Hunting for effectors-elicitors in the fungal wheat pathogen Mycosphaerella graminicola with a quantitative proteomic approach using comparative (label-free) LC-MSE (PR7.3)

Septoria tritici blotch caused by the haploid ascomycete Mycosphaerella graminicola (Fuckel) J. Schröt. in Cohn, is the most important wheat disease in Europe. Despite the recent identification of 15 resistance genes and their potential application in breeding, disease control is currently achieved mainly by fungicides. The genome of M. graminicola has been sequenced and finished by the US. Department of Energy- Joint Genome Institute and together with the high quality of the genome annotation, the high-density genetic linkage maps, including 11 quantitative trait loci involved in species specificity as well as in cultivar-specificity (Ware et al. 2006; Wittenberg et al. 2007) this provides an excellent foundation for proteomic studies that focus on the pathogen side of the interaction. The lifestyle of M. graminicola is significantly different from other cereal pathogens. It is a hemibiotroph with an initial symptomless biotrophic and intercellular phase that is followed by a necrotrophic phase resulting in disease symptoms starting from approximately 14 days after infection. The switch from biotrophy to necrotrophy is poorly understood and we therefore have started experiments using established protocols for apoplast analysis from the Cladosporium-tomato pathosystem. Extracellular liquid (apoplast) was collected from (in)compatible interactions at several time points after inoculation. Proteins were extracted and digested with trypsin. The complex peptide digests were separated and detected with nano-UPLC-QTOF operating in an alternating mode of low and high collision energy. With this approach peptide abundances can be quantitatively compared between multiple complex protein samples. In the initial experiment 18 LCMS traces (6 samples in triplicate) were compared providing highly detailed quantification and identification. Using the identification algorithm 3150 peaks were identified, resulting in 1926 unique peptide sequences that were identified and quantified. We identified several proteins of M. graminicola that appear to be secreted in specific stages of the infection to the apoplast and currently study these candidate effectors of the M.graminicola-wheat interaction