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.     

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.     

Enhanced resistance to blast fungus and bacterial blight in transgenic rice constitutively expressing OsSBP, a rice homologue of mammalian selenium-binding proteins

The rice Oryza sativa selenium-binding protein homologue (OsSBP) gene encodes a homologue of mammalian selenium-binding proteins, and it has been isolated as one of the genes induced by treating a plant with a cerebroside elicitor from rice blast fungus. The possible role of OsSBP in plant defense was evaluated by using a transgenic approach. Plants overexpressing OsSBP showed enhanced resistance to a virulent strain of rice blast fungus as well as to rice bacterial blight. The expression of defense-related genes and the accumulation of phytoalexin after infection by rice blast fungus were accelerated in the OsSBP overexpressors. A higher level of H(2)O(2) accumulation and reduced activity of such scavenging enzymes as ascorbate peroxidase and catalase were seen when the OsSBP-overexpressing plants were treated with the protein phosphatase 1 inhibitor, calyculin A. These results suggest that the upregulation of OsSBP expression conferred enhanced tolerance to different pathogens, possibly by increasing plant sensitivity to endogenous defense responses. Additionally, the OsSBP protein might have a role in modulating the defense mechanism to biotic stress in rice.     

Cerebroside mediated elicitation of defense response in chilli (Capsicum annuum L.) against Colletotrichum capsici infection

Abstract

Enhancing the host resistance by using naturally occurring elicitors derived from pathogenic organisms is emerging as an ecofriendly approach in plant disease management. Cerebrosides, categorized as glycosphingolipids, were isolated and partially purified from the wilt causing fungus (Fusarium oxysporum f. sp. lycopersici). Cerebroside treatment significantly reduced the anthracnose disease incidence under greenhouse conditions. Cerebroside elicitors were found to stimulate the early H₂O₂ accumulation followed by the production of plant defense-related enzymes such as Phenylalanine ammonia lyase (PAL), Peroxidase (POX), Polyphenol oxidase (PPO), and Lipoxygenase (LOX) when applied to chilli (Capsicum annuum L.) plants by spray treatment and also induced the accumulation of capsidiol. Defense-related enzyme activities were increased by the elicitor treatment and an high level in activity was maintained during the experimental period. Under greenhouse conditions, the cerebroside elicitors effectively protected chilli plants against infection by anthracnose causing organism, Colletotrichum capsici.     

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     

Two purified oligosaccharide elicitors,N-acetylchitohepatose and tetraglucosyl glucitol, derived fromMagnaporthe grisea cell walls, synergistically activate biosynthesis of phytoalexin in suspension-cultured rice cells

Two purified oligosaccharide elicitors generatable from fungal cell walls, N-acetylchitoheptaose and a tetraglucosyl glucitol from rice blast fungus (Magnaporthe grisea), synergistically activated phytoalexin biosynthesis in cultured rice cells. Inhibition experiments for the binding of radiolabeled N-acetylchitooligosaccharide elicitor to the plasma membrane from rice cells indicate that the two elicitors are recognized by different receptors. These results also indicate the presence of a positive interaction between the signal transduction cascade downstream of each elicitor/receptor, which enhances resistance against pathogens.     

Identification and characterization of 2'-deoxyuridine from the supernatant of conidial suspensions of rice blast fungus as an infection-promoting factor in rice plants

We previously detected infection-promoting activity in the supernatant of the conidial suspension (SCS) of the rice blast fungus. In the present study, a molecule carrying the activity was purified and identified as 2'-deoxyuridine (dU). The infection-promoting activity of dU was strictly dependent on its chemical structure and displayed characteristics consistent with those of the SCS. Notably, the activity of dU was exclusively detected during interactions between rice and virulent isolates of the fungus, the number of susceptible lesions in leaf blades was increased by dU, and nonhost resistance in rice plants was not affected by treatment with dU. In addition, the expression of pathogensis-related genes, accumulation of H(2)O(2), and production of phytoalexins in rice in response to inoculation with virulent fungal isolates was not suppressed by dU. The infection-promoting activity of dU was not accompanied by elevated levels of endogenous abscissic acid, which is known to modify plant-pathogen interactions, and was not detected in interactions between oat plants and a virulent oat blast fungus isolate. Taken together, these results demonstrate that dU is a novel infection-promoting factor that acts specifically during compatible interactions between rice plants and rice blast fungus in a mode distinct from that of toxins and suppressors.     

Induction of resistance against rice blast fungus in rice plants treated with a potent elicitor, N-acetylchitooligosaccharide

The mode of action of a potent elicitor, N-acetylchitooligosaccharide, in rice plants was examined. In intact seedlings, no significant uptake of the elicitor via the roots was observed within 3 h, whereas rapid uptake was observed in excised leaves. Rapid and transient expression of an elicitor-responsive gene, EL2, was induced in the leaves of intact seedlings sprayed with the elicitor or in the roots and leaves of intact seedlings by immersing roots in the elicitor solution. Histochemical analysis indicated that EL2 was expressed in cells exposed to the elicitor of root and leaves. In seedlings treated with the elicitor for 1 d or longer, hyphal growth of rice blast fungus was significantly delayed, and an accumulation of auto-fluorescence around the infection site was observed. Two defense-related genes, PR-1 and PR-10 (PBZ1), were induced in a systemic and local manner by elicitor treatment, in correlation with the induction of resistance against rice blast fungus. N-Acetylchitoheptaose did not inhibit the hyphal growth of the fungi. These results indicate the occurrence of systemic signal transmission from N-acetylchitooligosaccharide in rice plants.     

Stemar-13-ene synthase, a diterpene cyclase involved in the biosynthesis of the phytoalexin oryzalexin S in rice

In suspension-cultured rice cells, diterpenoid phytoalexins are produced in response to exogenously applied elicitors. We isolated a cDNA encoding a diterpene cyclase, OsDTC2, from suspension-cultured rice cells treated with a chitin elicitor. The OsDTC2 cDNA was overexpressed in Escherichia coli as a fusion protein with glutathione S-transferase, and the recombinant OsDTC2 was indicated to function as stemar-13-ene synthase that converted syn-copalyl diphosphate to stemar-13-ene, a putative diterpene hydrocarbon precursor of the phytoalexin oryzalexin S. The level of OsDTC2 mRNA in suspension-cultured rice cells began to increase 3 h after addition of the elicitor and reached the maximum after 8 h. The expression of OsDTC2 was also induced in UV-irradiated rice leaves. In addition, we indicated that stemar-13-ene accumulated in the chitin-elicited suspension-cultured rice cells and the UV-irradiated rice leaves.     

Elicitation of Diterpene Biosynthesis in Rice (Oryza sativa L.) by Chitin

Cell-free extracts of UV-irradiated rice (Oryza sativa L.) leaves have a much greater capacity for the synthesis from geranylgeranyl pyrophosphate of diterpene hydrocarbons, including the putative precursors of rice phytoalexins, than extracts of unstressed leaves (KA Wickham, CA West [1992] Arch Biochem Biophys 293: 320-332). An elicitor bioassay was developed on the basis of these observations in which 6-day-old rice cell suspension cultures were incubated for 40 hours with the substance to be tested, and an enzyme extract of the treated cells was assayed for its diterpene hydrocarbon synthesis activity as a measure of the response to elicitor. Four types of cell wall polysaccharides and oligosaccharide fragments that have elicitor activity for other plants were tested. Of these, polymeric chitin was the most active; a suspension concentration of approximately 7 micrograms per milliliter gave 50% of the maximum response in the bioassay. Chitosan and a branched β-1,3-glucan fraction from Phytophthora megasperma f. sp. glycinea cell walls were only weakly active, and a mixture of oligogalacturonides was only slightly active. A crude mycelial cell wall preparation from the rice pathogen, Fusarium moniliforme, gave a response comparable to that of chitin, and this activity was sensitive to predigestion of the cell wall material with chitinase before the elicitor assay. N-Acetylglucosamine, chitobiose, chitotriose, and chitotetrose were inactive as elicitors, whereas a mixture of chitin fragments solubilized from insoluble chitin by partial acid hydrolysis was highly active. Constitutive chitinase activity was detected in the culture filtrate and enzyme extract of cells from a 6-day-old rice cell culture; the amount of chitinase activity increased markedly in both the culture filtrate and cell extracts after treatment of the culture with chitin. We propose on the basis of these results that soluble chitin fragments released from fungal cell walls through the action of constitutive rice chitinases serve as biotic elicitors of defense-related responses in rice.     

Tangeretin inhibits fungal ferroptosis to suppress rice blast

Flavonoids are polyphenolic secondary metabolites that function as signaling molecules, allopathic compounds, phytoalexins, detoxifying agents and antimicrobial defensive compounds in plants. Blast caused by the fungus Magnaporthe oryzae is a serious disease affecting rice cultivation. In this study, we revealed that a natural flavonoid, tangeretin, substantially delays the formation of M. oryzae appressoria and blocks the development of blast lesions on rice plants. Our data suggest that tangeretin has antioxidant activity that interferes with conidial cell death/ferroptosis, which is critical for M. oryzae pathogenicity. Tangeretin showed a ferroptosis inhibition efficacy comparable to the well-established liproxstatin-1. Furthermore, overexpression of the NADPH oxidases NOX1 or NOX2 significantly decreased sensitivity toward tangeretin treatment, suggesting Nox-mediated lipid peroxidation as a possible target for tangeretin in regulating redox signaling and ferroptosis in M. oryzae. Our nursery and field tests showed that application of tangeretin can effectively mitigate overall disease symptoms and prevent leaf blast. Our study reveals the plant-derived fungal ferroptosis inhibitor tangeretin as a potential and novel antifungal agrochemical for the sustainable prevention of the devastating blast disease in important cereal crops.     

Accumulation of salicylic acid,jasmonic acid and phytoalexins in rice, <Emphasis Type="Italic">Oryza sativa</Emphasis>, infested by the white-backed planthopper, <Emphasis Type="Italic">Sogatella furcifera</Emphasis> (Hemiptera: Delphacidae)

In order to clarify the mechanism of induced resistance to blast disease in rice, Oryza sativa, that had been previously infested by the white-backed planthopper, Sogatella furcifera Horváth, we first investigated the accumulation of salicylic acid (SA) and jasmonic acid (JA) in rice plants infested by the planthopper. The results confirmed that infestation of S. furcifera strongly stimulates the production of SA and JA in rice. These results indicate that both salicylate- and jasmonate-mediated pathways (SA and JA pathways), which are involved in the general defense system in plants, were activated in rice infested by S. furcifera. Further results confirmed that S. furcifera infestation induces accumulation of a major rice diterpenoid phytoalexin, momilactone A, and a flavonoid phytoalexin, sakuranetin, which are well known as antimicrobial chemicals, particularly in blast disease caused by the blast fungus, Magnaporthe oryzae B. Couch. All these results strongly suggest the following hypothetical mechanism of induced-resistance to M. oryzae in rice infested by S. furcifera. First, S. furcifera releases some elicitor-active compounds, which might be produced in the salivary glands, into the rice plant during feeding. Next, the defense signal systems, SA- and JA-mediated pathways, are activated by the elicitor. Finally, phytoalexins are induced in rice as antimicrobial compounds mainly through activation of the JA-mediated pathway.     

Active Oxygen Radicals Induce Peroxidase Activity in Rice Blade Tissues

The relationship between active oxygen radicals and peroxidase induction on disease resistance in rice blades was investigated. Nitric oxide was produced in the whole blade stimulated by blast fungus elicitor. The induction of peroxidase activity was detected in active oxygen radical-treated rice blades 1 hour after treatment and thereafter. These results suggest that active oxygen radicals produced by stimulation with the elicitor could trigger peroxidase induction.     

Active oxygen radicals induce peroxidase activity in rice blade tissues

The relationship between active oxygen radicals and peroxidase induction on disease resistance in rice blades was investigated. Nitric oxide was produced in the whole blade stimulated by blast fungus elicitor. The induction of peroxidase activity was detected in active oxygen radical-treated rice blades 1 hour after treatment and thereafter. These results suggest that active oxygen radicals produced by stimulation with the elicitor could trigger peroxidase induction.     

Phytoalexin Elicitor Activity of Carbohydrates from Phytophthora megasperma f.sp. glycinea and Other Sources

Three unique classes of carbohydrates were isolated from the hyphal cell walls of Phytophthora megasperma f.sp. glycinea (Pmg) and compared with other substances for their activity as elicitors of the phytoalexin glyceollin in soybean tissues. Glucomannans extracted from cell walls with soybean β-1,3-endoglucanase were purified and proved to be the most active elicitors yet reported. They were approximately 10 times more active in soybean cotyledons than the heterogeneous β-glucan elicitor fraction extracted from Pmg walls. In addition, the glucomannan fraction gave race-specific elicitor activity in soybean hypocotyls. Pronase was found to be a suitable reagent for the mild extraction of glycopeptides from Pmg cell walls. All of the carbohydrates isolated from Pmg cell walls possessed significant elicitor activity, but other glucans, a glucomannan and mannan from other sources, were much less active. Chitin and chitosan, reported to function as elicitors in other plants, had low activity in soybean cotyledons. Arachidonic acid was inactive, despite its previously observed elicitor activity in potato tubers. The results indicated that, for Pmg, the carbohydrate elicitor most probably involved in the initiation of phytoalexinmediated defense during fungus infection of soybean plants is the glucomannan fraction liberated by endoglucanase.     

Proteomic analysis of differentially expressed proteins induced by rice blast fungus and elicitor in suspension-cultured rice cells

We used two-dimensional electrophoresis (2-DE) and other proteomic approaches to identify proteins expressed in suspension-cultured rice cells in response to the rice blast fungus, Magnaporthe grisea. Proteins were extracted from suspension-cultured cells at 24 and 48 h after rice blast fungus inoculation or treatment with elicitor or other signal molecules such as jasmonic acid (JA), salicylic acid, and H(2)O(2). The proteins were then polyethylene glycol fractionated before separation by 2-DE. Fourteen protein spots were induced or increased by the treatments, which we analyzed by N-terminal or internal amino acid sequencing. Twelve proteins from six different genes were identified. Rice pathogen-related protein class 10 (OsPR-10), isoflavone reductase like protein, beta-glucosidase, and putative receptor-like protein kinase were among those induced by rice blast fungus; these have not previously been reported in suspension-cultured rice cells. Six isoforms of probenazole-inducible protein (PBZ1) and two isoforms of salt-induced protein (SalT) that responded to blast fungus, elicitor, and JA were also resolved on a 2-DE gel and identified by proteome analysis. The expression level of these induced proteins both in suspension-cultured cells and in leaves of whole plants was analyzed by Western blot. PBZ1, OsPR-10, and SalT proteins from incompatible reactions were induced earlier and to a greater extent than those in compatible reactions. Proteome analysis can thus distinguish differences in the timing and amount of protein expression induced by pathogens and other signal molecules in incompatible and compatible interactions.     

Plants interact with microbial polysaccharides

Plants are resistant to almost all of the microorganisms with which they come in contact. In response to invasion by a fungus, bacterium, or a virus, many plants produce low molecular weight compounds, phytoalexins, which inhibit the growth of microorganisms. Phytoalexins are produced whether or not the invading microorganism is a pathogen. The production of phytoalexins appears to be a widespread mechanism by which plants attempt to defend themselves against pests. Molecules of microbial origin which trigger phytoalexin accumulation in plants are called elicitors. Structural polysaccharides from the mycelial walls of several fungi elicit phytoalexin accumlation in plants. Approximately 10 ng of the polysaccharide elicits the accumulation in plants of more than sufficient amounts of phytoalexin to stop the growth of microorganisms in vitro. The best characterized elicitors have been demonstrated to be β-1,3-glucans with branches to the 6 position of some of the glucosyl residues. Oligosaccharides, produced by partial acid hydrolysis of the mycelial wall glucans, are exceptionally active elicitors. The smallest oligosaccharide which is still an effective elicitor is composed of about 8 sugar residues. Bacteria also elicit phytoalexin accumulation in plants, but the Rhizobium symbionts of legumes presumably have a mechanism which allows them to avoid either eliciting phytoalexin accumulation or the effects of the phytoalexins if they are accumulated. The lectins of legumes bind to the lipopolysaccharides of their symbiont, but not of their non-symbiont, Rhizobium. It is not known whether the lectin-lipopolysaccharide interaction is involved with the establishment of symbiosis. However, evidence will be presented that suggests that lectins are, in fact, enzymes capable of modifying the structurs of the lipopolysaccharides of their symbiont, but not of their non-symbiont, Rhizobium. It will also be shown that the lipopolysaccharides isolated from different Rhizobium species and from different strains of individual Rhizobium species have different sugar compositions. Thus, the different strains of a single Rhizobium species are as different from one another as the different species of Salmonella and other gram-negative bacteria. This conclusion is substantiated by experiments demonstrating that antibodies to the lipopolysaccharide from a single Rhizobium strain can differentiate that strain from other strains of the same species as well as from other Rhizobium species. The role in symbiosis of the strain-specific O-antigens is unknown.     

Differences in the recognition of glucan elicitor signals between rice and soybean: beta-glucan fragments from the rice blast disease fungus Pyricularia oryzae that elicit phytoalexin biosynthesis in suspension-cultured rice cells

Partial acid/enzymatic hydrolysis of the beta-(1-->3, 1-->6)-glucan from the cell walls of the rice blast disease fungus Pyricularia oryzae (Magnaporthe grisea) released elicitor-active fragments that induced phytoalexin biosynthesis in suspension-cultured rice cells. From the digestion of the glucan by an endo-beta-(1-->3)-glucanase, one highly elicitor-active glucopentaose was purified as a reduced compound, tetraglucosyl glucitol. The structure of this tetraglucosyl glucitol as well as two other related tetraglucosyl glucitols was elucidated as follows: (1) Glcbeta(1-->3)Glcbeta(1-->3)(Glcbeta(1-->6)) Glcbeta(1-->3)Glucitol (most active fragment); (2) Glcbeta(1-->3)(Glcbeta(1-->6))Glcbeta(1-->3)Glcbeta (1-->3)Glucitol; and (3) Glcbeta(1-->6) Glcbeta(1-->3)Glcbeta(1-->3)Glcbeta(1-->3)Glucitol. However, a synthetic hexa-beta-glucoside, known as a minimal structural element for the phytoalexin elicitor for soybean cotyledon cells, did not induce phytoalexin biosynthesis in the rice cells. Conversely, the beta-glucan fragment from P. oryzae did not induce phytoalexin biosynthesis in the soybean cotyledon cells, indicating differences in the recognition of glucooligosaccharide elicitor signals in these two plants. Because rice cells have been shown to recognize chitin fragments larger than pentamers as potent elicitors, these results also indicate that the rice cells can recognize at least two types of oligosaccharides from fungal cell walls as signal molecules to initiate defense response.