Kenji Umemura

Cerebrosides A and C, Sphingolipid Elicitors of Hypersensitive Cell Death and Phytoalexin Accumulation in Rice Plants

When plants interact with certain pathogens, they protect themselves by generating various chemical and physical barriers called the hypersensitive response. These barriers are induced by molecules called elicitors that are produced by pathogens. In the present study, the most active elicitors of the hypersensitive response in rice were isolated from the rice pathogenic fungus Magnaporthe grisea, and their structures were identified as cerebrosides A and C, sphingolipids that were previously isolated as inducers of cell differentiation in the fungusSchizophyllum commune. Treatment of rice leaves with cerebroside A induced the accumulation of antimicrobial compounds (phytoalexins), cell death, and increased resistance to subsequent infection by compatible pathogens. The degradation products of cerebroside A (fatty acid methyl ester, sphingoid base, and glucosyl sphingoid base) showed no elicitor activity. Hydrogenation of the 8E-double bond in the sphingoid base moiety or the 3E-double bond in the fatty acid moiety of cerebroside A did not alter the elicitor activity, whereas hydrogenation of the 4E-double bond in the sphingoid base moiety led to a 12-fold decrease in elicitor activity. Furthermore, glucocerebrosides from Gaucher’s spleen consisting of (E)-4-sphingenine and cerebrosides from rice bran mainly consisting of (4E,8E)-4,8-sphingadienine and (4E,8Z)-4,8-sphingadienine showed no elicitor activity. These results indicate that the methyl group at C-9 and the 4E-double bond in the sphingoid base moiety of cerebrosides A and C are the key elements determining the elicitor activity of these compounds. This study is the first to show that sphingolipids have elicitor activity in plants.

Proteomics of Rac GTPase signaling reveals its predominant role in elicitor-induced defense response of cultured rice cells.

We have previously shown that a human small GTPase Rac homolog, OsRac1, from rice (Oryza sativa) induces cascades of defense responses in rice plants and cultured cells. Sphingolipid elicitors (SEs) have been similarly shown to activate defense responses in rice. Therefore, to systematically analyze proteins whose expression levels are altered by OsRac1 and/or SE treatment, we performed a differential display analysis of proteins by the use of two-dimensional gel electrophoresis and mass spectrometry. A total of 271 proteins whose expression levels were altered by constitutively active (CA)-OsRac1 or SE were identified. Interestingly, of 100 proteins that were up-regulated by a SE, 87 were also induced by CA-OsRac1, suggesting that OsRac1 plays a pivotal role in defense responses induced by SE in cultured rice cells. In addition, CA-OsRac1 induces the expression of 119 proteins. Many proteins, such as pathogenesis-related proteins, SGT1, and prohibitin, which are known to be involved in the defense response, were found among these proteins. Proteins involved in redox regulation, chaperones such as heat shock proteins, BiP, and chaperonin 60, proteases and protease inhibitors, cytoskeletal proteins, subunits of proteasomes, and enzymes involved in the phenylpropanoid and ethylene biosynthesis pathways were found to be induced by CA-OsRac1 or SE. Results of our proteomic analysis revealed that OsRac1 is able to induce many proteins in various signaling and metabolic pathways and plays a predominant role in the defense response in cultured rice cells.

Contribution of salicylic acid glucosyltransferase, OsSGT1, to chemically induced disease resistance in rice plants.

Systemic acquired resistance (SAR), a natural disease response in plants, can be induced chemically. Salicylic acid (SA) acts as a key endogenous signaling molecule that mediates SAR in dicotyledonous plants. However, the role of SA in monocotyledonous plants has yet to be elucidated. In this study, the mode of action of the agrochemical protectant chemical probenazole was assessed by microarray-based determination of gene expression. Cloning and characterization of the most highly activated probenazole-responsive gene revealed that it encodes UDP-glucose:SA glucosyltransferase (OsSGT1), which catalyzes the conversion of free SA into SA O-beta-glucoside (SAG). We found that SAG accumulated in rice leaf tissue following treatment with probenazole or 2,6-dichloroisonicotinic acid. A putative OsSGT1 gene from the rice cultivar Akitakomachi was cloned and the gene product expressed in Escherichia coli was characterized, and the results suggested that probenazole-responsive OsSGT1 is involved in the production of SAG. Furthermore, RNAi-mediated silencing of the OsSGT1 gene significantly reduced the probenazole-dependent development of resistance against blast disease, further supporting the suggestion that OsSGT1 is a key mediator of development of chemically induced disease resistance. The OsSGT1 gene may contribute to the SA signaling mechanism by inducing up-regulation of SAG in rice plants.

Possible Role of Phytocassane, Rice Phytoalexin, in Disease Resistance of Rice against the Blast Fungus Magnaporthe grisea

In addition to momilactone, phytocassanes A through E (diterpene phytoalexins) were detected in rice leaves in fields suffering from rice blast. Furthermore, phytocassane accumulation was most abundant at the edges of necrotic lesions, indicating that the phytoalexins prevent subsequent spread of the fungus from the infected site. In pot experiments the pattern of phytocassane accumulation in rice leaves in an incompatible interaction (infection with an avirulent race of Magnaporthe grisea) was more rapidly induced than in a compatible interaction (infection with a virulent race of M. grisea).

Cholic Acid, a Bile Acid Elicitor of Hypersensitive Cell Death, Pathogenesis-Related Protein Synthesis, and Phytoalexin Accumulation in Rice

When plants interact with certain pathogens, they protect themselves by generating various defense responses. These defense responses are induced by molecules called elicitors. Since long ago, composts fermented by animal feces have been used as a fertilizer in plant cultivation, and recently, have been known to provide suppression of plant disease. Therefore, we hypothesized that the compounds from animal feces may function as elicitors of plant defense responses. As a result of examination of our hypothesis, an elicitor of rice defense responses was isolated from human feces, and its structure was identified as cholic acid (CA), a primary bile acid in animals. Treatment of rice (Oryza sativa) leaves with CA induced the accumulation of antimicrobial compounds (phytoalexins), hypersensitive cell death, pathogenesis-related (PR) protein synthesis, and increased resistance to subsequent infection by virulent pathogens. CA induced these defense responses more rapidly than did fungal cerebroside, a sphingolipid elicitor isolated from the rice pathogenic fungus Magnaporthe grisea. Furthermore, fungal cerebroside induced both types of rice phytoalexins, phytocassanes and momilactones, whereas CA mainly induced phytocassanes, but not momilactones. In the structure-activity relationship analysis, the hydroxyl groups at C-7 and C-12, and the carboxyl group at C-24 of CA contributed to the elicitor activity. These results indicate that CA is specifically recognized by rice and is a different type of elicitor from fungal cerebroside. This report demonstrated that bile acid induced defense responses in plants.

Cerebroside elicitor confers resistance to fusarium disease in various plant species.

ABSTRACT In the rice blast fungus pathosystem, cerebroside, a compound categorized as a sphingolipid, was found in our previous study to be a non-racespecific elicitor, which elicits defense responses in rice. Here we describe that cerebroside C is produced in diverse strains of Fusarium oxysporum, a common soilborne agent of wilt disease affecting a wide range of plant species. In addition, some type of cerebroside elicitor involving cerebroside A, B, or C was detected in other soilborne phytopathogens, such as Pythium and Botrytis. Treatment of lettuce (Lactuca sativa), tomato (Lycopersicon esculentum), melon (Cucumis melo), and sweet potato (Ipomoea batatas) with cerebroside B resulted in resistance to infection by each pathogenic strain of F. oxysporum. Induction of pathogenesis-related genes and H(2)O(2) production by treatment with cerebroside B were observed in tomato root tissues. The cerebroside elicitor showed no antifungal activity against F. oxysporum in vitro, indicating that the cerebroside elicitor activates defense mechanisms to confer resistance to Fusarium disease. These results suggest that cerebroside functions as a non-race-specific elicitor in a wide range of plant-phytopathogenic fungus interactions. Additionally, cerebroside elicitor serves as a potential biologically derived control agent.

Negative feedback regulation of microbe-associated molecular pattern-induced cytosolic Ca2+ transients by protein phosphorylation

Microbe/pathogen-associated molecular patterns (MAMPs/PAMPs) often induce rises in cytosolic free Ca2+ concentration ([Ca2+]cyt) and protein phosphorylation. Though they are postulated to play pivotal roles in plant innate immunity, their molecular links and the regulatory mechanisms remain largely unknown. To investigate the regulatory mechanisms for MAMP-induced Ca2+ mobilization, we have established a transgenic rice (Oryza sativa) cell line stably expressing apoaequorin, and characterized the interrelationship among MAMP-induced changes in [Ca2+]cyt, production of reactive oxygen species (ROS) and protein phosphorylation. Oligosaccharide and sphingolipid MAMPs induced Ca2+ transients mainly due to plasma membrane Ca2+ influx, which were dramatically suppressed by a protein phosphatase inhibitor, calyculin A (CA). Hydrogen peroxide and hypo-osmotic shock triggered similar [Ca2+]cyt elevations, which were not affected by CA. MAMP-induced protein phosphorylation, which is promoted by CA, has been shown to be required for ROS production and MAPK activation, while it negatively regulates MAMPs-induced Ca2+ mobilization and may play a crucial role in temporal regulation of [Ca2+]cyt signature.

Role of the α subunit of heterotrimeric G-protein in probenazole-inducing defense signaling in rice

Abstract Certain cellular responses to stresses and stimuli are regulated by a G-protein-mediated signaling pathway. A rice dwarf mutant that is defective in the α subunit of the heterotrimeric G-protein was found to be fully protected from blast fungus by the plant activator probenazole (PBZ) despite the 1-day delay in induction of the PR-10 gene PBZ1 by PBZ. These results suggest that the signaling pathway for protection by PBZ is not via the G-protein, although G-protein is involved in the induction of PBZ1 by PBZ.

Probenazole (Oryzemate®) — A Plant Defense Activator

Most plants have the ability to fight off pathogens by invoking various defense systems, including systemic acquired resistance (SAR). Chemicals that activate SAR are called plant activators. Among the most effective known plant activators is probenazole (3-allyloxy-1,2-benzisothiazole-1,1- oxide; PBZ, Figure 1), developed by Meiji Seika Kaisha Ltd. Oryzemate® granule, containing PBZ, has been widely used against rice blast by Japanese farmers since 1975. In the past several years, it has been applied in some 25–30% of all paddy fields in Japan to control blast. Since PBZ activates plant defense systems, the study of PBZ’s mode of action has helped clarify how rice plant defense systems function. These studies suggest that PBZ is not only an essential protectant for rice cultivation, but also a useful probe in research into plant defense mechanisms.

Indole-3-acetic acid-induced oxidative burst and an increase in cytosolic calcium ion concentration in rice suspension culture

Indole-3-acetic acid (IAA) is the major natural auxin involved in the regulation of a variety of growth and developmental processes such as division, elongation, and polarity determination in growing plant cells. It has been shown that dividing and/or elongating plant cells accompanies the generation of reactive oxygen species (ROS) and a number of reports have suggested that hormonal actions can be mediated by ROS through ROS-mediated opening of ion channels. Here, we surveyed the link between the action of IAA, oxidative burst, and calcium channel activation in a transgenic cells of rice expressing aequorin in the cytosol. Application of IAA to the cells induced a rapid and transient generation of superoxide which was followed by a transient increase in cytosolic Ca2+ concentration ([Ca2+]c). The IAA-induced [Ca2+]c elevation was inhibited by Ca2+ channel blockers and a Ca2+ chelator. Furthermore, ROS scavengers effectively blocked the action of IAA on [Ca2+]c elevation. Graphical abstract Exogenously applied IAA induces the generation of superoxide followed by a rapid and transient increase in cytosolic calcium ion concentration.