Glucosylation of xenobiotics in maize, soybean and arabidopsis thaliana

Many natural products and xenobiotics become glucosylated in the course of their metabolism in plants. This reaction is catalysed by type 1 UDP-glucose dependent glucosyltransferases (GTs), a super-family of enzymes which differ in their substrate specificity and which are able to glucosylate hydroxy, amino and carboxylic acid groups to form conjugates with altered bioactivities as compared with the parent aglycones. This study has focused both on NGT and OGT enzymes active towards amino and hydroxy groups, respectively, present in natural products and pesticide metabolites in two major crops (Zea mays and Glycine max) and the model plant Arabidopsis thaliana. A sensitive radioactive enzyme assay was developed to monitor conjugating activity in vitro and the substrate specificity of N-GTs and O-GTs determined in the three plant species with respect to xenobiotic detoxification. 3,4-Dichloroaniline was found to be the optimal N-GT substrate and 2,4,5-trichlorophenol the preferred 0-GT substrate in all the species tested. In addition, O-GT activities were also determined with other phenols of both natural and synthetic origin. To confirm the importance of N-GTs and O-GTs in xenobiotic detoxification, plant metabolism studies were carried out with [(^14)C]-p-nitrophenol and [(^14)C]-3,4-dichloroaniline. In each case, O- I N-glucosylation was found to be a major route of detoxification respectively. To determine whether or not herbicide-safeners could enhance the glucosylation of xenobiotics as had been demonstrated for the S-glutathionylation of herbicides in cereals; soybean, maize and Arabidopsis were treated with a range of safeners. The plants were then either fed with radiolabelled [(^14)C]-3,4-dichloroaniline, or extracted and assayed for O-GT and N-GT activities. In all species, safener treatment had no significant effect on the rate of uptake of radioactivity following feeding with [(^14)C]-3,4-dichloroaniline. However, specific safeners were found to enliance N-GT and O-GT activities in etiolated shoots, roots or suspension cultures in all species where tested. Several attempts were made to clone GT enzymes from soybean and maize based on a combination of bioinformatic and PCR approaches, the latter using conserved blocks of sequence in type 1 plant GTs to amplify up partial cDNAs. Using PCR and analysis of soybean expressed tagged accessions, it was possible to assemble a full-length cDNA from soybean which encoded a GT resembling an arbutin synthase GT from Rauvolfia serpentina. Although the resulting GT (GmGT32_l) could be expressed as a recombinant polypeptide in E.coli, the resulting protein was inactive and accumulated in the insoluble inclusion bodies. In the case of maize, a GT termed Z/wRP was identified as a random sequenced clone from a proprietary maize cDNA library. However, ZmRP could not be translated into protein using bacterial expression systems. Instead, an alternative proteomics approach at isolating plant GTs involved in xenobiotic detoxification was undertaken in Arabidopsis, using suspension cultured cells as the starting material. The major N-GT conjugating activity towards 3,4-dichloroaniline was purified 9552-fold using a combination of hydrophobic interaction, ion exchange, and affinity chromatographies. The resulting 50 kDa polypeptide was digested with trypsin and the peptide fragments analysed by MALDI TOF MS. Database analysis unambiguously identified the Arabidopsis protein as UGT72B1 (NM 116337). Following the completion of the Ph.D. programme the activity of GT72B1 towards 3,4-dichloroaniline, 2,4,5-trichlorophenol and other xenobiotics was confirmed and an account of the studies earned out on Arabidopsis GTs published (Lao, et al., 2003), (Loutre, et al., 2003)

Improved detection of Rhodococcus coprophilus with a new quantitative PCR assay

Agricultural practices, such as spreading liquid manure or the utilisation of land as animal pastures, can result in faecal contamination of water resources. Rhodococcus coprophilus is used in microbial source tracking to indicate animal faecal contamination in water. Methods previously described for detecting of R. coprophilus in water were neither sensitive nor specific. Therefore, the aim of this study was to design and validate a new quantitative polymerase chain reaction (qPCR) to improve the detection of R. coprophilus in water. The new PCR assay was based on the R. coprophilus 16S rRNA gene. The validation showed that the new approach was specific and sensitive for deoxyribunucleic acid from target host species. Compared with other PCR assays tested in this study, the detection limit of the new qPCR was between 1 and 3 log lower. The method, including a filtration step, was further validated and successfully used in a field investigation in Switzerland. Our work demonstrated that the new detection method is sensitive and robust to detect R. coprophilus in surface and spring water. Compared with PCR assays that are available in the literature or to the culture-dependent method, the new molecular approach improves the detection of R. coprophilu

Rapid linkage disequilibrium decay in the Lr10 gene in wild emmer wheat (Triticum dicoccoides) populations

v2010o

Leaf rust resistance gene Lr1, isolated from bread wheat (Triticum aestivum L.) is a member of the large psr567 gene family

In hexaploid wheat, leaf rust resistance gene Lr1 is located at the distal end of the long arm of chromosome 5D. To clone this gene, an F-1-derived doubled haploid population and a recombinant inbred line population from a cross between the susceptible cultivar AC Karma and the resistant line 87E03-S2B1 were phenotyped for resistance to Puccinia triticina race 1-1 BBB that carries the avirulence gene Avr1. A high-resolution genetic map of the Lr1 locus was constructed using microsatellite, resistance gene analog (RGA), BAC end (BE), and low pass (LP) markers. A physical map of the locus was constructed by screening a hexaploid wheat BAC library from cultivar Glenlea that is known to have Lr1. The locus comprised three RGAs from a gene family related to RFLP marker Xpsr567. Markers specific to each paralog were developed. Lr1 segregated with RGA567-5 while recombinants were observed for the other two RGAs. Transformation of the susceptible cultivar Fielder with RGA567-5 demonstrated that it corresponds to the Lr1 resistance gene. In addition, the candidate gene was also confirmed by virus-induced gene silencing. Twenty T (1) lines from resistant transgenic line T (0)-938 segregated for resistance, partial resistance and susceptibility to Avr1 corresponding to a 1:2:1 ratio for a single hemizygous insertion. Transgene presence and expression correlated with the phenotype. The resistance phenotype expressed by Lr1 seemed therefore to be dependant on the zygosity status. T (3)-938 sister lines with and without the transgene were further tested with 16 virulent and avirulent rust isolates. Rust reactions were all as expected for Lr1 thereby providing additional evidence toward the Lr1 identity of RGA567-5. Sequence analysis of Lr1 indicated that it is not related to the previously isolated Lr10 and Lr21 genes and unlike these genes, it is part of a large gene family

Rapid linkage disequilibrium decay in the Lr10 gene in wild emmer wheat (Triticum dicoccoides) populations

INTRODUCTION: Recombination is a key evolutionary factor enhancing diversity. However, the effect of recombination on diversity in inbreeding species is expected to be low. To estimate this effect, recombination and diversity patterns of Lr10 gene were studied in natural populations of the inbreeder species, wild emmer wheat (Triticum dicoccoides). Wild emmer wheat is the progenitor of most cultivated wheats and it harbors rich genetic resources for disease resistance. Lr10 is a leaf rust resistance gene encoding three domains: a coiled-coil, nucleotide-binding site, and leucine-rich repeat (CC-NBS-LRR). RESULTS: Lr10 was sequenced from 58 accessions representing 12 diverse habitats in Israel. Diversity analysis revealed a high rate of synonymous and non-synonymous substitutions (d (S) = 0.029, d (N) = 0.018, respectively) in the NBS-LRR domains. Moreover, in contrast to other resistance genes, in Lr10 the CC domain was more diverse than the NBS-LRR domains (d (S) = 0.069 vs. 0.029, d (N) = 0.094 vs. 0.018) and was subjected to positive selection in some of the populations. Seventeen recombination events were detected between haplotypes, especially in the CC domain. Linkage disequilibrium (LD) analysis has shown a rapid decay from r (2) = 0.5 to r (2) = 0.1 within a 2-kb span. CONCLUSION: These results suggest that recombination is a diversifying force for the R-gene, Lr10, in the selfing species T. dicoccoides. This is the first report of a short-range LD decay in wild emmer wheat