Expression and function of sex pheromones and receptors in the homothallic ascomycete Gibberella zeae.
In heterothallic ascomycete fungi, idiomorphic alleles at the MAT locus control two sex pheromone-receptor pairs that function in the recognition and chemoattraction of strains with opposite mating types. In the ascomycete Gibberella zeae, the MAT locus is rearranged such that both alleles are adjacent on the same chromosome. Strains of G. zeae are self-fertile but can outcross facultatively. Our objective was to determine if pheromones retain a role in sexual reproduction in this homothallic fungus. Putative pheromone precursor genes (ppg1 and ppg2) and their corresponding pheromone receptor genes (pre2 and pre1) were identified in the genomic sequence of G. zeae by sequence similarity and microsynteny with other ascomycetes. ppg1, a homolog of the Saccharomyces alpha-factor pheromone precursor gene, was expressed in germinating conidia and mature ascospores. Expression of ppg2, a homolog of the a-factor pheromone precursor gene, was not detected in any cells. pre2 was expressed in all cells, but pre1 was expressed weakly and only in mature ascospores. ppg1 or pre2 deletion mutations reduced fertility in self-fertilization tests by approximately 50%. Deltappg1 reduced male fertility and Deltapre2 reduced female fertility in outcrossing tests. In contrast, Deltappg2 and Deltapre1 had no discernible effects on sexual function. Deltapre2 double mutants had the same phenotype as the Deltappg1 and Deltapre2 single mutants. Thus, one of the putative pheromone-receptor pairs (ppg1/pre2) enhances, but is not essential for, selfing and outcrossing in G. zeae whereas no functional role was found for the other pair (ppg2/pre1).

title: FgVelB globally regulates sexual reproduction, mycotoxin production and pathogenicity in the cereal pathogen Fusarium graminearum.
abstract: The velvet genes are conserved in ascomycetous fungi and function as global regulators of differentiation and secondary metabolism. Here, we characterized one of the velvet genes, designated FgVelB, in the plant-pathogenic fungus Fusarium graminearum, which causes fusarium head blight in cereals and produces mycotoxins within plants. FgVelB-deleted (ΔFgVelB) strains produced fewer aerial mycelia with less pigmentation than those of the wild-type (WT) during vegetative growth. Under sexual development conditions, the ΔFgVelB strains produced no fruiting bodies but retained male fertility, and conidiation was threefold higher compared with the WT strain. Production of trichothecene and zearalenone was dramatically reduced compared with the WT strain. In addition, the ΔFgVelB strains were incapable of colonizing host plant tissues. Transcript analyses revealed that FgVelB was highly expressed during the sexual development stage, and may be regulated by a mitogen-activated protein kinase cascade. Microarray analysis showed that FgVelB affects regulatory pathways mediated by the mating-type loci and a G-protein alpha subunit, as well as primary and secondary metabolism. These results suggest that FgVelB has diverse biological functions, probably by acting as a member of a possible velvet protein complex, although identification of the FgVelB-FgVeA complex and the determination of its roles require further investigation.

title: Genetic diversity and fitness of Fusarium graminearum populations from rice in Korea.
abstract: Fusarium graminearum is an important fungal pathogen of cereal crops and produces mycotoxins, such as the trichothecenes nivalenol and deoxynivalenol. This species may be subdivided into a series of genetic lineages or phylogenetic species. We identified strains of F. graminearum from the Republic of Korea to lineage, tested their ability to produce nivalenol and deoxynivalenol, and determined the genetic composition and structure of the populations from which they were recovered. Based on amplified fragment length polymorphism (AFLP), PCR genotyping, and chemical analyses of trichothecenes, all 249 isolates from southern provinces belonged to lineage 6, with 241 having the nivalenol genotype and 8 having the deoxynivalenol genotype. In the eastern Korea province, we recovered 84 lineage 6 isolates with the nivalenol genotype and 23 lineage 7 isolates with the deoxynivalenol genotype. Among 333 lineage 6 isolates, 36% of the AFLP bands were polymorphic, and there were 270 multilocus haplotypes. Genetic identity among populations was high (>0.972), and genotype diversity was low (30 to 58%). To test the adaptation of lineage 6 to rice, conidial mixtures of strains from lineages 3, 6, and 7 were inoculated onto rice plants and then recovered from the rice grains produced. Strains representing lineages 6 and 7 were recovered from inoculated spikelets at similar frequencies that were much higher than those for the strain representing lineage 3. Abundant perithecia were produced on rice straw, and 247 single-ascospore isolates were recovered from 247 perithecia. Perithecia representing lineage 6 (87%) were the most common, followed by those representing lineage 7 (13%), with perithecia representing lineage 3 not detected. These results suggest that F. graminearum lineage 6 may have a host preference for rice and that it may be more fit in a rice agroecosystem than are the other lineages present in Korea.

title: Shifting fungal reproductive mode by manipulation of mating type genes: obligatory heterothallism of Gibberella zeae.
abstract: Fungi capable of sexual reproduction use heterothallic (self-sterile) or homothallic (self-fertile) mating strategies. In most ascomycetes, a single mating type locus, MAT, with two alternative forms (MAT1-1 and MAT1-2) called idiomorphs, controls mating ability. In heterothallic ascomycetes, these alternative idiomorphs reside in different nuclei. In contrast, most homothallic ascomycetes carry both MAT1-1 and MAT1-2 in a single nucleus, usually closely linked. An example of the latter is Gibberella zeae, a species that is capable of both selfing and outcrossing. G. zeae is a devastating cereal pathogen of ubiquitous geographical distribution, and also a producer of mycotoxins that threaten human and animal health. We asked whether G. zeae could be made strictly heterothallic by manipulation of MAT. Targeted gene replacement was used to differentially delete MAT1-1 or MAT1-2 from a wild-type haploid MAT1-1; MAT1-2 strain, resulting in MAT1-1; mat1-2, mat1-1; MAT1-2 strains that were self-sterile, yet able to cross to wild-type testers and, more importantly, to each other. These results indicated that differential deletion of MAT idiomorphs eliminates selfing ability of G. zeae, but the ability to outcross is retained. They also indicated that both MAT idiomorphs are required for self-fertility. To our knowledge, this is the first report of complete conversion of fungal reproductive strategy from homothallic to heterothallic by targeted manipulation of MAT. Practically, this approach opens the door to simple and efficient procedures for obtaining sexual recombinants of G. zeae that will be useful for genetic analyses of pathogenicity and other traits, such as the ability to produce mycotoxins.

title: Identification and functional characterization of genes involved in the sexual reproduction of the ascomycete fungus Gibberella zeae.
abstract: We previously reported that G protein alpha subunit 1 (GPA1) is essential for sexual reproduction in the homothallic ascomycete fungus Gibberella zeae. In this study we performed microarray analyses on a GPA1 deletion mutant of G. zeae (Δgpa1) to identify genes involved in the sexual reproduction of this fungus. In the Δgpa1 strain, 645 genes were down-regulated and 550 genes were up-regulated during sexual reproduction when compared to the wild-type strain. One hundred of the down-regulated genes were selected for further investigation based on orthologous group clusters and differences in transcript levels. Quantitative real time-PCR was used to determine transcriptional profiles of these genes at various sexual and vegetative stages. We observed that transcript levels of 78 of these genes were dramatically increased in the wild-type strain during sexual reproduction compared to levels observed during vegetative growth, and were down-regulated in Δgpa1 compared to the wild-type strain. We deleted 57 of these genes and found that four of the deletion mutants lost self-fertility and five produced fewer perithecia compared to the wild-type strain. Two mutants produced wild-type numbers of perithecia, but maturation of perithecia and ascospores was delayed. In all we identified 11 genes that are involved in sexual reproduction of G. zeae and present evidence that some of these genes function at distinct stages during sexual reproduction in the fungus.

title: Population structure of and mycotoxin production by Fusarium graminearum from maize in South Korea.
abstract: Fusarium graminearum (Gibberella zeae) is an important pathogen of wheat, maize, barley, and rice in South Korea, and harvested grain often is contaminated with trichothecenes such as deoxynivalenol and nivalenol. In this study, we examined 568 isolates of F. graminearum collected from maize at eight locations in South Korea. We used amplified fragment length polymorphisms (AFLPs) to identify four lineages (2, 3, 6, and 7); lineage 7 was the most common (75%), followed by lineage 6 (12%), lineage 3 (12%), and lineage 2 (1%). The genetic identity among populations was high (>0.98), and the effective migration rate between locations was higher than that between lineages. Female fertility varied by lineage: all lineage 7 isolates were fertile, while 70%, 26%, and 14% of the isolates in lineages 6, 3, and 2, respectively, were fertile. All lineage 3 and lineage 7 isolates produced deoxynivalenol, whereas most lineage 2 and 6 isolates produced nivalenol. Genotypic diversity in lineage 3 and lineage 6 populations is similar to that found in previously described Korean rice populations, but genotypic diversity in lineage 7 is much lower, even though similar levels of gene flow occur between lineage 7 populations. We conclude that lineage 7 was relatively recently introduced into South Korea, perhaps accompanying imported maize seeds.

title: Receptor-activity modifying protein 1 expression is increased in the striatum following repeated L-DOPA administration in a 6-hydroxydopamine lesioned rat model of Parkinson's disease.
abstract: Abnormalities in signaling by G-protein coupled receptors (GPCRs) within the striatum are involved in the pathophysiology of Parkinson's disease (PD) and L-DOPA induced dyskinesia (LID). Receptor activity modifying proteins (RAMPs) are single transmembrane accessory proteins crucial for both trafficking and defining the phenotype of GPCRs. In the CNS, RAMP1 mRNA is predominantly expressed in striatum, cortex, and olfactory tubercles. In the present study, expression of RAMP1 mRNA is increased in the striatum (68-77%), following repeated L-DOPA administration, in the 6-hydroxydopamine-lesioned rat. These data are the first to describe regulation of RAMP1 expression in the CNS and suggest that changes in RAMP1 activity are involved in the pathophysiology of LID.

title: Establishing a case for improved food phenolic analysis
abstract: Future phenolic research findings using a multidisciplinary approach will ensure profitability and sustainability of specialty crop industries, while also improving the nutritional and economic choices available to increasingly health- and environmentally conscious consumers. Recent examples of phenolics used in commercial and research scenarios, and new phenolic research discoveries are discussed. Despite being a heavily researched topic, there remains a need to identify, develop, and define analyses targeted for specific quality-related plant metabolites.

title: Erasing genomic imprinting memory in mouse clone embryos produced from day 11.5 primordial germ cells.
abstract: Genomic imprinting is an epigenetic mechanism that causes functional differences between paternal and maternal genomes, and plays an essential role in mammalian development. Stage-specific changes in the DNA methylation patterns of imprinted genes suggest that their imprints are erased some time during the primordial germ cell (PGC) stage, before their gametic patterns are re-established during gametogenesis according to the sex of individuals. To define the exact timing and pattern of the erasure process, we have analyzed parental-origin-specific expression of imprinted genes and DNA methylation patterns of differentially methylated regions (DMRs) in embryos, each derived from a single day 11.5 to day 13.5 PGC by nuclear transfer. Cloned embryos produced from day 12.5 to day 13.5 PGCs showed growth retardation and early embryonic lethality around day 9.5. Imprinted genes lost their parental-origin-specific expression patterns completely and became biallelic or silenced. We confirmed that clones derived from both male and female PGCs gave the same result, demonstrating the existence of a common default state of genomic imprinting to male and female germlines. When we produced clone embryos from day 11.5 PGCs, their development was significantly improved, allowing them to survive until at least the day 11.5 embryonic stage. Interestingly, several intermediate states of genomic imprinting between somatic cell states and the default states were seen in these embryos. Loss of the monoallelic expression of imprinted genes proceeded in a step-wise manner coordinated specifically for each imprinted gene. DNA demethylation of the DMRs of the imprinted genes in exact accordance with the loss of their imprinted monoallelic expression was also observed. Analysis of DNA methylation in day 10.5 to day 12.5 PGCs demonstrated that PGC clones represented the DNA methylation status of donor PGCs well. These findings provide strong evidence that the erasure process of genomic imprinting memory proceeds in the day 10.5 to day 11.5 PGCs, with the timing precisely controlled for each imprinted gene. The nuclear transfer technique enabled us to analyze the imprinting status of each PGC and clearly demonstrated a close relationship between expression and DNA methylation patterns and the ability of imprinted genes to support development.

title: Sucrose-based fabrication of 3D-networked, cylindrical microfluidic channels for rapid prototyping of lab-on-a-chip and vaso-mimetic devices.
abstract: We present a new fabrication scheme for 3D-networked, cylindrical microfluidic (MF) channels based on shaping, bonding, and assembly of sucrose fibers. It is a simple, cleanroom-free, and environment-friendly method, ideal for rapid prototyping of lab-on-a-chip devices. Despite its simplicity, it can realize complex 3D MF channel architectures such as cylindrical tapers, internal loops, end-to-side junctions, tapered junctions, and stenosis. The last two will be of special use for realizing vaso-mimetic MF structures. It also enables molding with polymers incompatible with high-temperature processing.

title: Multiple-Capillary Measurement of RBC Speed, Flux, and Density with Optical Coherence Tomography
abstract: As capillaries exhibit heterogeneous and fluctuating dynamics even during baseline, a technique measuring red blood cell (RBC) speed and flux over many capillaries at the same time is needed. Here, we report that optical coherence tomography can capture individual RBC passage simultaneously over many capillaries located at different depths. Further, we demonstrate the ability to quantify RBC speed, flux, and linear density. This technique will provide a means to monitor microvascular flow dynamics over many capillaries at different depths at the same time.