Cloning and Characterization of the <Emphasis Type="Italic">BLR2</Emphasis>, the Homologue of the Blue-Light Regulator of <Emphasis Type="Italic">Neurospora crassa</Emphasis> WC-2, in the Phytopathogenic Fungus <Emphasis Type="Italic">Bipolaris oryzae</Emphasis>

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. The DDBJ accession number for the sequence reported in this paper is AB282674.     

Deficiency of the melanin biosynthesis genes SCD1 and THR1 affects sclerotial development and vegetative growth,but not pathogenicity,in Sclerotinia sclerotiorum

The fungus Sclerotinia sclerotiorum is a necrotrophic plant pathogen causing significant damage on a broad range of crops. This fungus produces sclerotia that serve as the long‐term survival structures in the life cycle and the primary inoculum in the disease cycle. Melanin plays an important role in protecting mycelia and sclerotia from ultraviolet radiation and other adverse environmental conditions. In this study, two genes, SCD1 encoding a scytalone dehydratase and THR1 encoding a trihydroxynaphthalene reductase, were disrupted by target gene replacement, and their roles in mycelial growth, sclerotial development and fungal pathogenicity were investigated. Phylogenetic analyses indicated that the deduced amino acid sequences of SCD1 and THR1 were similar to the orthologues of Botrytis cinerea. Expression of SCD1 was at higher levels in sclerotia relative to mycelia. THR1 was expressed at similar levels in mycelia and sclerotia at early stages, but was up‐regulated in sclerotia at the maturation stage. Disruption of SCD1 or THR1 did not change the pathogenicity of the fungus, but resulted in slower radial growth, less biomass, wider angled hyphal branches, impaired sclerotial development and decreased resistance to ultraviolet light.     

Sugar homeostasis mediated by cell wall invertase GRAIN INCOMPLETE FILLING 1 (GIF1) plays a role in pre‐existing and induced defence in rice

Sugar metabolism and sugar signalling are not only critical for plant growth and development, but are also important for stress responses. However, how sugar homeostasis is involved in plant defence against pathogen attack in the model crop rice remains largely unknown. In this study, we observed that the grains of gif1, a loss‐of‐function mutant of the cell wall invertase gene GRAIN INCOMPLETE FILLING 1 (GIF1), were hypersusceptible to postharvest fungal pathogens, with decreased levels of sugars and a thinner glume cell wall in comparison with the wild‐type. Interestingly, constitutive expression of GIF1 enhanced resistance to both the rice bacterial pathogen Xanthomonas oryzae pv. oryzae and the fungal pathogen Magnaporthe oryzae. The GIF1‐overexpressing (GIF1‐OE) plants accumulated higher levels of glucose, fructose and sucrose compared with the wild‐type plants. More importantly, higher levels of callose were deposited in GIF1‐OE plants during pathogen infection. Moreover, the cell wall was much thicker in the infection sites of the GIF1‐OE plants when compared with the wild‐type plants. We also found that defence‐related genes were constitutively activated in the GIF1‐OE plants. Taken together, our study reveals that sugar homeostasis mediated by GIF1 plays an important role in constitutive and induced physical and chemical defence.     

Osa‐miR1873 fine‐tunes rice immunity against Magnaporthe oryzae and yield traits

MicroRNAs (miRNAs) are known to fine‐tune growth, development, and stress‐induced responses. Osa‐miR1873 is a rice‐specific miRNA targeting LOC_Os05g01790. Here, we show that Osa‐miR1873 fine‐tunes rice immunity against Magnaporthe oryzae and yield traits via LOC_Os05g01790. Osa‐miR1873 was significantly upregulated in a susceptible accession but downregulated in a resistance accession at 24 h post‐inoculation (hpi) of M. oryzae. Overexpressing Osa‐miR1873 enhanced susceptibility to M. oryzae and compromised induction of defense responses. In contrast, blocking Osa‐miR1873 through target mimicry compromised susceptibility to M. oryzae and enhanced induction of defense responses. Altered expression of Osa‐miR1873 also resulted in some defects in yield traits, including grain numbers and seed setting rate. Moreover, overexpression of the target gene LOC_Os05g01790 increased rice blast disease resistance but severely penalized growth and yield. Taken together, we demonstrate that Osa‐miR1873 fine‐tunes the rice immunity‐growth trade‐off via LOC_Os05g01790, and blocking Osa‐miR1873 could improve blast disease resistance without significant yield penalty. Thus, the Osa‐miR1873‐LOC_Os05g01790 regulatory module is valuable in balancing yield traits and blast resistance.     

Ultraviolet‐B‐driven pigmentation and genetic diversity of benthic macroinvertebrates from high‐altitude Andean streams

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Accumulation of Indole‐3‐Acetic Acid in Rice sl Mutant Leaves Infected with Bipolaris oryzae

Rice leaves accumulate serotonin in response to infection by Bipolaris oryzae. The leaves of the sl mutant, which is deficient in the gene encoding tryptamine 5‐hydroxylase, accumulate tryptamine instead of serotonin upon infection by B. oryzae. Because tryptamine is a possible precursor of indole‐3‐acetic acid (IAA), we investigated the accumulation of IAA in sl leaves infected with B. oryzae. Liquid chromatography coupled with tandem mass spectrometry analysis indicated that IAA accumulated at approximately 1.5 μmol/gFW in the leaves of sl mutant. This accumulation was suppressed by 95% by the treatment with the tryptamine decarboxylase inhibitor, (S)‐α‐(fluoromethyl)tryptophan, at 100 μm , indicating that tryptamine served as the precursor of IAA. The accumulation of IAA was not reproduced by treatment with CuCl₂ or by exogenous feeding of tryptamine. Furthermore, inoculation of Magnaporthe grisea induced only a lower level of IAA accumulation. On the other hand, B. oryzae produced IAA in culture media containing tryptamine. These findings strongly suggested that the metabolism of tryptamine by B. oryzae was responsible for IAA accumulation in the leaves of the sl mutant. Serotonin added to the culture media was also converted into 5‐hydroxyindole‐3‐acetic acid (5HIAA) at a rate similar to that of tryptamine. Considering that wild‐type rice leaves accumulate serotonin for defensive purposes, reducing the concentration of serotonin by conversion into 5HIAA may be significant as a detoxification process in the interaction between B. oryzae and rice.     

A Strobilurin Fungicide Relieves Bipolaris oryzae‐Induced Oxidative Stress in Rice

Although strobilurins are one of the most effective and broad spectrum classes of systemic fungicides, they may also increase plant stress tolerance by modulating the activity of antioxidant enzymes. To address this issue, the effect of azoxystrobin (Az) on the activity of antioxidant enzymes and on the concentrations of antioxidant metabolites and oxidative stress‐related compounds was studied in rice plants (cv. Metica‐1) either inoculated or not with Bipolaris oryzae, the causal agent of brown spot (BS). The Az minimally affected the enzyme activities, but consistently increased the glutathione reduced (GSH) concentrations in the noninoculated plants. The activities of superoxide dismutase, peroxidase, ascorbate peroxidase, glutathione peroxidase, glutathione reductase and glutathione‐S‐transferase were increased upon B. oryzae infection, but such increases were greatly limited in the Az‐sprayed plants. Catalase activity decreased in the inoculated plants compared to the noninoculated plants regardless of fungicide treatment. The GSH concentration increased in response to the B. oryzae infection, and the Az‐sprayed plants sustained higher levels of GSH at advanced stages of fungal infection than did the nonsprayed plants. The inoculated plants exhibited an extensive oxidative stress as evidenced by higher concentrations of hydrogen peroxide and malondialdehyde compared to the noninoculated plants, but lower and later increases were recorded in the Az‐sprayed plants than in the nonsprayed plants. Therefore, Az greatly reduces B. oryzae‐induced oxidative stress by limiting BS development rather than by activating antioxidant enzymes. The GSH, however, seems to be Az‐modulated, and this may partially explain the constrained oxidative stress observed in the Az‐sprayed plants.     

OsASR2 regulates the expression of a defence‐related gene,Os2H16, by targeting the GT‐1 cis‐element

The GT‐1 cis‐element widely exists in many plant gene promoters. However, the molecular mechanism that underlies the response of the GT‐1 cis‐element to abiotic and biotic stresses remains elusive in rice. We previously isolated a rice short‐chain peptide‐encoding gene, Os2H16, and demonstrated that it plays important roles in both disease resistance and drought tolerance. Here, we conducted a promoter assay of Os2H16 and identified GT‐1 as an important cis‐element that mediates Os2H16 expression in response to pathogen attack and osmotic stress. Using the repeated GT‐1 as bait, we characterized an abscisic acid, stress and ripening 2 (ASR2) protein from yeast‐one hybridization screening. Sequence alignments showed that the carboxy‐terminal domain of OsASR2 containing residues 80–138 was the DNA‐binding domain. Furthermore, we identified that OsASR2 was specifically bound to GT‐1 and activated the expression of the target gene Os2H16, as well as GFP driven by the chimeric promoter of 2 × GT‐1‐35S mini construct. Additionally, the expression of OsASR2 was elevated by pathogens and osmotic stress challenges. Overexpression of OsASR2 enhanced the resistance against Xanthomonas oryzae pv. oryzae and Rhizoctonia solani, and tolerance to drought in rice. These results suggest that the interaction between OsASR2 and GT‐1 plays an important role in the crosstalk of the response of rice to biotic and abiotic stresses.     

Cyclone‐based spore trapping,quantitative real‐time polymerase chain reaction and high resolution melting analysis for monitoring airborne inoculum of Magnaporthe oryzae

Rice blast, caused by Magnaporthe oryzae, is one of the most devastating diseases in rice worldwide. We aimed to develop an integrated approach for convenient collection, quantification and characterisation of M. oryzae spores (airborne inoculum) in the field. We developed an easy‐to‐use cyclone‐based spore trap (the AirSampler) and a standard procedure for handling a small amount of airborne spores. Using a specific primer pair or a probe designed for the single‐copy gene mif23, SYBR Green and TaqMan assays could quantify 10 and 4 copy numbers, respectively, of M. oryzae DNA. During 2012 and 2013, the AirSampler and SYBR Green quantitative real‐time polymerase chain reaction were used to monitor temporal dynamics of M. oryzae spores in nursery fields of rice showing symptoms of blast disease. During four cropping seasons, the new techniques could detect M. oryzae spores before the appearance of rice blast symptoms. The amount of spores was low in the early season, then increased, with high fluctuations during the mid‐season and decreased to low levels at the heading stage in the late season. To improve the handling and storage of spore samples, we tested the effect of different treatments on the preservation of spore DNA. DNA loss was reduced with samples protected from ultraviolet B radiation, suspended in CTAB buffer, kept at room temperature or 4°C and used for DNA extraction in 2 weeks. Finally, we demonstrated that the high resolution melting analysis could be used for rapid determination of A, D, A + D and C alleles of the avirulence gene pex31 (Avr‐Pik/kp/km) in M. oryzae.     

Free‐Radical‐Scavenging,Antityrosinase, and Cellular Melanogenesis Inhibitory Activities of Synthetic Isoflavones

In this study, we examined the potential of synthetic isoflavones for application in cosmeceuticals. Twenty‐five isoflavones were synthesized and their capacities of free‐radical‐scavenging and mushroom tyrosinase inhibition, as well as their impact on cell viability of B16F10 murine melanoma cells and HaCaT human keratinocytes were evaluated. Isoflavones that showed significant mushroom tyrosinase inhibitory activities were further studied on reduction of cellular melanin formation and antityrosinase activities in B16F10 melanocytes in vitro. Among the isoflavones tested, 6‐hydroxydaidzein ( 2 ) was the strongest scavenger of both ABTS ^{. +} and DPPH ^. radicals with SC₅₀ values of 11.3±0.3 and 9.4±0.1 μM , respectively. Texasin ( 20 ) exhibited the most potent inhibition of mushroom tyrosinase (IC₅₀ 14.9±4.5 μM ), whereas retusin ( 17 ) showed the most efficient inhibition both of cellular melanin formation and antityrosinase activity in B16F10 melanocytes, respectively. In summary, both retusin ( 17 ) and texasin ( 20 ) exhibited potent free‐radical‐scavenging capacities as well as efficient inhibition of cellular melanogenesis, suggesting that they are valuable hit compounds with potential for advanced cosmeceutical development.     

Red‐Light‐Induced Resistance to Brown Spot Disease Caused by Bipolaris oryzae in Rice

In this study, the protective effect of red light against the brown spot disease caused by the fungus Bipolaris oryzae in rice was investigated. Lesion formation was significantly inhibited on detached leaves that were inoculated with B. oryzae and kept under red for 48 h, but it was not inhibited when the leaves were kept under natural light or in the dark. The protective effect was also observed in intact rice plants inoculated with B. oryzae; the plants survived under red light, but most of them were killed by infection under natural light or dark condition. Red light did not affect fungal infection in onion epidermis cells or heat‐shocked leaves of rice, and it did not affect cellulose digestion ability; this suggested that the protective effect is due to red‐light‐induced resistance. In addition, the degree of protection increased as the red light dosage increased, regardless of the order of the red light and natural light period, indicating that red‐light‐induced resistance is time dependent. Feeding of detached leaves with a tryptophan decarboxylase inhibitor, s‐α‐fluoromethyltryptophan (0.1 mm ), for 24 h inhibited the development of resistance in response to red light irradiation. Suppression of resistance was also observed in leaves treated with a phenylalanine ammonia‐lyase inhibitor, α‐aminooxy acetic acid (0.5 mm ). These results suggest that the tryptophan and phenylpropanoid pathways are involved in the red‐light‐induced resistance of rice to B. oryzae.