Confirming a major QTL and finding additional loci responsible for field resistance to brown spot (Bipolaris oryzae) in rice.

Brown spot is a devastating rice disease. Quantitative resistance has been observed in local varieties (e.g., 'Tadukan'), but no economically useful resistant variety has been bred. Using quantitative trait locus (QTL) analysis of recombinant inbred lines (RILs) from 'Tadukan' (resistant) × 'Hinohikari' (susceptible), we previously found three QTLs (qBS2, qBS9, and qBS11) that conferred resistance in seedlings in a greenhouse. To confirm their effect, the parents and later generations of RILs were transplanted into paddy fields where brown spot severely occurred. Three new resistance QTLs (qBSfR1, qBSfR4, and qBSfR11) were detected on chromosomes 1, 4, and 11, respectively. The 'Tadukan' alleles at qBSfR1 and qBSfR11 and the 'Hinohikari' allele at qBSfR4 increased resistance. The major QTL qBSfR11 coincided with qBS11 from the previous study, whereas qBSfR1 and qBSfR4 were new but neither qBS2 nor qBS9 were detected. To verify the qBSfR1 and qBSfR11 'Tadukan' resistance alleles, near-isogenic lines (NILs) with one or both QTLs in a susceptible background ('Koshihikari') were evaluated under field conditions. NILs with qBSfR11 acquired significant field resistance; those with qBSfR1 did not. This confirms the effectiveness of qBSfR11. Genetic markers flanking qBSfR11 will be powerful tools for marker-assisted selection to improve brown spot resistance.

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.

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.

RNA-mediated gene silencing in the phytopathogenic fungus Bipolaris oryzae.

The Ascomycetous fungus Bipolaris oryzae is the causal agent of brown leaf spot disease in rice and is a model for studying photomorphogenetic responses by near-UV radiation. Targeted gene disruption (knockout) for functional analysis of photomorphogenesis-related genes in B. oryzae can be achieved by homologous recombination with low efficiency. Here, the applicability of RNA silencing (knockdown) as a tool for targeting endogenous genes in B. oryzae is reported. A polyketide synthase gene (PKS1), involved in fungal DHN melanin biosynthesis pathways, was targeted by gene silencing as a marker. The silencing vector encoding hairpin RNA of the PKS1 fragment was constructed in a two-step PCR-based cloning, and introduced into the B. oryzae genomic DNA. Silencing of the PKS1 gene resulted in albino phenotypes and reduction of PKS1 mRNA expression. These results demonstrate the applicability of targeted gene silencing as a useful reverse-genetics approach in B. oryzae.

Characterization of the BLR1 gene encoding a putative blue-light regulator 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 BLR1, a gene that encodes a putative blue-light regulator similar to Neurospora crassa white-collar 1 (WC-1). The deduced amino acid sequence of BLR1 showed high degrees of similarity to other fungal blue-light regulator protein. Disruption of the BLR1 gene demonstrated that this gene is essential for conidial development after conidiophore formation and for near-UV radiation-enhanced photolyase gene expression.

A MAP kinase gene, BMK1, is required for conidiation and pathogenicity in the rice leaf spot pathogen Bipolaris oryzae.

We isolated and characterized BMK1, a gene encoding a mitogen-activated protein kinase (MAPK), from the rice leaf spot pathogen Bipolaris oryzae. The deduced amino acid sequence showed significant homology with Fus3/Kss1 MAPK homologues from other phytopathogenic fungi. The BMK1 disruptants showed impaired hyphal growth, no conidial production, and loss of virulence against rice leaves, indicating that the BMK1 is essential for conidiation and pathogenicity in B. oryzae.

Disruption of SRM1, a mitogen-activated protein kinase gene, affects sensitivity to osmotic and ultraviolet stressors in the phytopathogenic fungus Bipolaris oryzae.

Mitogen-activated protein kinases (MAPKs) play key roles in biological processes including differentiation, growth, proliferation, survival, and stress responses. We isolated and characterized the SRM1 gene, which encodes an MAPK related to yeast High-osmolarity glycerol 1 (Hog1), from the rice leaf pathogen Bipolaris oryzae. The deduced amino sequence of the SRM1 gene showed significant homology with Hog1-type MAPK homologues from other phytopathogenic fungi and contained a TGY motif for phosphorylation. The B. oryzae mutants with disruption of the SRM1 gene (Deltasrm1) showed growth inhibition under hyperosmotic, hydrogen peroxide, and UV exposure conditions. The Deltasrm1 mutants showed moderate resistance to dicarboximide and phenylpyrrole fungicides. The Deltasrm1 mutations caused a defect in the expression of the gene that encodes antioxidant enzyme catalase (CAT2) under UV and hyperosmotic conditions. Furthermore, the transcriptional patterns of the three melanin biosynthesis genes (PKS1, THR1, and SCD1) and of a gene of unknown function, uvi-1, which are specifically induced by near-ultraviolet (NUV) radiation, gradually decreased in comparison with the wild-type expression patterns. These results suggest that Srm1 contributes to responses to not only osmostress but also to hydrogen peroxide and UV stress, whereas Srm1 does not appear to regulate directly the expression of genes related to NUV-induced photomorphogenesis.

Insertional mutagenesis and characterization of a polyketide synthase gene (PKS1) required for melanin biosynthesis in Bipolaris oryzae.

A polyketide synthase gene named PKS1, involved in the melanin biosynthesis pathway of the phytopathogenic fungus Bipolaris oryzae, was isolated using restriction enzyme-mediated integration. Sequence analysis showed that the PKS1 encodes a putative protein that has 2155 amino acids and significant similarity to other fungal polyketide synthases. Targeted disruption of the PKS1 gene showed that it is necessary for melanin biosynthesis in B. oryzae. Northern blot analysis showed that PKS1 transcripts were specifically enhanced by near-ultraviolet radiation (300-400 nm) and that its temporal transcriptional patterns were similar to those of THR1 and SCD1 genes involved in the melanin biosynthesis pathway of B. oryzae.

Cloning, functional characterization, and near-ultraviolet radiation-enhanced expression of a photolyase gene (PHR1) from the phytopathogenic fungus Bipolaris oryzae.

Photolyase is a DNA repair enzyme that can absorb blue/ultraviolet A light as energy and split a pyrimidine dimer induced by ultraviolet radiation. We isolated and characterized PHR1, a gene encoding photolyase, from the phytopathogenic fungus Bipolaris oryzae. Sequence analysis showed that PHR1 encodes a putative protein that has 634 amino acids, a molecular mass of 72.6 kDa, and 51.3-55.5% sequence identity to other fungal photolyases. Complementation of the photoreactivation-deficient Escherichia coli mutant by PHR1 cDNA demonstrated that the PHR1 gene from B. oryzae encodes a functional photolyase. Real-time PCR analysis showed that the PHR1 transcripts were specifically enhanced by near-ultraviolet radiation (300-400 nm) and by sunlight.

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

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 185 amino acids, a molecular weight of 21 kDa 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 (300-400 nm) radiation.

Expression of THR1, a 1,3,8-trihydroxynaphthalene reductase gene involved in melanin biosynthesis in the phytopathogenic fungus Bipolaris oryzae, is enhanced by near-ultraviolet radiation.

1,3,8-Trihydroxynaphthalene (1,3,8-THN) reductase is involved in the production of fungal dihydroxynaphthalene (DHN) melanin. We isolated and characterized THR1, a gene encoding 1,3,8-THN reductase, from the phytopathogenic fungus Bipolaris oryzae. Sequence analysis showed that THR1 encodes a putative protein of 267 amino acids having a molecular weight of 28.5 kDa and 68-98% sequence identity to other fungal 1,3,8-THN reductases. Targeted disruption of the THR1 gene showed that it is essential for melanin biosynthesis in B. oryzae. Northern blot analysis showed that THR1 transcripts are constitutively expressed during normal growth but are specifically enhanced by near-ultraviolet (NUV) radiation in a dose-dependent manner. These results indicate that THR1 expression is transcriptionally enhanced by NUV radiation in B. oryzae.

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.