Syringolin A: action on plants, regulation of biosynthesis, and phylogenetic occurrence of structurally related compounds

Syringolin A, the product of a mixed non-ribosomal peptide/polyketide synthetase, is secreted by Pseudomonas syringae pv. syringae under in planta conditions and is one of the molecular determinants recognized by nonhost plant species. Spray application of syringolin A onto powdery mildew-infected wheat and Arabidopsis has the remarkable effect of reprogramming epidermal cells that are colonized by the powdery mildew fungi Blumeria graminis f. sp. tritici and Erysiphe cichoracearum, respectively, in a compatible interaction to undergo hypersensitive cell death. No hypersensitive cell death is observed if the compound is applied onto uninfected plants. Transcriptome analyses in wheat and Arabidopsis with regard to powdery mildew inoculation and/or syringolin A spraying lead to a hypothesis about how syringolin A may accomplish to induce the hypersensitive reaction (HR) in colonized cells. The model is supported by transcriptome analyis of an Arabidopsis mutant in which HR is not induced upon syringolin A spraying of powdery mildew-infected plants. Cloning of the syringolin A synthetase genes has allowed us to build a detailed model of syringolin A synthesis based on the gene structure. This model in turn enabled us to clone the genes responsible for the synthesis of glidobactins (syn. cepafungins), antibiotics with a structure related to syringolin A that were reported to have antitumor activity, from an unknown species belonging to the order Burkholderiales. Comparisons to the approximately 700 complete eubacterial genomic sequences known resulted in the identification of a small but very intriguing group of pathogenic bacteria postulated to produce glidobacting-like molecules

Activity enhancement of the synthetic syrbactin proteasome inhibitor hybrid and biological evaluation in tumor cells

Syrbactins belong to a recently emergent class of bacterial natural product inhibitors that irreversibly inhibit the proteasome of eukaryotes by a novel mechanism. The total syntheses of the syrbactin molecules syringolin A, syringolin B, and glidobactin A have been achieved, which allowed the preparation of syrbactin-inspired derivatives, such as the syringolin A-glidobactin A hybrid molecule (SylA-GlbA). To determine the potency of SylA-GlbA, we employed both in vitro and cell culture-based proteasome assays that measure the subcatalytic chymotrypsin-like (CT-L), trypsin-like (T-L), and caspase-like (C-L) activities. We further studied the inhibitory effects of SylA-GlbA on tumor cell growth using a panel of multiple myeloma, neuroblastoma, and ovarian cancer cell lines and showed that SylA-GlbA strongly blocks the activity of NF-kappa B. To gain more insights into the structure-activity relationship, we cocrystallized SylA-GlbA in complex with the proteasome and determined the X-ray structure. The electron, density map displays covalent binding of the Thr1 O-gamma atoms of all active sites to the macrolactam ring of the ligand via ether bonds formation, thus providing insights into the structure-activity relationship for the improved affinity of SylA-GlbA for the CT-L activity compared to those of the natural compounds SylA and GlbA. Our study revealed that the novel synthetic syrbactin compound represents one of the most potent proteasome inhibitors analyzed to date and therefore exhibits promising properties for improved drug development as an anticancer therapeutic

Analysis of epidermis- and mesophyll-specific transcript accumulation in powdery mildew-inoculated wheat leaves.

Powdery mildew is an important disease of wheat caused by the obligate biotrophic fungus Blumeria graminis f. sp. tritici. This pathogen invades exclusively epidermal cells after penetrating directly through the cell wall. Because powdery mildew colonizes exclusively epidermal cells, it is of importance not only to identify genes which are activated, but also to monitor tissue specificity of gene activation. Acquired resistance of wheat to powdery mildew can be induced by a previous inoculation with the non-host pathogen B. graminis f. sp. hordei, the causal agent of barley powdery mildew. The establishment of the resistant state is accompanied by the activation of genes. Here we report the tissue-specific cDNA-AFLP analysis and cloning of transcripts accumulating 6 and 24 h after the resistance-inducing inoculation with B. graminis f. sp. hordei. A total of 25,000 fragments estimated to represent about 17,000 transcripts were displayed. Out of these, 141 transcripts, were found to accumulate after Bgh inoculation using microarray hybridization analysis. Forty-four accumulated predominantly in the epidermis whereas 76 transcripts accumulated mostly in mesophyll tissue