Yusuke Yoshida

Conidiation rhythm and light entrainment in superoxide dismutase mutant in Neurospora crassa

Conidial formation in the filamentous fungus Neurospora crassa is regulated by nutritional conditions, light, and the circadian clock. We found that a sod-1 mutant, with a defective superoxide dismutase catalyzing the conversion of superoxide to hydrogen peroxide, had a slightly shorter period length than the wild type and clear conidial banding similar to a mutant of band (bd). However, unlike the bd mutant, the sod-1 mutant could sustain conidial banding with light pulses on a nutrient-rich medium, which involved an enhancement of the light-induced transcription of frequency (frq). sod-1 was hypersensitive to entrainment of the conidiation rhythm by light in race tubes. Furthermore, a frq10; sod-1 double mutant showed conidiation rhythm in darkness and could be synchronized to light/dark cycles by the masking effect of light. These genetic analyses suggested that intracellular reactive oxygen species (ROS) act on circadian conidiation via multiple circadian clocks and output pathways.

Loss of Catalase-1 (Cat-1) results in decreased conidial viability enhanced by exposure to light in Neurospora crassa

Light is one of the most important factors inducing morphogenesis in Neurospora crassa. The reception of light triggers the generation of reactive oxygen species (ROS) including hydrogen peroxide (H2O2). Catalase-1 (Cat-1) is one of three catalases known to detoxify H2O2 into water and oxygen. We reported that the photomorphogenetic characteristics of mutants in nucleoside diphosphate kinase-1 (NDK-1), a light signal transducer, are severely affected, and NDK-1 interacted with Cat-1 in a yeast two-hybrid assay. To disclose the function of Cat-1, we created a Cat-1 loss-of-function mutant (cat-1RIP) by the repeat induced point-mutation (RIPing) method. No Cat-1 activity was detected in the mutant strain. Forty guanines were replaced with adenines in the cat-1 gene of cat-1RIP, which caused 30 amino acid substitutions. The mutant strain grew normally, but its conidia and mycelia were more sensitive to H2O2 than those of the wild type. The lack of Cat-1 activity also caused a significant reduction in the conidial germination rate. Furthermore, light enhanced this reduction in cat-1RIP more than that in the wild type. Introduction of cat-1 into the mutant reversed all of these defective phenotypes. These results indicate that Cat-1 plays an important role in supporting the survival of conidia under oxidative and light-induced stress.

Photomorphogenetic characteristics are severely affected in nucleoside diphosphate kinase-1 (ndk-1)-disrupted mutants in Neurospora crassa

We previously demonstrated that the NDK-1 (Nucleoside Diphosphate Kinase-1) point mutant, ndk-1P72H, displays a defective phenotype in light-induced perithecial polarity in Neurospora crassa. To investigate the biological function of NDK-1 in detail, we isolated two ndk-1 mutants, ndk-1RIP-1 and ndk-1RIP-2, using the RIPing (repeat induced point mutation) method. Notably, we detected no accumulation of ndk-1RIP-1 mRNA and truncated NDK-1RIP-2 protein. The ndk-1RIP mutants exhibited altered morphogenesis; (1) aerial hypha was not formed with no conidium formation, (2) the mutants exhibited colonial, and very slow mycelial growth on a solid medium and by shaking culture in a liquid medium, (3) light-induced carotenoid accumulation in mutant mycelia is reduced to less than half that by wild type, (4) the mutants exhibited spiral growth of mycelia, and (5) female sterility with defective protoperithecium formation. The morphogenetic processes of 1, 3, and 5 are light induced in the wild type. Moreover, despite only 10–20% of total nucleoside diphosphate kinase activity, the accumulation of relevant transcripts in the ndk-1RIP mutants, such as al-1 and al-2, was similar to that of wild type.

Novel members of the phosphate regulon in Escherichia coli O157:H7 identified using a whole-genome shotgun approach

Escherichia coli PhoB protein is the transcriptional activator of the phosphate (pho) regulon genes involved in phosphate utilization. To gain further insight into the potential roles of PhoB in the phosphate starvation response, we attempted to identify PhoB-regulated promoters using a random shotgun library of E. coli O157:H7 genomic fragments that were fused to a promoterless lacZ reporter gene on a low-copy-number plasmid. Using this approach, numerous chromosomal regions containing phosphate-starvation-inducible (psi) promoters, including nearly all known pho regulon promoters, were identified. β-Galactosidase and electrophoretic mobility shift assays showed that transcription from the 22 identified psi promoters was directly regulated by PhoB. PhoB-binding sites within the promoter regions were identified by DNase I footprinting. The genes for yoaI, rpsG, galP, rnr, udp, sstT, ybiM, and vgrE were located downstream of these promoters, indicating that these genes are members of the pho regulon. Surprisingly, the other 14 promoters were located within sense or antisense strands of open reading frames (ORFs), and/or at a distance from ORFs. Our results suggest that PhoB has broader roles in gene regulation and RNA expression in E. coli strains than was previously supposed. Our shotgun-library cloning approach represents a powerful tool for identifying promoters activated or repressed by transcriptional regulators that respond to environmental stimuli.

ROS resistance in Pisum sativum cv. Alaska: the involvement of nucleoside diphosphate kinase in oxidative stress responses via the regulation of antioxidants.

This study investigated the reactive oxygen species (ROS) tolerance mechanism of a paraquat-resistant Pisum sativum line (R3-1) compared with the wild type (WT). Physiological and biochemical analyses showed significant differences in the phenotypes, such as delayed leaf and floral development, superior branching, and greater biomass and yields in the R3-1 line, as well as an increased level of antioxidant pigments and a lower rate of cellular lipid peroxidation in the resistant R3-1. Additionally, the phosphorylation of crude proteins showed distinguishable differences in band mobility and intensity between the R3-1 and WT plants. cDNA cloning and sequence analysis of NDPKs, which were candidate phosphorylated proteins, revealed that two of the deduced amino acids in NDPK2 (IL12L and Glu205Lys) and one in NDPK3 (P45S) were mutated in R3-1. Using glutathione S-transferase–NDPK fusion constructs, we found that the precursor recombinant R3-1 NDPK2 showed an increased level of activity and autophosphorylation in R3-1 plants compared to WT plants. Native PAGE analysis of the crude proteins revealed that NDPK and catalase (CAT) activity co-existed in the same area of the gel. In a yeast two-hybrid assay, the N-terminal region of NDPK2 showed an interaction with the full-length CAT1 protein. Furthermore, we found that WT showed a decreased level of CAT activity compared with R3-1 under illumination and/or on media containing ROS-releasing reagents. Taken together, these results suggest that there is a strong interaction between NDPK2 and CAT1 in R3-1 plants, which possibly plays a vital role in the antioxidant defense against ROS.

Identification of PhoB binding sites of the yibD and ytfK promoter regions in Escherichia coli.

By using a lacZ operon fusion genomic library of the Escherichia coli 0157:H7 Sakai, we identified phosphate-starvation-inducible (psi) promoters located upstream of the yibD and ytfK genes. They have been previously proposed to belong to the phosphate regulon (pho regulon) by Beak and Lee (2006), based on the DNA array and in vivo transcriptional experiments. However, the direct interaction of these promoters with the activator protein of the pho regulon, PhoB, has not been determined. We determined the binding regions of PhoB in these promoter regions by DNase I footprinting. Both regions contained two pho boxes similar to the consensus sequence for PhoB binding.

Global warming, plant paraquat resistance, and light signal transduction through nucleoside diphosphate kinase as a paradigm for increasing food supply

Light signal transduction was studied in extracts of mycelia of the fungus Neurospora crassa, and the third internodes of dark-grown Pisum sativum cv Alaska. Both processes increased the phosphorylation of nucleoside diphosphate kinase (NDPK). NDPK may function as a carrier of reduction equivalents, as it binds NADH, thereby providing electrons to transform singlet oxygen to superoxide by catalases (CAT). As the C-termini of NDPK interact with CAT which receive singlet oxygen, emitted from photoreceptors post light perception (which is transmitted to ambient triplet oxygen), we hypothesize that this may increase phospho-NDPK. Singlet oxygen, emitted from the photoreceptor, also reacts with unsaturated fatty acids in membranes thereby forming malonedialdehyde, which in turn could release ions from, e.g., the thylacoid membrane thereby reducing the rate of photosynthesis. A mutant of Alaska pea, which exhibited two mutations in chloroplast NDPK-2 and one mutation in mitochondrial localized NDPK-3, was resistant to reactive oxygen species including singlet oxygen and showed an increase in the production of carotenoids, anthocyanine, and thereby could reduce the concentration of singlet oxygen. The reduction of the concentration of singlet oxygen is predicted to increase the yield of crop plants, such as Alaska pea, soybean, rice, wheat, barley, and sugarcane. This approach to increase the yield of crop plants may contribute not only to enhance food supply, but also to reduce the concentration of CO2 in the atmosphere.

Paraquat-resistant lines in Pisum sativum cv. Alaska: biochemical and phenotypic characterization

In plants, the oxygen generated by photosynthesis can be excited to form reactive oxygen species (ROS) under excessive sunlight. Excess ROS including singlet oxygen (1O2) inhibit the growth, development and photosynthesis of plants. To isolate ROS-resistant crop plants, we used paraquat (PQ), a generator of O2·− as a source of screening and mutagen, and obtained two PQ-resistant lines in Pisum sativum, namely R3-1 and R3-2. Both lines showed greater resistance to PQ than their wild type (WT) siblings with respect to germination, root growth, and shoot growth. Biochemical analysis showed differences in these lines, in which ROS-scavenging enzymes undergo changes with a distinguishable increase in Mn-SOD. We further observed that the cytosolic catalases (CATs) in leaves in both lines were shifted in a native-PAGE analysis compared with that of the WT, indicating that the release of bound 1O2 was enhanced. Phenotypic analysis revealed distinguishable differences in leaf development, and in flowering time and position. In addition, R3-1 and R3-2 showed shorter individual internode lengths, dwarf plant height, and stronger branching compared with the WT. These results suggested that PQ-induced ROS-resistant Pisum have the potential pleiotropic effects on flowering time and stem branching, and that ROS including 1O2 plays not only important roles in plant growth and development as a signal transducer, but also appears as a strong inhibitor for crop yield.

Light Signal Transduction Coupled with Reactive Oxygen Species in Neurospora crassa

The development of the filamentous fungus Neurospora crassa is subject to light regulation at various stages of its life cycle.The light regulated processes include the following. (1) Induction of carotenoid synthesis in the mycelia. (2) Development of aerial hyphae and conidium from mycelia. (3) Phase shift of circadian rhythm of conidiation. (4) Protoperithecium formation under nitrogen limited condition. (5) Formation of perithecium beak under directional light. (6) Bending of beak toward directional light.