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.

An efficient method for the extraction of DNA and RNA from fungi is described. Urediosporelings and sporidia of two basidiomycete species and mycellia from several species of Ascomycetes and Oomycetes were homogenized in a lysis buffer containing sodium dodecyl sulfate followed by cetyltrimethylammonium bromide extraction of carbohydrates in 1.4 M NaCl, leaving nucleic acids in the supernatant. After chloroform – isoamyl alcohol extraction of proteins, nucleic acids were precipitated with ethanol. Total nucleic acids prepared in this way contained nuclear, ribosomal, and mitochondrial DNA as well as double-stranded and single-stranded RNA. DNA was eluted from agarose gels and digested with endonucleases, labelled by nick translation, and used for hybridization without nonspecific background signal. A method is also described for RNase digestion of single-stranded and double-stranded RNA in agarose gels. Key words: DNA extraction, double-stranded RNA, cetyltrimethylammonium bromide, sodium dodecyl sulfate.

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.

No abstract

No abstract