Phosphoproteome Analysis Links Protein Phosphorylation to Cellular Remodeling and Metabolic Adaptation during Magnaporthe oryzae Appressorium Development

Abstract

The rice pathogen, Magnaporthe oryzae, undergoes a complex developmental process leading to formation of an appressorium prior to plant infection. In an effort to better understand phosphoregulation during appressorium development, a mass spectrometry based phosphoproteomics study was undertaken. A total of 2924 class I phosphosites were identified from 1514 phosphoproteins from mycelia, conidia, germlings and appressoria of the wild type and a protein kinase A (PKA) mutant. Phosphoregulation during appressorium development was observed for 448 phosphosites on 320 phosphoproteins. In addition, a set of candidate PKA targets was identified encompassing 253 phosphosites on 227 phosphoproteins. Network analysis incorporating regulation from transcriptomic, proteomic and phosphoproteomic data revealed new insights into the regulation of the metabolism of conidial storage reserves and phospholipids, autophagy, actin dynamics and cell wall metabolism during appressorium formation. In particular, protein phosphorylation appears to play a central role in the regulation of autophagic recycling and actin dynamics during appressorium formation. Changes in phosphorylation were observed in multiple components of the cell wall integrity pathway providing evidence that this pathway is highly active during appressorium development. Several transcription factors were phosphoregulated during appressorium formation including the bHLH domain transcription factor MGG_05709. Functional analysis of MGG_05709 provided further evidence for the role of protein phosphorylation in regulation of glycerol metabolism and the metabolic reprogramming characteristic of appressorium formation. The data presented here represent a comprehensive investigation of the M. oryzae phosphoproteome and provide key insights on the role of protein phosphorylation during infection-related development.

The basic helix–loop–helix transcription factor Crf1 is required for development and pathogenicity of the rice blast fungus by regulating carbohydrate and lipid metabolism

“…Franck et al . reported the involvement of MGG_05709 ( CRF1 ) in glycerol metabolism and virulence, but did not report the role of CRF1 in lipid and carbohydrate metabolism, conidial cell death, or appressorium turgor production (Franck et al ., ). In addition to glycerol metabolism, we found defects of Δ crf1 in carbohydrate metabolism, lipid catabolism (Figs.…”

Sequential Phosphopeptide Enrichment for Phosphoproteome Analysis of Filamentous Fungi: A Test Case Using Magnaporthe oryzae

“…A representative setup involves a 15 cm x 75 μm nanoLC analytical column containing 3 μm C18 resin coupled to a high-resolution ion trap (Orbitrap Q-Exactive) mass spectrometer as described in our previous work [15]. Phosphopeptides are typically eluted from the nanoLC with a linear gradient of 5% to 40% acetonitrile (ACN) over 3 hours using mobile phase A and mobile phase B listed in materials.…”

“…Resolving power is set at 70,000FWHM at 200 m/z for MS acquisition and 17,500FWHM at 200 m/z for MS/MS acquisition. AGC settings for MS and MS/MS is 1E6 and 2E4, respectively (see [15]). The nanoLC setup may need modification for more complex samples and other mass spectrometers and may require longer columns with smaller chromatographic beads to improve phosphopeptide resolving power.…”

“…High confidence sites are defined as having a location probability greater than or equal to 0.75 and a score difference greater than or equal to five. The Perseus module of MaxQuant is used for quantification and to examine differential changes in protein phosphorylation as described in [15]. Further statistical tests can be performed using the JMP software package (SAS).…”

“…Using this approach to enrich phosphopeptides and using a nanoLC MS/MS high-resolution ion trap (Q-Exactive) mass spectrometer with data processed in MaxQuant, we identified and were able to examine changes in phosphorylation at 2,924 phosphosites on 1,914 phosphoproteins in conidia, mycelia and during conidial germination and appressorium formation of M. oryzae [15]. The protocol presented here is applicable to any fungal species or tissues and requires only 250 μg of protein input.…”