Phosphorylation-mediated Regulatory Networks in Mycelia of Pyricularia oryzae Revealed by Phosphoproteomic Analyses

Rui-Jin Wang,Junbo Peng,Qing X Li,You-Liang Peng

doi:10.1074/mcp.m116.066670

Abstract

Protein phosphorylation is known to regulate pathogenesis, mycelial growth, conidiation and stress response in Pyricularia oryzae However, phosphorylation mediated regulatory networks in the fungal pathogen remain largely to be uncovered. In this study, we identified 1621 phosphorylation sites of 799 proteins in mycelia of P. oryzae, including 899 new p-sites of 536 proteins and 47 new p-sites of 31 pathogenicity-related proteins. From the sequences flanking the phosphorylation sites, 19 conserved phosphorylation motifs were identified. Notably, phosphorylation was detected in 7 proteins that function upstream of Pmk1, but not in Pmk1 and its downstream Mst12 and Sfl1 that have been known to regulate appressorium formation and infection hyphal growth of P. oryzae Interestingly, phosphorylation was detected at the site Ser240 of Pmp1, which is a putative protein phosphatase highly conserved in filamentous fungi but not characterized. We thus generated Δpmp1 deletion mutants and dominant allele PMP1S240D mutants. Phenotyping analyses indicated that Pmp1 is required for virulence, conidiation and mycelial growth. Further, we observed that phosphorylation level of Pmk1 in mycelia was significantly increased in the Δpmp1 mutant, but decreased in the PMP1S240D mutant in comparison with the wild type, demonstrating that Pmp1 phosphorylated at Ser240 is important for regulating phosphorylation of Pmk1. To our surprise, phosphorylation of Mps1, another MAP kinase required for cell wall integrity and appressorium formation of P. oryzae, was also significantly enhanced in the Δpmp1 mutant, but decreased in the PMP1S240D mutant. In addition, we found that Pmp1 directly interacts with Mps1 and the region AA180-230 of Pmp1 is required for the interaction. In summary, this study sheds new lights on the protein phosphorylation mediated regulatory networks in P. oryzae.

Highlights

From the ‡State Key Laboratory of Agrobiotechnology and MOA Key Laboratory for Monitoring and Green Management of Crop Pests, China Agricultural University, Beijing 100193, China; §Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, HI 96822
Phosphopeptides from mycelia of three duplicates were enriched with strong cation exchange (SCX)/Immobilized Metal Affinity Chromatography (IMAC) approach (Fig. 1A) and detected with neutral loss triggered ETD strategy on amaZon Speed ETD mass spectrometer (Fig. 1B)
A total of 8087 phosphopeptides from 1187 proteins were identified with p Ͻ 0.05 and protein score Ն 39.0, and the false discovery rate (FDR) at the peptide level were 0.40%, 0.50%, and 0.55% for the three biological duplicates calculated with the target and decoy strategy (Fig. 1C, supplemental Table S1)

Summary

Introduction

From the ‡State Key Laboratory of Agrobiotechnology and MOA Key Laboratory for Monitoring and Green Management of Crop Pests, China Agricultural University, Beijing 100193, China; §Department of Molecular Biosciences and Bioengineering, University of Hawaii at Manoa, Honolulu, HI 96822. We found that the protein phosphatase Pmp1 is phosphorylated at the site Ser240 in mycelia and is required to regulate phosphorylation of Pmk1 and Mps1, two MAPK kinases previously reported to be essential to pathogenicity of P. oryzae. An early study identified 1393 p-sites of 831 proteins in mycelia of the P. oryzae strain 70 –15 [29].

Results

Conclusion