Mitogen-activated protein kinase (MAPK) cascades support the flow of extracellular signals to intracellular target molecules and ultimately drive a diverse array of physiological functions in cells, tissues, and organisms by interacting with other proteins. Yet, our knowledge of the global physical MAPK interactome in plants remains largely fragmented. Here, we utilized the yeast twohybrid system and coimmunoprecipitation, pull-down, bimolecular fluorescence complementation, subcellular localization, and kinase assay experiments in the model crop rice (Oryza sativa) to systematically map what is to our knowledge the first plant MAPK-interacting proteins. We identified 80 nonredundant interacting protein pairs (74 nonredundant interactors) for rice MAPKs and elucidated the novel proteome-wide network of MAPK interactors. The established interactome contains four membrane-associated proteins, seven MAP2Ks (for MAPK kinase), four MAPKs, and 59 putative substrates, including 18 transcription factors. Several interactors were also validated by experimental approaches (in vivo and in vitro) and literature survey. Our results highlight the importance of OsMPK1, an ortholog of tobacco (Nicotiana benthamiana) salicyclic acid-induced protein kinase and Arabidopsis (Arabidopsis thaliana) AtMPK6, among the rice MAPKs, as it alone interacts with 41 unique proteins (51.2% of the mapped MAPK interaction network). Additionally, Gene Ontology classification of interacting proteins into 34 functional categories suggested MAPK participation in diverse physiological functions. Together, the results obtained essentially enhance our knowledge of the MAPK-interacting protein network and provide a valuable research resource for developing a nearly complete map of the rice MAPK interactome.

Secreted proteins control a multitude of biological and physiological processes in multicellular organisms such as plants. Identification of secreted proteins in reference plants like Arabidopsis and rice under normal growth conditions and adverse environmental conditions will help better understand the secretory pathways. Here, we have performed a systematic in planta and in vitro analyses of proteins secreted by rice leaves (in planta) and seed callus suspension-cultured cells (SCCs; in vitro), respectively, using a combination of biochemical and two-dimensional gel electrophoresis (2-DGE) coupled with liquid chromatography mass spectrometry analyses. Secreted proteins prepared from either leaves or SCCs medium were essentially free from contamination of intracellular proteins as judged by biochemical and Western blot analyses. 2-DGE analyses of secreted proteins collectively identified 222 protein spots with only 6 protein spots common to both in planta and in vitro derived data sets. Data were used to establish high-resolution and high-density 2-D gel reference maps for both in planta and in vitro secreted proteins. Identified proteins belonged to 11 (in planta) and 6 (in vitro) functional classes. Proteins involved in carbon metabolism (33%) and cell wall metabolism having plant defense mechanism (18%) were highly represented in the in planta secreted proteins accounting for 51% of total identified proteins, whereas proteins of cell wall metabolism having plant defense mechanism (64%) were predominant in the in vitro secreted proteins. Interestingly, secreted proteins possessing signal peptides were significantly lower in an in planta (27%) prepared secreted protein population than in vitro (76%) as predicted by SignalP prediction tool, implying the notion that plant might possess yet unidentified secretory pathway(s) in addition to the classical endoplasmic reticulum/Golgi pathway. Taken together, this systematic study provides evidence for (i) significant difference in protein population secreted in planta and in vitro suggesting both approaches are complementary, (ii) identification of many novel and previously known secreted proteins, and (iii) the presence of large number of functionally diverse proteins secreted in planta and in vitro.

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Plant disease resistance occurs as a hypersensitive response (HR) at the site of attempted pathogen invasion. This specific event is initiated in response to recognition of pathogen-associated molecular pattern (PAMP) and subsequent PAMP-triggered immunity (PTI) and effector-triggered immunity (ETI). Both PTI and ETI mechanisms are tightly connected with reactive oxygen species (ROS) production and disease resistance that involves distinct biphasic ROS production as one of its pivotal plant im-mune responses. This unique oxidative burst is strongly dependent on the resistant cultivars because a monophasic ROS burst is a hallmark of the susceptible cultivars. However, the cause of the differential ROS burst remains unknown. In the study here, we revealed the plausible underlying mechanism of the differential ROS burst through functional understanding of the Magnaporthe oryzae (M. oryzae) AVR effector, AVR-Pii. We performed yeast two-hybrid (Y2H) screening using AVRPii as bait and isolated rice NADP-malic enzyme2 (Os-NADP-ME2) as the rice target protein. To our surprise, deletion of the rice Os-NADP-ME2 gene in a resistant rice cultivar disrupted innate immunity against the rice blast fungus. Malic enzyme activity and inhibition studies demonstrated that AVR-Pii proteins specifically inhibit in vitro NADP-ME activity. Overall, we demonstrate that rice blast fungus, M. oryzae attenuates the host ROS burst via AVR-Pii-mediated inhibition of Os-NADP-ME2, which is indispensable in ROS metabolism for the innate immunity of rice. This characterization of the regulation of the host oxidative burst will help to elucidate how the products of AVR genes function associated with virulence of the pathogen.

Mitogen-activated protein kinase (MAPK) signaling cascades are evolutionarily conserved fundamental signal transduction pathways. A MAPK cascade consists of many distinct MAPKKK-MAPKK-MAPK modules linked to various upstream receptors and downstream targets through sequential phosphorylation and activation of the cascade components. These cascades collaborate in transmitting a variety of extracellular signals and in controlling cellular responses and processes such as growth, differentiation, cell death, hormonal signaling, and stress responses. Although MAPK proteins play central roles in signal transduction pathways, our knowledge of MAPK signaling in hormonal responses in rice has been limited to a small subset of specific upstream and downstream interacting targets. However, recent studies revealing direct MAPK and MAPKK interactions have provided the basis for elucidating interaction specificities, functional divergence, and functional modulation during hormonal responses. In this review, we highlight current insights into MAPKK-MAPK interaction patterns in rice, with emphasis on the biological significance of these interacting pairs in SA (salicylic acid), JA (jasmonic acid), ET (ethylene), and ABA (abscisic acid) responses, and discuss the challenges in understanding functional signal transduction networks mediated by these hormones.

Rice Oryza sativa accelerated cell death and resistance 1 (OsACDR1) encodes a putative Raf-like mitogen-activated protein kinase kinase kinase (MAPKKK). We had previously reported upregulation of the OsACDR1 transcript by a range of environmental stimuli involved in eliciting defense-related pathways. Here we apply biochemical, gain and loss-of-function approaches to characterize OsACDR1 function in rice. The OsACDR1 protein showed autophosphorylation and possessed kinase activity. Rice plants overexpressing OsACDR1 exhibited spontaneous hypersensitive response (HR)-like lesions on leaves, upregulation of defense-related marker genes and accumulation of phenolic compounds and secondary metabolites (phytoalexins). These transgenic plants also acquired enhanced resistance to a fungal pathogen (Magnaporthe grisea) and showed inhibition of appressorial penetration on the leaf surface. In contrast, loss-offunction and RNA silenced OsACDR1 rice mutant plants showed downregulation of defense-related marker genes expressions and susceptibility to M. grisea. Furthermore, transient expression of an OsACDR1:GFP fusion protein in rice protoplast and onion epidermal cells revealed its localization to the nucleus. These results indicate that OsACDR1 plays an important role in the positive regulation of disease resistance in rice.

Identifying and exploiting three informative DNA variant segregation types in pooled genome sequencing analysis of the weeping phenotype in Malus empowers an effective genetic mapping strategy in out-crossing woody species.

Diverse abiotic and biotic stresses have marked effects on plant growth and productivity. To combat such stresses, plants have evolved complex but not well understood responses. Common effects upon perception of environmental stress are differential expression of the plant proteome and the synthesis of novel regulatory proteins for protection from and acclimation to stress conditions. Plants respond differently in terms of activation of stress-responsive signaling pathways depending upon the type and nature of the stresses to which they are exposed. Progress in proteomics and systems biology approaches has made it possible to identify the novel proteins and their interactions that function in abiotic stress responses. This will enable elucidation of the functions of individual proteins and their roles in signaling networks. Proteomic analysis of the responses to various stress conditions is performed most commonly using 2D gel electrophoresis and high-throughput identification by LC-MS/MS. Because of recent developments in proteomics techniques, numerous proteomics studies of rice under abiotic stress conditions have been performed. In this review, proteomics studies addressing rice responses to the major environmental stresses--including cold, heat, drought, salt, heavy metals, minerals, UV radiation, and ozone--are discussed. Unique or common protein responses to these stress conditions are summarized and interpreted according to their possible physiological responses in each stress. Additionally, proteomics studies on various plant systems under various abiotic stress conditions are compared to provide deeper understanding of specific and common proteome responses in rice and other plant systems, which will further contribute to the identification of abiotic stress tolerance factor at protein level. Functional analysis of stress-responsive proteins will provide new research objectives with the aim of achieving stable crop productivity in the face of the increasing abiotic stress conditions caused by global climate change.