Makoto Ueno

Cloning, functional analysis and expression of a scytalone dehydratase gene (SCD1) involved in melanin biosynthesis of the phytopathogenic fungus Bipolaris oryzae

Abstract Scytalone dehydratase is involved in the production of fungal dihydroxynaphthalene melanin. We isolated and characterized SCD1, a gene encoding scytalone dehydratase, from the phytopathogenic fungus Bipolaris oryzae. Sequence analysis showed that SCD1 encodes a putative protein that has 185xa0amino acids, a molecular weight of 21xa0kDa and 51–75% sequence identity to other fungal scytalone dehydratases. Targeted disruption of SCD1 showed that this gene is necessary for melanin biosynthesis in B. oryzae. Northern blot analysis showed that SCD1 transcripts are specifically enhanced by near-ultraviolet (300–400xa0nm) radiation.

Characterization of the BMR1 gene encoding a transcription factor for melanin biosynthesis genes in the phytopathogenic fungus Bipolaris oryzae

We isolated and characterized Bipolaris melanin regulation 1 gene (BMR1) encoding a transcription factor for melanin biosynthesis genes in the phytopathogenic fungus Bipolaris oryzae. Sequence analysis showed that the BMR1 gene encodes a putative protein of 1012 amino acids that has 99% sequence similarity to transcription factor Cmr1 of Cochliobolus heterostrophus. The predicted B. oryzae Bmr1 protein has two DNA-binding motifs, two Cys2His2 zinc finger domains, and a Zn(II)2Cys6 binuclear cluster domain at the N-terminal region of Bmr1. Targeted disruption of the BMR1 gene showed that BMR1 is essential for melanin biosynthesis in B. oryzae. The overexpression of the BMR1 gene led to more dark colonies than in the wild-type strain under dark conditions. Real-time PCR analysis showed that the BMR1 expression of the overexpression transformant was about 10-fold that of the wild type under dark conditions and of the expression of three melanin biosynthesis genes. These results indicated that BMR1 encodes the transcription factor of melanin biosynthesis genes in B. oryzae.

Cloning and Characterization of the BLR2, the Homologue of the Blue-Light Regulator of Neurospora crassa WC-2, in the Phytopathogenic Fungus Bipolaris oryzae

Bipolaris oryzae is a filamentous ascomycetous fungus that causes brown leaf spot disease in rice. We isolated and characterized BLR2, a gene that encodes a putative blue-light regulator similar to Neurospora crassa white collar-2 (WC-2). The deduced amino acid sequence of the BLR2 showed significant homology to other fungal blue-light regulator proteins in the Per-Arnt-Sim (PAS) protein–protein interaction domain, nuclear localization signal, and GATA zinc finger DNA-binding domains. The BLR2-silenced transformants hardly produced conidia in the subsequent dark condition after near-ultraviolet (NUV) irradiation. Furthermore, the BLR2-silenced transformants suppressed the photolyase (PHR1) gene expression enhanced by NUV irradiation. These results indicate that BLR2 is necessary not only for conidial formation, but also for NUV radiation-enhanced photolyase gene expression in B. oryzae.

Cloning and expression analysis of two opsin-like genes in the phytopathogenic fungus Bipolaris oryzae

Two opsin-like genes, OPS1 and OPS2, were identified from a subtracted cDNA library for the identification of near-UV (NUV) radiation-enhanced genes using suppression subtractive hybridization methods in the brown leaf spot fungus Bipolaris oryzae. The OPS1 and OPS2 genes encode predicted proteins of 306 and 304 amino acids, respectively. Real-time PCR analysis showed that the OPS1 transcript is expressed weakly in mycelia under dark conditions but shows enhanced expression after NUV irradiation. By contrast, the OPS2 transcript is constitutively expressed at a high level in mycelia under dark conditions but is only weakly enhanced after NUV irradiation. These enhancement patterns of OPS1 and OPS2 gene expression after NUV irradiation did not occur in the blue-light regulator 1 (BLR1)-deficient mutant, suggesting that NUV radiation-enhanced gene expression of OPS1 and OPS2 could be controlled by the BLR1 in B. oryzae.

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.

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.

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.

Indole derivatives enhance resistance against the rice blast fungus Magnaporthe oryzae

In leaves pretreated with an indole derivative [indole-3-acetic acid (IAA) tryptamine, or tryptophan], blast lesion formation was suppressed compared to those treated with distilled water (DW) as a control. Phenylalanine ammonia-lyase (PAL) activity and PAL expression were significantly enhanced in the IAA- or tryptophan-pretreated leaves, but not in tryptamine-pretreated leaves. This induction of resistance was inhibited by pretreatment with a PAL inhibitor, α-aminooxyacetic acid, in IAA- and in tryptophan-treated leaves, but not in tryptamine-treated leaves. This study strongly shows that the indole derivatives IAA tryptamine and tryptophan can enhance a disease resistance mechanism that is supported by different metabolic pathways.

Effects of some indole-related compounds on the infection behavior of Magnaporthe grisea

Based on their effect on the infection behavior of Magnaporthe grisea, indole-related compounds were classified into three groups. The first group, including tryptophan, indole-3-butyric acid, indole-3-pyruvic acid, and indole-3-acetamide, did not inhibit infection behavior such as spore germination, appressorium formation, or infection hypha formation in M. grisea. The second, including indole acetic acid, indole-3-acetonitrile, oxindole, and tryptamine inhibited all stages of infection behaviors in a dose-dependent manner. The third, including gramine and indole, did not inhibit spore germination or appressorium formation, whereas it did inhibit infection hypha formation in a dose-dependent manner. These results suggest that endogenous or exogenously applied indole-related compounds in the second and third groups may contribute to protection in blast-susceptible plants such as rice and barley.

Suppressor- and Elicitor-Activities of Magnaporthe Grisea Toxin in Rice Leaves

The rice blast fungus Magnaporthe grisea (Hebert) Barr (anamorph Pyricularia oryzae Cavara) causes the most serious disease of rice. Many genetic studies have demonstrated that blast resistance of rice plants is controlled by several major gene pairs, and that resistance against the rice blast fungus differs among rice cultivars (Kiyosawa, 1974). Resistance or susceptibility of rice plants to M. grisea is determined by race-cultivar combinations (Yamada et al., 1976). Further, the fungus can also infect other Graminae such as barley, Italian ryegrass, and corn (Asuyama, 1965; Yaegashi 1981). Thus, the rice blast fungus offers an excellent system for studying molecular determinants of host species specificity and cultivar specificity. Host specific-toxins (Nishimura and Kohmoto, 1983; Otani et al., 1995; Scheffer and Yoder, 1983; Walton, 1996; Yoder, 1980) or suppressors (Doke et al, 1980; Oku et al, 1987) are well known as pathogenicity factors of fungal pathogens. In the M. grisea-plant system, however, the pathogenicity factors involved in host susceptibility have not heretofore been demonstrated.