Phosphoproteome profiles of the phytopathogenic fungi Alternaria brassicicola and Botrytis cinerea during exponential growth in axenic cultures

Abstract

This study describes the gel-free phosphoproteomic analysis of the phytopathogenic fungi Alternaria brassicicola and Botrytis cinerea grown in vitro under nonlimiting conditions. Using a combination of strong cation exchange and IMAC prior to LC-MS, we identified over 1350 phosphopeptides per fungus representing over 800 phosphoproteins. The preferred phosphorylation sites were found on serine (>80%) and threonine (>15%), whereas phosphorylated tyrosine residues were found at less than 1% in A. brassicicola and at a slightly higher ratio in B. cinerea (1.5%). Biological processes represented principally among the phoshoproteins were those involved in response and transduction of stimuli as well as in regulation of cellular and metabolic processes. Most known elements of signal transduction were found in the datasets of both fungi. This study also revealed unexpected phosphorylation sites in histidine kinases, a category overrepresented in filamentous ascomycetes compared to yeast. The data have been deposited to the ProteomeXchange database with identifier PXD000817 (http://proteomecentral.proteomexchange.org/dataset/PXD000817).

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

“…A total of 10 Class I phosphosites were observed on tyrosine residues of which six were observed on CMGC family kinases including phosphotyrosine in a TEY motif on PMK1. Interestingly, functional tyrosine kinases have not yet been identified in filamentous fungi although evidence for tyrosine phosphorylation has been reported in phosphoproteome analysis of other fungi in addition to that reported here 52-55 . Nearly one half of the phosphoproteins contained a single phosphorylation site and phosphoproteins containing one to three class I phosphosites accounted for the vast majority of phosphoproteins detected (Supplementary Figure 2B).…”

“…In a comparison of the two strains analyzed, 376 and 109 phosphosites were identified in only the wild type and Δ cpkA strains respectively (Figure 2B). Included in the 1514 phosphoproteins identified here are 571 proteins not previously identified in recent global proteome analyses of M. oryzae [52,53] bringing the total number of protein identifications for the M. oryzae proteome to 4111 proteins which represents 31.6% of the predicted protein coding genes in M. oryzae ( Magnaporthe comparative Sequencing Project, Broad Institute of Harvard and MIT (http://www.broadinstitute.org/)).…”

“…With regards to fungal species, multiple phosphoproteomics datasets are available for the yeast, S. cerevisiae 4, 38-46 . In addition to S. cerevisiae , phosphoproteomics data sets are available for the single celled yeasts, Schizosaccharomyces pombe 38, 47 and Candida albicans 38 , the basidomycete, Cryptococcus neoformans 48 and the filamentous ascomycetes, Fusarium graminearum 49, 50 , Aspergillus nidulans 51 , Alternaria brassicicola and Botrytis cinerea 52-55 . Recently, phosphoproteome data from six life stages of the Oomycete Phytophthora infestans was published 56 .…”

Proteomic profiling of Botrytis cinerea conidial germination

“…Therefore, proteomic analysis has an increasing importance in the search of virulence factors in plant pathogenic fungi (Garrido et al 2010;Gonzalez-Fernandez and Jorrin-Novo 2012). Recently, an increasing number of B. cinerea proteomics approaches has been published, ranging from total proteome analyses to various subproteome studies based on gel or gel-free techniques (Fernández-Acero et al 2006Shah et al 2009a, b;Espino et al 2010;Cherrad et al 2012;Li et al 2012;Gonzalez-Fernandez et al 2013;Davanture et al 2014). These advances have been facilitated due to the recent publication of two different genome databases of B. cinerea (http://www.broadinstitute.org/ annotation/genome/botrytis_cinerea/MultiHome.html and https://urgi.versailles.inra.fr/Species/Botrytis).…”

Analysis of the Candida albicans Phosphoproteome

“…In the fungus Fusarium graminearum, 2,902 putative phosphopeptides on 1,496 different proteins were identified (23). Phosphoproteomics data sets for the fungi Cryptococcus neoformans (24), Aspergillus nidulans (25), Alternaria brassicicola (26), Botrytis cinerea (26), Neurospora crassa (27), and Schizosaccharomyces pombe (28) have also been published.…”