Comparative Secretome Investigation of Magnaporthe oryzae Proteins Responsive to Nitrogen Starvation

Abstract

Magnaporthe oryzae is a fungal pathogen that causes blast disease in rice. During its early infection process, during which starvation of nutrients, including nitrogen, prevails before establishment of successful infection, the fungally secreted proteins play an important role in the pathogenicity and stress response. In this study, M. oryzae-secreted proteins were investigated in an N-deficient minimal medium using two-dimensional gel electrophoresis (2-DGE) coupled with mass spectrometry analysis (MALDI-TOF-MS and mu LC-ESI-MS/MS). The 2-DGE analysis of secreted proteins detected 89 differentially expressed protein spots (14 downregulated and 75 upregulated) responsive to N starvation. Eighty five of the protein spots were identified by mass spectrometry analyses. Identified proteins were mainly cell wall hydrolase enzymes (22.4%), protein and lipid hydrolases (24.7%), reactive oxygen species detoxifying proteins (22.4%), and proteins with unknown function (14.1%), suggesting early production of prerequisite proteins for successful infection of the host. SignalP analysis predicted the presence of signal peptides in 67% of the identified proteins, suggesting that in addition to the classical Golgi/endoplasmic reticulum secretory pathway, M. oryzae might possess other, as yet undefined, secretory pathways. Those nonclassical or leaderless secretion proteins accounted for 25.9% of the total identified proteins by TatP and SecretomeP predictions. Semiquantitative reverse transcriptase polymerase chain reaction of seven randomly selected N-responsive secreted proteins also revealed a good correlation between RNA and protein levels. Taken together, the establishment of the M. oryzae secretome that is responsive to N starvation provides the first evidence of the secretion of 60 unreported and 25 previously known proteins. This developed protein inventory could be exploited to improve our understanding of the secretory mechanisms of M. oryzae and its invasive growth process in rice tissue.