Organization and molecular evolution of a disease-resistance gene cluster in coffee trees

<p>Abstract</p> <p>Background</p> <p>Most disease-resistance (R) genes in plants encode NBS-LRR proteins and belong to one of the largest and most variable gene families among plant genomes. However, the specific evolutionary routes of NBS-LRR encoding genes remain elusive. Recently in coffee tree (<it>Coffea arabica</it>), a region spanning the <it>S</it>_<it>H</it><it>3 </it>locus that confers resistance to coffee leaf rust, one of the most serious coffee diseases, was identified and characterized. Using comparative sequence analysis, the purpose of the present study was to gain insight into the genomic organization and evolution of the <it>S</it>_<it>H</it><it>3 </it>locus.</p> <p>Results</p> <p>Sequence analysis of the <it>S</it>_<it>H</it><it>3 </it>region in three coffee genomes, E^aand C^asubgenomes from the allotetraploid <it>C. arabica </it>and C^cgenome from the diploid <it>C. canephora</it>, revealed the presence of 5, 3 and 4 R genes in E^a, C^a, and C^cgenomes, respectively. All these R-gene sequences appeared to be members of a CC-NBS-LRR (CNL) gene family that was only found at the <it>S</it>_<it>H</it><it>3 </it>locus in <it>C. arabica</it>. Furthermore, while homologs were found in several dicot species, comparative genomic analysis failed to find any CNL R-gene in the orthologous regions of other eudicot species. The orthology relationship among the <it>S</it>_<it>H</it><it>3</it>-CNL copies in the three analyzed genomes was determined and the duplication/deletion events that shaped the <it>S</it>_<it>H</it><it>3 </it>locus were traced back. Gene conversion events were detected between paralogs in all three genomes and also between the two sub-genomes of <it>C. arabica</it>. Significant positive selection was detected in the solvent-exposed residues of the <it>S</it>_<it>H</it><it>3</it>-CNL copies.</p> <p>Conclusion</p> <p>The ancestral <it>S</it>_<it>H</it><it>3</it>-CNL copy was inserted in the <it>S</it>_<it>H</it><it>3 </it>locus after the divergence between Solanales and Rubiales lineages. Moreover, the origin of most of the <it>S</it>_<it>H</it><it>3</it>-CNL copies predates the divergence between <it>Coffea </it>species. The <it>S</it>_<it>H</it><it>3</it>-CNL family appeared to evolve following the birth-and-death model, since duplications and deletions were inferred in the evolution of the <it>S</it>_<it>H</it><it>3 </it>locus. Gene conversion between paralog members, inter-subgenome sequence exchanges and positive selection appear to be the major forces acting on the evolution of <it>S</it>_<it>H</it><it>3</it>-CNL in coffee trees.</p

Stable isotope-free relative and absolute quantitation of protein phosphorylation stoichiometry by MS

Qualitative and quantitative information are crucial to a detailed understanding of the function of protein phosphorylation. MS is now becoming a quantitative approach to analyze protein phosphorylation. All methods that have been described either require the elaborate/expensive use of stable isotopes to compare a limited number of samples or do not provide phosphorylation stoichiometries. Here, we present stable isotope-free MS strategies that allow relative and absolute quantitation of phosphorylation stoichiometries. By using the developed methods, we can normalize to robustly account for run-to-run variations and variations in amounts of starting material. This procedure monitors the unmodified proteolytic peptides derived from the protein of interest and identifies peptides that are suitable for normalization purposes. Also, we can determine changes in phosphorylation stoichiometry by monitoring the changes in the normalized ion currents of the phosphopeptide(s) of interest. Absolute phosphorylation stoichiometry are measured by monitoring the ion currents of a phosphopeptide and its unmodified cognate as the signal intensity changes of both peptide species are correlated. The method is applicable to multiply phosphorylated species (for which one more sample with varying phosphorylation stoichiometry than number of phosphorylation sites is required to correct for the differences in the ionization/detection efficiencies of the phosphopeptide, its partially phosphorylated and unphosphorylated cognates). Last, we can quantitate species with ragged ends resulting from incomplete proteolysis and measure phosphorylation stoichiometries of single samples by controlled dephosphorylation. These approaches were validated and subsequently applied to the phosphorylation of the yeast transcription factor Pho4