When the rice blast fungus attacks rice, fungal proteins are secreted into the plant apoplast to facilitate infection. The rice plant recognizes such secreted proteins, which result in the induction of defense responses. However, the molecular mechanisms of how rice plant recognizes secreted proteins remain elusive. Here, we report that a small, secreted protein, Magnaporthe oryzae snodprot1 homolog (MSP1), is recognized by rice plants and triggers host cell death and defense responses. Furthermore, pre-treatment of rice with Domain II, elicitor-active epitope of MSP1, induces resistance to the pathogen KJ301. We demonstrated that secretion of MSP1 into the apoplast is prerequisite for triggering cell death and activating defense-related gene expression, suggesting that it is recognized by a receptor in the host plasma membrane. Through comprehensively analysis of transcriptional profile in rice leaves and suspension cultured cells (SCCs) in response to exogenous MSP1 and Domain II treatment using 60K Agilent microarray chip, we found that 27 signaling genes, such as F-box(6), MAPK(4), protein kinase(11), transcription factor(6), were up-regulated in leaves and SCCs and six protein kinases were targeted into plasma membrane. Thus, we suggest that some of these genes may act as receptor of MSP1 in response to exogenous MSP1 treatment. Expression pattern of candidate genes was further checked in response to different environment cues using open rice data. These results demonstrate that these genes may be also involved in the signaling in response to cold stress, root-JA treatment and brown plant hopper (BPH) attack.

Understanding the host defense mechanisms in response to brown leaf spot disease caused by Cochliobolus miyabeanus is very important for production of resistant plant. In this study, two-dimensional gel electrophoresis (2-DGE) in conjunction with mass spectrometry was utilized to unravel changes of stress inducible proteins in rice leaves infected with C. miyabeanus. For this purpose, we firstly observed disease developmental process of C. miyabeanus in rice using trypan blue, anilin blue, acid fuchsin staining, and DAB staining for ROS detection and expressional abundance of ROS related proteins in rice leaves inoculated was confirmed by Western blotting. Proteins were extracted by PEG fractionation and their expression patterns were analyzed by 2-DGE and subjected to image analysis using the ImageMaster 6.0 2D Platinum software, resulting in the identification of 86 differentially expressed protein spots with significantly changes (p<0.05) compared with control. MALDI-TOFTOF-MS analysis revealed that 69 proteins including 42 and 27 significantly up- and down-regulated proteins, respectively, were identified. Based on gene ontology analysis, identified proteins were classified according to their functional groups: metabolism (20%), oxygen-detoxifying (13%), protein stress/defense (24%). Thus, these results for the first time suggest that differentially induced proteins may play a key role for understanding host defense mechanisms during rice -C. miyabeanus interaction.

The rice blast disease caused by Magnaporthe oryzae (M. oryzae) is one of the most serious diseases of cultivated rice (Oryza sativa L.) in most rice-growing regions of the world. In order to investigate early responsible genes in rice in response to M. oryzae, we analyzed transcriptomics analysis using 300 K tilling microarray chip. The quality of RNA samples was initially validated by 4 defense related genes and phytoalexins measurement using RT-PCR and HPLC, respectively, which are well known defense markers. We determined that accumulation of 608 genes showed statistically significant changes in the level of transcription (>2 fold change, P<0.05). Among them, 261 genes were more up-regulated in incompatible interaction than that of compatible one. We further analyzed GO enrichment analysis of the 41 and 231 which were 2 fold up-regulated genes at 12h and 48h in incompatible interaction, respectively, using Rice Oligo nucleotide Array Database (http://ricearray.org). Furthermore, MapMan analysis (http://mapman.gabipd.org/) revealed that 21 and 85 genes including 18 receptor-like genes which were more induced in incompatible interaction compared to compatible interaction were found to be involved in biotic stress. Thus, this study suggests that early inducible genes including receptor-like protein kinases in incompatible interaction may play a key role in disease resistance against M. oryzae attacks.

Here, we first demonstrate that identification of rice brown spot disease fungus (Cochliobolus miyabeanus, C. miyabeanus) proteins is possible in infected tissues using in planta apoplastic proteome with non-destructive tissues. In planta apoplastic proteins from rice leaves inoculated with C. miyabeanus, CM2 (compatible race), were isolated by vacuum infiltration with CaCl2/Na-acetateextractionbuffer, separated by SDS-PAGE, and identified by MudPIT. Of the 529 proteins that were identified by MudPIT, a large proportion (490) was from the rice. Numerous carbohydrate metabolic process (48), oxidation and reduction (44), response to oxidative stress (20%) were identified and confirmed their expression at RNA levels using microarray. Bioinformatic analysis showed that 176 and 39 of these proteins have a signal peptide in rice and rice brown spot fungus, respectively, using Signal P. The large proportion of proteins interestingly identified from the in planta apoplast were involved inprotease, hydrophobin, and host cell wall hydrolysis (Xylanase, beta-glucosidase) derived from pathogen. Thus, we suggest that in planta rice apoplastic secretome will be an important clue to understand the rice-rice brown spot fungus interactions.