title: Comparative phosphoproteome analysis of Magnaporthe oryzae-responsive proteins in susceptible and resistant rice cultivars.
abstract: To explore the molecular mechanisms involved in the rice-Magnaporthe oryzae interaction, we conducted a time-course phosphoproteomic analysis of leaf samples from resistant and susceptible rice cultivars infected with M. oryzae, with emphasis on the initial biotrophic phase of the interaction. Phosphoproteomic profiling analysis led to the identification of 56 M. oryzae-regulated phosphoprotein spots. MALDI-TOF/TOF analysis unveiled 53 phosphoproteins belonging to 12 functional categories. Phosphorylation within 7 identified phosphoproteins was verified by mapping the phosphorylation sites by nanoLC-MS/MS. Although the phosphoproteins involved in photosynthesis, antioxidation, and protein folding showed similar changes in both compatible and incompatible interactions, differential regulation between the two interactions was documented for the phosphorylation status of defense-related proteins, signaling-related proteins, microtubule-associated proteins, energy-related enzymes, and amino acid synthesis-related proteins. Rice resistance is likely related to several rapidly and strongly triggered signal transduction cascades (e.g., Rac GTPases- and H2O2-mediated rice defense signaling) resulting in a multiple-level activation of defense responses. The six differentially expressed mRNA encoding proteins were measured by quantitative real-time PCR (qRT-PCR). This study provides useful clues for the further exploration of the sophisticated regulatory mechanisms of M. oryzae-rice interactions.
  
  
  CONCLUSIONS
  Although large-scale identification of phosphorylated proteins has been carried out in rice, there is little report on the rice phosphoproteome in response to pathogens in general and in response to M. oryzae in particular. In this study, a time-course comparative phosphoproteomics was performed to analyze the changes of phosphoprotein profiles in rice leaves during early compatible and incompatible interactions with M. oryzae by using a combination of PEG prefractionation, Al(OH)3-MOAC, 2-DE, Pro-Q DPS and mass spectrometry. Fifty-six M. oryzae-regulated phosphoproteins were identified successfully and phosphorylation within 7 identified phosphoproteins was verified by mapping the phosphorylation sites by nanoLC-MS/MS. Our study provides novel insights into the functions of proteins involved in rice resistance and the molecular mechanism of rice resistance against M. oryzae. Furthermore, we propose the working model for the signaling pathways of rice defense in response to M. oryzae.
authors.affiliation: Laboratory of Physiological Plant Pathology, South China Agricultural University, Guangzhou 510642, China; Guangdong Province Key Laboratory of Microbial Signals and Disease Control, South China Agricultural University, Guangzhou 510642, China.
authors.forename: Yunfeng
authors.initials: Y
authors.lastname: Li
authors.affiliation: Laboratory of Physiological Plant Pathology, South China Agricultural University, Guangzhou 510642, China; Guangdong Province Key Laboratory of Microbial Signals and Disease Control, South China Agricultural University, Guangzhou 510642, China.
authors.forename: Zhijian
authors.initials: Z
authors.lastname: Ye
authors.affiliation: College of Natural Resources and Environment, South China Agricultural University, Guangzhou 510642, China.
authors.forename: Yanfang
authors.initials: Y
authors.lastname: Nie
authors.affiliation: Laboratory of Physiological Plant Pathology, South China Agricultural University, Guangzhou 510642, China; Guangdong Province Key Laboratory of Microbial Signals and Disease Control, South China Agricultural University, Guangzhou 510642, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Department of Plant Pathology, Ohio State University, Columbus 43210, OH, USA.
authors.forename: Guo-Liang
authors.initials: GL
authors.lastname: Wang
authors.affiliation: Laboratory of Physiological Plant Pathology, South China Agricultural University, Guangzhou 510642, China; Guangdong Province Key Laboratory of Microbial Signals and Disease Control, South China Agricultural University, Guangzhou 510642, China. Electronic address: zzwang@scau.edu.cn.
authors.forename: Zhenzhong
authors.initials: Z
authors.lastname: Wang
mesh_terms: D060467:Disease Resistance; D005656:Fungal Proteins / Q000378:metabolism*; D054884:Host-Pathogen Interactions; D020082:Magnaporthe / Q000502:physiology*; D012275:Oryza / Q000378:metabolism / Q000382:microbiology; D010750:Phosphoproteins / Q000378:metabolism*; D010935:Plant Diseases / Q000382:microbiology; D010940:Plant Proteins / Q000378:metabolism*; D020543:Proteome / Q000378:metabolism*
chemical_list: D005656:Fungal Proteins; D010750:Phosphoproteins; D010940:Plant Proteins; D020543:Proteome
keywords: 2-DE; Magnaporthe oryzae; Phosphoproteome; Reactive oxygen species; Rice