Current status of Potyvirus in India

Potyvirus particles are flexuous rods of 700–900?nm in length and contain one positive sense single-stranded genomic RNA molecule of approximately 10?kb, which is encapsidated by a single type of coat protein. According to available NCBI database, Potyvirus infection is prominently present in Solanaceae, Leguminosae and Cucurbitaceae families in India. Potyviruses can induce a wide range of different symptoms in infected host plants including mosaic, stripe, mottling, vein clearing, vein banding, ringspots, necrotic or chlorotic lesions, flower breaking, stunting, wilting, and most commonly lead tostunting and yield losses. PCR-based methods for the detection and identification of potyviruses rely on degenerate primers designed for conserved regions. Potyvirus infection requires the interaction of host factors with viral proteins and RNA for its replication and systemic spread, i.e interaction between VPg and eIF4E is required for Potyvirus genome translation. Mutations in host translational initiation factor eIF4E cause the conformational shift in encoded proteins which are unable to bind with viral protein (VPg), resulting in broad-spectrum Potyvirus resistance.     

Molecular in silico structure and recombination analysis of betasatellite in Calotropis procera associated with begomovirus

The uncharacterised betasatellite of begomovirus associated with Calotropis procera was characterised by using molecular and in silico tools and techniques. Attempts to identify the presence of a DNA-β in the infected C. procera samples, using rolling circular amplification (RCA) followed by restriction digestion, produced a ca. 1.4 kb product, corresponding to that expected for a full-length amplicon from a betasatellite, which was sequenced (accession number HQ631430). During BLASTp, analysis of second reading frame of HQ631430 (HQ631430/2-f) against Protein Databank revealed 35% identity with Tryptophanyl–tRNA synthetase of Giardia lamblia (3FOC). Ramachandran plot of HQ631430/2-f.pdb had only 57.1% residues in the most favoured region while 3FOC.pdb had 94.2% residues in the most favoured region; therefore, only template 3FOC.pdb model could be placed in good quality category. The protein binding function was predicted for HQ631430/2-f as an important functional site of the model with 0.29 confidence level through 3d2GO. The Croton yellow vein mosaic betasatellite (GU111995 CroYVMB) serve as major parent and Croton yellow vein mosaic betasatellite-Panipat 8 (HM143908 PaLCuVM) as minor parent for HQ631430. Perhaps this is the first report of recombination in Croton yellow vein mosaic betasatellite (HQ631430).     

Mechanistic Insights into the Neutralization of Cytotoxic Abrin by the Monoclonal Antibody D6F10

Abrin, an A/B toxin obtained from the Abrus precatorius plant is extremely toxic and a potential bio-warfare agent. Till date there is no antidote or vaccine available against this toxin. The only known neutralizing monoclonal antibody against abrin, namely D6F10, has been shown to rescue the toxicity of abrin in cells as well as in mice. The present study focuses on mapping the epitopic region to understand the mechanism of neutralization of abrin by the antibody D6F10. Truncation and mutational analysis of abrin A chain revealed that the amino acids 74–123 of abrin A chain contain the core epitope and the residues Thr112, Gly114 and Arg118 are crucial for binding of the antibody. In silico analysis of the position of the mapped epitope indicated that it is present close to the active site cleft of abrin A chain. Thus, binding of the antibody near the active site blocks the enzymatic activity of abrin A chain, thereby rescuing inhibition of protein synthesis by the toxin in vitro. At 1∶10 molar concentration of abrin:antibody, the antibody D6F10 rescued cells from abrin-mediated inhibition of protein synthesis but did not prevent cell attachment of abrin. Further, internalization of the antibody bound to abrin was observed in cells by confocal microscopy. This is a novel finding which suggests that the antibody might function intracellularly and possibly explains the rescue of abrin’s toxicity by the antibody in whole cells and animals. To our knowledge, this study is the first report on a neutralizing epitope for abrin and provides mechanistic insights into the poorly understood mode of action of anti-A chain antibodies against several toxins including ricin.     

Identification and characterization of Neopestalotiopsis fungi associated with a novel leaf fall disease of rubber trees (Hevea brasiliensis) in Thailand

A novel leaf fall disease of rubber trees (Hevea brasiliensis) has been recently noted in Thailand. The fungal pathogens of this disease were identified based on both morphological and molecular characteristics as Neopestalotiopsis cubana and N. formicarum. Portions of internal transcribed spacer (ITS) region and the large subunit (LSU), translation elongation factor 1-α (TEF1-α) and β-tubulin (TUB) genes were PCR amplified with the primer pairs ITS1/ITS4, LR0R/LR5, EF1-728F/EF2 and T1/Bt2b, respectively. Sequencing of the PCR products and a phylogenetic tree based on the combined ITS, TEF1-α and TUB confirmed these pathogens as N. cubana and N. formicarum. Pathogenicity test results showed that the pathogens cause leaf spot and leaf fall similar to that observed in natural infections. This is the first report on the novel leaf fall disease of rubber trees in Thailand, with the results demonstrating that it is associated with N. cubana and N. formicarum.     

Genome‐wide analysis of polymorphisms identified domestication‐associated long low‐diversity region carrying important rice grain size/weight quantitative trait loci

Rice grain size and weight are major determinants of grain quality and yield and so have been under rigorous selection since domestication. However, the genetic basis for contrasting grain size/weight trait among Indian germplasms and their association with domestication‐driven evolution is not well understood. In this study, two long (LGG) and two short grain (SGG) genotypes were resequenced. LGG (LGR and PB 1121) differentiated from SGG (Sonasal and Bindli) by 504 439 single nucleotide polymorphisms (SNPs) and 78 166 insertion‐and‐deletion polymorphisms. The LRK gene cluster was different and a truncation mutation in the LRK8 kinase domain was associated with LGG. Phylogeny with 3000 diverse rice accessions revealed that the four sequenced genotypes belonged to the japonica group and were at the edge of the clades indicating them to be the potential source of genetic diversity available in Indian rice germplasm. Six SNPs were significantly associated with grain size/weight and the top four of these could be validated in mapping a population, suggesting this study as a valuable resource for high‐throughput genotyping. A contiguous long low‐diversity region (LDR) of approximately 6 Mb carrying a major grain weight quantitative trait loci (harbouring OsTOR gene) was identified on Chromosome 5. This LDR was identified as an evolutionary important site with significant positive selection and multiple selection sweeps, and showed association with many domestication‐related traits, including grain size/weight. The aus population retained more allelic variations in the LDR than the japonica and indica populations, suggesting it to be one of the divergence loci. All the data and analyses can be accessed from the RiceSzWtBase database.     

The catalase activity of diiron adenine deaminase

Adenine deaminase (ADE) from the amidohydrolase superfamily (AHS) of enzymes catalyzes the conversion of adenine to hypoxanthine and ammonia. Enzyme isolated from Escherichia coli was largely inactive toward the deamination of adenine. Molecular weight determinations by mass spectrometry provided evidence that multiple histidine and methionine residues were oxygenated. When iron was sequestered with a metal chelator and the growth medium supplemented with Mn²⁺ before induction, the post-translational modifications disappeared. Enzyme expressed and purified under these conditions was substantially more active for adenine deamination. Apo-enzyme was prepared and reconstituted with two equivalents of FeSO₄. Inductively coupled plasma mass spectrometry and Mössbauer spectroscopy demonstrated that this protein contained two high-spin ferrous ions per monomer of ADE. In addition to the adenine deaminase activity, [Fe^II/Fe^II]-ADE catalyzed the conversion of H₂O₂ to O₂ and H₂O. The values of k_cat and k_cat/K_m for the catalase activity are 200 s⁻¹ and 2.4 × 10⁴ M⁻¹ s⁻¹, respectively. [Fe^II/Fe^II]-ADE underwent more than 100 turnovers with H₂O₂ before the enzyme was inactivated due to oxygenation of histidine residues critical for metal binding. The iron in the inactive enzyme was high-spin ferric with g_ave = 4.3 EPR signal and no evidence of anti-ferromagnetic spin-coupling. A model is proposed for the disproportionation of H₂O₂ by [Fe^II/Fe^II]-ADE that involves the cycling of the binuclear metal center between the di-ferric and di-ferrous oxidation states. Oxygenation of active site residues occurs via release of hydroxyl radicals. These findings represent the first report of redox reaction catalysis by any member of the AHS.     

Validation and application of Drosophila melanogaster as an in vivo model for the detection of double strand breaks by neutral Comet assay

Comet assay under neutral conditions allows detection of DNA double-strand breaks (DSBs), which has consequence to genome instability and carcinogenesis. The present study aims to validate the neutral Comet assay for genotoxicity assessment in Drosophila melanogaster (Oregon R(+)) with three well known DSBs inducers i.e. cyclophosphamide (CP), bleomycin (BLM), cisplatin (CPT) and subsequently its efficacy in detecting DSBs in the organism exposed to a well known environmental chemical, chromium [Cr(VI)]. Third instar larvae of D. melanogaster were fed different concentrations of BLM, CPT and CP (50.0-200.0μg/ml) or Cr(VI) (5.0-20.0μg/ml) mixed standard Drosophila food for 48h. Neutral Comet assay was performed in cells of mid gut and brain from control and treated larvae. Our results show a dose-dependent increase in the migration of DNA in cells of the exposed organisms. A comparison among DNA lesions per mole number of the test chemical in the exposed groups showed that both BLM and CPT induce more DSBs than CP. Interestingly, Cr(VI) at 20.0μg/ml was found to induce significantly increased (p<0.001) DSBs in the exposed organism as compared to the control. The study while validating neutral Comet assay in D. melanogaster suggests its use for in vivo assessment of environmental chemical induced DSBs.     

Tracing the tracks of genotoxicity by trivalent and hexavalent chromium in Drosophila melanogaster

Mutagen sensitive strains (mus) in Drosophila are known for their hypersensitivity to mutagens and environmental carcinogens. Accordingly, these mutants were grouped in pre- and post-replication repair pathways. However, studying mutants belonging to one particular repair pathway may not be adequate for examining chemical-induced genotoxicity when other repair pathways may neutralize its effect. To test whether both pre-and post-replication pathways are involved and effect of Cr(III)- and Cr(VI)-induced genotoxicity in absence or presence of others, we used double mutant approach in D. melanogaster. We observed DNA damage as evident by changes in Comet assay DNA migration in cells of larvae of Oregon R(+) and single mutants of pre- (mei-9, mus201 and mus210) and post- (mei-41, mus209 and mus309) replication repair pathways and also in double mutants of different combinations (pre-pre, pre-post and post-post replication repair) exposed to increasing concentrations of Cr(VI) (0.0, 5.0, 10.0 and 20.0 μg/ml) for 48 h. The damage was greater in pre-replication repair mutants after exposure to 5.0 μg/ml Cr(VI), while effects on Oregon R(+) and post replication repair mutants were insignificant. Post-replication repair mutants revealed significant DNA damage after exposure to 20.0 μg/ml Cr(VI). Further, double mutants generated in the above repair categories were examined for DNA damage following Cr(VI) exposure and a comparison of damage was studied between single and double mutants. Combinations of double mutants generated in the pre-pre replication repair pathways showed an indifferent interaction between the two mutants after Cr(VI) exposure while a synergistic interaction was evident in exposed post-post replication repair double mutants. Cr(III) (20.0 μg/ml) exposure to these strains did not induce any significant DNA damage in their cells. The study suggests that both pre- and post-replication pathways are affected in Drosophila by Cr(VI) leading to genotoxicity, which may have consequences for metal-induced carcinogenesis.     

alpha-Synuclein-synaptosomal membrane interactions: implications for fibrillogenesis.

alpha-Synuclein exists in two different compartments in vivo-- correspondingly existing as two different forms: a membrane-bound form that is predominantly alpha-helical and a cytosolic form that is randomly structured. It has been suggested that these environmental and structural differences may play a role in aggregation propensity and development of pathological lesions observed in Parkinson's disease (PD). Such effects may be accentuated by mutations observed in familial PD kindreds. In order to test this hypothesis, wild-type and A53T mutant alpha-synuclein interactions with rat brain synaptosomal membranes were examined. Previous data has demonstrated that the A30P mutant has defective lipid binding and therefore was not examined in this study. Electron microscopy demonstrated that wild-type alpha-synuclein fibrillogenesis is accelerated in the presence of synaptosomal membranes whereas the A53T alpha-synuclein fibrillogenesis is inhibited under the same conditions. These results suggested that subtle sequence changes in alpha-synuclein could significantly alter interaction with membrane bilayers. Fluorescence and absorption spectroscopy using environment sensitive probes demonstrated variations in the inherent lipid properties in the presence and absence of alpha-synuclein. Addition of wild-type alpha-synuclein to synaptosomes did not significantly alter the membrane fluidity at either the fatty acyl chains or headgroup space, suggesting that synaptosomes have a high capacity for alpha-synuclein binding. In contrast, synaptosomal membrane fluidity was decreased by A53T alpha-synuclein binding with concomitant packing of the lipid headgroups. These results suggest that alterations in alpha-synuclein-lipid interactions may contribute to physiological changes detected in early onset PD.     

Covariation and composition of arthropod species across plant genotypes of evening primrose, Oenothera biennis

Genetic variation in plants has broad implications for both the ecology and evolution of species interactions. We addressed how a diverse community of arthropod species covary in abundance among plant genotypes of a native herbaceous plant ( Oenothera biennis ), and if these effects scale-up to shape the composition, diversity, and total abundance of arthropods over the entire lifetime of plants (two years). In a field experiment, we replicated 14 plant genotypes of O. biennis across five field habitats and studied the arthropod communities that naturally colonized plants. Genetic variation in O. biennis affected the abundance of 45% of the eleven common species in 2002, and 75% of sixteen common species in 2003. We examined the strength of correlations in mean abundance of arthropod species among plant genotypes and found that species responded independently to variation among genotypes in the first year of the study, whereas species formed positively covarying clusters of taxa in the second year (r_mean=0.35). The strength of these correlations did not consistently correspond to either taxonomy or functional attributes of the different species. The effects of plant genetic variation on the abundance and covariation of multiple arthropod species was associated with cascading effects on higher levels of community organization, as plant genotype and habitat interacted to affect the species composition, diversity, and total abundance of arthropods in both 2002 and 2003, though the specific effects varied across years. Our results suggest that plants may employ generalized resistance strategies effective against multiple herbivores, but such strategies are unlikely to be effective against entire functional groups of species. Moreover, we show that genotypic variation in plants is an important ecological factor that affects multiple levels of community organization, but the effects of plant genotype vary in both space and time.