Functional Characterization of a c-type Lysozyme from Indian Shrimp Fenneropenaeus indicus

Lysozyme gene from Fenneropenaeus indicus was cloned, expressed in Escherichia coli and characterized. The cDNA consists of 477 base pairs and encodes amino acid sequence of 159 residues. F. indicus lysozyme had high identity (98 %) with Fenneropenaeus merguiensis and Fenneropenaeus chinensis and exhibits low to moderate identities with lysozymes of other invertebrates and vertebrates. This lysozyme is presumed to be chicken types as it possesses two catalytic and eight cysteine residues that are conserved across c-type lysozymes and a c-terminal extension, which is a characteristic of lysozymes from marine invertebrates. Further, the antimicrobial properties of the recombinant lysozyme from F. indicus were determined in comparison with recombinant hen egg white lysozyme. This exhibited high activity against a Gram-negative pathogenic bacterium Salmonella typhimurium and two fungal strains Pichia pastoris and Saccharomyces cerevisiae in turbidimetric assay. Distribution of lysozyme gene and protein in tissues of shrimps infected with white spot syndrome virus revealed that the high levels of lysozyme are correlated with low and high viral load in abdominal muscle and tail, respectively. In conclusion, lysozyme from F. indicus has a broad spectrum of antimicrobial properties, which once again emphasizes its role in shrimp innate immune response.     

Coupling Between Silicon Waveguide and Metal-Dielectric-Metal Plasmonic Waveguide with Lens-Funnel Structure

Various photonic integrated components have been implemented by ultra-thin silicon-on-insulator (SOI) waveguides; therefore, it is desirable to couple ultra-thin SOI waveguides to plasmonic waveguides. In this paper, we present an ultra-thin SOI waveguide to a metal-dielectric-metal plasmonic waveguide based on a lens-funnel structure consisting of truncated Luneburg lens and metallic parabolic funnel. The lens is implemented by varying the guiding layer thickness. The effect of different parameters of the coupler’s geometry is studied using the finite-difference time-domain method. The 1.13-μm-long coupler improves the average coupling efficiency in the C-band from 66.4 to 82.1%. The numerical simulations indicate that the coupling efficiency is higher than 69% in the entire O, E, S, C, L, and U bands of optical communication.

     

Effects of temperature on the growth of spring ephemerals: Crocus vernus

Spring ephemerals of deciduous forests appear shortly after snow melt and senesce shortly after the overstorey canopy has closed. During this short period, they take advantage of the high light conditions, but also of the low-temperature regime that favours growth in these species. This better growth at cooler temperatures has been attributed to longer leaf life duration. The objective of this study was to test if temperature effect on growth in spring ephemerals can be explained solely by the leaf life duration, or if there is also a direct temperature effect on the underground organ growth. Crocus vernus (L.) Hill was exposed to two temperature regimes, 12/8 and 18/14°C (day/night), for sequential harvests and to four differential air to soil temperature regimes: 12/12, 12/18, 18/12 and 18/18°C (air/soil temperature). Dry mass, leaf nitrogen, starch concentration and cell size of the new corm were determined. Final dry mass and cell size were higher at the lower temperature regime while leaf lasted longer than at the higher temperature regime. Although both air and soil temperatures had an impact on the different growth parameters measured, the impact of soil temperatures was greater and it also influenced leaf life duration. At higher temperature, corm growth stopped before the first visual sign of leaf senescence, suggesting that corm growth controls leaf life duration in C. vernus rather than the opposite and that crocus growth becomes rapidly sink limited at higher temperatures.     

Symbiotic diversity of Ensifer meliloti strains recovered from various legume species in Tunisia

Ensifer meliloti (formerly Sinorhizobium meliloti) was first considered as a specific microsymbiont of Medicago, Melilotus and Trigonella. However, strains of E. meliloti were recovered from root nodules of various legume species and their symbiotic status still remains unclear. Here, we further investigate the specificity of these strains. A collection of 47 E. meliloti strains isolated in Tunisia from root nodules of Medicago truncatula, Medicago sativa, Medicago ciliaris, Medicago laciniata, Medicago marina, Medicago scutellata, Phaseolus vulgaris, Cicer arietinum, Argyrolobium uniflorum, Lotus creticus, Lotus roudairei, Ononis natrix, Retama raetam, Genista saharae, Acacia tortilis, Hedysarum carnosum and Hippocrepis bicontorta were examined by REP-PCR fingerprinting, PCR-RFLPs of the 16S-23S rDNA IGS, the nifH gene and nifD-K intergenic spacer, and sequencing of 16S rRNA and nodA genes. Their nodulation range was also assessed by cross-inoculation experiments. No clear correlation was found between chromosomal backgrounds and host plants of origin. The nodulation polyvalence of the species E. meliloti was associated with a high symbiotic heterogeneity. On the basis of PCR-RFLP data from the nifH gene and nifD-K intergenic spacer, E. meliloti strains isolated from non-Medicago legumes harboured distinct genes and possessed wider host ranges. Some strains did not nodulate Medicago species. On the basis of nodA phylogeny, the majority of the Tunisian strains, including strains from Medicago, harboured distinct nodA alleles more related to those found in E. medicae than those found in E. meliloti. However, more work is still needed to characterize this group further. The diversity observed among M. laciniata isolates, which was supported by nodA phylogeny, nifH typing and the efficiency profile on M. ciliaris, indicated that what was thought to be bv. medicaginis is certainly heterogeneous.     

Evolutionary History of GS3, a Gene Conferring Grain Length in Rice

Unlike maize and wheat, where artificial selection is associated with an almost uniform increase in seed or grain size, domesticated rice exhibits dramatic phenotypic diversity for grain size and shape. Here we clone and characterize GS3, an evolutionarily important gene controlling grain size in rice. We show that GS3 is highly expressed in young panicles in both short- and long-grained varieties but is not expressed in leaves or panicles after flowering, and we use genetic transformation to demonstrate that the dominant allele for short grain complements the long-grain phenotype. An association study revealed that a C to A mutation in the second exon of GS3 (A allele) was associated with enhanced grain length in Oryza sativa but was absent from other Oryza species. Linkage disequilibrium (LD) was elevated and there was a 95.7% reduction in nucleotide diversity (θ_π) across the gene in accessions carrying the A allele, suggesting positive selection for long grain. Haplotype analysis traced the origin of the long-grain allele to a Japonica-like ancestor and demonstrated introgression into the Indica gene pool. This study indicates a critical role for GS3 in defining the seed morphologies of modern subpopulations of O. sativa and enhances the potential for genetic manipulation of grain size in rice.SEED size and seed number are the major determinants of crop yield in both the cereals and the grain legumes. Seed size was also a target of artificial selection during domestication, where large seeds are generally favored due to ease of harvesting and enhanced seedling vigor (Harlan et al. 1972). In rice, traits related to grain size and appearance have a large impact on market value and play a pivotal role in the adoption of new varieties (Champagne et al. 1999; Juliano 2003). However, different grain quality traits are prized by different local cultures and cuisines and, unlike other cereals such as wheat, barley, and maize that are sold largely in processed forms, the physical properties of rice grains are immediately obvious to consumers (Fitzgerald et al. 2009). Thus, rice offers a unique opportunity to investigate the genetics and evolutionary history of seed size and shape.Cultivated rice (Oryza sativa) was domesticated in Asia from the wild progenitor O. rufipogon Griff. and/or O. nivara Sharma (Ishii et al. 1988; Oka 1988; Dally and Second 1990; Nakano et al. 1992; Chen et al. 1993). Classical studies of the subpopulation structure of O. sativa have identified two primary subspecies or varietal groups, namely Indica and Japonica (Oka 1988; Wang and Tanksley 1989; Sun et al. 2002). Studies that have dated the divergence between the Indica and the Japonica groups indicate that it predates rice domestication by at least 100,000 years (Ma and Bennetzen 2004; Vitte et al. 2004; Zhu and Ge 2005), suggesting that at least two genetically distinct gene pools of O. rufipogon were cultivated and subsequently domesticated.Isozyme and DNA studies revealed that there is additional genetic structure within these two groups, with three subpopulations composing the Japonica varietal group (temperate japonica, tropical japonica, and aromatic, written all in lowercase) and two subpopulations composing the Indica group (indica and aus) (Second 1985; Glaszmann 1987; Garris et al. 2005; Caicedo et al. 2007). While there is great diversity of seed size and shape both within and between the different subpopulations of O. sativa, each subpopulation is popularly associated with a characteristic seed morphology. Temperate japonica varieties are known for their short, round grains, indica and aus for slender grains, and within the aromatic subpopulation [hereafter referred to as Group V varieties, according to the isozyme group designation (Glaszmann 1987)] the group of basmati varieties is highly valued for their very long, slender grains (Juliano and Villareal 1993). Identification of the genes that control the range of seed size variation in rice will offer opportunities to study the evolutionary history and phenotypic diversification of the five subpopulations within O. sativa and also provide valuable targets for genetic manipulation.In rice, four genes contributing to seed or grain size have been identified and characterized. The first, grain size 3 (GS3), was isolated from an indica × indica population and found to encode a novel protein with several conserved domains including a phosphatidylethanolamine-binding protein (PEBP)-like domain, a transmembrane region, a putative tumor necrosis factor receptor/nerve growth factor receptor (TNFR/NGFR) family domain, and a von Willebrand factor type C (VWFC) domain (Fan et al. 2006). A second gene, grain weight 2 (GW2), was found to encode an unknown RING-type protein with E3 ubiquitin ligase activity (Song et al. 2007). The third, grain incomplete filling 1 (GIF1), encodes a cell-wall invertase required for carbon partitioning during early grain filling (Wang et al. 2008). Finally, the recently characterized seed width 5 (SW5) has no apparent homolog in the database but was shown to interact with polyubiquitin in a yeast two-hybrid assay; thus it likely acts in the ubiquitin–proteasome pathway to regulate cell division during seed development (Shomura et al. 2008; Weng et al. 2008).Many genes controlling seed size have also been identified in Arabidopsis and tomato, providing a framework for assembling the genetic pathway that determines this trait in dicotyledonous plants (Chaudhury et al. 2001; Jofuku et al. 2005; Ohto et al. 2005; Sundaresan 2005; Schruff et al. 2006; Yoine et al. 2006; Roxrud et al. 2007; Li et al. 2008; Xiao et al. 2008; Orsi and Tanksley 2009; Zhou et al. 2009). Several of these genes show maternal control by regulating endosperm and/or ovule development (Garcia et al. 2003; Jofuku et al. 2005; Li et al. 2008; Ohto et al. 2005; Xiao et al. 2008).Numerous studies have identified rice QTL associated with grain weight and grain length [www.gramene.org (Ni et al. 2009)]. Ten of these studies identified a seed size QTL located in the pericentromeric region of rice chromosome 3, using both inter- and intraspecific crosses (Li et al. 1997; Yu et al. 1997; Redona and Mackill 1998; Xiao et al. 1998; Kubo et al. 2001; Moncada et al. 2001; Brondani et al. 2002; Xing et al. 2002; Thomson et al. 2003; Li et al. 2004). In interspecific crosses, the wild accessions always contributed the dominant allele for small seed size at this locus. Comparative mapping of QTL controlling seed weight in rice, maize, and sorghum further suggested that orthologous seed size genes at this locus might be associated with domestication in all three crops (Paterson et al. 1995).In the current study, we used positional cloning and transformation to demonstrate that the GS3 gene underlies both the gw3.1 QTL (Thomson et al. 2003; Li et al. 2004) and the lk3 QTL (Kubo et al. 2001). In transformation experiments, we demonstrated for the first time that the dominant allele for small grain size complements the long-grain phenotype and we characterized the spatial expression patterns of the gene at different developmental stages. We undertook an association analysis to examine the relationship between the alleles at GS3 and the observed variation for grain length/size in both wild and cultivated rice. Finally, we examined sequence haplotypes across the GS3 region to look for evidence of selection and to identify the origin of the mutation leading to increased grain length in O. sativa.     

Global Gene Expression Profiles Suggest an Important Role for Nutrient Acquisition in Early Pathogenesis in a Plant Model of Pseudomonas aeruginosa Infection

Although Pseudomonas aeruginosa is an opportunistic pathogen that does not often naturally infect alternate hosts, such as plants, the plant-P. aeruginosa model has become a widely recognized system for identifying new virulence determinants and studying the pathogenesis of the organism. Here, we examine how both host factors and P. aeruginosa PAO1 gene expression are affected in planta after infiltration into incompatible and compatible cultivars of tobacco (Nicotiana tabacum L.). N. tabacum has a resistance gene (N) against tobacco mosaic virus, and although resistance to PAO1 infection is correlated with the presence of a dominant N gene, our data suggest that it is not a factor in resistance against PAO1. We did observe that the resistant tobacco cultivar had higher basal levels of salicylic acid and a stronger salicylic acid response upon infiltration of PAO1. Salicylic acid acts as a signal to activate defense responses in plants, limiting the spread of the pathogen and preventing access to nutrients. It has also been shown to have direct virulence-modulating effects on P. aeruginosa. We also examined host effects on the pathogen by analyzing global gene expression profiles of bacteria removed from the intracellular fluid of the two plant hosts. We discovered that the availability of micronutrients, particularly sulfate and phosphates, is important for in planta pathogenesis and that the amounts of these nutrients made available to the bacteria may in turn have an effect on virulence gene expression. Indeed, there are several reports suggesting that P. aeruginosa virulence is influenced in mammalian hosts by the availability of micronutrients, such as iron and nitrogen, and by levels of O₂.     

Assessment of genetic and functional diversity of phosphate solubilizing fluorescent pseudomonads isolated from rhizospheric soil

Background  

Phosphorus is an essential macronutrient for the growth of plants. However, in most soils a large portion of phosphorus becomes insoluble and therefore, unavailable to plants. Knowledge on biodiversity of phosphate-solubilizing fluorescent pseudomonads is essential to understand their ecological role and their utilization in sustainable agriculture.     

Development of antigen detection ELISA for the diagnosis of brugian and bancroftian filariasis using antibodies to recombinant filarial antigens Bm-SXP-1 and Wb-SXP-1

Antibodies specific to recombinant filarial antigens Wb-SXP-1 and Bm-SXP-1 have been used to develop a sandwich ELISA for the detection of circulating filarial antigen (CFA) in sera from patients with lymphatic filariasis caused by Wuchereria bancrofti of Brugia malayi. In patients with W. bancrofti infections, a high proportion of microfilaria (mf) positive (MF) and low proportions of patients with chronic pathology (CP) and endemic normals (EN) showed the presence of CFA. Similarly in patients with brugian infections a high proportion of mf positive individuals contained CFA while none of the patients with chronic pathology or endemic normals showed the presence of CFA. Sera from patients with other parasitic infections (OPI) like O. volvulus, Loa loa, Ascaris lumbricoides and from individuals residing in areas non-endemic to filariasis did not exhibit any reactivity. This assay shows promise for the detection of microfilaremic infections in lymphatic filariasis and its usefulness as a diagnostic tool especially in B. malayi infections, needs to be further evaluated.     

Molecular diagnostic toolkit for <Emphasis Type="Italic">Rhizophagus irregularis</Emphasis> isolate DAOM-197198 using quantitative PCR assay targeting the mitochondrial genome

Rhizophagus irregularis (previously named Glomus irregulare) is one of the most widespread and common arbuscular mycorrhizal fungal (AMF) species. It has been recovered worldwide in agricultural and natural soils, and the isolate DAOM-197198 has been utilized as a commercial inoculant for two decades. Despite the ecological and economical importance of this taxon, specific markers for quantification of propagules by quantitative real-time PCR (qPCR) are extremely limited and none have been rigorously validated for quality control of manufactured products such as biofertilizers. From the sequencing of 14 complete AMF mitochondrial (mt) genomes, a qPCR assay using a hydrolysis probe designed in the single copy cox3-rnl intergenic region was tested and validated to specifically and accurately quantify the spores of R. irregularis isolate DAOM-197198. Specificity tests were performed using standard PCR and qPCR, and results clearly showed that the primers specifically amplified the isolate DAOM-197198, yielding a PCR product of 106 bp. According to the qPCR analyses on spores produced in vitro, the average copy number of mt genomes per spore was 3172?±?304 SE (n?=?6). Quantification assays were successfully undertaken on known and unknown samples in liquid suspensions and commercial dry formulations to show the accuracy, precision, robustness, and reproducibility of the qPCR assay. This study provides a powerful molecular toolkit specifically designed to quantify spores of the model AMF isolate DAOM-197198. The approach of molecular toolkit used in our study could be applied to other AMF taxa and will be useful to research institutions and governmental and industrial laboratories running routine quality control of AMF-based products.     

Fluorescence energy transfer in one dimension: Frequency-domain fluorescence study of DNA–fluorophore complexes

The theory for the salt dependence of the free energy, entropy, and enthalpy of a polyelectrolyte in the PB (PB) model is extended to treat the nonspecific salt dependence of polyelectrolyte–ligand binding reactions. The salt dependence of the binding constant (K) is given by the difference in osmotic pressure terms between the react ants and the products. For simple 1-1 salts it is shown that this treatment is equivalent to the general preferential interaction model for the salt dependence of binding [C. Anderson and M. Record (1993) Journal of Physical Chemistry, Vol. 97, pp. 7116–7126]. The salt dependence, entropy, and enthalpy are compared for the PB model and one specific form of the preferential interaction coefficient model that uses counterion condensation/limiting law (LL) behavior. The PB and LL models are applied to three ligand–polyelectrolyte systems with the same net ligand charge: a model sphere–cylinder binding reaction, a drug–DNA binding reaction, and a protein–DNA binding reaction. For the small ligands both the PB and limiting law models give (ln K vs. In [salt]) slopes close in magnitude to the net ligand charge. However, the enthalpy/entropy breakdown of the salt dependence is quite different. In the PB model there are considerable contributions from electrostatic enthalpy and dielectric (water reorientation) entropy, compared to the predominant ion cratic (release) entropy in the limiting law model. The relative contributions of these three terms in the PB model depends on the ligand: for the protein, ion release entropy is the smallest contribution to the salt dependence of binding. The effect of three approximations made in the LL model is examined: These approximations are (1) the ligand behaves ideally, (2) the preferential interaction coefficient of the polyelectrolyte is unchanged upon ligand binding, and (3) the polyelectrolyte preferential interaction coefficient is given by the limiting law/counterion-condensation value. Analysis of the PB model shows that assumptions 2 and 3 break down at finite salt concentrations. For the small ligands the effects on the slope cancel, however, giving net slopes that are similar in the PB and LL models, but with a different entropy/enthalpy breakdown. For the protein ligand the errors from assumptions 2 and 3 in the LL model do not cancel. In addition, the ligand no longer behaves ideally due to its complex structure and charge distribution. Thus for the protein the slope is no longer related simply to the net ligand charge, and the PB model gives a much larger slope than the LL model. Additionally, in the PB model most of the salt dependence of the protein binding comes from the change in ligand activity, i.e. from nonspecific anion effects, in contrast to the small ligand case. While the absolute binding is sensitive to polyelectrolyte length, little length effect is seen on the salt dependence for the small ligands at 0.1M salt, and for lengths > 60 Å. Almost no DNA length dependenceis seen in the salt dependence of the protein binding, since this is determined primarily by the protein, not the DNA. © 1995 John Wiley & Sons, Inc.