrane to allow the capsid and/or the genome to be delivered to the interior of the cell (reviewed in reference 60).The Caliciviridae are small nonenveloped viruses containing a positive-sense RNA genome (∼7 to 8 kb). Several important disease-causing members of the Caliciviridae, including human noroviruses and rabbit hemorrhagic disease virus, cannot be propagated in tissue culture systems (19, 56). This has slowed progress on studies of the mechanisms of cellular entry of these viruses. In contrast, feline caliciviruses (FCVs) propagate readily in tissue culture, and two cell surface receptor molecules, feline junctional adhesion molecule A (fJAM-A) and α2,6 sialic acid, are known (29, 55).The FCV receptor, fJAM-A, is a type I transmembrane glycoprotein (molecular size of 36 to 41 kDa) member of the immunoglobulin superfamily (IgSF); it consists of an amino-terminal signal peptide, an extracellular domain (composed of two Ig-like domains—a membrane-distal D1 and a membrane-proximal D2), a transmembrane domain, and a short cytoplasmic domain that contains a type II PDZ domain-binding motif (11, 30). We have previously shown that the D1 domain of the fJAM-A ectodomain is necessary and sufficient for FCV binding and that preincubation of FCV with soluble fJAM-A (sfJAM-A) results in virus neutralization (35). Additional roles that fJAM-A might play in FCV entry, however, have not been investigated.Caliciviruses are composed of 180 copies of a single capsid protein. Atomic resolution structures of recombinant virus-like particles of Norwalk virus (genus Norovirus) and native San Miguel sea lion virus (SMSV) virions (genus Vesivirus) indicate that the virion consists of 90 dimers of the capsid protein arranged in T=3 icosahedral symmetry (5, 41). Each capsid monomer contains three structural domains—an N-terminal arm (NTA), the shell (S), and a protruding domain (P) that is further subdivided into P1 and P2 subdomains. The distal subdomain, P2, is structurally conserved between Norwalk virus and SMSV, but there is little sequence conservation. In the primary sequence of the FCV capsid, there are two hypervariable regions that contain neutralizing epitopes (18, 34, 58). The corresponding hypervariable regions (HVRs) of the SMSV capsid structure map to surface-exposed loops. Surface residues at the dimeric interface between two capsid monomers are conserved within individual calicivirus genera, and it has been suggested that this interface is involved in receptor binding (5). A cryo-electron microscopy (cryo-EM) reconstruction of the FCV vaccine strain F9 complexed with the ectodomain of fJAM-A (modeled on the crystal structures of SMSV and human JAM-A, respectively) shows that fJAM-A engages the top of the P2 domain and that binding causes a rotation in the P dimer (1). However, the relatively low resolution and the lack of atomic resolution structures of FCV and fJAM-A prevented precise identification of residues on the viral capsid that contact fJAM-A.A classical approach for identifying virus residues that directly bind or modulate the binding of a receptor is to select for mutant viruses resistant to neutralization with soluble receptors (6, 23, 46). Soluble receptor-resistant (srr) mutants of poliovirus revealed that both surface-exposed and internal residues regulate receptor attachment and conformational changes in the capsid (6, 42). Here, we report 24 srr mutants and the location of their capsid mutations on a 3.6-Å structure of FCV. In addition, we describe the growth kinetics and receptor-binding properties of a subpanel of eight srr mutants and examine changes in capsid hydrophobicity concurrent with the interaction of FCV capsids with sfJAM-A.     

Identification of novel α7 nAChR positive allosteric modulators with the use of pharmacophore in silico screening methods

The pharmacophore model of in house potent and selective α7 nAChR positive allosteric modulators is reported. The model was used to fish out commercially-available compounds from corporate 3D databases. As a result, novel α7 positive modulator chemotypes were identified. A rat full PK profile of a representative compound is also described.     

High temperature effects on photosynthate partitioning and sugar metabolism during ear expansion in maize (Zea mays L.) genotypes

Short hot and dry spells before, or during, silking have an inordinately large effect on maize (Zea mays L.; corn) grain yield. New high yielding genotypes could be developed if the mechanism of yield loss were more fully understood and new assays developed. The aim here was to determine the effects of high temperature (35/27 °C) compared to cooler (25/18 °C) temperatures (day/night). Stress was applied for a 14 d-period during reproductive stages prior to silking. Effects on whole plant biomass, ear development, photosynthesis and carbohydrate metabolism were measured in both dent and sweet corn genotypes. Results showed that the whole plant biomass was increased by the high temperature. However, the response varied among plant parts; in leaves and culms weights were slightly increased or stable; cob weights decreased; and other ear parts of dent corn also decreased by high temperature. Photosynthetic activity was not affected by the treatments. The ¹³C export rate from an ear leaf was decreased by the high temperature treatment. The amount of ¹³C partitioning to the ears decreased more than to other plant parts by the high temperature. Within the ear decreases were greatest in the cob than the shank within an ear. Sugar concentrations in both hemicellulose and cellulose fractions of cobs in sweet corn were decreased by high temperature, and the hemicellulose fraction in the shank also decreased. In dent corn there was no reduction of sugar concentration except in the in cellulose fraction, suggesting that synthesis of cell-wall components is impaired by high temperatures. The high temperature treatment promoted the growth of vegetative plant parts but reduced ear expansion, particularly suppression of cob extensibility by impairing hemicellulose and cellulose synthesis through reduction of photosynthate supply. Therefore, plant biomass production was enhanced and grain yield reduced by the high temperature treatment due to effects on sink activity rather than source activity. Heat resistant ear development can be targeted for genetic improvement     

Genetic and physical localization of the soybean Rpg1-b disease resistance gene reveals a complex locus containing several tightly linked families of NBS-LRR genes

Alleles or tightly linked genes at the soybean (Glycine max L. Merr.) Rpg1 locus confer resistance to strains of Pseudomonas syringae pv. glycinea that express the avirulence genes avrB or avrRpm1. We have previously mapped Rpg1-b (the gene specific for avrB) to a cluster of resistance genes (R genes) with diverse specificities in molecular linkage group F. Here, we describe the high-resolution physical and genetic mapping of Rpg1-b to a 0.16-cM interval encompassed by two overlapping BAC clones spanning approximately 270 kilobases. Rpg1-b is part of a complex locus containing numerous genes related to previously characterized coiled coil-nucleotide binding site-leucine rich repeat (CC-NBS-LRR)-type R genes that are spread throughout this region. Phylogenetic and Southern blot analyses group these genes into four distinct subgroups, some of which are conserved in the common bean, Phaseolus vulgaris, indicating that this R gene cluster may predate the divergence of Phaseolus and Glycine. Members from different subgroups are physically intermixed and display a high level of polymorphism between soybean cultivars, suggesting that this region is rearranging at a high frequency. At least five CC-NBS-LRR-type genes cosegregate with Rpg1-b in our large mapping populations.     

Fanconi anemia group C protein prevents apoptosis in hematopoietic cells through redox regulation of GSTP1.

The Fanconi anemia group C protein (FANCC) plays an important role in hematopoiesis by ensuring the survival of hematopoietic progenitor cells through an unknown mechanism. We investigated the function of FANCC by identifying FANCC-binding proteins in hematopoietic cells. Here we show that glutathione S-transferase P1-1 (GSTP1) interacts with FANCC, and that overexpression of both proteins in a myeloid progenitor cell line prevents apoptosis following factor deprivation. FANCC increases GSTP1 activity after the induction of apoptosis. GSTP1 is an enzyme that catalyzes the detoxification of xenobiotics and by-products of oxidative stress, and it is frequently upregulated in neoplastic cells. Although FANCC lacks homology with conventional disulfide reductases, it functions by preventing the formation of inactivating disulfide bonds within GSTP1 during apoptosis. The prevention of protein oxidation by FANCC reveals a novel mechanism of enzyme regulation during apoptosis and has implications for the treatment of degenerative diseases with thiol reducing agents.     

Species delimitation of the liquorice tribe (Leguminosae: Glycyrrhizeae) based on phylogenomic and machine learning analyses

The liquorice tribe Glycyrrhizeae is a leguminous herbaceous group of plants comprised of the genera Glycyrrhiza and Glycyrrhizopsis. Some Glycyrrhiza taxa contain glycyrrhizin, a pharmacologically significant sweet substance that also has applications in crafting industrial materials. Here, we utilized an expanded taxon sampling of Glycyrrhizeae to reconstruct the phylogenetic relationships in the tribe based on genome skimming data, including whole chloroplast genomes, nuclear ribosomal DNA, and low-copy nuclear DNA. We also launched machine learning analysis (MLA) for one species pair with controversial taxonomic boundary. The integrated results indicated Glycyrrhizopsis should be split from Glycyrrhiza, while the former genus Meristotropis should be treated as part of Glycyrrhiza. Glycyrrhizopsis includes two species, Glycyrrhizopsis asymmetrica and Glycyrrhizopsis flavescens, and we recognize 13 species in Glycyrrhiza: Glycyrrhiza acanthocarpa, Glycyrrhiza astragalina, Glycyrrhiza bucharica, Glycyrrhiza echinata, Glycyrrhiza foetida, Glycyrrhiza glabra, Glycyrrhiza gontscharovii, Glycyrrhiza lepidota, Glycyrrhiza macedonica, Glycyrrhiza pallidiflora, Glycyrrhiza squamulosa, Glycyrrhiza triphylla, and Glycyrrhiza yunnanensis. We propose a broader G. glabra that includes former Glycyrrhiza aspera, G. glabra s.s., Glycyrrhiza inflata, and Glycyrrhiza uralensis, and represents the glycyrrhizin-contained medicinal group. Our ancestral state inferences show the ancestor of Glycyrrhiza lacked glycyrrhizin, and the presence of glycyrrhizin evolved twice within Glycyrrhiza during the last one million years. Our integrative phylogenomics-MLA study not only provides new insights into long-standing taxonomic controversies of Glycyrrhizeae, but also represents a useful approach for future taxonomic studies on other plant taxa.     

Rearrangements of integral membrane components during in vitro aging of sheep erythrocyte membranes

In vitro aged sheep erythrocytes and sheep erythrocyte ghosts spontaneously release vesicles that consist of long protrusions affixed to flattened headlike structures. The intramembranous particles seen on the protoplasmic face of freeze fracture electron micrographs of vesicle protrusions are arranged in paired particle rows. On the equivalent fracture face of headlike structures, the particle density is low; if particles are present, they are clustered along the rim of the flattened headlike structure and at the junction with the protrusion. The released vesicles are depleted of the intramembranous particles seen on the exoplasmic face of ghost but retain almost exclusively particles of the protoplasmic face. Correspondingly, the exoplasmic face of ghosts that have released vesicles reveals a 28 percent higher density of intramembranous particles than that of fresh ghosts. Purified vesicles are depleted of spectrin but retain integral membrane proteins, with one of an apparent mol wt of 160,000 accounting for nearly 50 percent of the total protein (Lutz, H.U.,R. Barber, and R.F. McGuire. 1976. J. Biol. Chem. 251:3500-3510). When vesicles are modified with the cleavable cross-linking reagent [(35)S]dithiobis (succinimidyl propionate)at 0 degrees C, the 160,000 mol wt protein is rapidly converted to disulfide-linked dimers and higher oligomers. Exposure of intact ghosts to the reagent in the same way fails to yield equivalent polymers. A comparison of the morphological and biochemical aspects of ghosts and vesicles suggest that a marked rearrangement of membrane proteins accompanies the supramolecular redistribution of intramembranous particles during spontaneous vesiculation. The results also suggest that the paired particles of the protoplasmic face of vesicle protrusions are arranged in paired helices and contain the 160,000 mol wt protein as dimers.