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.

SummaryThe pollutant 3,4-dichloroaniline (DCA) was rapidly detoxified by glucosylation in Arabidopsis thaliana root cultures, with the N-b-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.

SUMMARYComparative 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.

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 (= 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 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. AbstractIn 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 class of protein (CC-NBS-LRR).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 ( = 0.029). The CC domain was found to be the most diverse domain and subject to positive selection. Superimposing the diversity on the CC 3D structure has shown that some of the variable and positively selected residues are solvent exposed and may interact with other proteins. The LRR domain was relatively conserved, but had a hotspot of amino acid variation between two haplotypes in the ninth repeat. This repeat is longer than the other LRRs and the 3D modeling suggests that an extensive alpha helix structure is 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 while 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.