Profiling the total and nuclear proteomes of host-pathogen interactions of the wheat Thatcher near-isogenic line for Lr2a resistance gene with virulent and avirulent races of Puccinia triticina

Wheat leaf rust is a foliar disease caused by an obligate parasite fungus Puccinia triticina (Pt). Many races of Pt are classified based on their ability to overcome certain host resistance genes. In this work we studied host-pathogen responses of wheat ‘Thatcher’ NIL bearing the Lr2a gene that confers resistance to Pt Race 161 (avirulent) but is overcome by Pt Race 9 (virulent) using quantitative proteomic approaches. Profiling of the total avirulent or virulent interactomes at day 2 and day 5 post-infection was performed using 8-plex iTRAQ, pre-fractionation of peptides by high pH HPLC and LC-MS/MS analysis. Nuclei-enriched fractions were prepared using iterative differential and Percoll density centrifugations, followed by SDS-PAGE pre-fractionation combined with iTRAQ analysis. Nuclei were visualized with DNA-binding dyes and fluorescent microscopy imaging. Protein identifications were derived from IWGSC RefSeq and ContigEST wheat databases, and race-specific Pt proteogenomic databases, and quantitative analysis was performed with Scaffold Q+. All four types of core histones and a linker histone were used as nuclei marker proteins. A subtractive proteomic approach was used for the analysis of total and nuclear proteomes from the two organisms. The functional classification of identified proteins was conducted using the Mercator functional annotation pipeline and the MapMan BIN ontology. A total of 2976 proteins in 2155 clusters were identified with high confidence for day 2 post infection experiments. A total of 3178 proteins in 2655 clusters were identified with high confidence for day 5 post infection experiments. At day 2, only wheat proteins showed significant fold change values while no Pt proteins showed statistically significant values. A set of 192 up- and 47 down-regulated wheat proteins were responding at day 2 post-infection with 58 and 183 proteins changing in race 161 and race 9 interaction, respectively. Another set of 34 up- and 450 down-regulated wheat proteins were responding at day 5 post-infection with 170 and 314 proteins changing in race 161 and race 9 interaction, respectively. Among those, many wheat proteins belong to stress, redox-responding, and pathogenesis-related categories. At day 5 post-infection a number of unique fungal Pt high confidence proteins were quantified, including 25 and 46 significantly up-regulated for the race 161 and race 9, respectively. For the fungal identified proteins significantly enriched functional terms were protein turnover, RNA metabolism, stress, amino acid and secondary metabolism, redox response and miscellaneous enzymes.