Many of the world’s most devastating crop diseases are caused by fungal pathogens which elaborate specialized infection structures to invade plant tissue. Here we present a quantitative mass spectrometry-based phosphoproteomic analysis of infection-related development by the rice blast fungus Magnaporthe oryzae, which threatens global food security. We mapped 8,005 phosphosites on 2,062 fungal proteins, revealing major re-wiring of phosphorylation-based signaling cascades during fungal infection. Comparingme phosphosite conservation across 41 fungal species reveals phosphorylation signatures specifically associated with biotrophic and hemibiotrophic fungal infection. We then used parallel reaction monitoring to identify phosphoproteins directly regulated by the Pmk1 MAP kinase that controls plant infection by M. oryzae. We define 33 substrates of Pmk1 and show that Pmk1-dependent phosphorylation of a newly identified regulator, Vts1, is required for rice blast disease. Defining the phosphorylation landscape of infection therefore identifies potential therapeutic interventions for control of plant diseases.

Peptide quantitation was performed using Parallel Reaction Monitoring (PRM) as described previously 105. Briefly, mass to charge ratios (m/z) corresponding to selected phospho-peptides were monitored and filtered by the first quadrupole and fragment ions were scanned out in the orbitrap mass analyzer over the duration of the elution profile. The PRM assay also included a selection of control peptides having similar relative intensities in each sample and used to measure relative phospho-peptide content (Supplementary Table 1). Raw data were peak picked and searched against the data bases on the Mascot server as described above and combined with chromatographic profiles in Skyline 106 to determine individual peptide intensities. Extracted phospho-peptides intensity were normalized against the summed control peptide intensities to correct for differences in phospho-peptide yield. The assay was performed once for each of three biological replicates and results were subjected to differential phosphosite analysis.

Protein extraction and phosphopeptide enrichment Spores and appressoria samples were lyophilized and resuspended in extraction buffer (Urea 8M, NaCl 150 mM, Tris pH 8 100 mM, EDTA 5 Mm, aprotinin 1 µg/mL, leupeptin 2 µg/mL) for mechanical disruption using GenoGrinder 2010 (Thermo Scientific) in cold conditions (1 min at 1200 rpm). The homogenate was centrifuged for 10 min at 16,000 × g (Eppendorf 5415D microcentrifuge). The supernatant was used for phosphopeptide enrichment. Sample preparation started from 1.5 mg of protein extract (determined using the Bradford assay) dissolved in ammonium bicarbonate buffer containing 8 M urea. First, the protein extracts were reduced with 5 mM Tris (2-carboxyethyl) phosphine (TCEP) for 30 min at 30°C with gentle shaking, followed by alkylation of cysteine residues with 40mM iodoacetamide at room temperature for 1 hour. Subsequently, the samples were diluted to a final concentration of 1.6 M urea with 50mM ammonium bicarbonate and digested overnight with trypsin (Promega; 1:100 enzyme to substrate ratio). Peptide digests were purified using C18 SepPak columns as described before 91. Phosphopeptides were enriched using titanium dioxide (TiO2, GL Science) with phthalic acid as a modifier as described previously 92. Phosphopeptides were eluted by a pH-shift to pH 10.5 and immediately purified using C18 microspin columns (The Nest Group Inc., 5 – 60 µg loading capacity). After purification, all samples were dried in a Speedvac, stored at -80°C and re-suspended in 2% Acetonitril (AcN) with 0.1% trifluoroacetic acid (TFA) just before the mass spectrometric measurement.