RESUMEN
In recent years Acidovorax avenae subsp. avenae was identified as a major cause of bacterial etiolation and decline (BED) in turfgrasses and has become a growing economical concern for the turfgrass industry. The symptoms of BED resemble those of "bakanae," or foolish seedling disease, of rice (Oryzae sativa), in which the gibberellins produced by the infecting fungus, Fusarium fujikuroi, contribute to the symptom development. Additionally, an operon coding for the enzymes necessary for bacterial gibberellin production was recently characterized in plant-pathogenic bacteria belonging to the γ-proteobacteria. We therefore investigated whether this gibberellin operon might be present in A. avenae subsp. avenae. A homolog of the operon has been identified in two turfgrass-infecting A. avenae subsp. avenae phylogenetic groups but not in closely related phylogenetic groups or strains infecting other plants. Moreover, even within these two phylogenetic groups, the operon presence is not uniform. For that reason, the functionality of the operon was examined in one strain of each turfgrass-infecting phylogenetic group (A. avenae subsp. avenae strains KL3 and MD5). All nine operon genes were functionally characterized through heterologous expression in Escherichia coli and enzymatic activities were analyzed by liquid chromatography-tandem mass spectrometry and gas chromatography-mass spectrometry. All enzymes were functional in both investigated strains, thus demonstrating the ability of phytopathogenic ß-proteobacteria to produce biologically active GA4. This additional gibberellin produced by A. avenae subsp. avenae could disrupt phytohormonal balance and be a leading factor contributing to the pathogenicity on turf grasses. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
Asunto(s)
Comamonadaceae , Giberelinas , Filogenia , Poaceae , Comamonadaceae/genética , PlantasRESUMEN
Type I fatty acid synthases (FASs) are known from higher eukaryotes and fungi. We report the discovery of FasT, a rare type I FAS from the cyanobacterium Chlorogloea sp. CCALA695. FasT possesses an unusual off-loading domain, which was heterologously expressed in E. coli and found to act as an α-oxoamine synthase (AOS) inâ vitro. Similar to serine palmitoyltransferases from sphingolipid biosynthesis, the AOS off-loading domain catalyzes a decarboxylative Claisen condensation between l-serine and a fatty acyl thioester. While the AOS domain was strictly specific for l-serine, thioesters with saturated fatty acyl chains of six carbon atoms and longer were tolerated, with the highest activity observed for stearoyl-coenzyme A (C18 ). Our findings suggest a novel route to α-amino ketones via the direct condensation of iteratively produced long-chain fatty acids with l-serine by a FAS with a cis-acting AOS off-loading domain.
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Escherichia coli , Serina C-Palmitoiltransferasa , Ácidos Grasos , SerinaRESUMEN
Optical imaging of gene expression by fluorescence in situ hybridisation (FISH) in insects is often impeded by their pigmented cuticle. As most chemical bleaching agents are incompatible with FISH, we developed an RNA interference (RNAi)-based method for clearing cuticular pigmentation which enables the use of whole-mount body appendages for RNA FISH (termed RNA-i-FISH). Silencing laccase2 or tyrosine hydroxylase in two leaf beetles species (Chrysomela populi and Phaedon cochleariae) cleared their pigmented cuticle and decreased light absorbance. Subsequently, intact appendages (palps, antennae, legs) from RNAi-cleared individuals were used to image the expression and spatial distribution of antisense mRNA of two chemosensory genes encoding gustatory receptor and odorant-binding protein. Imaging did not work for RNAi controls because the pigmentation was retained, or for FISH controls (sense mRNA). Several bleaching agents were incompatible with FISH, because of degradation of RNA, lack of clearing efficacy or long incubation times. Overall, silencing pigmentation genes is a significant improvement over bleaching agents, enabling FISH in intact insect appendages.
Asunto(s)
Escarabajos/genética , Silenciador del Gen , Hibridación Fluorescente in Situ/métodos , Pigmentación/genética , Interferencia de ARN/fisiología , Animales , Extremidades/fisiología , Hibridación Fluorescente in Situ/instrumentación , Pigmentos Biológicos/análisisRESUMEN
Tubuloclustin [N-(7-adamant-2-yloxy-7-oxoheptanoyl)-N-deacetylcolchicine], a highly cytotoxic anti-tubulin compound is known for its ability to promote microtubule disassembly followed by the formation of tubulin clusters of unique morphology. Three series of antimitotic agents related to tubuloclustin were designed and synthesized in order to enhance the molecular diversity of "tubuloclustin-like" family of compounds. The series of compounds with modified adamantane moiety was highly potent in cytotoxic effect on human lung carcinoma A549 cells (EC50 = 6-400 nM) and was active in affecting the microtubule arrays and induction of strong tubulin clusterization. In two other sets of compounds, the colchicine moiety of tubuloclustin was replaced by podophyllotoxin or combretastatin A-4. All combretastatin A-4 derivatives displayed noticeable cytotoxic activity ([Formula: see text]) but their effect on microtubules depended on the position of the linker attachment. Podophyllotoxin derivatives were also toxic to A549 cells ([Formula: see text]) and caused both microtubule depolymerization and some tubulin clustering. The data obtained gave additional evidence that the whole panel of C7-colchicine, podophyllotoxin and combretastatin derivatives could manifest clustering effect, and the strength of this effect correlated with cytotoxic activity of the compounds.