NodMutDB: a database for genes and mutants involved in symbiosis

SUMMARY: Functional genomics research is producing large amounts of data on the functions of individual genes related to symbiosis. We have developed a relational database, NodMutDB (Nodulation Mutant Database), to provide a comprehensive resource for depositing, organizing and retrieving information on symbiosis-related genes, mutants and published literature. NodMutDB brings together new studies and existing mutant-based literature to facilitate our understanding of how genes function in symbiotic processes in both Rhizobia and their host plants. AVAILABILITY: http://nodmutdb.vbi.vt.edu CONTACT: cmao@vbi.vt.edu SUPPLEMENTARY INFORMATION: Database schema and data curation model are available at http://nodmutdb.vbi.vt.edu.     

High-resolution mapping of genes involved in plant stage-specific partial resistance of barley to leaf rust

Partial resistance quantitative trait loci (QTLs) Rphq11 and rphq16 against Puccinia hordei isolate 1.2.1 were previously mapped in seedlings of the mapping populations Steptoe/Morex and Oregon Wolfe Barleys, respectively. In this study, QTL mapping was performed at adult plant stage for the two mapping populations challenged with the same rust isolate. The results suggest that Rphq11 and rphq16 are effective only at seedling stage, and not at adult plant stage. The cloning of several genes responsible for partial resistance of barley to P. hordei will allow elucidation of the molecular basis of this type of plant defence. A map-based cloning approach requires to fine-map the QTL in a narrow genetic window. In this study, Rphq11 and rphq16 were fine-mapped using an approach aiming at speeding up the development of plant material and simplifying its evaluation. The plant materials for fine-mapping were identified from early plant materials developed to produce QTL-NILs. The material was first selected to carry the targeted QTL in heterozygous condition and susceptibility alleles at other resistance QTLs in homozygous condition. This strategy took four to five generations to obtain fixed QTL recombinants (i.e., homozygous resistant at the Rphq11 or rphq16 QTL alleles, homozygous susceptible at the non-targeted QTL alleles). In less than 2 years, Rphq11 was fine-mapped into a 0.2-cM genetic interval and a 1.4-cM genetic interval for rphq16. The strongest candidate gene for Rphq11 is a phospholipid hydroperoxide glutathione peroxidase. Thus far, no candidate gene was identified for rphq16.     

Candidate plant gene homologues in grapevine involved in Agrobacterium transformation

The grapevine (Vitis vinifera) genome was analyzed in silico for homologues of plant genes involved in Agrobacterium transformation in Arabidopsis thaliana and Nicotiana spp. Grapevine homologues of the glucomannan 4-betamannosyltransferase 9 gene CslA-09 involved in bacterial attachment to the cell wall, homologues of reticulon-like proteins BTI1, 2, 3 and RAB8 GTPases, both involved in T-DNA transfer to the host cell, homologues of VirE2 interacting protein VIP1 that contributes to the targeting of T-DNA into the nucleus and to its integration, and homologues of the histone protein H2A, which promotes the expression of T-DNA encoded genes, were selected. Sequences homologous to the arabinogalactan-protein AtAGP17 were not found in the grape genome. Seventeen selected candidates were tested by semiquantitative RT-PCR analysis for changes in their expression levels upon inoculation with Agrobacterium tumefaciens C58. Of the tested homologues, the expression of VvRab8a, VvVip1a and two histone genes (VvHta2 and VvHta10) increased significantly, therefore we hypothesize that these might be involved in Agrobacterium transformation of V. vinifera.     

Nonsense-mediated mRNA decay in barley mutants allows the cloning of mutated genes by a microarray approach.

We have previously described a microarray approach to identify and clone genes from mutants of higher organisms. In the method cDNA of two mutants with similar phenotype are competitively hybridized to DNA clones arrayed on a glass slide. Clones corresponding to an mRNA that is not expressed in one of the strains due to a mutation will be specifically highlighted in the hybridization, which provides a possibility to identify and eventually clone the mutated gene. The approach is dependent on mutations that affect the amount of mRNA. Nonsense mutations, which prematurely terminate translation, can be such mutations as a surveillance system known as nonsense-mediated decay (NMD) has been developed by organisms to reduce the abundance of mRNA with nonsense codons. In the present study, we have analysed the barley (Hordeum vulgare L.) magnesium chelatase mutants xantha-f26, xantha-f27 and xantha-f40 in order to investigate the presence of NMD in barley, as well as the importance of the position of the stop codon for NMD. Both nonsense-mutants xantha-f27 and xantha-f40, but not the missense mutant xantha-f26, showed NMD. This was not expected for xantha-f27 as its mutation is in the last exon of the gene. We conclude the NMD expands the number of mutants that can be used for gene cloning by our described microarray approach.     

177 T-cell vaccine design for Streptococcus pneumoniae: an in silico approach

Streptococcus pneumoniae (pneumococcus) remains an important cause of meningitis, bacteremia, acute otitis media, community acquired pneumonia associated with significant morbidity, and mortality world wide. Conjugated polysaccharide, glycoconjugated, and capsular polysaccharide based vaccines were existent for pneumococcal disease but are still specific and restricted to serotypes of S. pneumoniae. Proteome of eight serotypes of S. pneumoniae was retrieved and identified in common proteins (Munikumar et al., 2012). 18 membrane proteins were distinguished from 1657 common proteins of eight serotypes of S. pneumoniae. Implementing comparative genomic approach and subtractive genomic approach, three membrane proteins were predicted as essential for bacterial survival and non-homologous to human (Munikumar et al., 2012; Umamaheswari et al., 2011). ProPred server was used to propose four promiscuous T-cell epitopes from three membrane proteins and validated through published positive control, SYFPEITHI and immune epitope database (Munikumar et al., in press). The four epitopes docked into peptide binding region of predominant HLA-DRB alleles with good binding affinity in Maestro v9.2. The T-cell epitope 89-VVYLLPILI-97 and HLA-DRB5^?0101 docking complex was with best XPG score (?13.143?kcal/mol). Further, the stability of the complex was checked through molecular dynamics simulations in Desmond v3.3. The simulation results had revealed that the complex was stable throughout 5000?ps (Munikumar et al., in press). Thus, the epitope would be the ideal candidate for T-cell driven subunit vaccine design against selected serotypes of S. pneumoniae.     

Recessiveness and dominance in barley mutants deficient in Mg-chelatase subunit D, an AAA protein involved in chlorophyll biosynthesis

Mg-chelatase catalyzes the insertion of Mg2+ into protoporphyrin IX at the first committed step of the chlorophyll biosynthetic pathway. It consists of three subunits: I, D, and H. The I subunit belongs to the AAA protein superfamily (ATPases associated with various cellular activities) that is known to form hexameric ring structures in an ATP-dependant fashion. Dominant mutations in the I subunit revealed that it functions in a cooperative manner. We demonstrated that the D subunit forms ATP-independent oligomeric structures and should also be classified as an AAA protein. Furthermore, we addressed the question of cooperativity of the D subunit with barley (Hordeum vulgare) mutant analyses. The recessive behavior in vivo was explained by the absence of mutant proteins in the barley cell. Analogous mutations in Rhodobacter capsulatus and the resulting D proteins were studied in vitro. Mixtures of wild-type and mutant R. capsulatus D subunits showed a lower activity compared with wild-type subunits alone. Thus, the mutant D subunits displayed dominant behavior in vitro, revealing cooperativity between the D subunits in the oligomeric state. We propose a model where the D oligomer forms a platform for the stepwise assembly of the I subunits. The cooperative behavior suggests that the D oligomer takes an active part in the conformational dynamics between the subunits of the enzyme.     

How plant architecture affects light absorption and photosynthesis in tomato: towards an ideotype for plant architecture using a functional-structural plant model

Background and Aims

Manipulation of plant structure can strongly affect light distribution in the canopy and photosynthesis. The aim of this paper is to find a plant ideotype for optimization of light absorption and canopy photosynthesis. Using a static functional structural plant model (FSPM), a range of different plant architectural characteristics was tested for two different seasons in order to find the optimal architecture with respect to light absorption and photosynthesis.

Methods

Simulations were performed with an FSPM of a greenhouse-grown tomato crop. Sensitivity analyses were carried out for leaf elevation angle, leaf phyllotaxis, leaflet angle, leaf shape, leaflet arrangement and internode length. From the results of this analysis two possible ideotypes were proposed. Four different vertical light distributions were also tested, while light absorption cumulated over the whole canopy was kept the same.

Key Results

Photosynthesis was augmented by 6 % in winter and reduced by 7 % in summer, when light absorption in the top part of the canopy was increased by 25 %, while not changing light absorption of the canopy as a whole. The measured plant structure was already optimal with respect to leaf elevation angle, leaflet angle and leaflet arrangement for both light absorption and photosynthesis while phyllotaxis had no effect. Increasing the length : width ratio of leaves by 1·5 or increasing internode length from 7 cm to 12 cm led to an increase of 6–10 % for light absorption and photosynthesis.

Conclusions

At high light intensities (summer) deeper penetration of light in the canopy improves crop photosynthesis, but not at low light intensities (winter). In particular, internode length and leaf shape affect the vertical distribution of light in the canopy. A new plant ideotype with more spacious canopy architecture due to long internodes and long and narrow leaves led to an increase in crop photosynthesis of up to 10 %.     

Candidate genes for behavioural ecology

In spite of millions of years of evolutionary divergence, the conservation of gene function is common across distant lineages. As such, genes that are known to influence behaviour in one organism are likely to influence similar behaviours in other organisms. Recent studies of the evolution of behaviour and morphological adaptation support this notion. Thus, the candidate gene approach offers great potential to expand our understanding of behavioural ecology. Changes in the expression of candidate genes can reveal their contribution to behavioural variation and/or phenotypic plasticity. Knowledge of gene function also enables experimental manipulation of behaviour in the lab and in the field. The candidate gene approach provides an accessible and useful tool for generating insights about animals that are not typically associated with genetic experimentation.     

Production and study of Bacillus subtilis mutants for genes involved in nucleoside catabolism]

By means of selection for a low thymine requirement the mutants fo thymine auxotrophs for deoxyriboaldolase (dra) and phosphodeoxyribomutase (drm) genes were obtained. Besides the mutants for pyrimidinenucleoside phosphorylase gene (pdp) were olso isolated using selection on the fluorodeoxyuridine resistance. The latter enzyme provides for pyrimidine nucleosides catabolism (thymidine, uridine) in Bacilli, as well as the conversion of exogenous thymine to thymidine in thymine auxotrophs. The data obtained when studying the deo-enzymes activities in various types of the mutants and also under the condition of induction by thymidine and acetoaldehyde are in accordance with the assumption that deoxyriboso-5-phosphate is an inductor of the deo-enzymes in Bacillus subtilis. The genes dra and pdp were tightly linked as it had been shown by the transformation experiments; in contrast, no linkage was revealed between dra and drm or pdp and drm. A secondary mutation (adn), not linked with dra and blocking the ability of bacteria to catabolise adenosine (purine nucleoside phosphorylase activity remains constant) was found in some dra-mutants.     

Nitrate reductase-deficient mutants in barley : immunoelectrophoretic characterization

Nitrate reductase-deficient barley (Hordeum vulgare L.) mutants were assayed for the presence of a functional molybdenum cofactor determined from the activity of the molybdoenzyme, xanthine dehydrogenase, and for nitrate reductase-associated activities. Rocket immunoelectrophoresis was used to detect nitrate reductase cross-reacting material in the mutants. The cross-reacting material levels of the mutants ranged from 8 to 136% of the wild type and were correlated with their nitrate reductase-associated activities, except for nar 1c, which lacked all associated nitrate reductase activities but had 38% of the wild-type cross-reacting material. The cross-reacting material of two nar 1 mutants, as well as nar 2a, Xno 18, Xno 19, and Xno 29, exhibited rocket immunoprecipitates that were similar to the wild-type enzyme indicating structural homology between the mutant and wild-type nitrate reductase proteins. The cross-reacting materials of the seven remaining nar 1 alleles formed rockets only in the presence of purified wild-type nitrate reductase, suggesting structural modifications of the mutant cross-reacting materials. All nar 1 alleles and Xno 29 had xanthine dehydrogenase activity indicating the presence of functional molybdenum cofactors. These results suggest that nar 1 is the structural gene for nitrate reductase. Mutants nar 2a, Xno 18, and Xno 19 lacked xanthine dehydrogenase activity and are considered to be molybdenum cofactor deficient mutants. Cross-reacting material was not detected in uninduced wild-type or mutant extracts, suggesting that nitrate reductase is synthesized de novo in response to nitrate.     

Molecular dynamics simulations of RNA: an in silico single molecule approach

RNA molecules are now known to be involved in the processing of genetic information at all levels, taking on a wide variety of central roles in the cell. Understanding how RNA molecules carry out their biological functions will require an understanding of structure and dynamics at the atomistic level, which can be significantly improved by combining computational simulation with experiment. This review provides a critical survey of the state of molecular dynamics (MD) simulations of RNA, including a discussion of important current limitations of the technique and examples of its successful application. Several types of simulations are discussed in detail, including those of structured RNA molecules and their interactions with the surrounding solvent and ions, catalytic RNAs, and RNA-small molecule and RNA-protein complexes. Increased cooperation between theorists and experimentalists will allow expanded judicious use of MD simulations to complement conceptually related single molecule experiments. Such cooperation will open the door to a fundamental understanding of the structure-function relationships in diverse and complex RNA molecules. .     

Molecular inhibition of telomerase recruitment using designer peptides: an in silico approach

Telomere holds special mechanism for solving end repair problems and maintaining genomic stability. Protection of telomeres 1 (POT1) which belongs to shelterin family is identified as a key protein that recruits telomerase by interacting with telomere repeat binding factors (TRB1-3). Since, deciphering the mechanism through which POT assembles telomerase is of great interest, computational approaches have been undertaken to understand the mechanism in a well- developed model system – Arabidopsis thaliana. As a first step, an untraditional approach was mediated to locate the active site residues on modeled AtPOT1b protein by interaction studies using molecular docking. To keep in trend with the recent developments, peptide construction and validation was promoted as the next step via molecular dynamics simulation studies. Finally, the validated peptides based on propensity score was evaluated for its efficacy as a potent inhibitor for POT and TRB1-3 interactions. The best peptide, namely, (1-2-d) out of 30 designed peptides, was proved to be vital inhibitor by weakening the interacting complexes.