World Dissemination of the Cereal-Cyst Nematode (Heterodera avenae) and Its Potential as a Pathogen of Wheat

World distribution of the cereal-cyst nematode is herein reviewed. It is suggested that Heterodera avenae originated in Europe and has been widely disseminated, largely by the activities of Man but also by wind movement of cysts. So far, it may not have spread to some major wheat-growing regions of the New World, but a non-friable soil structure limits population level and disease. Yield loss could result from the introduction of new cultivars to developing countries where H . avenae has not been detected or where existing cultivars possess tolerance.     

Oviposition Choice of Two Fall Armyworm (Lepidoptera: Noctuidae) Host Strains

Fall armyworm, Spodoptera frugiperda (J. E. Smith), is a noctuid species that is composed of two morphologically identical sympatric host strains (corn and rice) that differ in their distribution among plant hosts. In an effort to explain observations of host fidelity in the field, ovipositional preference of the two strains on corn (Zea mays L.) or pasture grass (Cynodon nlemfuensis Vanderyst var. nlemfuensis) was determined using two greenhouse bioassays. In the first bioassay, corn strain females placed more eggmasses on the screen enclosure than on corn plants while grass plants contained an intermediate number of eggmasses. Rice strain females placed most of their eggmasses on grass plants. In the second bioassay, corn strain females placed an equal number of eggmasses on corn and grass plants in comparison to rice strain females which placed >3.5× more eggmasses on grass plants than on corn plants. Individual eggs as part of the eggmasses were also counted on plants and on the screen enclosure. Corn strain females equally placed eggs on the two host plants and on the screen enclosures, however rice strain females placed more eggs on grass plants compared to corn plants or the screen enclosure. This is the first report of consistent differential oviposition between corn and rice strain fall armyworm females.     

Arabidopsis actin depolymerizing factor4 modulates the stochastic dynamic behavior of actin filaments in the cortical array of epidermal cells

Actin filament arrays are constantly remodeled as the needs of cells change as well as during responses to biotic and abiotic stimuli. Previous studies demonstrate that many single actin filaments in the cortical array of living Arabidopsis thaliana epidermal cells undergo stochastic dynamics, a combination of rapid growth balanced by disassembly from prolific severing activity. Filament turnover and dynamics are well understood from in vitro biochemical analyses and simple reconstituted systems. However, the identification in living cells of the molecular players involved in controlling actin dynamics awaits the use of model systems, especially ones where the power of genetics can be combined with imaging of individual actin filaments at high spatial and temporal resolution. Here, we test the hypothesis that actin depolymerizing factor (ADF)/cofilin contributes to stochastic filament severing and facilitates actin turnover. A knockout mutant for Arabidopsis ADF4 has longer hypocotyls and epidermal cells when compared with wild-type seedlings. This correlates with a change in actin filament architecture; cytoskeletal arrays in adf4 cells are significantly more bundled and less dense than in wild-type cells. Several parameters of single actin filament turnover are also altered. Notably, adf4 mutant cells have a 2.5-fold reduced severing frequency as well as significantly increased actin filament lengths and lifetimes. Thus, we provide evidence that ADF4 contributes to the stochastic dynamic turnover of actin filaments in plant cells.     

Cry1F Resistance in Fall Armyworm Spodoptera frugiperda: Single Gene versus Pyramided Bt Maize

Evolution of insect resistance to transgenic crops containing Bacillus thuringiensis (Bt) genes is a serious threat to the sustainability of this technology. However, field resistance related to the reduced efficacy of Bt maize has not been documented in any lepidopteran pest in the mainland U.S. after 18 years of intensive Bt maize planting. Here we report compelling evidence of field resistance in the fall armyworm, Spodoptera frugiperda (J.E. Smith), to Cry1F maize (TC 3507) in the southeastern region of the U.S. An F₂ screen showed a surprisingly high (0.293) Cry1F resistance allele frequency in a population collected in 2011 from non-Bt maize in south Florida. Field populations from non-Bt maize in 2012–2013 exhibited 18.8-fold to >85.4-fold resistance to purified Cry1F protein and those collected from unexpectedly damaged Bt maize plants at several locations in Florida and North Carolina had >85.4-fold resistance. In addition, reduced efficacy and control failure of Cry1F maize against natural populations of S. frugiperda were documented in field trials using Cry1F-based and pyramided Bt maize products in south Florida. The Cry1F-resistant S. frugiperda also showed a low level of cross-resistance to Cry1A.105 and related maize products, but not to Cry2Ab2 or Vip3A. The occurrence of Cry1F resistance in the U.S. mainland populations of S. frugiperda likely represents migration of insects from Puerto Rico, indicating the great challenges faced in achieving effective resistance management for long-distance migratory pests like S. frugiperda.     

Phylogenetic Patterns of Codon Evolution in the ACTIN-DEPOLYMERIZING FACTOR/COFILIN (ADF/CFL) Gene Family

The actin-depolymerizing factor/cofilin (ADF/CFL) gene family encodes a diverse group of relatively small proteins. Once known strictly as modulators of actin filament dynamics, recent research has demonstrated that these proteins are involved in a variety of cellular processes, from signal transduction to the cytonuclear trafficking of actin. In both plant and animal lineages, expression patterns of paralogs in the ADF/CFL gene family vary among tissue types and developmental stages. In this study we use computational approaches to investigate the evolutionary forces responsible for the diversification of the ADF/CFL gene family. Estimating the rate of non-synonymous to synonymous mutations (dN/dS) across phylogenetic lineages revealed that the majority of ADF/CFL codon positions were under strong purifying selection, with rare episodic events of accelerated protein evolution. In both plants and animals these instances of accelerated evolution were ADF/CFL subclass specific, and all of the sites under selection were located in regions of the protein that could serve in new functional roles. We suggest these sites may have been important in the functional diversification of ADF/CFL proteins.     

Micropatterning of polymer brushes: grafting from dewetting polymer films for biological applications

In this novel platform, a micropatterned polymer brush was obtained by grafting poly(poly(ethylene glycol) methyl ether methacrylate) (poly(PEGMA)) from a thin macroinitiator film using atom transfer radical polymerization (ATRP). A pattern of holes was formed in the macroinitiator film by taking advantage of its spontaneous dewetting above the glass transition temperature from a bottom polystyrene film, driven by unfavorable intermolecular forces. Patterning by dewetting can be achieved at length-scales from a few hundred nanometers to several tens of micrometers, by simply thermally annealing the bilayer above the glass transition temperature of the polymer. This approach is substrate-independent, as polymer films can be cast onto surfaces of different size, shape, or material. As a demonstration of its potential, proteins, and individual cells were attached on targeted bioadhesive polystyrene areas of the micropatterns within poly(PEGMA) protein-repellent brushes. We anticipate this approach will be suitable for the patterning of brushes, especially for biomedical applications such as in the study of single cells and of cell cocultures.     

Variations in the Composition of Gelling Agents Affect Morphophysiological and Molecular Responses to Deficiencies of Phosphate and Other Nutrients

Low inorganic phosphate (Pi) availability triggers an array of spatiotemporal adaptive responses in Arabidopsis (Arabidopsis thaliana). There are several reports on the effects of Pi deprivation on the root system that have been attributed to different growth conditions and/or inherent genetic variability. Here we show that the gelling agents, largely treated as inert components, significantly affect morphophysiological and molecular responses of the seedlings to deficiencies of Pi and other nutrients. Inductively coupled plasma-mass spectroscopy analysis revealed variable levels of elemental contaminants not only in different types of agar but also in different batches of the same agar. Fluctuating levels of phosphorus (P) in different agar types affected the growth of the seedlings under Pi-deprivation condition. Since P interacts with other elements such as iron, potassium, and sulfur, contaminating effects of these elements in different agars were also evident in the Pi-deficiency-induced morphological and molecular responses. P by itself acted as a contaminant when studying the responses of Arabidopsis to micronutrient (iron and zinc) deficiencies. Together, these results highlighted the likelihood of erroneous interpretations that could be easily drawn from nutrition studies when different agars have been used. As an alternative, we demonstrate the efficacy of a sterile and contamination-free hydroponic system for dissecting morphophysiological and molecular responses of Arabidopsis to different nutrient deficiencies.Plant development is a dynamic and complex process often subject to biotic and abiotic stresses. On encountering nutrient stress, plants undergo an array of adaptive changes. Phosphorus (P) is an essential plant macronutrient, but low availability of soluble inorganic phosphate (Pi) is common in many natural and agricultural ecosystems (Marschner, 1995; Schachtman and Shin, 2007). Arabidopsis (Arabidopsis thaliana) is used as a model system to answer many of the fundamental questions related to responses of plants to Pi stress. To minimize variation caused by macroenvironmental and microenvironmental conditions, Pi-deficiency responses traditionally have been studied by growing Arabidopsis aseptically in continuously shaken liquid culture or on solid medium in petri plates. Although the use of liquid culture is a convenient technique for generating bulk tissue for assaying biochemical and molecular responses to Pi deprivation (Karthikeyan et al., 2002; Misson et al., 2005), continuous swirling to provide aeration makes the study of root hair development and root system architecture (RSA) all but impossible. Therefore, growth of seedlings on vertically oriented petri plates containing solidified nutrient medium with sufficient (1.0–2.5 mm) or limiting (0–10 μm) Pi would be ideal for morphophysiological and molecular studies (López-Bucio et al., 2002; Jain et al., 2007a).Agar and phytagel, extracted from red algae and bacteria, respectively, are commonly used gelling agents (http://www.sigmaaldrich.com/sigma/product). Ideally, a gelling agent would be an inert constituent of the plant growth medium. Studies show, however, that the agent itself causes variations in plant growth responses on otherwise identical nutrient media (Nowak and Asiedu, 1992; Scholten and Pierik, 1998a, 1998b; Beruto et al., 1999). Differences in the performance of gelling agents have been attributed to their variable physiochemical characteristics, such as nutrient diffusion rate, elemental and organic impurities, and gel strength (Debergh, 1983; Nairn et al., 1995). While the elemental contaminants in gelling agents used with nutrient-rich media may not significantly affect the growth of Arabidopsis seedlings, they could make a significant difference under nutrient-deficient conditions. It has been shown that a low concentration of Pi (25 μm) in the medium negated the inhibitory effect of Pi deprivation on shoot growth (López-Bucio et al., 2002). Furthermore, the Pi-deficiency response is also influenced by the interaction of Pi with other elements and compounds. For instance, primary roots of Pi-starved seedlings failed to enter the determinant growth phase, a hallmark of the Pi-deficiency response, when the medium was also deprived of iron (Fe; Sánchez-Calderón et al., 2005; Svistoonoff et al., 2007; Ward et al., 2008). Incidents of cross talk between P and other macroelements and microelements (potassium [K] and zinc [Zn]) have also been reported in crop species (Huang et al., 2000; Wang et al., 2002). Therefore, gel medium contaminants other than P could have a significant bearing on overall Pi-deficiency responses.Inherent genetic variability of Arabidopsis ecotypes is thought to be the likely cause of the large variations noted in morphological responses during Pi-deprivation studies (Chevalier et al., 2003). Some attribute the divergent reports to differences in experimental design (Al-Ghazi et al., 2003; Reymond et al., 2006). For example, photosynthetically active radiation ranging from 30 to 300 μmol m⁻² s⁻¹ has been used to grow Arabidopsis (Williamson et al., 2001; López-Bucio et al., 2002; Al-Ghazi et al., 2003; Chevalier et al., 2003; Jain et al., 2007a). Since photosynthates play a pivotal role in the regulation of the Pi-starvation responses (Liu et al., 2005; Jain et al., 2007a; Karthikeyan et al., 2007), variations in photosynthetically active radiation could be a contributory factor to these discrepancies.To further address disparities in Pi-starvation responses, we investigated the effects of the elemental composition of different batches and types of agar on morphophysiological and molecular responses of Pi-deprived Arabidopsis seedlings. The growth responses of Pi-deprived plants were correlated with P contamination levels of the gelling medium. Also, the effects of Fe contamination in agar were demonstrated under P−Fe− condition on morphological and molecular responses of the seedlings. This study also highlights the fact that P acts as a contaminant in influencing the responses of seedlings to micronutrient (Fe and Zn) deficiencies.     

Arabidopsis Actin-Depolymerizing Factor AtADF4 Mediates Defense Signal Transduction Triggered by the Pseudomonas syringae Effector AvrPphB

The actin cytoskeleton has been implicated in plant defenses against pathogenic fungi and oomycetes with limited, indirect evidence. To date, there are no reports linking actin with resistance against phytopathogenic bacteria. The dynamic behavior of actin filaments is regulated by a diverse array of actin-binding proteins, among which is the Actin-Depolymerizing Factor (ADF) family of proteins. Here, we demonstrate that actin dynamics play a role in the activation of gene-for-gene resistance in Arabidopsis (Arabidopsis thaliana) following inoculation with the phytopathogenic bacterium Pseudomonas syringae pv tomato. Using a reverse genetics approach, we explored the roles of Arabidopsis ADFs in plant defenses. AtADF4 was identified as being specifically required for resistance triggered by the effector AvrPphB but not AvrRpt2 or AvrB. Recombinant AtADF4 bound to monomeric actin (G-actin) with a marked preference for the ADP-loaded form and inhibited the rate of nucleotide exchange on G-actin, indicating that AtADF4 is a bona fide actin-depolymerizing factor. Exogenous application of the actin-disrupting agent cytochalasin D partially rescued the Atadf4 mutant in the AvrPphB-mediated hypersensitive response, demonstrating that AtADF4 mediates defense signaling through modification of the actin cytoskeleton. Unlike the mechanism by which the actin cytoskeleton confers resistance against fungi and oomycetes, AtADF4 is not involved in resistance against pathogen entry. Collectively, this study identifies AtADF4 as a novel component of the plant defense signaling pathway and provides strong evidence for actin dynamics as a primary component that orchestrates plant defenses against P. syringae.The actin cytoskeleton has been implicated in plant defenses against pathogenic fungi and oomycetes (Hardham et al., 2007). Evidence largely comes from studies using actin cytoskeleton-disrupting agents, such as cytochalasins. Treatments with a variety of cytochalasins were shown to increase the penetration rate of both adapted and nonadapted pathogens in multiple plant-pathogen systems, thereby implicating the actin cytoskeleton as having a role in basal defenses and nonhost resistance (Kobayashi et al., 1997; Yun et al., 2003; Shimada et al., 2006; Miklis et al., 2007). The actin cytoskeleton may also play a role in race-specific resistance (Skalamera and Heath, 1998). To date, no reports linking actin dynamics with resistance against phytopathogenic bacteria have been published.While the actin cytoskeleton as a virulence target of plant pathogens has not been documented, it was well characterized in mammalian pathosystems, particularly in studies investigating macrophage interactions with the pathogenic bacterium Yersinia pestis (Mattoo et al., 2007). Yersinia species deliver a suite of effectors into the target host cell, and at least four of them (YopE, YpkA/YopO, YopT, and YopH) are involved in rearrangement of the actin cytoskeleton (Aepfelbacher and Heesemann, 2001). YopT, a Cys protease, targets a plasma membrane-localized Rho GTPase in affected phagocytes (Aepfelbacher and Heesemann, 2001). Cleavage of the GTPase by YopT releases the prenylated protein from the plasma membrane and disrupts the actin cytoskeleton, effectively shutting down phagocytosis, preventing elimination of the pathogen (Iriarte and Cornelis, 1998; Shao et al., 2002). Similarly, microbial pathogens also usurp host processes for the benefit of infection, disease, and death. Listeria species hijack the host''s cytoskeleton to move around inside the infected cell through the induction of directed polymerization of actin (Pistor et al., 1994). Salmonella injects into host cells two actin-binding proteins (SipA and SipC) as well as other regulators of actin dynamics to enhance phagocytic uptake and intracellular propagation (Galan and Zhou, 2000). In short, either by preventing polymerization or by promoting it, pathogens have evolved strategies to modify the host actin cytoskeleton for purposes of evading detection or eliciting disease and death.Dynamic actin cytoskeleton rearrangements are regulated by a pool of actin-binding proteins, which sense environmental changes and modulate the cytoskeleton through various biochemical activities (Hussey et al., 2006; Staiger and Blanchoin, 2006). Among the proteins that regulate these dynamic processes are the Actin-Depolymerizing Factor (ADF) family of proteins (Maciver and Hussey, 2002). In general, ADFs bind both monomeric (G-) and filamentous (F-) actin to increase actin dynamics. They function by severing F-actin to generate more ends for polymerization and by increasing the dissociation rate of actin monomers from the pointed ends (Maciver, 1998; Maciver and Hussey, 2002). Plant ADFs play roles in pollen tube growth (Chen et al., 2003), root formation (Thomas and Schiefelbein, 2002), and cold acclimation (Ouellet et al., 2001). There is also one report linking ADFs with plant defenses (Miklis et al., 2007). In that study, ectopic expression of barley (Hordeum vulgare) HvADF3 and several isovariants of Arabidopsis (Arabidopsis thaliana) ADFs in barley epidermal cells was shown to compromise penetration resistance to powdery mildew fungi (Miklis et al., 2007).The Arabidopsis-Pseudomonas syringae interaction provides an ideal model plant-pathogen system to study plant defense signaling. Like Yersinia species, P. syringae delivers effector proteins into the host cells via the type III secretion system and relies on these proteins for pathogenesis (Alfano and Collmer, 2004). However, once these proteins (Avr) are recognized either directly or indirectly by plant resistance (R) proteins, plant immune responses are activated (Jones and Dangl, 2006). Exciting progress has been made toward understanding the indirect recognition of several pairs of Avr-R proteins; the best examples include AvrB/AvrRPM1-RPM1, AvrRpt2-RPS2, and AvrPphB-RPS5. During activation of defense mediated by AvrB/AvrRPM1-RPM1 and AvrRpt2-RPS2, the phosphorylation or elimination of a third protein, RIN4, is essential (Mackey et al., 2002; Axtell and Staskawicz, 2003). In the case of AvrPphB-RPS5 recognition, the AvrPphB Cys protease of the same family as YopT (Shao et al., 2002) cleaves the plant protein kinase PBS1, inducing a conformational change in RPS5, which in turn leads to the activation of resistance (Ade et al., 2007). Although these studies have greatly enhanced our understanding of how pathogen effectors initiate plant defense responses, the ultimate signaling processes associated with the activation of resistance remain largely unknown, due to the limited number of genetic loci identified in these pathways. In this work, we hypothesize that actin-binding proteins play a role during plant-bacteria interactions based on the functional and structural similarity between AvrPphB and YopT.There are 11 ADFs in the Arabidopsis genome (Ruzicka et al., 2007). We utilized a reverse genetics approach to identify the putative roles these proteins play in plant resistance against the bacterial pathogen P. syringae pv tomato (Pst). AtADF4 was identified as a novel signaling component in the AvrPphB-RPS5-mediated defense signal transduction pathway. Loss of AtADF4 confers on Arabidopsis enhanced susceptibility to P. syringae expressing AvrPphB. Further subcellular localization and biochemical analyses, as well as pharmacological studies, suggest that AtADF4 functions as a bona fide actin-depolymerizing factor through modifying the actin cytoskeleton. Unlike the documented mechanism by which the actin cytoskeleton plays roles in resistance against fungi and oomycetes, the resistance against P. syringae mediated by AtADF4 is not involved in hindering pathogen entry.     

Antibodies designed as effective cancer vaccines

Antigen/antibody complexes can efficiently target antigen presenting cells to allow stimulation of the cellular immune response. Due to the difficulty of manufacture and their inherent instability complexes have proved inefficient cancer vaccines. However, anti-idiotypic antibodies mimicking antigens have been shown to stimulate both antibody and T cell responses. The latter are due to T cell mimotopes expressed within the complementarity-determining regions (CDRs) of antibodies that are efficiently presented to dendritic cells in vivo. Based on this observation we have designed a DNA vaccine platform called ImmunoBody™, where cytotoxic T lymphocyte (CTL) and helper T cell epitopes replace CDR regions within the framework of a human IgG1 antibody. The ImmunoBody™ expression system has a number of design features which allow for rapid production of a wide range of vaccines. The CDR regions of the heavy and light chain have been engineered to contain unique restriction endonuclease sites, which can be easily opened, and oligonucleotides encoding the T cell epitopes inserted. The variable and constant regions of the ImmunoBody™ are also flanked by restriction sites, which permit easy exchange of other IgG subtypes. Here we show a range of T cell epitopes can be inserted into the ImmunoBody™ vector and upon immunization these T cell epitopes are efficiently processed and presented to stimulate high frequency helper and CTL responses capable of anti-tumor activity.Key words: DNA vaccines, cancer vaccines, melanoma, CTL, helper T cells