Secretome analysis of differentially induced proteins in rice suspension‐cultured cells triggered by rice blast fungus and elicitor

Abstract

Secreted proteins were investigated in rice suspension-cultured cells treated with rice blast fungus Magnaporthe grisea and its elicitor using biochemical and 2-DE coupled with MS analyses followed by their in planta mRNA expression analysis. M. grisea and elicitor successfully interacted with suspension-cultured cells and prepared secreted proteins from these cultures were essentially intracellular proteins free. Comparative 2-D gel analyses identified 21 differential protein spots due to M. grisea and/or elicitor over control. MALDI-TOF-MS and microLC-ESI-MS/MS analyses of these protein spots revealed that most of assigned proteins were involved in defense such as nine chitinases, two germin A/oxalate oxidases, five domain unknown function 26 (DUF 26) secretory proteins, and beta-expansin. One chitin binding chitinase protein was isolated using chitin binding beads and strong enzymatic activity was identified in an in-gel assay. Interestingly, their protein abundance correlated well at transcript levels in elicitor-treated cultures as judged by semi-quantitative RT-PCR. Each identified differentially expressed protein group was compared at transcript levels in rice leaves inoculated with incompatible (KJ401) and compatible (KJ301) races of M. grisea. Time-course profiling revealed their inductions were stronger and earlier in incompatible than compatible interactions. Identified secreted proteins and their expression correlation at transcript level in suspension-cultured cells and also in planta suggest that suspension-cultured cells can be useful to investigate the secretome of rice blast-pathogen interactions.

Tissue-Adapted Invasion Strategies of the Rice Blast Fungus Magnaporthe oryzae

“…Modeling the dynamics of the 2245 features across the two treatments and three time points allowed features to be grouped into six distinct expression profiles (profiles I to VI), of which the largest group, termed profile I, corresponded to 720 features that showed a strong increase in accumulation at 2 DAI with a reduction at the 4 and 6 DAI time points ( Figure 5B; see Supplemental Data Set 1 online). Within this category were a large proportion of genes representative of basal defense with some of them already well characterized during leaf infection (Table 1); for example, an endochitinase (Chit1; Kim et al, 2009), an elicitor-inducible shikimate kinase (SK2; Kasai et al, 2005), certain members of the WRKY family (WRKY71, Chujo et al, 2008;WRKY53, Chujo et al, 2009), or noteworthy, the previously described chitin receptor (chitin-elicitor binding protein, CEBiP; Kaku et al, 2006), a Ser/Thr protein kinase previously reported to be associated with defense against biotrophic rust fungi in other cereals (ORK10; Cheng et al, 2002b), and NAC4, a transcriptional activator involved in the initiation of hypersensitive response-associated cell death (Kaneda et al, 2009). The distinct gene expression pattern was independently confirmed by real-time RT-PCR (see Supplemental Figure 1 online).…”

Effect of Phytohormones and Chemical Inhibitors on Pathogenesis-related Genes Identified by Differential Hybridization in Rice Suspension Culture Cells

“…2). In previous reports, OsRLK and OsTLP were identified as secreted proteins in rice SCCs treated with the rice blast fungus Magnaporthe grisea and its elicitor using secretome analysis (Kim et al, 2009) and also highly induced in the incompatible interaction, compared to the compatible interaction (Kim et al, 2004a). OsPR-10 was expressed in developmental tissues, including the flower and root (Kim et al, 2008b) and was also expressed under environmental stresses such as rice blast fungus/fungal elicitor, wounding, senescence, and phytohormone application (Kim et al, 2003b(Kim et al, , 2004a(Kim et al, , 2008b.…”

Use of intercellular washing fluid to investigate the secreted proteome of the rice–Magnaporthe interaction

“…Several studies have examined proteins induced during Magnaporthe-rice interactions for example (Kim et al 2009). The difficulty in obtaining a complete picture of proteins involved in the Magnaporthe-rice interaction results from the complexity of the interaction.…”

“…A number of proteomic studies have identified Magnaporthe or rice proteins induced during infection (Kim et al 2003(Kim et al , 2004(Kim et al , 2009, although in rice there is a general lack of information on extracellular proteins. The examination of sub-cellular proteomes can give more specific and relevant information about biological processes than whole extract studies e.g.…”

“…In biotrophic interactions such as the Magnaporthe-rice example it is useful to think of a specialized tissue derived from the infection, with its own proteome. In a previous work (Kim et al 2009) the interaction secretome was examined by using proteins secreted into the culture medium of rice calli treated with Magnaporthe or elicitor. This approach is limited to those proteins which are freely secreted into the extracellular environment and stable in the culture medium.…”