Proteome of monoclonal antibody-purified haustoria from Puccinia triticina Race-1.

Puccinia triticina causes leaf rust, a disease that causes annual yield losses in wheat. It is an obligate parasite that invades the host leaf and forms intracellular structures called haustoria, which obtain nutrients and suppress host immunity using secreted proteins called effectors. Since effector proteins act at the frontier between plant and pathogen and help determine the outcome of the interaction, it is critical to understand their functions. Here, we used a direct proteomics approach to identify effector candidates from P. triticina Race 1 haustoria isolated with a specific monoclonal antibody. Haustoria were >95% pure and free of host contaminants. Using high resolution MS we have identified 1192 haustoria proteins. These were quantified using normalized spectral counts and spanned a dynamic range of three orders of magnitude, with unknown proteins and metabolic enzymes as the most highly represented. The dataset contained 140 candidate effector proteins, based on the presence of a signal peptide and the absence of a known function for the protein. Some of these candidates were significantly enriched with cysteine, with up to 13 residues per protein and up to 6.8% cysteine in composition.

Proteome analysis of wheat leaf rust fungus, Puccinia triticina, infection structures enriched for haustoria.

Puccinia triticina (Pt) is a representative of several cereal-infecting rust fungal pathogens of major economic importance world wide. Upon entry through leaf stomata, these fungi establish intracellular haustoria, crucial feeding structures. We report the first proteome of infection structures from parasitized wheat leaves, enriched for haustoria through filtration and sucrose density centrifugation. 2-D PAGE MS/MS and gel-based LC-MS (GeLC-MS) were used to separate proteins. Generated spectra were compared with a partial proteome predicted from a preliminary Pt genome and generated ESTs, to a comprehensive genome-predicted protein complement from the related wheat stem rust fungus, Puccinia graminis f. sp. tritici (Pgt) and to various plant resources. We identified over 260 fungal proteins, 16 of which matched peptides from Pgt. Based on bioinformatic analyses and/or the presence of a signal peptide, at least 50 proteins were predicted to be secreted. Among those, six have effector protein signatures, some are related and the respective genes of several seem to belong to clusters. Many ribosomal structural proteins, proteins involved in energy, general metabolism and transport were detected. Measuring gene expression over several life cycle stages of ten representative candidates using quantitative RT-PCR, all were shown to be strongly upregulated and four expressed solely upon infection.

Phosphoproteomic profiling of wheat callus labelled in vivo.

Callus is an important intermediate tissue for the propagation of plants through tissue culture, as it can form new shoots, and hence plants, through somatic embryogenesis, a process whose underlying biochemical mechanisms are poorly understood. The involvement of kinases suggests that signalling through phosphorylation cascades may play an important role. In this report methods for labelling wheat (Triticum aestivum L.) callus phosphoproteins in vivo through the uptake of [(32)P]orthophosphate and subsequently revealing the phosphoproteome of this tissue by two-dimensional electrophoresis and autoradiography are described. Labelled proteins from 2D gels were identified by tandem mass spectrometry analysis using the 'Mascot' search engine, and by de novo sequencing in combination with BLAST and MS-BLAST, using a cross-species protein identification approach. Eight putative phosphoproteins from 10 spots were identified, confirming the potential utility of this method; three of these have not previously shown to be phosphorylated. Furthermore, the phosphorylation site for one of the proteins was identified. Protein synthesis functions were tentatively assigned to five of the phosphoproteins, one reactive oxygen species (ROS)-detoxifying enzyme ascorbate peroxidase, and two could not be identified reliably from current database matches.