Quantitative Phosphoproteome Analysis of the Interaction Between Fusarium graminearum and Triticum aestivum.

Quantitative proteomics is a powerful method for distinguishing protein abundance changes in a biological system across conditions. In addition to recent advances in computational power and bioinformatics methods, improvements to sensitivity and resolution of mass spectrometry (MS) instrumentation provide an innovative approach for studying host-pathogen interaction dynamics and posttranslational modifications. In this protocol, we provide a workflow for state-of-the-art MS-based proteomics to assess changes in phosphorylated protein abundance upon interaction between the worldwide cereal crop, Triticum aestivum (wheat), and the global cereal crop fungal pathogen, Fusarium graminearum, during infection. This protocol mimics a time course of infection of T. aestivum by F. graminearum in the greenhouse, and the harvested samples undergo Fe-NTA phosphoenrichment combined with label-free quantification (LFQ) for detection by liquid-chromatography (LC)-coupled with tandem MS/MS. Our approach provides an in-depth view of changes in phosphorylation from both the host and pathogen perspectives in a single experiment across infection time points and different host cultivars.

Label-Free Quantitative Proteomic Profiling of Fusarium Head Blight in Wheat.

To distinguish protein abundance changes in biological systems under different conditions, mass spectrometry-based proteomics provides a powerful tool to detect and quantify such responses. Improvements in mass spectrometry instrumentation sensitivity and resolution, along with advanced bioinformatics enable new strategies to study host-pathogen interactions. This protocol uses the state-of-the-art MS-based proteomics to assess infection of the global fungal pathogen Fusarium graminearum, on the world-wide cereal crop Triticum aestivum, resulting in the devastating disease of Fusarium head blight (FHB). Here, host infection is mimicked by inoculating F. graminearum onto T. aestivum cultivars (e.g., FHB-resistant and -susceptible) in the growth room under controlled environment, followed by sample harvesting at different time points (e.g., 24 and 120 h post-inoculation) to assess temporal responses to infection. The collected samples are processed using our in-house pipeline for total protein extraction and quantified via label-free methods by liquid-chromatography-coupled with tandem MS/MS. From this experiment, we define dual perspectives of infection considering dynamic protein abundance changes in both the pathogen and host simultaneously, allowing us to identify strategies used by the pathogen to evade the host defense responses and those used by the host to protect from severe infection.