The gene for albicidin detoxification from Pantoea dispersa encodes an esterase and attenuates pathogenicity of Xanthomonas albilineans to sugarcane

Albicidin phytotoxins are pathogenicity factors in a devastating disease of sugarcane known as leaf scald, caused by Xanthomonas albilineans. A gene (albD) from Pantoea dispersa has been cloned and sequenced and been shown to code for a peptide of 235 amino acids that detoxifies albicidin. The gene shows no significant homology at the DNA or protein level to any known sequence, but the gene product contains a GxSxG motif that is conserved in serine hydrolases. The AlbD protein, purified to homogeneity by means of a glutathione S-transferase gene fusion system, showed strong esterase activity on p-nitrophenyl butyrate and released hydrophilic products during detoxification of albicidins. AlbD hydrolysis of p-nitrophenyl butyrate and detoxification of albicidins required no complex cofactors. Both processes were strongly inhibited by phenylmethylsulfonyl fluoride, a serine enzyme inhibitor. These data strongly suggest that AlbD is an albicidin hydrolase. The enzyme detoxifies albicidins efficiently over a pH range from 5.8 to 8.0, with a broad temperature optimum from 15 to 35°C. Expression of albD in transformed X. albilineans strains abolished the capacity to release albicidin toxins and to incite disease symptoms in sugarcane. The gene is a promising candidate for transfer into sugarcane to confer a form of disease resistance.

Subgenome chromosome walking in wheat: A 450-kb physical contig in Triticum monococcum L. spans the Lr10 resistance locus in hexaploid wheat (Triticum aestivum L.)

For many agronomically important plant genes, only their position on a genetic map is known. In the absence of an efficient transposon tagging system, such genes have to be isolated by map-based cloning. In bread wheat Triticum aestivum, the genome is hexaploid, has a size of 1.6 × 1010 bp, and contains more than 80% of repetitive sequences. So far, this genome complexity has not allowed chromosome walking and positional cloning. Here, we demonstrate that chromosome walking using bacterial artificial chromosome (BAC) clones is possible in the diploid wheat Triticum monococcum (Am genome). BAC end sequences were mostly repetitive and could not be used for the first walking step. New probes corresponding to rare low-copy sequences were efficiently identified by low-pass DNA sequencing of the BACs. Two walking steps resulted in a physical contig of 450 kb on chromosome 1AmS. Genetic mapping of the probes derived from the BAC contig demonstrated perfect colinearity between the physical map of T. monococcum and the genetic map of bread wheat on chromosome 1AS. The contig genetically spans the Lr10 leaf rust disease resistance locus in bread wheat, with 0.13 centimorgans corresponding to 300 kb between the closest flanking markers. Comparison of the genetic to physical distances has shown large variations within 350 kb of the contig. The physical contig can now be used for the isolation of the orthologous regions in bread wheat. Thus, subgenome chromosome walking in wheat can produce large physical contigs and saturate genomic regions to support positional cloning.