Design,synthesis, docking,Hirshfeld surface analysis and DFT calculations of 2-methylxanthen-9-with the FtsZ protein from Staphylococcus aureus

It is of interest to document the design, synthesis, docking, Hirshfeld surface analysis and DFT calculations of 2-methylxanthen-9-with the FtsZ protein (PDB ID: 3VOB) from Staphylococcus aureus for antimicrobial applications. We report the quantitative structure function data in this context.     

Detecting single nucleotide polymorphisms using DNA arrays for plant pathogen diagnosis

The lack of a rapid and reliable means for routine pathogen identification has been one of the main limitations in plant disease management, and has pushed the development of culture-independent, molecular approaches. Currently, DNA array technology is the most suitable technique for high-throughput detection and identification, as well as quantification, of multiple pathogens in a single assay. Closely related pathogens that may have completely different host ranges or pathogenicity often differ in only a single to a few base pairs in genes that may be targeted for identification. Therefore, the ability to discriminate single nucleotide polymorphisms (SNPs) should be pursued in any diagnostic assay. In this paper, we demonstrate the utility of DNA array technology to detect SNPs while accounting for specific criteria such as the position of the mismatch, the sequence of the oligonucleotide, and the length and amount of labeled amplicons that are hybridized. When disregarding mismatches at the extreme ends of the oligonucleotides, cross hybridization to single mismatch oligonucleotides is rare when processing environmental samples that contain genetic material from unknown sources. In addition to plant pathology, this study is relevant for any field of research where DNA arrays are used to detect mutations or polymorphisms, ranging from human diagnostics to environmental microbiology and microbial ecology.     

Mycorrhizal associations and reproductive isolation in three closely related Orchis species

Background and Aims

The maintenance of species boundaries in sympatric populations of closely related species requires some kind of reproductive isolation that limits gene flow among species and/or prevents the production of viable progeny. Because in orchids mycorrhizal fungi are needed for seed germination and subsequent seedling establishment, orchid–mycorrhizal associations may be involved in acting as a post-mating barrier.

Methods

We investigated the strength of post-mating barriers up to the seed germination stage acting between three closely related Orchis species (Orchis anthropophora, O. militaris and O. purpurea) and studied the role of mycorrhizal fungi in hybridization by burying seed packets of pure and hybrid seeds. After retrieval and assessment of seed germination, the fungi associating with protocorms originating from hybrid and pure seeds were determined and compared with those associating with adult individuals using DNA array technology.

Results

Whereas pre-zygotic post-mating barriers were rather weak in most crosses, post-zygotic post-mating barriers were stronger, particularly when O. purpurea was crossed with O. anthropophora. Germination trials in the field showed that seed germination percentages of hybrid seeds were in most cases lower than those originating from pure crosses. In all species pair combinations, total post-mating reproductive isolation was asymmetric. Protocorms associated with a smaller range of fungal symbionts than adult plants, but there was considerable overlap in mycorrhizal associations between protocorms and their respective parents.

Conclusions

Our results suggest that mycorrhizal associations contribute little to reproductive isolation. Pre-mating barriers are probably the main factors determining hybridization rates between the investigated species.     

Cloning and expression in Escherichia coli of the Azospirillum brasilense Sp7 gene encoding ampicillin resistance

The Azospirillum brasilense ATCC 29145 gene coding for beta-lactamase was cloned in Escherichia coli. The gene was expressed in E. coli from its own promoter as a 30-kilodalton protein, conferring resistance to high levels of beta-lactam antibiotics. The DNA sequence containing the beta-lactamase gene was found to be highly amplified in the Azospirillum genome, scattered in the chromosomal as well as in the plasmidic DNA.     

Interactions of antifungal plant defensins with fungal membrane components

Plant defensins are small, basic, cysteine-rich peptides that are generally active against a broad spectrum of fungal and yeast species at micromolar concentrations. Some of these defensins interact with fungal-specific lipid components in the plasmamembrane. Structural differences of these membrane components between fungal and plant cells probably account for the selective activity of plant defensins against fungal pathogens and their nonphytotoxic properties. This review will focus on different classes of complex lipids in fungal membranes and on the selective interaction of plant defensins with these complex lipids.     

Miconazole induces changes in actin cytoskeleton prior to reactive oxygen species induction in yeast

The antifungal compound miconazole inhibits ergosterol biosynthesis and induces reactive oxygen species (ROS) in susceptible yeast species. To further uncover the mechanism of miconazole antifungal action and tolerance mechanisms, we screened the complete set of haploid Saccharomyces cerevisiae gene deletion mutants for mutants with an altered miconazole sensitivity phenotype. We identified 29 S. cerevisiae genes, which when deleted conferred at least 4-fold hypersensitivity to miconazole. Major functional groups encode proteins involved in tryptophan biosynthesis, membrane trafficking including endocytosis, regulation of actin cytoskeleton, and gene expression. With respect to the antifungal activity of miconazole, we demonstrate an antagonism with tryptophan and a synergy with a yeast endocytosis inhibitor. Because actin dynamics and induction of ROS are linked in yeast, we further focused on miconazole-mediated changes in actin cytoskeleton organization. In this respect, we demonstrate that miconazole induces changes in the actin cytoskeleton, indicative of increased filament stability, prior to ROS induction. These data provide novel mechanistic insights in the mode of action of a ROS-inducing azole.     

The influence of matrix attachment regions on transgene expression in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> wild type and gene silencing mutants

Many studies in both animal and plant systems have shown that matrix attachment regions (MARs) can increase the expression of flanking transgenes. However, our previous studies revealed no effect of the chicken lysozyme MARs (chiMARs) on transgene expression in the first generation transgenic Arabidopsis thaliana plants transformed with a β-glucuronidase gene (uidA) unless gene silencing mutants were used as genetic background for transformation. In the present study, we investigated why chiMARs do not influence transgene expression in transgenic wild-type Arabidopsis plants. We first studied the effect of chiMARs on transgene expression in the progeny of primary transformants harboring chiMAR-flanked T-DNAs. Our data indicate that chiMARs do not affect transgene expression in consecutive generations of wild-type A. thaliana plants. Next, we examined whether these observed results in A. thaliana transformants are influenced by the applied transformation method. The results from in vitro transformed A. thaliana plants are in accordance with those from in planta transformed A. thaliana plants and again reveal no influence of chiMARs on transgene expression in A. thaliana wild-type transformants. The effect of chiMARs on transgene expression is also examined in in vitro transformed Nicotiana tabacum plants, but as for A. thaliana, the transgene expression in tobacco transformants is not altered by the presence of chiMARs. Taken together, our results show that the applied method or the plant species used for transformation does not influence whether and how chiMARs have an effect on transgene expression. Finally, we studied the effect of MARs (tabMARs) of plant origin (tobacco) on the transgene expression in A. thaliana wild-type plants and suppressed gene silencing (sgs2) mutants. Our results clearly show that similar to chiMARs, the tobacco-derived MARs do not enhance transgene expression in a wild-type background but can be used to enhance transgene expression in a mutant impaired in gene silencing. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users. Miguel F.C. De Bolle, Katleen M.J. Butaye Contributed equally to this work