Prophenoloxidase Activation Is Required for Survival to Microbial Infections in Drosophila

The melanization reaction is a major immune response in Arthropods and involves the rapid synthesis of melanin at the site of infection and injury. A key enzyme in the melanization process is phenoloxidase (PO), which catalyzes the oxidation of phenols to quinones, which subsequently polymerize into melanin. The Drosophila genome encodes three POs, which are primarily produced as zymogens or prophenoloxidases (PPO). Two of them, PPO1 and PPO2, are produced by crystal cells. Here we have generated flies carrying deletions in PPO1 and PPO2. By analyzing these mutations alone and in combination, we clarify the functions of both PPOs in humoral melanization. Our study shows that PPO1 and PPO2 are responsible for all the PO activity in the hemolymph. While PPO1 is involved in the rapid early delivery of PO activity, PPO2 is accumulated in the crystals of crystal cells and provides a storage form that can be deployed in a later phase. Our study also reveals an important role for PPO1 and PPO2 in the survival to infection with Gram-positive bacteria and fungi, underlining the importance of melanization in insect host defense.     

Proteomic signature of Lactococcus lactis NCDO763 cultivated in milk

We have compared the proteomic profiles of L. lactis subsp. cremoris NCDO763 growing in the synthetic medium M17Lac, skim milk microfiltrate (SMM), and skim milk. SMM was used as a simple model medium to reproduce the initial phase of growth of L. lactis in milk. To widen the analysis of the cytoplasmic proteome, we used two different gel systems (pH ranges of 4 to 7 and 4.5 to 5.5), and the proteins associated with the cell envelopes were also studied by two-dimensional electrophoresis. In the course of the study, we analyzed about 800 spots and identified 330 proteins by mass spectrometry. We observed that the levels of more than 50 and 30 proteins were significantly increased upon growth in SMM and milk, respectively. The large redeployment of protein synthesis was essentially associated with an activation of pathways involved in the metabolism of nitrogenous compounds: peptidolytic and peptide transport systems, amino acid biosynthesis and interconversion, and de novo biosynthesis of purines. We also showed that enzymes involved in reactions feeding the purine biosynthetic pathway in one-carbon units and amino acids have an increased level in SMM and milk. The analysis of the proteomic data suggested that the glutamine synthetase (GS) would play a pivotal role in the adaptation to SMM and milk. The analysis of glnA expression during growth in milk and the construction of a glnA-defective mutant confirmed that GS is an essential enzyme for the development of L. lactis in dairy media. This analysis thus provides a proteomic signature of L. lactis, a model lactic acid bacterium, growing in its technological environment.     

Characterization of proteins secreted during maize microspore culture: arabinogalactan proteins (AGPs) stimulate embryo development

To study molecules secreted from cultured plant cells that promote development, maize microspores were transferred into culture and the conditioned media were collected over time and analysed. Electrophoresis indicated that both non-glycosylated and glycosylated proteins including arabinogalactan proteins (AGPs) appeared in the medium and their concentration increased during the time of culture. The development of embryos was correlated with the presence of specific extracellular proteins, using an experimental system based on a tunicamycin inhibition test. In addition, a precise protein analysis was conducted using MALDI-TOF and ESI-MS-MS techniques. These approaches have allowed the identification of 5 other types of proteins: a cell wall invertase, two thaumatin isoforms, one 1-3 beta-glucanase and two chitinase isoforms. Altogether these experiments and results open ways for research aimed at understanding which molecules stimulate embryo formation. Moreover, AGPs may be used to stimulate the development of microspores (pollen embryogenesis) prepared from non-responsive genotypes.     

Effect of Inoculum Size on In Vitro Susceptibility Testing with Lincomycin

There is disagreement in the literature as to whether lincomycin is primarily a bacteriostatic or a bactericidal agent against gram-positive cocci and also regarding the levels of activity of this agent against susceptible microorganisms. These questions were examined in a study of the effect of inoculum size on the results of tube dilution susceptibility determinations with lincomycin against 49 clinical isolates of Staphylococcus aureus and 25 strains of streptococci and pneumococci. Lincomycin was both highly active and bactericidal when tested against 40 strains of S. aureus with inocula containing a maximum of 10(4) cells per ml [median minimal inhibitory concentration (MIC), 0.78 mug/ml; median minimal bactericidal concentration (MBC), 1.56 mug/ml]. With inocula of 10(5) cells per ml, lincomycin was primarily bacteriostatic (median MIC, 1.56 mug/ml; median MBC, 12.5 mug/ml). There were further decreases in inhibitory levels and significant losses of bactericidal activity when inocula containing more than 10(7) cells were tested (median MIC, 3.13 mug/ml; median MBC > 100 mug/ml). Similar measurements with streptococci and pneumococci revealed a lesser effect of inoculum size. The mean MBC value for alpha-hemolytic streptococci increased from 0.40 to 1.05 mug/ml with an increase in inocula from 10(4) to 10(6) cells per ml, but without a marked increase in MIC values. Similar results were obtained for beta-hemolytic streptococci and pneumococci.     

A QTL detected in an interspecific pear population confers stable fire blight resistance across different environments and genetic backgrounds

Fire blight, caused by the bacterium Erwinia amylovora (Burrill) Winslow et al., is one of the most serious diseases of pear. The development of pear cultivars with a durable resistance is extremely important for effective control of fire blight and is a key objective of most pear breeding programs throughout the world. We phenotyped seedlings from the interspecific pear population PEAR3 (PremP003, P. × bretschneideri × P. communis) × ‘Moonglow’ (P. communis) for fire blight resistance at two different geographic locations, in France and New Zealand, respectively, employing two local E. amylovora isolates. Using a genetic map constructed with single nucleotide polymorphism (SNP) and microsatellite (SSR) markers previously developed for this segregating population, we detected a major quantitative trait locus (QTL) on linkage group (LG)2 of ‘Moonglow’ (R ² = 12.9–34.4 %), which was stable in both environments. We demonstrated that this QTL co-localizes with another major QTL for fire blight resistance previously detected in ‘Harrow Sweet’ and that the two favorable (i.e., resistant) alleles were not identical by descent. We also identified some smaller effect (R ² = 8.1–14.8 %) QTLs derived from the susceptible parent PEAR3. We propose SNP and SSR markers linked to the large effect QTL on LG2 as candidates for marker-assisted breeding for fire blight resistance in pear.     

Development of analytical tools for evaluating the effect of T-DNA chimeric integration on transgene expression in vegetatively propagated plants

T-DNA chimeric integration and unexpected transgene expression are relevant constraints affecting transgenic plants. This study aims to properly investigate the occurrence of these events and to what extent they may be related. The final goal is to develop an effective screening tool for earlier selection of proper transgenic lines. A strategy based on qPCR and Southern blot was adopted for evaluating gus and Egfp chimerism degree in transgenic Vitis vinifera cv ‘Chardonnay’. Of nine transgenic lines, one had a very high chimerism value, which was shown to be associated with minimal transgene expression. The evaluation of the gus gene over time and space on a line selected as a model showed that transgene’s chimerism was stable and uniform throughout plant tissues whilst its expression was highly variable. Transgene chimerism issue was investigated in detail and useful hints were given for selecting the most favorable transgenic plants and for proper planning of in vitro and ex vitro experiments.     

Identification of a major quantitative trait locus for resistance to fire blight in the wild apple species Malus fusca

Fire blight, caused by the Gram-negative bacterium Erwinia amylovora, is the most important bacterial disease affecting apple (Malus × domestica) and pear (Pyrus communis) production. The use of antibiotic treatment, though effective to some degree, is forbidden or strictly regulated in many European countries, and hence an alternative means of control is essential. The planting of fire blight-resistant cultivars seems to be a highly feasible strategy. In this study, we explored a segregating population derived from a cross between the wild apple species Malus fusca and the M. × domestica cultivar Idared. F1 progenies used for mapping were artificially inoculated with Erwinia amylovora strain Ea222_JKI at a concentration of 10⁹ cfu/ml in three different years. The averages of percentage lesion length of all replicates of each genotype were used as numerical traits for statistical analysis. A Kruskal–Wallis analysis was used to determine marker–phenotype association and revealed a linkage group with Diversity Arrays Technology (DArT) markers significantly linked with fire blight. After locating the positions of the DArT markers on the Golden Delicious genome, simple sequence repeat (SSR) markers were developed from chromosome 10 to replace the DArT markers and to determine the quantitative trait locus (QTL) region. Multiple QTL mapping (MQM) revealed a strong QTL (Mfu10) on linkage group 10 of M. fusca explaining about 65.6 % of the phenotypic variation. This is the first report on a fire blight resistance QTL of M. fusca.     

Structure and expression analysis of rice paleo duplications

Having a well-known history of genome duplication, rice is a good model for studying structural and functional evolution of paleo duplications. Improved sequence alignment criteria were used to characterize 10 major chromosome-to-chromosome duplication relationships associated with 1440 paralogous pairs, covering 47.8% of the rice genome, with 12.6% of genes that are conserved within sister blocks. Using a micro-array experiment, a genome-wide expression map has been produced, in which 2382 genes show significant differences of expression in root, leaf and grain. By integrating both structural (1440 paralogous pairs) and functional information (2382 differentially expressed genes), we identified 115 paralogous gene pairs for which at least one copy is differentially expressed in one of the three tissues. A vast majority of the 115 paralogous gene pairs have been neofunctionalized or subfunctionalized as 88%, 89% and 96% of duplicates, respectively, expressed in grain, leaf and root show distinct expression patterns. On the basis of a Gene Ontology analysis, we have identified and characterized the gene families that have been structurally and functionally preferentially retained in the duplication showing that the vast majority (>85%) of duplicated have been either lost or have been subfunctionalized or neofunctionalized during 50–70 million years of evolution.