Sekiguchi Lesion Research Articles

Rice sl-MH-1 mutant induces cell death and confers blast resistance via the synergistic roles of signaling systems

Serotonin is ubiquitous across all forms of life and functions in responses to biotic and abiotic stresses. In rice, the conversion of tryptamine to serotonin is catalyzed by Sekiguchi lesion (SL). Previous studies have identified an sl mutation (a null mutation of SL) in several rice varieties and confirmed its increase of resistance and cell death. However, a systematic understanding of the reprogrammed cellular processes causing cell death and resistance is lacking. We performed a multi-omics analysis to clarify the fundamental mechanisms at the protein, gene transcript, and metabolite levels. We found that cell death and Magnaporthe oryzae (M. oryzae) infection of the sl-MH-1 mutant activated plant hormone signal transduction involving salicylic acid (SA), jasmonic acid (JA), and abscisic acid (ABA) in multiple regulatory layers. We characterized the dynamic changes of several key hormone levels during disease progression and under the cell death conditions and showed that SA and JA positively regulated rice cell death and disease resistance. SL-overexpressing lines confirmed that the sl-MH-1 mutant positively regulated rice resistance to M. oryzae. Our studies shed light on cell death and facilitate further mechanistic dissection of programmed cell death in rice.

Mutation Types of CYP71P1 Cause Different Phenotypes of Mosaic Spot Lesion and Premature Leaf Senescence in Rice.

Lesion mimic mutants (LMMs) are ideal materials for studying programmed cell death and defense response in plants. Here we report investigations on two LMMs (msl-1 and msl-2) from the indica rice cultivar JG30 treated by ethyl methyl sulfone. Both of the mutants showed similar mosaic spot lesions at seedling stage, but they displayed different phenotypes along with development of the plants. At tillering stage, larger orange spots appeared on leaves of msl-2, while only small reddish-brown spots exhibit on leaves of msl-1. At heading stage, the msl-2 plants were completely dead, while the msl-1 plants were still alive even if showed apparent premature senility. For both the mutants, the mosaic spot lesion formation was induced by light; DAB and trypan blue staining showed a large amount of hydrogen peroxide accumulated at the lesion sites, accompanied by a large number of cell death. Consequently, reactive oxygen species were enriched in leaves of the mutants; SOD and CAT activities in the scavenging enzyme system were decreased compared with the wild type. In addition, degraded chloroplasts, decreased photosynthetic pigment content, down-regulated expression of genes associated with chloroplast synthesis/photosynthesis and up-regulated expression of genes related to senescence were detected in the mutants, but the abnormality of msl-2 was more serious than that of msl-1 in general. Genetic analysis and map-based cloning revealed that the lesion mimic and premature senescence traits of both the mutants were controlled by recessive mutated alleles of the SL (Sekiguchi lesion) gene, which encodes the CYP71P1 protein belonging to cytochrome P450 monooxygenase family. The difference of mutation sites and mutation types (SNP-caused single amino acid change and SNP-caused early termination of translation) led to the different phenotypes in severity between msl-1 and msl-2. Taken together, this work revealed that the CYP71P1 is involved in regulation of both premature senescence and cell death in rice, and its different mutation sites and mutation types could cause different phenotypes in terms of severity.

Loss function of SL (sekiguchi lesion) in the rice cultivar Minghui 86 leads to enhanced resistance to (hemi)biotrophic pathogens

BackgroundSerotonin, originally identified as a neurotransmitter in mammals, functions as an antioxidant to scavenge cellular ROS in plants. In rice, the conversion of tryptamine to serotonin is catalyzed by SL (sekiguchi lesion), a member of cytochrome P450 monooxygenase family. The sl mutant, originated from rice cultivar Sekiguchi-asahi, exhibits spontaneous lesions, whereas its immune responses to pathogens have not been clearly characterized.ResultsHere we identified three allelic mutants of SL in an indica rice restore line Minghui 86 (MH86), named as sl-MH-1, − 2 and − 3, all of which present the typical lesions under normal growth condition. Compared with those in MH86, the serotonin content in sl-MH-1 is dramatically decreased, whereas the levels of tryptamine and L-trytophan are significantly increased. The sl-MH-1 mutant accumulates high H2O2 level at its lesion sites and is more sensitive to exogenous H2O2 treatment than the wild type. When treated with the reductant vitamin C (Vc), the lesion formation on sl-MH-1 leaves could be efficiently suppressed. In addition, sl-MH-1 displayed more resistant to both the blast fungus and blight bacteria, Pyricularia oryzae (P. oryzae, teleomorph: Magnaporthe oryzae) and Xanthomonas oryzae pv. Oryzae (Xoo), respectively. The pathogen-associated molecular patterns (PAMPs)-triggered immunity (PTI) responses, like reactive oxygen species (ROS) burst and callose deposition, were enhanced in sl-MH-1. Moreover, loss function of SL resulted in higher resting levels of the defense hormones, salicylic acid and jasmonic acid. The RNA-seq analysis indicated that after P. oryzae infection, transcription of the genes involved in reduction-oxidation regulation was the most markedly changed in sl-MH-1, compared with MH86.ConclusionsOur results indicate that SL, involving in the final step of serotonin biosynthesis, negatively regulates rice resistance against (hemi)biotrophic pathogens via compromising the PTI responses and defense hormones accumulation.

Tryptamine and sakuranetin accumulation in Sekiguchi lesions associated with the light-enhanced resistance of the lesion mimic mutant of rice to Magnaporthe oryzae

In a study on the correlation between tryptamine levels and sakuranetin accumulation in the Sekiguchi lesion mutant (SLM) of rice, accumulation was found to be light dependent after inoculation with Magnaporthe oryzae. It was also induced by treatment with tryptamine under white light, but not in the dark. Light-dependent sakuranetin accumulation induced by tryptamine treatment or by infection with M. oryzae was significantly inhibited in the leaves pretreated with metalaxyl, a monoamine oxidase inhibitor. These results suggest that light-dependent accumulation of tryptamine induces sakuranetin production in the SLM of rice.

Sekiguchi Lesion Gene Encodes a Cytochrome P450 Monooxygenase That Catalyzes Conversion of Tryptamine to Serotonin in Rice

Serotonin is a well known neurotransmitter in mammals and plays an important role in various mental functions in humans. In plants, the serotonin biosynthesis pathway and its function are not well understood. The rice sekiguchi lesion (sl) mutants accumulate tryptamine, a candidate substrate for serotonin biosynthesis. We isolated the SL gene by map-based cloning and found that it encodes CYP71P1 in a cytochrome P450 monooxygenase family. A recombinant SL protein exhibited tryptamine 5-hydroxylase enzyme activity and catalyzed the conversion of tryptamine to serotonin. This pathway is novel and has not been reported in mammals. Expression of SL was induced by the N-acetylchitooligosaccharide (chitin) elicitor and by infection with Magnaporthe grisea, a causal agent for rice blast disease. Exogenously applied serotonin induced defense gene expression and cell death in rice suspension cultures and increased resistance to rice blast infection in plants. We also found that serotonin-induced defense gene expression is mediated by the RacGTPase pathway and by the G alpha subunit of the heterotrimeric G protein. These results suggest that serotonin plays an important role in rice innate immunity.

Tryptamine Pathway‐mediated DNA Fragmentation is Involved in Sekiguchi Lesion Formation for Light‐enhanced Resistance in Lesion Mimic Mutant of Rice to Magnaporthe grisea Infection

AbstractIn this study, the correlation between tryptamine pathway‐mediated Sekiguchi lesion formation and DNA fragmentation in the sl mutant infected with Magnaporthe grisea under light was investigated. DNA fragmentation was observed beginning at 12 h after M. grisea inoculation, and then increased with increasing time after inoculation under light, but not in the dark. However, in cycloheximide (CHX)‐ or heat shock (HS)‐pretreated leaves of the sl mutant, Sekiguchi lesion formation and DNA fragmentation were significantly inhibited, but not in chloramphenicol (CP)‐pretreated leaves. Tryptamine accumulation and enzyme activity such as tryptophan decarboxylase (TDC) and monoamine oxidase (MAO) were also inhibited, even under light in the CHX‐ or HS‐pretreated leaves, but not in the CP‐pretreated ones. DNA fragmentation and TDC gene expression were induced in the leaves by the treatment with tryptamine or H2O2 under light, but not in the presence of the MAO inhibitor and H2O2 scavenger. The suppressive and inducible effects of biotic and abiotic agents on the induction of DNA fragmentation in the leaves of the sl mutant agree well with those effects on the activation of the tryptamine pathway involved in Sekiguchi lesion formation. These results suggested that tryptamine pathway‐mediated DNA fragmentation induces Sekiguchi lesion formation, which is responsible for light‐enhanced resistance in the sl mutant infected with M. grisea under light.

Effect of a Photo-synthetic Inhibitor on Tryptamine Pathway-mediated Sekiguchi Lesion Formation in Lesion Mimic Mutant of Rice Infected withMagnaporthe 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, H 2 O 2 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.

The tryptophan pathway is involved in the defense responses of rice against pathogenic infection via serotonin production

The upregulation of the tryptophan (Trp) pathway in rice leaves infected by Bipolaris oryzae was indicated by: (i) enhanced enzyme activity of anthranilate synthase (AS), which regulates metabolic flux in the Trp pathway; (ii) elevated levels of the AS (OASA2, OASB1, and OASB2) transcripts; and (iii) increases in the contents of anthranilate, indole, and Trp. The measurement of the contents of Trp-derived metabolites by high-performance liquid chromatography coupled with tandem mass spectrometry revealed that serotonin and its hydroxycinnamic acid amides were accumulated in infected leaves. Serotonin accumulation was preceded by a transient increase in the tryptamine content and by marked activation of Trp decarboxylase, indicating that enhanced Trp production is linked to the formation of serotonin from Trp via tryptamine. Feeding of radiolabeled serotonin to inoculated leaves demonstrated that serotonin is incorporated into the cell walls of lesion tissue. The leaves of a propagating-type lesion mimic mutant (sl, Sekiguchi lesion) lacked both serotonin production and deposition of unextractable brown material at the infection sites, and showed increased susceptibility to B. oryzae infection. Treating the mutant with serotonin restored deposition of brown material at the lesion site. In addition, the serotonin treatment suppressed the growth of fungal hyphae in the leaf tissues of the sl mutant. These findings indicated that the activation of the Trp pathway is involved in the establishment of effective physical defenses by producing serotonin in rice leaves.

Effect of glyphosate on tryptamine production and Sekiguchi lesion formation in rice infected with Magnaporthe grisea

When exposed to light, the Sekiguchi lesion (sl) rice mutant has an enhanced resistance to Magnaporthe grisea infection responsible for Sekiguchi lesion formation and tryptamine accumulation. Glyphosate [N-(phosphonomethyl) glycine] pretreatment suppressed Sekiguchi lesion formation and tryptamine accumulation in the sl mutant after M. grisea infection even under light. This inhibition by glyphosate was blocked by the supply of exogenous tryptophan, but not by exogenous phenylalanine. In glyphosate-pretreated leaves, 5-enol-pyruvyl-shikimate-3-phosphate synthase gene expression and tryptophan biosynthesis were significantly suppressed. During tryptophan starvation, catalase activity was maintained at a high level even under light, leading to the suppression of H2O2 generation and DNA fragmentation. These results show a strong relationship between the tryptophan and tryptamine pathways in the induction of light-enhanced resistance to M. grisea infection in the sl mutant.

Effect of Photosynthetic Inhibitors on Induction of Tryptamine Pathway‐mediated Resistance in Lesion Mimic Mutant of Rice Infected with Magnaporthe grisea

AbstractRice cv. Sekiguchi‐asahi was identified as a lesion mimic mutant from cv. Asahi. This mutant showed light‐enhanced resistance to Magnaporthe grisea infection, inducing Sekiguchi lesion (sl) formation and tryptamine accumulation. To elucidate the role of chloroplasts on the activation of tryptamine pathway, the effect of some photosynthetic inhibitors on light‐dependant Sekiguchi lesion formation, tryptamine accumulation, and enzyme activities was investigated. When detached leaf blades which had been pretreated with Linuron, 1, 10‐Phenanthroline or Poly‐l‐lysine were inoculated with M. grisea, respectively, Sekiguchi lesion formation and typtamine accumulation were suppressed even under light, suppressing gene expression of tryptophan decarboxylase and monoamine oxidase activity. On the other hand, catalase activity was significantly inhibited in control leaves infected with M. grisea under light, but kept at high level in chemical‐pretreated leaves. These results suggested that photosynthetic activity in chloroplasts was involved in inhibition of antioxidant sysytem and activation of tryptamine pathway responsible for light‐enhanced resistance in sl mutant to M. grisea infection.

Effects of Light Quality on Induction of Tryptamine‐mediated Resistance in Lesion Mimic Mutant of Rice Infected with Magnaporthe grisea

AbstractThe effective wavelength of light on Sekiguchi lesion formation, tryptamine accumulation, enzyme activities and hydrogen peroxide (H2O2) generation involved in light‐induced resistance of rice (cv. Sekiguchi‐asahi) infected with Magnaporthe grisea was determined by using band‐pass filters (BPF). When the M. grisea‐inoculated leaves were irradiated by BPF‐filtered lights from daylight fluorescent lamps, Sekiguchi lesion development was enhanced by increasing the wavelength of visible light. The longer the radiation wavelength, the greater the tryptamine accumulation, the tryptophan decarboxylase and monoamine oxidase activities, and the H2O2 generation. By contrast, the longer the radiation wavelength of radiation, the greater the inhibition of catalase (CAT) activity. Among the visible lights, red light was the most effective for tryptamine pathway activation and CAT inhibition involved in light‐induced resistance.

DNA fragmentation in Sekiguchi lesion mutants of rice infected with Magnaporthe grisea

Light-dependent activation of the tryptamine pathway in Sekiguchi lesion (sl) mutants (Sekiguchi-asahi, Sekiguchi-koshihikari, and Sekiguchi-sasanishiki) inoculated with Magnaporthe grisea, was demonstrated by a significant increase in tryptophan decarboxylase and monoamine oxidase activities and tryptamine accumulation. Terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling (TUNEL) staining and gel analysis indicated DNA fragmentation in cells of leaf tissues with Sekiguchi lesions of the three sl mutants. Furthermore, increased DNase activity was also light-dependent in the sl mutants after inoculation with M. grisea. DNA fragmentation was inhibited in leaves when Sekiguchi lesion formation was suppressed by cycloheximide and heat shock pretreatments. These data suggest that Sekiguchi lesion formation in the sl mutants is an apoptosis-like response and that its response is induced by light-dependent activation of the tryptamine pathway, which is responsible for light-enhanced resistance.

Increased tryptophan decarboxylase and monoamine oxidase activities induce Sekiguchi lesion formation in rice infected with Magnaporthe grisea.

Sekiguchi lesion (sl)-mutant rice infected with Magnaporthe grisea showed increased light-dependent tryptophan decarboxylase (TDC) and monoamine oxidase (MAO) activities. TDC and MAO activities were observed before the penetration of M. grisea to rice cells and maintained high levels even after Sekiguchi lesion formation. Light-dependent expression of TDC gene was observed in leaves inoculated with M. grisea before Sekiguchi lesion formation. Spore germination fluid (SGF) of M. grisea also induced Sekiguchi lesion formation accompanied by increased enzymes activities and tryptamine accumulation. Sekiguchi lesion was also induced by treatments with tryptamine and beta-phenylethylamine, which are substrates for MAO, but was not induced by non-substrates such as indole-3-propionic acid, (+/-)-phenylethylamine and tryptophan under light. Light-dependent induction of Sekiguchi lesion by tryptamine was significantly inhibited in the presence of MAO inhibitors, metalaxyl and semicarbazide, and H2O2-scavengers, ascorbic acid and catalase. H2O2 in M. grisea-infected leaves with and without Sekiguchi lesions was demonstrated directly in situ by strong 3,3'-diaminobenzidine (DAB) staining. On the other hand, H2O2 induced Sekiguchi lesions on leaves of cv. Sekiguchi-asahi under light, but not in darkness. This difference was associated with the decrease of catalase activity in infected leaves under light and the absence of decrease in darkness. We hypothesize that the H2O2-induced breakdown of cellular organelles such as chloroplasts and mitochondria in mesophyll cells may cause high TDC and MAO activities and the development of Sekiguchi lesion, and that the sl gene products in wild-type rice may function as a suppressor of organelle breakdown caused by chemical or environmental stress.

Light‐Dependent Accumulation of Tryptamine in the Rice Sekiguchi Lesion Mutant Infected with Magnaporthe grisea

AbstractAn antifungal compound with an infection‐inhibiting activity was isolated from a rice Sekiguchi lesion (sl) mutant and identified as the indole alkaloid tryptamine. Tryptamine inhibited not only spore germination and appressorium formation of Magnaporthe grisea at high concentrations (> 600 μ g/ml) but also the infection hypha formation in onion cells at low concentrations (150‐300 μ g/ml). Tryptamine is a normal compound of the rice sl‐mutant but accumulates further in rice with a mutant‐type response (Sekiguchi lesion formation) to inoculation with M. grisea spores. A mutant type of response is involved in light‐enhanced resistance. The accumulation of tryptamine was not induced, however, in rice with a wild‐type response (blast lesion formation). This study strongly suggests that tryptamine plays an important role as a possible factor in light‐enhanced resistance in the rice sl‐mutants.

Light-Enhanced Resistance toMagnaporthe griseaInfection in the Rice Sekiguchi Lesion Mutants

The rice sl mutant showed two types of responses to Magnaporthe grisea infection by light treatments. One was an sl-mutant-type response characterized by Sekiguchi lesion expression under light waves of 400-700 nm, and the other was a wild-type response characterized by blast and/or necrotic spot lesion expression in the dark or at wavelength between 290 and 330 nm. There was a large difference in the resistance to M. grisea infection between the mutant- and wild-type responses in the rice sl mutant. When the mutant-type response was induced in the rice sl mutant, the disease resistance was enhanced relative to that in the wild-type response. Enhanced resistance was demonstrated by two components: (a) the number of Sekiguchi lesions was reduced relative to that of blast or necrotic lesions; (b) sporulation of M. grisea was not induced in Sekiguchi lesions. The enhanced resistance was dependent on light of 400-700 nm.

The Effects of Light and Photo‐ and Protein‐synthetic Inhibitors on the Sekiguchi Lesion Formation by Magnaporthe grisea in Rice cv. Sekiguchi‐asahi*

AbstractThe significance of light irradiation in Sekiguchi lesion (SL) formation by infection with Magnaporthe grisea on rice cv. Sekiguchi‐asahi was investigated. When the leaf blades of cv. Sekiguchi‐asahi inoculated with M. grisea spores were kept under different wavelengths of light. SLs were formed under visible light regardless of the compatibility between fungal race and cv. Sekiguchi‐asahi. On the contrary, typical blast and/or nectrotic spot lesions were formed under near ultraviolet radiation from the black light fluorescent lamps and near infrared radiation from infrared fluorescent lamps. The effective wavelength for light‐dependent SL formation was 400–700 nm. Furthermore, the longer the wavelength of radiation, the bigger were the SLs. Such light‐dependent induction of the SL was suppressed by pretreatment of 3‐(3,4‐dichlorophenyl)‐l,l‐dimethylurea (DCMU) and cycloheximide (CY). These results suggested that photosynthetic and protein synthetic activities were involved in SL formation.

Spontaneous Occurrence of the Sekiguchi Lesion in Two American Rice Lines: Its Induction, Inheritance, and Utilization

Spontaneous Occurrence of the Sekiguchi Lesion in Two American Rice Lines: Its Induction, Inheritance, and Utilization