Molecular modeling and docking characterization of CzR1, a CC-NBS-LRR R-gene from Curcuma zedoaria Loeb. that confers resistance to Pythium aphanidermatum

Plant NBS-LRR R-genes recognizes several pathogen associated molecular patterns (PAMPs) and limit pathogen infection through a multifaceted defense response. CzR1, a coiled-coil-nucleotide-binding-site-leucine-rich repeat R-gene isolated from Curcuma zedoaria L exhibit constitutive resistance to different strains of P. aphanidermatum. Majority of the necrotrophic oomycetes are characterized by the presence of carbohydrate PAMPs β-glucans in their cell walls which intercat with R-genes. In the present study, we predicted the 3D (three dimensional) structure of CzR1 based on homology modeling using the homology module of Prime through the Maestro interface of Schrodinger package ver 2.5. The docking investigation of CzR1 with β-glucan using the Glide software suggests that six amino acid residues, Ser186, Glu187, Ser263, Asp264, Asp355 and Tyr425 act as catalytic residues and are involved in hydrogen bonding with ligand β-(1,3)-D-Glucan. The calculated distance between the carboxylic oxygen atoms of Glu187–Asp355 pair is well within the distance of 5Å suggesting a positive glucanase activity of CzR1. Elucidation of these molecular characteristics will help in in silico screening and understanding the structural basis of ligand binding to CzR1 protein and pave new ways towards a broad spectrum rhizome rot resistance development in the cultivated turmeric.     

A novel arsenate reductase from the bacterium Thermus thermophilus HB27: Its role in arsenic detoxification

Microorganisms living in arsenic-rich geothermal environments act on arsenic with different biochemical strategies, but the molecular mechanisms responsible for the resistance to the harmful effects of the metalloid have only partially been examined. In this study, we investigated the mechanisms of arsenic resistance in the thermophilic bacterium Thermus thermophilus HB27. This strain, originally isolated from a Japanese hot spring, exhibited tolerance to concentrations of arsenate and arsenite up to 20 mM and 15 mM, respectively; it owns in its genome a putative chromosomal arsenate reductase (TtarsC) gene encoding a protein homologous to the one well characterized from the plasmid pI258 of the Gram + bacterium Staphylococcus aureus. Differently from the majority of microorganisms, TtarsC is part of an operon including genes not related to arsenic resistance; qRT-PCR showed that its expression was four-fold increased when arsenate was added to the growth medium. The gene cloning and expression in Escherichia coli, followed by purification of the recombinant protein, proved that TtArsC was indeed a thioredoxin-coupled arsenate reductase with a k_cat/K_M value of 1.2 × 10⁴ M^− 1 s^− 1. It also exhibited weak phosphatase activity with a k_cat/K_M value of 2.7 × 10^− 4 M^− 1 s^− 1. The catalytic role of the first cysteine (Cys7) was ascertained by site-directed mutagenesis. These results identify TtArsC as an important component in the arsenic resistance in T. thermophilus giving the first structural–functional characterization of a thermophilic arsenate reductase.     

Puromycin resistance gene as an effective selection marker for ciliate Tetrahymena

A puromycin-N-acetyltransferase gene (pac) is widely used as a selection marker for eukaryotic gene manipulation. However, it has never been utilized for molecular studies in the ciliate Tetrahymena thermophila, in spite of the limited number of selection markers available for this organism. To utilize pac as a maker gene for T. thermophila, the nucleotide sequence of the pac gene was altered to accord with the most preferred codon-usage in T. thermophila. This codon-optimized pac gene expressed in T. thermophila conferred a resistance to transformed cells against 2000 μg/ml of puromycin dihydrochloride, whereas the growth of wild-type cells was completely inhibited by 200 μg/ml. Furthermore, an expression cassette constructed with the codon-optimized pac and an MTT1 promoter was effectively utilized for experiments to tag endogenous proteins of interest by fusing the cassette into the target gene locus. These results indicate that pac can be used as a selection marker in molecular studies of T. thermophila.     

MH-DAB gene polymorphism and disease resistance to Flavobacterium columnare in grass carp (Ctenopharyngodon idellus)

In this study, the association between MH-DAB gene polymorphism and disease resistance was evaluated by challenging grass carp (Ctenopharyngodon idellus) with Flavobacterium columnare. Eight genotypes and six alleles were found, and named by common nomenclature. The genotypes AA, BB, EE, and DE, and the alleles Ctid-DAB1*0101, Ctid-DAB1*0201 and Ctid-DAB1*0401 were more preponderant in fish. The genotype BB was associated with higher resistance to F. columnare, as well as two alleles Ctid-DAB*0101 and Ctid-DAB*0201. Allele Ctid-DAB*0102 has decreased resistance to F. columnare. The expression of MH-DAB gene was decreased in the liver, kidney, and intestine but not in the spleen, gill, and skin at 2 days post infection (dpi), versus to that in the control group. MH-DAB gene expression was up-regulated in most tissues but remained at normal levels in the intestine at 15 days post infection. Our data suggested that MH-DAB polymorphism can be used as a potential genetic marker for disease resistance breeding of grass carp in the future.     

Evaluating type III polyketide synthase (PKS) and terpene synthase (TPS) expression in incompatible interactions of Zingiber zerumbet to Pythium myriotylum Drechsler

Abstract

Pythium species are aggressive soil-borne necrotrophic oomycetes causing soft-rot disease of ginger. Disease severity indices determined following infection with P. myriotylum Drechsler in ginger cultivar, Zingiber officinale cv. Varada and a wild congener, Z. zerumbet at varying zoospore concentrations (10⁴–10¹² spores/ml) revealed high disease severity (100%) in ginger cultivar whereas Z. zerumbet displayed resistance. Absence of positive correlation between Z. zerumbet resistance and polyphenolic content indicates role of polyketides and zerumbone in preventing pathogen ingress, as reported earlier. Towards elucidating this, Z. zerumbet specific polyketide synthase (PKS) and terpene synthase (TPS) gene sequences designated ZzTPS and ZzPKS respectively were characterised. Phylogenetic analysis clustered ZzTPS with TPS-b sub-family and ZzPKS with non-chalcone forming PKS. ZzTPS and ZzPKS showed biphasic expression with first at 6?hours post infection (hpi) and then at 8 hpi, indicative of rapid induction followed by reinforcement to sustain resistance mechanisms in the wild taxon.     

Biocontrol activity of the extract prepared from Zingiber zerumbet for the management of rhizome rot in Zingiber officinale caused by Pythium myriotylum

Zingiber zerumbet Smith or wild ginger is remarkable for its inherent resistance to Pythium spp., which cause soft rot disease in Zingiber officinale Rosc. In the present study, various concentrations of extract prepared from Z. zerumbet were screened for its activity against Pythium myriotylum. Microscopic observation of P. myriotylum in presence of Z. zerumbet extract has confirmed the complete lysis of pathogen within 10 h. However, the same treatment with Z. officinale extract was found to have partial antifungal effect even after 24 h due to inability of its metabolites to prevent the growth of P. myriotylum. Due to the antifungal activity, extract from Z. zerumbet was subjected to GC–MS and LC-QTOF-MS which has identified Zerumbone with m/z 219 as the major compound. Further, in vivo study and the subsequent microscopic analysis have confirmed the applicability of extract from Z. zerumbet as a phytomedicine to control rhizome rot in ginger.     

Association between osteoporosis and polymorphisms of the bone Gla protein, estrogen receptor 1, collagen 1-A1 and calcitonin receptor genes in Turkish postmenopausal women

In this study, we have investigated the association between osteoporosis and osteocalcin (BGLAP) − 298 C>T, estrogen receptor 1 (ER1) 397 T>C, collagen type1 alpha 1 (Col1A1) 2046 G>T and calcitonin receptor (CALCR) 1340 T>C polymorphisms. Genomic DNA was obtained from 266 persons (158 osteoporotic and 108 healthy controls). Genomic DNA was extracted from EDTA-preserved peripheral venous blood of patients and controls by a salting-out method and analyzed by PCR-RFLP. As a result, there was no statistically significant difference in the genotype and allele frequencies of patients and controls for BGLAP − 298 C>T, Col1A1 2046 G>T, ER1 397 T>C and CALCR 1340 T>C polymorphisms. However, ER1 CC genotype compared with TT + TC genotypes was found to increase the two fold the risk of osteoporosis [p = 0.039, OR = 2.156, 95% CI (1.083–4.293)] and CALCR CC genotype compared with TT + TC genotypes was found to have protective effect against osteoporosis [p = 0.045, OR = 0.471, 95% CI (0.237–0.9372)]. In the combined genotype analysis, ER1/CALCR TCCC combined genotype was estimated to have protective effect against osteoporosis [p = 0.0125, OR = 0.323, 95% CI (0.1383–0.755)] whereas BGLAP/Col1A1 CCTT and ER1/CALCR CCTT combined genotypes were estimated as risk factors for osteoporosis in Turkish population (p = 0.027, p = 0.009 respectively).     

Bacterial resistance modifying tetrasaccharide agents from Ipomoea murucoides

As part of an ongoing project to identify oligosaccharides which modulate bacterial multidrug resistance, the CHCl₃-soluble extract from flowers of a Mexican arborescent morning glory, Ipomoea murucoides, through preparative-scale recycling HPLC, yielded five lipophilic tetrasaccharide inhibitors of Staphylococcusaureus multidrug efflux pumps, murucoidins XII-XVI (1-5). The macrocyclic lactone-type structures for these linear hetero-tetraglycoside derivatives of jalapinolic acid were established by spectroscopic methods. These compounds were tested for in vitro antibacterial and resistance modifying activity against strains of Staphylococcus aureus possessing multidrug resistance efflux mechanisms. Only murucoidin XIV (3) displayed antimicrobial activity against SA-1199B (MIC 32 μg/ml), a norfloxacin-resistant strain that over-expresses the NorA MDR efflux pump. The four microbiologically inactive (MIC > 512 μg/ml) tetrasaccharides increased norfloxacin susceptibility of this strain by 4-fold (8 μg/ml from 32 μg/ml) at concentrations of 25 μg/ml, while murucoidin XIV (3) exerted the same potentiation effect at a concentration of 5 μg/ml.     

The mitochondrial genome of the Russian wheat aphid Diuraphis noxia: Large repetitive sequences between trnE and trnF in aphids

To characterize aphid mitochondrial genome (mitogenome) features, we sequenced the complete mitogenome of the Russian wheat aphid, Diuraphis noxia. The 15,784-bp mitogenome with a high A + T content (84.76%) and strong C skew (− 0.26) was arranged in the same gene order as that of the ancestral insect. Unlike typical insect mitogenomes, D. noxia possessed a large tandem repeat region (644 bp) located between trnE and trnF. Sequencing partial mitogenome of the cotton aphid (Aphis gossypii) further confirmed the presence of the large repeat region in aphids, but with different repeat length and copy number. Another motif (58 bp) tandemly repeated 2.3 times in the control region of D. noxia. All repeat units in D. noxia could be folded into stem-loop secondary structures, which could further promote an increase in copy numbers. Characterization of the D. noxia mitogenome revealed distinct mitogenome architectures, thus advancing our understanding of insect mitogenomic diversities and evolution.     

Genetic structure,polymorphism identification of LGP2 gene and their relationship with the resistance/susceptibility to GCRV in grass carp, Ctenopharyngodon idella

Laboratory of genetics and physiology 2 (LGP2) is an actual detector and regulator during RNA viral infection in innate immunity. In this study, 5′-flanking region and all introns of LGP2 in grass carp (Ctenopharyngodon idella) were excavated. The genomic CiLGP2 (C. idella LGP2) was 8062 bp in length, with a 364 bp 5′-flanking region, twelve exons and eleven introns. Besides, the promoter activity of the upstream region before initiator codon was identified. By sequencing, six single nucleotide polymorphisms (SNPs) and one 20-bp insertion/deletion polymorphism were detected in CiLGP2. With a challenge experiment, the genotype and allele distributions of these seven polymorphisms were examined. Analytic result revealed only the − 1392 C/G, 494 A/T and 4403 C/T loci were significantly associated with the resistance/susceptibility to grass carp reovirus (GCRV) (P < 0.05). To further identify these correlations, another independent challenge test was performed. The analytic result based on the cumulative mortality demonstrated that the stock in − 1392 GG genotype was more susceptible to GCRV than that in CC genotype, while the stocks in 494 TT genotype and 4403 TT genotype were more resistant to GCRV than that in AA and CC genotype stocks, respectively (P < 0.05). Those significant SNPs might be potential gene markers for the future molecular selection of C. idella strains that are resistant to GCRV.     

Correlation between grass carp (Ctenopharyngodon idella) resistance to grass carp reovirus and the genetic insert-deletion polymorphisms in promoter and intron of RIG-I gene

RIG-I (retinoic acid-inducible gene I) is an essential cytosolic pathogen recognition receptor that binds to a variety of viral RNA or DNA to induce type I interferons. In the present study, insert–deletion polymorphisms in promoter and introns of CiRIG-I (Ctenopharyngodon idella RIG-I) were explored, their associations with resistance/susceptibility to grass carp reovirus (GCRV) were analyzed. To this end, genomic sequence of CiRIG-I gene was obtained, and twenty pairs of primers were prepared for the detection of insert–deletion polymorphisms. Five insert–deletion mutations were found, a 2-bp mutation and an 8-bp mutation existed in the promoter and other three sizes in 74 bp, 146 bp and 53 bp were sited in the intron 8. After a challenge experiment, only the genotype and allele of − 740 insert–deletion mutation in the promoter and allele of 6804 insert–deletion mutation were significantly associated with resistance/susceptibility to GCRV among the five mutations (P < 0.05). To further identify this correlation, another independent challenge test was carried out. The result revealed that the cumulative mortality in ins/ins genotype individuals (43.75%) at − 740 insert–deletion mutation was significantly lower than that in ins/del (72.09%) and del/del (74.19%) genotypes (P < 0.05). Linkage disequilibrium and haplotype analysis showed 6610 insert–deletion mutation and 6804 insert–deletion mutation were linkage disequilibrium. The haplotype ins–ins (6610ins–6804ins) was significantly susceptible to GCRV, and ins–del (6610ins–6804del) was significantly resistant to GCRV (P < 0.05). Those could be potential gene markers for the future molecular selection of strains that are resistant to GCRV.     

In sacco degradation kinetics of fresh and field-cured peanut (Arachis hypogaea L.) forage harvested at different maturities

There is interest in growing peanut (Arachis hypogaea L.) for forage, but little is known about the nutritive value and forage quality of modern cultivars. The objective of this study was to compare the chemical composition and in sacco degradation kinetics of three cultivars of peanuts (cv. ‘C99-R’, ‘Georgia-01R’, and ‘York’) at either stage 2 or 8 maturities when fresh and field-cured. Herbage yield was at least 3000 kg DM/ha for all cultivars at both maturities. Crude protein (CP) was greater (P < 0.0001) at R2 stage than at R8 stage; whereas, neutral detergent fiber (aNDF), acid detergent fiber, and Lignin (sa) were greater (P < 0.01) at R8 than R2 maturity stages. Water soluble carbohydrate and acid detergent insoluble nitrogen was not different (P > 0.07) among cultivars, maturity stage, or harvest forms. In vitro true digestibility was greatest (P < 0.02) for C99-R and least for York. Undegradable intake protein concentration was greatest (P < 0.04) in York and least for C99-R. Maturity had a greater effect on the degradation kinetics than harvest form or cultivar. The dry matter (DM) and CP in the soluble wash fraction (A) and insoluble but degradable fraction (B) and the effective ruminal degradability were greater among all cultivars and both harvest forms of the R2 maturity stage than the R8. The undegradable DM, aNDF, and CP in the undegradable fraction were greatest (P < 0.002) for all three cultivars at R8 maturity. The rate of degradation of DM and CP in the B fraction was faster (P < 0.001) at R2 stage than at R8 stage; whereas, rate of aNDF degradation was not different (P > 0.09) among treatments. Lag of DM, aNDF, or CP degradation was not different (P > 0.1) among treatments. The cultivars C99-R and Georgia-01R are recommended for further feeding trials.     

Null genotypes of GSTM1 and GSTT1 contribute to increased risk of diabetes mellitus: A meta-analysis

Diabetes mellitus (DM) is a common disease which results from various causes including genetic and environmental factors. Glutathione S-Transferase M1 (GSTM1) and Glutathione S-Transferase T1 (GSTT1) genes are polymorphic in human and the null genotypes lead to the absence of enzyme function. Many studies assessed the associations between GSTM1/GSTT1 null genotypes and DM risk but reported conflicting results. In order to get a more precise estimate of the associations of GSTM1/GSTT1 null genotypes with DM risk, we performed this meta-analysis. Published literature from PubMed, Embase and China Biology Medicine (CBM) databases was searched for eligible studies. Pooled odds ratios (OR) and corresponding 95% confidence intervals (95%CI) were calculated using a fixed- or random-effects model. 11 publications (a total of 2577 cases and 4572 controls) were finally included into this meta-analysis. Meta-analyses indicated that null genotypes of GSTM1/GSTT1 and dual null genotype of GSTM1–GSTT1 were all associated with increased risk of DM (GSTM1: OR _{random-effects} = 1.60, 95%CI 1.10–2.34, P_OR = 0.014; GSTT1: OR _{random-effects} = 1.47, 95%CI 1.12–1.92, P_OR = 0.005; GSTM1–GSTT1: OR _{fixed-effects} = 1.83, 95%CI 1.30–2.59, P_OR = 0.001). Subgroup by ethnicity suggested significant associations between null genotypes of GSTM1 and GSTT1 and DM risk among Asians (GSTM1: OR _{random-effects} = 1.77, 95%CI 1.24–2.53, P_OR = 0.002; GSTT1: OR _{random-effects} = 1.58, 95%CI 1.09–2.27, P_OR = 0.015). This meta-analysis suggests null genotypes of GSTM1/GSTT1 and dual null genotype of GSTM1–GSTT1 are all associated with increased risk of DM, and null genotypes of GSTM1/GSTT1 and dual null genotype of GSTM1–GSTT1 are potential biomarkers of DM.     

Presence of Nosema ceranae in honeybees (Apis mellifera) in Uruguay

The microsporidium Nosema ceranae is an emergent pathogen of European honeybees Apis mellifera. Using a PCR-RFLP diagnosis, 29 samples of infected honeybees obtained in 2007-2008 (N = 26), 2004 (N = 2) and before 1990 (N = 1) were analyzed for the presence of Nosema apis and N. ceranae. Only N. ceranae was found in all samples, indicating that this species dispersed to Uruguay (and likely the region) at some time before 1990. The presence of N. ceranae in Uruguay is not associated with an increase of Nosemosis, and its role in colony loss seems to be irrelevant.     

Field-acclimated Gossypium hirsutum cultivars exhibit genotypic and seasonal differences in photosystem II thermostability

Previous investigations have demonstrated that photosystem II (PSII) thermostability acclimates to prior exposure to heat and drought, but contrasting results have been reported for cotton (Gossypium hirsutum). We hypothesized that PSII thermotolerance in G. hirsutum would acclimate to environmental conditions during the growing season and that there would be differences in PSII thermotolerance between commercially-available U.S. cultivars. To this end, three cotton cultivars were grown under dryland conditions in Tifton Georgia, and two under irrigated conditions in Marianna Arkansas. At Tifton, measurements included PSII thermotolerance (T₁₅, the temperature causing a 15% decline in maximum quantum yield), leaf temperatures, air temperatures, midday (1200 to 1400 h) leaf water potentials (Ψ_MD), leaf-air vapor pressure deficit (VPD), actual quantum yield (Φ_PSII) and electron transport rate through PSII (ETR) on three sample dates. At Marianna, T₁₅ was measured on two sample dates. Optimal air and leaf temperatures were observed on all sample dates in Tifton, but PSII thermotolerance increased with water deficit conditions (Ψ_MD = −3.1 MPa), and ETR was either unaffected or increased under water-stress. Additionally, T₁₅ for PHY 499 was ∼5 °C higher than for the other cultivars examined (DP 0912 and DP 1050). The Marianna site experienced more extreme high temperature conditions (20–30 days T_max ≥ 35 °C), and showed an increase in T₁₅ with higher average T_max. When average T₁₅ values for each location and sample date were plotted versus average daily T_max, strong, positive relationships (r² from .954 to .714) were observed between T_max and T₁₅. For all locations T₁₅ was substantially higher than actual field temperature conditions. We conclude that PSII thermostability in G. hirsutum acclimates to pre-existing environmental conditions; PSII is extremely tolerant to high temperature and water-deficit stress; and differences in PSII thermotolerance exist between commercially-available cultivars.