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. coprophilus.

Ancient diversity of splicing motifs and protein surfaces in the wild emmer wheat (Triticum dicoccoides) LR10 coiled coil (CC) and leucine-rich repeat (LRR) domains.

In this study, we explore the diversity and its distribution along the wheat leaf rust resistance protein LR10 three-dimensional structure. Lr10 is a leaf rust resistance gene encoding a coiled coil-nucleotide-binding site-leucine-rich repeat (CC-NBS-LRR) class of protein. Lr10 was cloned and sequenced from 58 accessions representing diverse habitats of wild emmer wheat in Israel. Nucleotide diversity was very high relative to other wild emmer wheat genes (π= 0.029). The CC domain was found to be the most diverse domain and subject to positive selection. Superimposition of the diversity on the CC three-dimensional structure showed that some of the variable and positively selected residues were solvent exposed and may interact with other proteins. The LRR domain was relatively conserved, but showed a hotspot of amino acid variation between two haplotypes in the ninth repeat. This repeat was longer than the other LRRs, and three-dimensional modelling suggested that an extensive α helix structure was formed in this region. The two haplotypes also differed in splicing regulation motifs. In genotypes with one haplotype, an intron was alternatively spliced in this region, whereas, in genotypes with the other haplotype, this intron did not splice at all. The two haplotypes are proposed to be ancient and maintained by balancing selection.

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.

Two different CC-NBS-LRR genes are required for Lr10-mediated leaf rust resistance in tetraploid and hexaploid wheat.

Comparative study of disease resistance genes in crop plants and their relatives provides insight on resistance gene function, evolution and diversity. Here, we studied the allelic diversity of the Lr10 leaf rust resistance gene, a CC-NBS-LRR coding gene originally isolated from hexaploid wheat, in 20 diploid and tetraploid wheat lines. Besides a gene in the tetraploid wheat variety 'Altar' that is identical to the hexaploid wheat Lr10, two additional, functional resistance alleles showing sequence diversity were identified by virus-induced gene silencing in tetraploid wheat lines. In contrast to most described NBS-LRR proteins, the N-terminal CC domain of LR10 was found to be under strong diversifying selection. A second NBS-LRR gene at the Lr10 locus, RGA2, was shown through silencing to be essential for Lr10 function. Interestingly, RGA2 showed much less sequence diversity than Lr10. These data demonstrate allelic diversity of functional genes at the Lr10 locus in tetraploid wheat, and these new genes can now be analyzed for agronomic relevance. Lr10-based resistance is highly unusual both in its dependence on two, only distantly, related CC-NBS-LRR proteins, as well as in the pattern of diversifying selection in the N-terminal domain. This indicates a new and complex molecular mechanism of pathogen detection and signal transduction.

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.

3,4-Dichloroaniline is detoxified and exported via different pathways in Arabidopsis and soybean.

The metabolic fate of [UL-14C]-3,4-dichloroaniline (DCA) was investigated in Arabidopsis root cultures and soybean plants over a 48 h period following treatment via the root media. DCA was rapidly taken up by both species and metabolised, predominantly to N-malonyl-DCA in soybean and N-glucosyl-DCA in Arabidopsis. Synthesis occurred in the roots and the respective conjugates were largely exported into the culture medium, a smaller proportion being retained within the plant tissue. Once conjugated, the DCA metabolites in the medium were not then readily taken up by roots of either species. The difference in the routes of DCA detoxification in the two plants could be explained partly by the relative activities of the respective conjugating enzymes, soybean containing high DCA-N-malonyltransferase activity, while in Arabidopsis DCA-N-glucosyltransferase activity predominated. A pre-treatment of plants with DCA increased DCA-N-malonyltransferase activity in soybean but not in Arabidopsis, indicating differential regulation of this enzyme in the two plant species. This study demonstrates that DCA can undergo two distinct detoxification mechanisms which both lead to the export of conjugated metabolites from roots into the surrounding medium in contrast to the vacuolar deposition more commonly associated with the metabolism of xenobiotics in plants.

Isolation of a glucosyltransferase from Arabidopsis thaliana active in the metabolism of the persistent pollutant 3,4-dichloroaniline.

The pollutant 3,4-dichloroaniline (DCA) was rapidly detoxified by glucosylation in Arabidopsis thaliana root cultures, with the N-beta-d-glucopyranosyl-DCA exported into the medium. The N-glucosyltransferase (N-GT) responsible for this activity was purified from Arabidopsis suspension cultures and the resulting 50 kDa polypeptide analysed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) following tryptic digestion. The protein was identified as GT72B1. The GT was cloned and the purified recombinant enzyme shown to be highly active in conjugating DCA and 2,4,5-trichlorophenol, as well as several other chlorinated phenols and anilines, demonstrating both N-GT and O-GT activity. GT72B1 showed little activity towards natural products with the exception of the tyrosine catabolite 4-hydroxyphenylpyruvic acid. Both O-GT and N-GT activities were enhanced in both plants and cultures treated with herbicide safeners, demonstrating the chemical inducibility of this detoxification system in Arabidopsis.