Construction of gateway binary vector for selection with bialaphos or carboxin and GFP expression in fungi.

Genomic data has created a growing demand for tools and methodologies for studying the genes function, which can be realized through loss of function experiments (gene knockout) or by RNA silencing (knockdown). The develop-ment of binary vectors for Agrobacterium tumefaciens mediated transformation (ATMT) has the advantage of being independent of protoplast formation and can be used directly on a wide variety of fungal species and tissue types. The selection of transformants using bialaphos and carboxin has the advantages of low cost in the transformation and availability of different selectable markers, also allowing the analysis of several genes and combination of study by knockout or knockdown, using selectable markers in the same transformant. Thus, this study aimed to build two binary vectors containing reporter gene and selectable markers that confer resistance to carboxin and bialaphos. The cassettes were constructed using the Gateway system to two fragments. The gene encoding the GFP protein and PtoxA and PtrpCpromoters were cloned into pDONR P1-P5R plasmid. Genes that confer bialaphos and carboxin resistance, bar and cbxr respectively, were cloned into pDONR P5-P2 plasmid. The pPGW plasmid was used as des-tination vector. The gfp gene transcription is controlled by PtoxA promoter and the bar and cbxr genes transcriptions are controlled by PtrpC promoter. These binary vectors were named pGWGFP-BAR and pGWGFP-CBXR. The assembly of cassettes was confi rmed by sequencing, and the validation of vectors is being accomplished through transformation (ATMT) with the plant pathogens Mycosphaerella fi jiensis and Fusarium oxysporum f. sp. cubense

A novel approach to estimate the distribution, density and at-sea risks of a centrally-placed mobile marine vertebrate

This is the author accepted manuscript. The final version is available from Elsevier via the DOI in this record.Formulating management strategies for mobile marine species is challenging, as knowledge is required of distribution, density, and overlap with putative threats. As a step towards assimilating knowledge, ecological niche models may identify likely suitable habitats for species, but lack the ability to enumerate species densities. Traditionally, this has been catered for by sightings-based distance sampling methods that may have practical and logistical limitations. Here we describe a novel method to estimate at-sea distribution and densities of a marine vertebrate, using historic aerial surveys of Gabonese leatherback turtle (Dermochelys coriacea) nesting beaches and satellite telemetry data of females at sea. We contextualise modelled patterns of distribution with putative threat layers of boat traffic, including fishing vessels and large ship movements, using Vessel Monitoring System (VMS) and Automatic Identification System (AIS) data. We identify key at-sea areas in which protection for inter-nesting leatherback turtles could be considered within the coastal zone of Gabonese Exclusive Economic Zone (EEZ). Our approach offers a holistic technique that merges multiple datasets and methodologies to build a deeper and insightful knowledge base with which to manage known activities at sea. As such, the methodologies presented in this study could be applied to other species of sea turtles for cumulative assessments; and with adaptation, may have utility in defining critical habitats for other central-place foragers such as pinnipeds, or sea bird species. Although our analysis focuses on a single species, we suggest that putative threats identified within this study (fisheries, seismic activity, general shipping) likely apply to other mobile marine vertebrates of conservation concern within Gabonese and central African coastal waters, such as olive ridley sea turtles (Lepidochelys olivacea), humpback dolphins (Sousa teuszii) and humpback whales (Megaptera novaeangliae).We thank the following for support and funding: CARPE (Central African Regional Program for the Environment, Darwin Initiative, EAZA ShellShock Campaign, Gabon Sea Turtle Partnership with funding from the Marine Turtle Conservation Fund (United States Fish and Wildlife Service, U.S. Department of the Interior), Harvest Energy, Large Pelagics Research Centre at the University of Massachusetts (Boston), NERC, Vaalco Energy and the Wildlife Conservation Society. We are sincerely grateful to the field teams and logistics staff who assisted in the aerial and ground surveys and with field-site assistance. BJG and MJW receive funding from the Natural Environment Research Council (NE/J012319/1), the European Union and the Darwin Initiative

Identifying Regulators for EAG1 Channels with a Novel Electrophysiology and Tryptophan Fluorescence Based Screen

Ether-à-go-go (EAG) channels are expressed throughout the central nervous system and are also crucial regulators of cell cycle and tumor progression. The large intracellular amino- and carboxy- terminal domains of EAG1 each share similarity with known ligand binding motifs in other proteins, yet EAG1 channels have no known regulatory ligands.Here we screened a library of small biologically relevant molecules against EAG1 channels with a novel two-pronged screen to identify channel regulators. In one arm of the screen we used electrophysiology to assess the functional effects of the library compounds on full-length EAG1 channels. In an orthogonal arm, we used tryptophan fluorescence to screen for binding of the library compounds to the isolated C-terminal region.Several compounds from the flavonoid, indole and benzofuran chemical families emerged as binding partners and/or regulators of EAG1 channels. The two-prong screen can aid ligand and drug discovery for ligand-binding domains of other ion channels

Whole-Genome and Chromosome Evolution Associated with Host Adaptation and Speciation of the Wheat Pathogen Mycosphaerella graminicola

The fungus Mycosphaerella graminicola has been a pathogen of wheat since host domestication 10,000–12,000 years ago in the Fertile Crescent. The wheat-infecting lineage emerged from closely related Mycosphaerella pathogens infecting wild grasses. We use a comparative genomics approach to assess how the process of host specialization affected the genome structure of M. graminicola since divergence from the closest known progenitor species named M. graminicola S1. The genome of S1 was obtained by Illumina sequencing resulting in a 35 Mb draft genome sequence of 32X. Assembled contigs were aligned to the previously sequenced M. graminicola genome. The alignment covered >90% of the non-repetitive portion of the M. graminicola genome with an average divergence of 7%. The sequenced M. graminicola strain is known to harbor thirteen essential chromosomes plus eight dispensable chromosomes. We found evidence that structural rearrangements significantly affected the dispensable chromosomes while the essential chromosomes were syntenic. At the nucleotide level, the essential and dispensable chromosomes have evolved differently. The average synonymous substitution rate in dispensable chromosomes is considerably lower than in essential chromosomes, whereas the average non-synonymous substitution rate is three times higher. Differences in molecular evolution can be related to different transmission and recombination patterns, as well as to differences in effective population sizes of essential and dispensable chromosomes. In order to identify genes potentially involved in host specialization or speciation, we calculated ratios of synonymous and non-synonymous substitution rates in the >9,500 aligned protein coding genes. The genes are generally under strong purifying selection. We identified 43 candidate genes showing evidence of positive selection, one encoding a potential pathogen effector protein. We conclude that divergence of these pathogens was accompanied by structural rearrangements in the small dispensable chromosomes, while footprints of positive selection were present in only a small number of protein coding genes

Stem rust resistance in wheat is suppressed by a subunit of the mediator complex

Stem rust is an important disease of wheat that can be controlled using resistance genes. The gene SuSr-D1 identified in cultivar 'Canthatch' suppresses stem rust resistance. SuSr-D1 mutants are resistant to several races of stem rust that are virulent on wild-type plants. Here we identify SuSr-D1 by sequencing flow-sorted chromosomes, mutagenesis, and map-based cloning. The gene encodes Med15, a subunit of the Mediator Complex, a conserved protein complex in eukaryotes that regulates expression of protein-coding genes. Nonsense mutations in Med15b.D result in expression of stem rust resistance. Time-course RNAseq analysis show a significant reduction or complete loss of differential gene expression at 24h post inoculation in med15b.D mutants, suggesting that transcriptional reprogramming at this time point is not required for immunity to stem rust. Suppression is a common phenomenon and this study provides novel insight into suppression of rust resistance in wheat. Stem rust is an important disease of wheat and resistance present in some cultivars can be suppressed by the SuSr-D1 locus. Here the authors show that SuSr-D1 encodes a subunit of the Mediator Complex and that nonsense mutations are sufficient to abolish suppression and confer stem rust resistance