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 elicitors found in diverse phytopathogens activate defense responses in rice plants

Cerebrosides A and C, compounds categorized as glycosphingolipids, were isolated in our previous study from the rice blast fungus (Magnaporthe grisea) as novel elicitors which induce the synthesis of rice phytoalexins. In this paper, these cerebroside elicitors showed phytoalexin-inducing activity when applied to plants by spray treatment and also induced the expression of pathogenesis-related (PR) proteins in rice leaves. This elicitor activity of the cerebrosides showed the structural specificity as that for the induction of phytoalexins. Ceramides prepared from the cerebrosides by removal of glucose also showed the elicitor activity even in lower level compared to the cerebrosides. In field experiments, the cerebroside elicitors effectively protected rice plants against the rice blast fungus, an economically devastating agent of disease of rice in Japan. The cerebrosides elicitors protected rice plants from other disease as well and were found to occur in a wide range of different phytopathogens, indicating that cerebrosides function as general elicitors in a wide variety of rice-pathogen interactions.     

Parsing a multifunctional biosynthetic gene cluster from rice: Biochemical characterization of CYP71Z6 & 7

Rice (Oryza sativa) contains a biosynthetic gene cluster associated with production of at least two groups of diterpenoid phytoalexins, the antifungal phytocassanes and antibacterial oryzalides. While cytochromes P450 (CYP) from this cluster are known to be involved in phytocassane production, such mono-oxygenase activity relevant to oryzalide biosynthesis was unknown. Here we report biochemical characterization demonstrating that CYP71Z6 from this cluster acts as an ent-isokaurene C2-hydroxylase that is presumably involved in the biosynthesis of oryzalides. Our results further suggest that the closely related and co-clustered CYP71Z7 likely acts as a C2-hydroxylase involved in a latter step of phytocassane biosynthesis. Thus, CYP71Z6 & 7 appear to have evolved distinct roles in rice diterpenoid metabolism, offering insight into plant biosynthetic gene cluster evolution.     

Probenazole-induced accumulation of salicylic acid confers resistance to Magnaporthe grisea in adult rice plants

Probenazole (PBZ) is the active ingredient of Oryzemate, an agrochemical which is used for the protection of rice plants from Magnaporthe grisea (blast fungus). While PBZ was reported to function upstream of salicylic acid (SA) in Arabidopsis, little is known about the mechanism of PBZ-induced resistance in rice. The role of SA in blast fungus resistance is also unclear. The recommended application period for Oryzemate is just before the Japanese rainy season, at which time rice plants in the field have reached the 8-leaf stage with adult traits. Thus, the involvement of SA in PBZ-induced resistance was studied in compatible and incompatible blast fungus-rice interactions at two developmentally different leaf morphology stages. Pre-treatment of inoculated fourth leaves of young wild-type rice plants at the 4-leaf stage with PBZ did not influence the development of whitish expanding lesions (ELs) in the susceptible interaction without the accumulation of SA and pathogenesis-related (PR) proteins. However, PBZ pre-treatment increased accumulation of SA and PR proteins in the eighth leaves of adult plants at the 8-leaf stage, resulting in the formation of hypersensitive reaction (HR) lesions (HRLs). Exogenous SA induced resistance in adult but not young plants. SA concentrations in blast fungus-inoculated young leaves were essentially the same in compatible and incompatible interactions, suggesting that PBZ-induced resistance in rice is age-dependently regulated via SA accumulation.     

稻瘟菌侵染诱导水稻凝集素基因的表达

利用mRNA差异显示技术（DDRT-PCR）,从非亲和性稻瘟菌生理小种131侵染的水稻品种爱知旭（Oryza sati-vaL.cv.Aichi-asahi)叶片中分离了8个诱导差异表达的cDNA片段,对这8个差示片段进行了回收,重扩增和克隆,以其中一个长度为321碱基并与甘露糖结合水稻凝集素和水稻盐诱导蛋白基因高度同源的差示片段为探针。筛选水稻非亲和性cDNA文库,获得12个阳性克隆。序列测定和数据库查询表明该基因的cDNA与水稻凝集素基因的cDNA及盐诱导蛋白基因的cDNA核苷酸同源笥高达96%。推定的氨基酸序列与甘露糖结合水稻凝集素的氨基酸序列一致。与水稻盐诱导蛋白仅相差2个氨基酸。Southern杂交显示该基因在水稻基因组中有两个同源拷贝数。Northern杂交表明非亲和性稻瘟菌侵染可强烈诱导该基因表达。因此推则该基因参与了水稻对稻瘟菌侵染的防御反应。     

The rice (Oryza sativa) blast lesion mimic mutant, blm, may confer resistance to blast pathogens by triggering multiple defense-associated signaling pathways.

Here we characterized a rice (Oryza sativa L.) blast lesion mimic (blm) mutant, identified previously in an N-methyl-N-nitrosourea-mutagenized population of the cultivar Hwacheong (wild type). The rice blm displayed spontaneous necrotic lesion formation on the leaves during development under long-day condition and temperature shift from 28 to 24 degrees C in the absence of obvious stress/disease, and provided us with a highly reproducible and convenient experimental system in the growth chamber to study blm. The blm phenotype resembled to the cell death of hypersensitive reaction (HR), and subsequent, two-dimensional gel electrophoresis (2-DGE) revealed induction of many leaf proteins; prominent among them were the three pathogenesis-related (PR) marker proteins of class 5 (one spot) and 10 (two spots). Interestingly, the rice blm manifested HR against all races tested of the rice blast fungus (Magnaporthe grisea), providing high resistance in a non-race specific manner. It was also observed that blm was highly resistant to hydrogen peroxide treatment. Using 2-DGE immunoblotting, we identified the presence of 4 new spots cross-reacting with a superoxide dismutase (SOD) antibody, only in blm, suggesting the expression of potentially new SOD protein (isoforms) during lesion formation. In the leaves of blm, autofluorescent compounds accumulated in and around the site of lesion progression. Moreover, enhanced levels of two major rice phytoalexins, sakuranetin and momilactone A were also observed in the leaves of blm. These results indicate that blm confers broad-spectrum resistance to multiple pathogens, and so, it could be hypothesized that the BLM gene product may control the HR-like cell death and its associated multiple defense signaling pathways, as evidenced by induction of known hallmark features (proteins/metabolites) linked with the defense responses, in rice.     

Molecular characterization of a pathogenesis-related protein 8 gene encoding a class III chitinase in rice

A cDNA encoding a class III chitinase (Oschib1) was isolated from a cDNA library constructed from rice leaves infected with the blast fungus Magnaporthe grisea. The cDNA contains an open reading frame of 861 nucleotides encoding 286 amino acid residues with a pI of 5.06. The deduced amino acid sequence of Oschibl has a high level of similarity with class IIIb chitinases of Gladiolus gandavensis (46%) and Tulipa bakeri (49%). A high level of Oschibl mRNA was detected after inoculation with M. grisea or Xanthomonas oryzae pv. oryzae. Expression of Oschib1 was induced more rapidly when an avirulent strain of M. grisea was inoculated (incompatible interaction) than when a virulent strain was used (compatible interaction). Expression of Oschibl was also induced by treatment of signaling molecules such as salicylic acid, ethylene, and methyl jasmonic acid, and by treatment with H2O2 or CuSO4. The induction patterns of Oschibl expression suggest that Oschib1 may be involved in defense response against pathogen infections and may be classified as a member of pathogenesis-related protein 8 in rice.     

Arachidonic and Linoleic Acids Elicit Isoflavonoid Phytoalexin Accumulation in Phaseolus vulgaris (French bean)

Arachidonic and linoleic, but not α-linolenic or γ-linolenic acids, induced necrosis and the accumulation of isoflavonoid phytoalexins in leaves of French bean (Phaseolus vulgaris L.) cv. ‘Red Mexican'. The levels of phytoalexins which accumulated were similar to those found in an incompatible interaction between cv. ‘Red Mexican’and an avirulent race 1 isolate of Pseudomonas syringae pv. phaseolicola. Maximum amounts of phytoalexins accumulated in response to 1.6 mM fattyacids, as opposed to 3.3 or 0.6 mM. The most severe tissue necrosis was not accompanied by the highest levels of phytoalexin accumulation. In contrast to leaves, only trace amounts of phytoalexins were elicited in hypocotyls. The possible significance of, these results is discussed in terms of mechanisms leading to plant cell death and phytoalexin accumulation.     

Effect of a Photo-synthetic Inhibitor on Tryptamine Pathway-mediated Sekiguchi Lesion Formation in Lesion Mimic Mutant of Rice Infected with Magnaporthe grisea

A lesion-mimic mutant of rice (cv. Sekiguchi-asahi) showed enhanced resistance to Magnaporthe grisea infection, thereby inducing Sekiguchi lesion ( sl ) formation and tryptamine accumulation under light. Both Sekiguchi lesion formation and tryptamine accumulation in leaves infected with M. grisea were inhibited by pretreatment with the photosynthetic inhibitor, 3-(3, 4-Dichlorophenyl)-1,1-dimethylurea (DCMU), which suppressed the gene expression of tryptophan decarboxylase ( TDC ), monoamine oxidase activity, H₂O₂ generation and DNA fragmentation. Catalase activity was inhibited by M. grisea infection under light, but magnitude of the inhibition was reduced in leaves pretreated with DCMU. Furthermore, tryptophan accumulated in M. grisea- infected leaves under light but not in DCMU-pretreated ones. Interestingly, such DCMU inhibition was reduced in the presence of tryptophan. Our studies suggest that chloroplasts function as the inhibitor of anti-oxidant system such as catalase activity and the supplier of a precursor of tryptamine and tryptophan in the sl mutant infected with M. grisea .     

Elicitor activity of cerebroside,a sphingolipid elicitor,in cell suspension cultures of rice

Cerebrosides, compounds categorized as glycosphingolipids, were found to occur in a wide range of phytopathogens as novel elicitors and to induce the effective disease resistance for rice plants in our previous study. Here, we showed that cerebroside elicitors lead to the accumulation of phytoalexins and pathogenesis-related (PR) protein in cell suspension cultures of rice with the structural specificity similar to that for the rice whole plants. This elicitor activity of the cerebroside was greater than jasmonic acid (JA) and chitin oligomer (which is known to be an elicitor for cell suspension cultures of rice). Treatment of cell suspension cultures with cerebroside and chitin oligomer resulted in a synergetic induction of phytoalexins, suggesting that cerebroside and carbohydrate elicitors, such as glucan and chitin elicitor, enhance the defense signals of rice in vivo. Induction of phytoalexins by the treatment with cerebroside elicitor was markedly inhibited by LaCl(3) and GdCl(3), Ca(2+ )channel blockers. It is possible that Ca(2+) may be involved in the signaling pathway of elicitor activity of cerebroside.     

Jasmonoyl-l-isoleucine is required for the production of a flavonoid phytoalexin but not diterpenoid phytoalexins in ultraviolet-irradiated rice leaves

Rice produces low-molecular-weight antimicrobial compounds known as phytoalexins, in response to not only pathogen attack but also abiotic stresses including ultraviolet (UV) irradiation. Rice phytoalexins are composed of diterpenoids and a flavonoid. Recent studies have indicated that endogenous jasmonyl-l-isoleucine (JA-Ile) is not necessarily required for the production of diterpenoid phytoalexins in blast-infected or CuCl₂-treated rice leaves. However, JA-Ile is required for the accumulation of the flavonoid phytoalexin, sakuranetin. Here, we investigated the roles of JA-Ile in UV-induced phytoalexin production. We showed that UV-irradiation induces the biosynthesis of JA-Ile and its precursor jasmonic acid. We also showed that rice jasmonate biosynthesis mutants produced diterpenoid phytoalexins but not sakuranetin in response to UV, indicating that JA-Ile is required for the production of sakuranetin but not diterpenoid phytoalexins in UV-irradiated rice leaves.     

Pathogen-induced production of the antifungal AFP protein from Aspergillus giganteus confers resistance to the blast fungus Magnaporthe grisea in transgenic rice

Rice blast, caused by Magnaporthe grisea, is the most important fungal disease of cultivated rice worldwide. We have developed a strategy for creating disease resistance to M. grisea whereby pathogen-induced expression of the afp (antifungal protein) gene from Aspergillus giganteus occurs in transgenic rice plants. Here, we evaluated the activity of the promoters from three maize pathogenesis-related (PR) genes, ZmPR4, mpi, and PRms, in transgenic rice. Chimeric gene fusions were prepared between the maize promoters and the beta-glucuronidase reporter gene (gus A). Histochemical assays of GUS activity in transgenic rice revealed that the ZmPR4 promoter is strongly induced in response to fungal infection, treatment with fungal elicitors, and mechanical wounding. The ZmPR4 promoter is not active in the seed endosperm. The mpi promoter also proved responsiveness to fungal infection and wounding but not to treatment with elicitors. In contrast, no activity of the PRms promoter in leaves of transgenic rice was observed. Transgenic plants expressing the afp gene under the control of the ZmPR4 promoter were generated. Transformants showed resistance to M. grisea at various levels. Our results suggest that pathogen-inducible expression of the afp gene in rice plants may be a practical way for protection against the blast fungus. Most agricultural crop species suffer from a vast array of fungal diseases that cause severe yield losses all over the world. Rice blast, caused by the fungus Magnaporthe grisea (Herbert) Barr (anamorph Pyricularia grisea), is the most devastating disease of cultivated rice (Oryza sativa L.), due to its     

Uncovering the complex metabolic network underlying diterpenoid phytoalexin biosynthesis in rice and other cereal crop plants

Rice (Oryza sativa) is a staple food crop and serves as a model cereal crop plant for scientific study. Phytochemical investigations of the agronomically devastating rice blast disease have identified a number of rice phytoalexins exhibiting significant direct anti-fungal activity against the causative agent, Magneporthe grisea. Current evidence strongly indicates that these phytoalexins, largely a family of labdane-related diterpenoids, are important as general antibiotics, and that similar phytoalexins are produced more broadly throughout the cereal crop family. From the extensive sequence information available for rice it has been possible to functionally identify the genes for the enzymes catalyzing the two consecutive cyclization reactions that initiate biosynthesis of these labdane-related diterpenoid phytoalexins. This has led to several insights into the underlying evolution of diterpene biosynthesis throughout the cereal crop family. The hydrocarbon olefins resulting from cyclization must be further elaborated to form bioactive natural products and, because not much is currently known, necessarily speculative biosynthetic pathways for these processes are presented. Given the significant antibiotic activity of the labdane-related diterpenoid phytoalexins from rice, and the presence of similar secondary metabolism throughout the cereal crop plant family, study of this type of biosynthesis will continue to be an area of active investigation.     

Inhibition of infection of the rice blast fungus by halisulfate 1, an isocitrate lyase inhibitor

Halisulfate 1, a sesterterpene sulfate and an isocitrate lyase (ICL) inhibitor that is isolated from tropical sponge Hippospongia spp., reduces both appressorium formation and infection of rice plants by the fungus Magnaporthe grisea. Rice plants infected with wild-type M. grisea Guy 11 exhibited significantly lower disease severity after halisulfate 1 treatment than without, and the treatment effect was comparable to the behavior of the Delta icl knockout mutant I-10. The protection observed upon applying halisulfate 1 to rice plants suggests that the ICL inhibitor may be a promising candidate for crop protection, particularly to protect rice plants against M. grisea.     

Molecular cloning and characterization of a cDNA encoding ent-cassa-12,15-diene synthase, a putative diterpenoid phytoalexin biosynthetic enzyme, from suspension-cultured rice cells treated with a chitin elicitor

We have isolated and characterized a cDNA encoding a novel diterpene cyclase, OsDTC1, from suspension-cultured rice cells treated with a chitin elicitor. OsDTC1 functions as ent-cassa-12,15-diene synthase, which is considered to play a key role in the biosynthesis of (-)-phytocassanes recently isolated as rice diterpenoid phytoalexins. The expression of OsDTC1 mRNA was also confirmed in ultraviolet (UV)-irradiated rice leaves. In addition, we identified ent-cassa-12,15-diene, a putative diterpene hydrocarbon precursor of (-)-phytocassanes, as an endogenous compound in the chitin-elicited suspension-cultured rice cells and the UV-irradiated rice leaves. The OsDTC1 cDNA isolated here will be a useful tool to investigate the regulatory mechanisms of the biosynthesis of (-)-phytocassanes in rice.