Brigitte Poppenberger

Detoxification of the Fusarium mycotoxin deoxynivalenol by a UDP-glucosyltransferase from Arabidopsis thaliana.

Plant pathogenic fungi of the genus Fusarium cause agriculturally important diseases of small grain cereals and maize. Trichothecenes are a class of mycotoxins produced by different Fusarium species that inhibit eukaryotic protein biosynthesis and presumably interfere with the expression of genes induced during the defense response of the plants. One of its members, deoxynivalenol, most likely acts as a virulence factor during fungal pathogenesis and frequently accumulates in grain to levels posing a threat to human and animal health. We report the isolation and characterization of a gene from Arabidopsis thaliana encoding a UDP-glycosyltransferase that is able to detoxify deoxynivalenol. The enzyme, previously assigned the identifier UGT73C5, catalyzes the transfer of glucose from UDP-glucose to the hydroxyl group at carbon 3 of deoxynivalenol. Using a wheat germ extract-coupled transcription/translation system we have shown that this enzymatic reaction inactivates the mycotoxin. This deoxynivalenol-glucosyltransferase (DOGT1) was also found to detoxify the acetylated derivative 15-acetyl-deoxynivalenol, whereas no protective activity was observed against the structurally similar nivalenol. Expression of the glucosyltransferase is developmentally regulated and induced by deoxynivalenol as well as salicylic acid, ethylene, and jasmonic acid. Constitutive overexpression in Arabidopsis leads to enhanced tolerance against deoxynivalenol.

Validation of a Candidate Deoxynivalenol-Inactivating UDP-Glucosyltransferase from Barley by Heterologous Expression in Yeast

Resistance to the virulence factor deoxynivalenol (DON) due to formation of DON-3-O-glucoside (D3G) is considered to be an important component of resistance against Fusarium spp. which produce this toxin. Multiple candidate UDP-glycosyltransferase (UGT) genes from different crop plants that are either induced by Fusarium spp. or differentially expressed in cultivars varying in Fusarium disease resistance have been described. However, UGT are encoded by a very large gene family in plants. The study of candidate plant UGT is highly warranted because of the potential relevance for developing Fusarium-spp.-resistant crops. We tested Arabidopsis thaliana genes closely related to a previously identified DON-glucosyltransferase gene by heterologous expression in yeast and showed that gene products with very high sequence similarity can have pronounced differences in detoxification capabilities. We also tested four candidate barley glucosyltransferases, which are highly DON inducible. Upon heterologous expression of full-length cDNAs, only one gene, HvUGT13248, conferred DON resistance. The conjugate D3G accumulated in the supernatant of DON-treated yeast transformants. We also present evidence that the product of the TaUGT3 gene recently proposed to encode a DON-detoxification enzyme of wheat does not protect yeast against DON.

Frequency and diversity of small cryptic plasmids in the genus Rahnella.

BackgroundRahnella is a widely distributed genus belonging to the Enterobacteriaceae and frequently present on vegetables. Although Rahnella has interesting agro-economical and industrial properties and several strains possess antibiotic resistances and toxin genes which might spread within microbial communities, little is known about plasmids of this genus. Thus, we isolated a number of Rahnella strains and investigated their complements of small plasmids.ResultsIn total 53 strains were investigated and 11 plasmids observed. Seven belonged to the ColE1 family; one was ColE2-like and three shared homology to rolling circle plasmids. One of them belonged to the pC194/pUB110 family and two showed similarity to poorly characterised plasmid groups. The G+C content of two rolling circle plasmids deviated considerably from that of Rahnella, indicating that their usual hosts might belong to other genera. Most ColE1-like plasmids formed a subgroup within the ColE1 family that seems to be fairly specific for Rahnella. Intriguingly, the multimer resolution sites of all ColE1-like plasmids had the same orientation with respect to the origin of replication. This arrangement might be necessary to prevent inappropriate synthesis of a small regulatory RNA that regulates cell division. Although the ColE1-like plasmids did not possess any mobilisation system, they shared large parts with high sequence identity in coding and non-coding regions. In addition, highly homologous regions of plasmids isolated from Rahnella and the chromosomes of Erwinia tasmaniensis and Photorhabdus luminescens could be identified.ConclusionsFor the genus Rahnella we observed plasmid-containing isolates at a frequency of 19%, which is in the average range for Enterobacteriaceae. These plasmids belonged to diffent groups with members of the ColE1-family most frequently found. Regions of striking sequence homology of plasmids and bacterial chromosomes highlight the importance of plasmids for lateral gene transfer (including chromosomal sequences) to distinct genera.

Identification of cis- and trans-acting elements in pHW126, a representative of a novel group of rolling circle plasmids.

pHW126, pIGRK, pIGMS31 and pRAO1 are the only known members of a novel and as yet uncharacterised family of rolling circle plasmids. pHW126 contains only two open reading frames, of which one shows homology to pMV158-family mobilisation proteins. Here we provide evidence that the second open reading frame encodes a replication protein (Rep). Mutation or deletion of this gene resulted in replication deficient constructs, but providing functional Rep from a compatible vector rescued these constructs, indicating that Rep acts in trans. An approximately 300 bp cis-acting region representing the origin of replication was identified upstream of the rep gene. The origin was identified to be composed of three parts: an accessory region, a conserved stretch and four perfect tandem repeats. The two latter elements were essential for replication. Constructs with a deletion of the accessory region could still replicate, but their loss rate was high, indicating that the accessory region is necessary for plasmid maintenance under non-selective conditions. Interestingly, pHW126 could replicate in all Enterobacteriaceae tested while Agrobacterium tumefaciens and Pseudomonas syringae were inappropriate hosts. Thus, pHW126 seems to have a rather limited host range.

The Natural Antibiotic Resistances of the Enterobacteriaceae Rahnella and Ewingella

In contrast, a number of bacteria are naturally resistant against some antibiotics. The molecular basis for natural resistance may be a general factor like the lack of the targeted pathway, a variant of the targeted molecule that is not inhibited by the antibiotic or a membrane limiting entry of the antibiotic into the cell. In addition natural resistance may also be mediated by a resistance gene belonging to the cell’s core genes. Such resistance genes are vertically inherited, shared by (nearly) all isolates of a species and co-evolve with their hosts. They are often encoded by the chromosome, are usually immobile and their expression level is tightly regulated or very low. The establishment of such a resistance requires a long lasting, usually mild selection pressure as it may be present in the soil, which contains many microorganisms producing antibiotics. Examples for this type of natural resistance are the chromosomally encoded β-lactamases found in several species of the Enterobacteriaceae (Naas et al., 2008), many of them colonising plants and soil.

Development and applications of a yeast-based bioassay for the mycotoxin zearalenone.

Zearalenone (ZON) is a non-steroidal estrogenic mycotoxin produced by plant pathogenic species ofFusarium. As a consequence of infection withF. culmorum andF. graminearum, ZON can be found in cereals and derived food products. Several countries have established monitoring programs and guidelines for ZON levels in grain intended for human consumption and animal feed. We have developed a sensitive yeast bioassay allowing detection of the estrogenic activity of ZON in cereal extracts without requiring further clean up steps. The high sensitivity makes this assay suitable for low cost monitoring of contamination of small grain cereals with estrogenicFusarium mycotoxins, but also attractive as a tool for basic research. We have successfully used yeast indicator strains to screen for mutants ofF. graminearum which no longer produce detectable amounts of ZON, and have identified a plant cDNA encoding a ZON detoxification enzyme.

First results of GEN-AU: Cloning of Deoxynivalenol- and Zearalenone-inactivating UDP-glucosyltransferase genes fromArabidopsis thaliana and expression in yeast for production of mycotoxin-glucosides

First results of the GEN-AU pilot project “Fusarium virulence and plant resistance mechanisms” are reported. Employing genetically engineered yeast strains we have been able to clone genes from the model plantArabidopsis thaliana encoding UDP-glucosyltransferases which can inactivate deoxynivalenol (DON) and zearalenone (ZON). The structure of the metabolites produced by the transformed yeast strains were determined by LC-MS/MS as DON-3O-glucoside and ZON-4O-glucoside, respectively. ZON and derivatives added to glucosyltransferase expressing yeast cultures are converted into the corresponding glucosides in very high yield, opening an efficient way to produce reference materials for these masked mycotoxins.