Comparative genomics and transcriptomics of lineages I, II, and III strains of Listeria monocytogenes.

BACKGROUND: Listeria monocytogenes is a food-borne pathogen that causes infections with a high-mortality rate and has served as an invaluable model for intracellular parasitism. Here, we report complete genome sequences for two L. monocytogenes strains belonging to serotype 4a (L99) and 4b (CLIP80459), and transcriptomes of representative strains from lineages I, II, and III, thereby permitting in-depth comparison of genome- and transcriptome -based data from three lineages of L. monocytogenes. Lineage III, represented by the 4a L99 genome is known to contain strains less virulent for humans. RESULTS: The genome analysis of the weakly pathogenic L99 serotype 4a provides extensive evidence of virulence gene decay, including loss of several important surface proteins. The 4b CLIP80459 genome, unlike the previously sequenced 4b F2365 genome harbours an intact inlB invasion gene. These lineage I strains are characterized by the lack of prophage genes, as they share only a single prophage locus with other L. monocytogenes genomes 1/2a EGD-e and 4a L99. Comparative transcriptome analysis during intracellular growth uncovered adaptive expression level differences in lineages I, II and III of Listeria, notable amongst which was a strong intracellular induction of flagellar genes in strain 4a L99 compared to the other lineages. Furthermore, extensive differences between strains are manifest at levels of metabolic flux control and phosphorylated sugar uptake. Intriguingly, prophage gene expression was found to be a hallmark of intracellular gene expression. Deletion mutants in the single shared prophage locus of lineage II strain EGD-e 1/2a, the lma operon, revealed severe attenuation of virulence in a murine infection model. CONCLUSION: Comparative genomics and transcriptome analysis of L. monocytogenes strains from three lineages implicate prophage genes in intracellular adaptation and indicate that gene loss and decay may have led to the emergence of attenuated lineages.

NcRNA-microchip analysis: a novel approach to identify differential expression of noncoding RNAs.

Epstein-Barr virus (EBV) infection of human B cells requires the presence of non-coding RNAs (ncRNAs), which regulate expression of viral and host genes. To identify differentially expressed regulatory ncRNAs involved in EBV infection, a specialized cDNA library, enriched for ncRNAs derived from EBV-infected cells, was subjected to deep-sequencing. From the deep-sequencing analysis, we generated a custom-designed ncRNA-microchip to investigate differential expression of ncRNA candidates. By this approach, we identified 25 differentially expressed novel host-encoded ncRNA candidates in EBV-infected cells, comprised of six non-repeat-derived and 19 repeat-derived ncRNAs. Upon EBV infection of B cells, we also observed increased expression levels of oncogenic miRNAs mir-221 and mir-222, which might contribute to EBV-related tumorigenesis, as well as decreased expression levels of RNase P RNA, a ribozyme involved in tRNA maturation. Thus, in this study we demonstrate that our ncRNA-microchip approach serves as a powerful tool to identify novel differentially expressed ncRNAs acting as potential regulators of gene expression during EBV infection.

Gene expression in Fusarium graminearum grown on plant cell wall.

Fusarium graminearum is a phytopathogenic filamentous fungus attacking a wide range of plants including Humulus lupulus (hop). Transcriptional analysis of F. graminearum grown on minimal media containing hop cell wall or glucose as the sole carbon source was performed by applying a highly stringent method combining microarrays and a subtracted cDNA library. In addition to genes coding for various cell wall degrading enzymes (CWDE), several metabolic pathways were induced in response to the plant cell wall substrate. Many genes participating in these pathways are probably involved in cellular transport. But the most interesting was that all the genes composing the 4-aminobutyrate-shunt (GABA-shunt) were also up-regulated in the presence of plant cell wall material and were present in the cDNA library. This study provides a description of a part of the fungal gene expression profile when it is in contact with raw biological materials, and helps in understanding the plant cell wall degradation and the infection process.

EDR2 negatively regulates salicylic acid-based defenses and cell death during powdery mildew infections of Arabidopsis thaliana.

BACKGROUND: The hypersensitive necrosis response (HR) of resistant plants to avirulent pathogens is a form of programmed cell death in which the plant sacrifices a few cells under attack, restricting pathogen growth into adjacent healthy tissues. In spite of the importance of this defense response, relatively little is known about the plant components that execute the cell death program or about its regulation in response to pathogen attack. RESULTS: We isolated the edr2-6 mutant, an allele of the previously described edr2 mutants. We found that edr2-6 exhibited an exaggerated chlorosis and necrosis response to attack by three pathogens, two powdery mildew and one downy mildew species, but not in response to abiotic stresses or attack by the bacterial leaf speck pathogen. The chlorosis and necrosis did not spread beyond inoculated sites suggesting that EDR2 limits the initiation of cell death rather than its spread. The pathogen-induced chlorosis and necrosis of edr2-6 was correlated with a stimulation of the salicylic acid defense pathway and was suppressed in mutants deficient in salicylic acid signaling. EDR2 encodes a novel protein with a pleckstrin homology and a StAR transfer (START) domain as well as a plant-specific domain of unknown function, DUF1336. The pleckstrin homology domain binds to phosphatidylinositol-4-phosphate in vitro and an EDR2:HA:GFP protein localizes to endoplasmic reticulum, plasma membrane and endosomes. CONCLUSION: EDR2 acts as a negative regulator of cell death, specifically the cell death elicited by pathogen attack and mediated by the salicylic acid defense pathway. Phosphatidylinositol-4-phosphate may have a role in limiting cell death via its effect on EDR2. This role in cell death may be indirect, by helping to target EDR2 to the appropriate membrane, or it may play a more direct role.

The role of plant cell wall polysaccharide composition in disease resistance.

The high degree of structural complexity of plant cell wall polysaccharides has led to suggestions that some components might function as latent signal molecules that are released during pathogen infections and elicit defensive responses by the plant. However, there has been a paucity of genetic evidence supporting the idea that variation in cell wall composition plays a role in the outcome of host-pathogen interactions. Recently, several genetic studies have provided new lines of evidence implicating cell wall polysaccharides as factors in host-pathogen interactions.

Genome-wide transcriptional responses of Fusarium graminearum to plant cell wall substrates.

We report a genome-wide transcriptomic study of Fusarium graminearum grown on four different substrates based on plant cell wall components. About 5% of the genes were differentially expressed in at least one condition. Analysis of upregulated cell wall-degrading enzymes highlights a sharp growth medium-specific adaptation process. In particular, the nature of the polysaccharides available for fungal growth induced a specific transcriptional response aiming at the targeted enzymatic degradation of the given polysaccharides.

Comparative genome-wide analysis of small RNAs of major Gram-positive pathogens: from identification to application.

In the recent years, the number of drug- and multi-drug-resistant microbial strains has increased rapidly. Therefore, the need to identify innovative approaches for development of novel anti-infectives and new therapeutic targets is of high priority in global health care. The detection of small RNAs (sRNAs) in bacteria has attracted considerable attention as an emerging class of new gene expression regulators. Several experimental technologies to predict sRNA have been established for the Gram-negative model organism Escherichia coli. In many respects, sRNA screens in this model system have set a blueprint for the global and functional identification of sRNAs for Gram-positive microbes, but the functional role of sRNAs in colonization and pathogenicity for Listeria monocytogenes, Staphylococcus aureus, Streptococcus pyogenes, Enterococcus faecalis and Clostridium difficile is almost completely unknown. Here, we report the current knowledge about the sRNAs of these socioeconomically relevant Gram-positive pathogens, overview the state-of-the-art high-throughput sRNA screening methods and summarize bioinformatics approaches for genome-wide sRNA identification and target prediction. Finally, we discuss the use of modified peptide nucleic acids (PNAs) as a novel tool to inactivate potential sRNA and their applications in rapid and specific detection of pathogenic bacteria.

Microfluidic-based enzymatic on-chip labeling of miRNAs.

Small noncoding RNAs (sncRNAs) have moved from oddity to recognized important players in gene regulation. Next generation sequencing approaches discover more and more such molecules from a variety of different groups, but flexible tools translating this sequence information into affordable high-throughput assays are missing. Here we describe a microfluidic primer extension assay (MPEA) for the detection of sncRNAs on highly flexible microfluidic microarrays which combines several beneficial parameters: it can effortless incorporate any new sequence information; it is sensitive enough to work with as little as 20ng of total RNA and has a high level of specificity owing to a combination of a conventional hybridization assay and an enzymatic elongation step. Importantly, no labeling step is needed before hybridization and - because of its high sensitivity - no amplification is required. Both aspects ensure that no bias is introduced by such processes. Although the assay is exemplified with miRNAs, the flexibility of the technology platform allows the analysis of any type of sncRNA, such as piRNAs.

Reproductive mode of grape phylloxera (Daktulosphaira vitifoliae, Homoptera: Phylloxeridae) in Europe: molecular evidence for predominantly asexual populations and a lack of gene flow between them.

The genetic structure of European grape phylloxera populations, Daktulosphaira vitifoliae (Homoptera: Phylloxeridae), was analyzed using 6 polymorphic microsatellite markers. Genetic diversity data of 6 populations originating from northern and southern European viticultural regions was assessed for geographic differences, and the structure of 2 additional populations was examined in more detail, focusing on specific host plant and habitat characteristics. To test for "signatures" of clonal reproduction, different population genetic measures were applied to the data obtained from these populations. A total of 195 multilocus genotypes were detected in 360 individuals tested. Significant deviations from Hardy-Weinberg equilibrium, negative FIS values (from -0.148 to -0.658 per population), and the presence of multicopy genotypes revealed that the current major reproductive mode at each of the locations tested was asexual. The high genotypic diversity detected within and among populations, however, together with the occurrence of unique D. vitifoliae genotypes, indicates sexual recombination events took place, probably prior to the multiple introductions into Europe. The absence of overlapping genotypes between the sampling sites suggests low migration rates among the populations studied and implies that the main mode of insect dispersal is through infested plant material carried by human agency. The specific features of European D. vitifoliae habitats are illustrated to discuss the role of habitat and life cycle in the genetic structure of this globally important pest aphid species.

A role for a flavin-containing mono-oxygenase in resistance against microbial pathogens in Arabidopsis.

Using activation tagging in the Arabidopsis Col-0 rps2-101C background, we identified a mutant (FMO1-3D) that showed virtually no symptoms after inoculation with virulent Pseudomonas syringae pv. tomato DC3000 bacteria. The dominant, gain-of-function phenotype of the FMO1-3D mutant is due to over-expression of a class 3 flavin-containing mono-oxygenase (FMO). We recapitulated the FMO1-3D mutant phenotype in independent transgenic Col-0 lines over-expressing the FMO1 cDNA under the control of the 35S CaMV promoter. The increased basal resistance observed in the FMO1-3D mutant was also effective against the taxonomically unrelated downy mildew-causing pathogen Hyaloperonospora parasitica. By investigating the progeny from crosses of the FMO1-3D mutant with the NahG transgenic line, we showed that the enhanced basal resistance phenotype was dependent on the accumulation of salicylic acid. FMO1-3D plants showed wild-type resistant reactions after inoculation with avirulent bacteria, indicating that the R-gene-mediated defence physiology was not compromised by FMO1 over-expression. Transcripts of the class 3 FMO1 gene accumulated within 6 h after inoculation of wild-type Col-0 plants with avirulent Pst + avrRpt2 cells. Moreover, a T-DNA insertion into the FMO1 gene resulted in enhanced susceptibility to virulent Pseudomonas and Hyaloperonospora parasitica, suggesting that expression of the FMO1 gene is a hitherto undescribed component of the plant's resistance repertoire. We discuss the possibility that the FMO may participate in the detoxification of virulence factors produced by pathogens.

Toward a systems approach to understanding plant cell walls.

One of the defining features of plants is a body plan based on the physical properties of cell walls. Structural analyses of the polysaccharide components, combined with high-resolution imaging, have provided the basis for much of the current understanding of cell walls. The application of genetic methods has begun to provide new insights into how walls are made, how they are controlled, and how they function. However, progress in integrating biophysical, developmental, and genetic information into a useful model will require a system-based approach.