Proteomic analysis of pathogen‐responsive proteins from rice leaves induced by rice blast fungus,Magnaporthe grisea

Abstract

Proteomic approaches using two-dimensional gel electrophoresis (2-DE) were adopted to identify proteins from rice leaf that are differentially expressed in response to the rice blast fungus, Magnaporthe grisea. Microscopic observation of inoculated leaf with M. grisea revealed that callose deposition and hypersensitive response was clearly visible in incompatible interactions but excessive invading hypha with branches were evident in compatible interactions. Proteins were extracted from leaves 24, 48, and 72 hours after rice blast fungus inoculation. Eight proteins resolved on the 2-DE gels were induced or increased in the inoculated leaf. Matrix-assisted laser desorption/ionization-time of flight analysis of these differentially displayed proteins showed them to be two receptor-like protein kinases (RLK), two beta-1.3-glucanases (Glu1, Glu2), thaumatin-like protein (TLP), peroxidase (POX 22.3), probenazole-inducible protein (PBZ1), and rice pathogenesis-related 10 (OsPR-10). Of these proteins, RLK, TLP, PBZ, and OsPR-10 proteins were induced more in the incompatible interactions than in compatible ones. A phytohormone, jasmonic acid also induced all eight proteins in leaves. To confirm whether the expression profile is equal to the 2-DE data, seven cDNA clones were used as probes in Northern hybridization experiments using total RNA from leaf tissues inoculated with incompatible and compatible rice blast fungal races. The genes encoding POX22.3, Glu1, Glu2, TLP, OsRLK, PBZ1, and OsPR-10 were activated in inoculated leaves, with TLP, OsRLK, PBZ1, and OsPR-10 being expressed earlier and more in incompatible than in compatible interactions. These results suggest that early and high induction of these genes may provide host plants with leading edges to defend themselves. The localization of two rice PR-10 proteins, PBZ1 and OsPR-10, was further examined by immunohistochemical analysis. PBZ1 accumulated highly in mesophyll cells under the attachment site of the appressorium. In contrast, OsPR-10 expression was mainly localized to vascular tissue.

A proteomic approach in analyzing heat‐responsive proteins in rice leaves

“…To compensate for subtle differences in sample loading, gel staining, and destaining, the volume of each spot (i.e., spot abundance) was normalized as a relative volume. Spots were detected and quantified with the BioRad PDQuest software (Version 7.2; BioRad, Hercules, CA, USA), on the basis of their relative volume, that is, the spot volume was divided by the total volume over the whole set of gel spots [16]. After automated detection and matching, manual editing was carried out.…”

“…5 ng/mL of trypsin (Promega, WI, USA) and incubated overnight at 377C. Tryptic-digested peptides were extracted according to the protocol described by Kim et al [16]. All samples were analyzed using a Voyager-DE STR MALDI-TOF mass spectrometer (PerSeptive Biosystems, Framingham, MA, USA).…”

“…In addition, these data also provided evidence that PEG-pellet fractions could be used as a powerful tool in evaluating the responses of multimeric proteins such as Rubisco or HSPs, in rice leaves. The advantages of the PEG fractionated method were outlined in our previous report [16].…”

“…We previously developed a prefractionation technique based on PEG, which provided a high-resolution of 2-DE images, as well as enriched low-abundance protein in rice samples [15][16][17][18]. In the present study, in order to investigate the responses of abundant and low-abundant proteins of rice leaves upon heat stress, PEG-fractionated supernatant and pellet samples were analyzed by 2-DE, thereby revealing a total of 48 differentially expressed proteins.…”

Identification and Molecular Properties of SUMO-Binding Proteins in Arabidopsis

“…For each sample, quantitation was performed with three analytical gels originating from three independent biological replicas. The silver-stained protein spots were excised from the gel, subjected to in-gel tryptic digestion (Promega, USA), and extracted as previously described (Kim et al, 2004). Peptide mass fingerprinting was carried out on a Voyager-DE STR MALDI-TOF mass spectrometer (PerSeptive Biosystems, USA) according to previously reported methods (Kim et al, 2004).…”