A reliable microplate assay for determination of B-galactosidase activity in Neurospora crassa strains bearing lacZ fusions

We have been using lacZ as a reporter gene in N. crassa. The standard -galactosidase assay can be labor intensive and time consuming when large numbers of strains are assayed simultaneously. We sought a technique to simplify the pipetting steps involved in assay preparation and in optical density reading. High reproducibility and rapid processing was obtained by adapting the standard test tube method to a microassay performed in a 96-well microplate

Identification of a gene from Neurospora crassa with similarity to a glucoamylase gene from Schwanniomyces occidentalis

Glucoamylases are important industrial enzymes used in the conversion of starches to syrups and in other fermentation processes. Previously, the gene encoding the major glucoamylase activity of N. crassa was characterized (Stone et al. 1993 Curr. Genet 24:205-211). Here we report the identification of a possible second glucoamylase (gla-2) that is similar to a member of a class of glucoamylases represented by the GAM1gene of S. occidentalis (Dohmen et al. 1990 Gene 95:111-121)

Magnaporthe oryzae CK2 Accumulates in Nuclei, Nucleoli, at Septal Pores and Forms a Large Ring Structure in Appressoria, and Is Involved in Rice Blast Pathogenesis

Magnaporthe oryzae (Mo) is a model pathogen causing rice blast resulting in yield and economic losses world-wide. CK2 is a constitutively active, serine/threonine kinase in eukaryotes, having a wide array of known substrates, and involved in many cellular processes. We investigated the localization and role of MoCK2 during growth and infection. BLAST search for MoCK2 components and targeted deletion of subunits was combined with protein-GFP fusions to investigate localization. We found one CKa and two CKb subunits of the CK2 holoenzyme. Deletion of the catalytic subunit CKa was not possible and might indicate that such deletions are lethal. The CKb subunits could be deleted but they were both necessary for normal growth and pathogenicity. Localization studies showed that the CK2 holoenzyme needed to be intact for normal localization at septal pores and at appressorium penetration pores. Nuclear localization of CKa was however not dependent on the intact CK2 holoenzyme. In appressoria, CK2 formed a large ring perpendicular to the penetration pore and the ring formation was dependent on the presence of all CK2 subunits. The effects on growth and pathogenicity of deletion of the b subunits combined with the localization indicate that CK2 can have important regulatory functions not only in the nucleus/nucleolus but also at fungal specific structures such as septa and appressorial pores

Gene Ontology annotation of the rice blast fungus, Magnaporthe oryzae

<p>Abstract</p> <p>Background</p> <p><it>Magnaporthe oryzae</it>, the causal agent of blast disease of rice, is the most destructive disease of rice worldwide. The genome of this fungal pathogen has been sequenced and an automated annotation has recently been updated to Version 6 <url>http://www.broad.mit.edu/annotation/genome/magnaporthe_grisea/MultiDownloads.html</url>. However, a comprehensive manual curation remains to be performed. Gene Ontology (GO) annotation is a valuable means of assigning functional information using standardized vocabulary. We report an overview of the GO annotation for Version 5 of <it>M. oryzae </it>genome assembly.</p> <p>Methods</p> <p>A similarity-based (i.e., computational) GO annotation with manual review was conducted, which was then integrated with a literature-based GO annotation with computational assistance. For similarity-based GO annotation a stringent reciprocal best hits method was used to identify similarity between predicted proteins of <it>M. oryzae </it>and GO proteins from multiple organisms with published associations to GO terms. Significant alignment pairs were manually reviewed. Functional assignments were further cross-validated with manually reviewed data, conserved domains, or data determined by wet lab experiments. Additionally, biological appropriateness of the functional assignments was manually checked.</p> <p>Results</p> <p>In total, 6,286 proteins received GO term assignment via the homology-based annotation, including 2,870 hypothetical proteins. Literature-based experimental evidence, such as microarray, MPSS, T-DNA insertion mutation, or gene knockout mutation, resulted in 2,810 proteins being annotated with GO terms. Of these, 1,673 proteins were annotated with new terms developed for Plant-Associated Microbe Gene Ontology (PAMGO). In addition, 67 experiment-determined secreted proteins were annotated with PAMGO terms. Integration of the two data sets resulted in 7,412 proteins (57%) being annotated with 1,957 distinct and specific GO terms. Unannotated proteins were assigned to the 3 root terms. The Version 5 GO annotation is publically queryable via the GO site <url>http://amigo.geneontology.org/cgi-bin/amigo/go.cgi</url>. Additionally, the genome of <it>M. oryzae </it>is constantly being refined and updated as new information is incorporated. For the latest GO annotation of Version 6 genome, please visit our website <url>http://scotland.fgl.ncsu.edu/smeng/GoAnnotationMagnaporthegrisea.html</url>. The preliminary GO annotation of Version 6 genome is placed at a local MySql database that is publically queryable via a user-friendly interface Adhoc Query System.</p> <p>Conclusion</p> <p>Our analysis provides comprehensive and robust GO annotations of the <it>M. oryzae </it>genome assemblies that will be solid foundations for further functional interrogation of <it>M. oryzae</it>.</p

Gene Ontology annotation of the rice blast fungus, Magnaporthe oryzae

Abstract Background Magnaporthe oryzae, the causal agent of blast disease of rice, is the most destructive disease of rice worldwide. The genome of this fungal pathogen has been sequenced and an automated annotation has recently been updated to Version 6 http://www.broad.mit.edu/annotation/genome/magnaporthe_grisea/MultiDownloads.html. However, a comprehensive manual curation remains to be performed. Gene Ontology (GO) annotation is a valuable means of assigning functional information using standardized vocabulary. We report an overview of the GO annotation for Version 5 of M. oryzae genome assembly. Methods A similarity-based (i.e., computational) GO annotation with manual review was conducted, which was then integrated with a literature-based GO annotation with computational assistance. For similarity-based GO annotation a stringent reciprocal best hits method was used to identify similarity between predicted proteins of M. oryzae and GO proteins from multiple organisms with published associations to GO terms. Significant alignment pairs were manually reviewed. Functional assignments were further cross-validated with manually reviewed data, conserved domains, or data determined by wet lab experiments. Additionally, biological appropriateness of the functional assignments was manually checked. Results In total, 6,286 proteins received GO term assignment via the homology-based annotation, including 2,870 hypothetical proteins. Literature-based experimental evidence, such as microarray, MPSS, T-DNA insertion mutation, or gene knockout mutation, resulted in 2,810 proteins being annotated with GO terms. Of these, 1,673 proteins were annotated with new terms developed for Plant-Associated Microbe Gene Ontology (PAMGO). In addition, 67 experiment-determined secreted proteins were annotated with PAMGO terms. Integration of the two data sets resulted in 7,412 proteins (57%) being annotated with 1,957 distinct and specific GO terms. Unannotated proteins were assigned to the 3 root terms. The Version 5 GO annotation is publically queryable via the GO site http://amigo.geneontology.org/cgi-bin/amigo/go.cgi. Additionally, the genome of M. oryzae is constantly being refined and updated as new information is incorporated. For the latest GO annotation of Version 6 genome, please visit our website http://scotland.fgl.ncsu.edu/smeng/GoAnnotationMagnaporthegrisea.html. The preliminary GO annotation of Version 6 genome is placed at a local MySql database that is publically queryable via a user-friendly interface Adhoc Query System. Conclusion Our analysis provides comprehensive and robust GO annotations of the M. oryzae genome assemblies that will be solid foundations for further functional interrogation of M. oryzae

Mesophilic and Thermophilic Conditions Select for Unique but Highly Parallel Microbial Communities to Perform Carboxylate Platform Biomass Conversion

The carboxylate platform is a flexible, cost-effective means of converting lignocellulosic materials into chemicals and liquid fuels. Although the platform's chemistry and engineering are well studied, relatively little is known about the mixed microbial communities underlying its conversion processes. In this study, we examined the metagenomes of two actively fermenting platform communities incubated under contrasting temperature conditions (mesophilic 40°C; thermophilic 55°C), but utilizing the same inoculum and lignocellulosic feedstock. Community composition segregated by temperature. The thermophilic community harbored genes affiliated with Clostridia, Bacilli, and a Thermoanaerobacterium sp, whereas the mesophilic community metagenome was composed of genes affiliated with other Clostridia and Bacilli, Bacteriodia, γ-Proteobacteria, and Actinobacteria. Although both communities were able to metabolize cellulosic materials and shared many core functions, significant differences were detected with respect to the abundances of multiple Pfams, COGs, and enzyme families. The mesophilic metagenome was enriched in genes related to the degradation of arabinose and other hemicellulose-derived oligosaccharides, and the production of valerate and caproate. In contrast, the thermophilic community was enriched in genes related to the uptake of cellobiose and the transfer of genetic material. Functions assigned to taxonomic bins indicated that multiple community members at either temperature had the potential to degrade cellulose, cellobiose, or xylose and produce acetate, ethanol, and propionate. The results of this study suggest that both metabolic flexibility and functional redundancy contribute to the platform's ability to process lignocellulosic substrates and are likely to provide a degree of stability to the platform's fermentation processes

Directional Selection from Host Plants Is a Major Force Driving Host Specificity in Magnaporthe Species

One major threat to global food security that requires immediate attention, is the increasing incidence of host shift and host expansion in growing number of pathogenic fungi and emergence of new pathogens. The threat is more alarming because, yield quality and quantity improvement efforts are encouraging the cultivation of uniform plants with low genetic diversity that are increasingly susceptible to emerging pathogens. However, the influence of host genome differentiation on pathogen genome differentiation and its contribution to emergence and adaptability is still obscure. Here, we compared genome sequence of 6 isolates of Magnaporthe species obtained from three different host plants. We demonstrated the evolutionary relationship between Magnaporthe species and the influence of host differentiation on pathogens. Phylogenetic analysis showed that evolution of pathogen directly corresponds with host divergence, suggesting that host-pathogen interaction has led to co-evolution. Furthermore, we identified an asymmetric selection pressure on Magnaporthe species. Oryza sativa-infecting isolates showed higher directional selection from host and subsequently tends to lower the genetic diversity in its genome. We concluded that, frequent gene loss or gain, new transposon acquisition and sequence divergence are host adaptability mechanisms for Magnaporthe species, and this coevolution processes is greatly driven by directional selection from host plants

Retromer Is Essential for Autophagy-Dependent Plant Infection by the Rice Blast Fungus

We thank Dr. Yizhen Deng at the Temasek Life sciences Laboratory (TLL) for providing the RFP-MoAtg8 plasmid. We would like to thank Drs. Zhenbiao Yang (University of California, Riverside) and Xianying Dou (Fujian Agriculture and Forestry University) for helpful discussions.Author Summary The rice blast fungus Magnaporthe oryzae utilizes key infection structures, called appressoria, elaborated at the tips of the conidial germ tubes to gain entry into the host tissue. Development of the appressorium is accompanied with autophagy in the conidium leading to programmed cell death. This work highlights the significance of the Vps35/retromer membrane-trafficking machinery in the regulation of autophagy during appressorium-mediated host penetration, and thus sheds light on a novel molecular mechanism underlying autophagy-based membrane trafficking events during pathogen-host interaction in rice blast disease. Our findings provide the first genetic evidence that the retromer controls the initiation of autophagy in filamentous fungi.Yeshttp://www.plosgenetics.org/static/editorial#pee

Characterization of the Bacillus subtilis pur operon

The Bacillus subtilis pur operon was characterized to better understand genetic organization and regulation in this gram positive bacterium. Over 13 kilobase pairs of DNA containing the pur operon were cloned and sequenced. Analysis of this sequence revealed 12 genes, eleven of which encode the nine enzymes required for de novo synthesis of IMP. The operon is organized as three overlapping sets of genes separated by intercistronic gaps followed by the last gene of the operon; purEKB-purC(orf)QLF-purMNH(J)-purD. Transcription of the operon is regulated by guanine and adenine. Guanine regulates transcription by a termination-antitermination mechanism in a 242 nucleotide mRNA leader. Regulation depends upon formation of RNA secondary structure in vivo. The structure of the RNA leader produced by guanine-regulated termination was deduced from analysis of intermediates in the decay of the terminated transcript. Adenine regulates transcription initiation by repression. Deletion analysis defined an element upstream of the promoter required for repression. A candidate repressor protein was partially purified from B. subtilis and was shown to bind to the control region of the pur operon. Expression and regulation throughout the operon was examined by using translational fusions of Escherichia coli lacZ to the first gene of each of the three clusters of overlapping genes. Measurements of $\beta$-galactosidase and mRNA provided preliminary data to suggest that a single promoter drives transcription of this operon. Regulation of transcription occurs only at the 5\sp\prime-end of the operon. Differential regulation of purC-lacZ enzyme suggested that a translational control may be present within the operon. Although levels of mRNA were similar at different sites throughout the operon, corresponding enzyme levels differed up to 7-fold. This difference in gene expression indicates that translational efficiencies may be important factors in determining the stoichiometry of the enzymes for de novo purine nucleotide synthesis