A codon-optimized green fluorescent protein for live cell imaging in Zymoseptoria tritici

AbstractFluorescent proteins (FPs) are powerful tools to investigate intracellular dynamics and protein localization. Cytoplasmic expression of FPs in fungal pathogens allows greater insight into invasion strategies and the host-pathogen interaction. Detection of their fluorescent signal depends on the right combination of microscopic setup and signal brightness. Slow rates of photo-bleaching are pivotal for in vivo observation of FPs over longer periods of time. Here, we test green-fluorescent proteins, including Aequorea coerulescens GFP (AcGFP), enhanced GFP (eGFP) from Aequorea victoria and a novel Zymoseptoria tritici codon-optimized eGFP (ZtGFP), for their usage in conventional and laser-enhanced epi-fluorescence, and confocal laser-scanning microscopy. We show that eGFP, expressed cytoplasmically in Z. tritici, is significantly brighter and more photo-stable than AcGFP. The codon-optimized ZtGFP performed even better than eGFP, showing significantly slower bleaching and a 20–30% further increase in signal intensity. Heterologous expression of all GFP variants did not affect pathogenicity of Z. tritici. Our data establish ZtGFP as the GFP of choice to investigate intracellular protein dynamics in Z. tritici, but also infection stages of this wheat pathogen inside host tissue

Epiphytic proliferation of Zymoseptoria tritici isolates on resistant wheat leaves

This is the author accepted manuscript. The final version is available on open access from the DOI in this recordThe wheat pathogen Zymoseptoria tritici is capable of a long period of pre-invasive epiphytic growth. Studies have shown that virulent isolates vary in the extent, duration and growth form of this epiphytic growth, and the fungus has been observed to undergo behaviours such as asexual reproduction by budding and vegetative fusion of hyphae on the leaf surface. This epiphytic colonisation has been investigated very little during interactions in which an isolate of Z. tritici is unable to colonise the apoplast, as occurs during avirulence. However, avirulent isolates have been seen to undergo sexual crosses in the absense of leaf penetration, and it is widely accepted that the main point of distinction between virulent and avirulent isolates occurs at the point of attempted leaf penetration or attempted apoplastic growth, which fails in the avirulent case. In this work, we describe extensive epiphytic growth in three isolates which are unable or have very limited ability to invade the leaf, and show that growth form is as variable as for fully virulent isolates. We demonstrate that during certain interactions, Z. tritici isolates rarely invade the leaf and form pycnidia, but induce necrosis and are able to achieve higher epiphytic biomass than virulent isolates during asymptomatic growth, and may undergo very extensive asexual reproduction on the leaf surface. These findings have implications for open questions such as whether and how Z. tritici obtains nutrients on the leaf surface and the nature of its interaction with wheat defences.UK Research and InnovationBiotechnology and Biological Sciences Research Council (BBSRC

Genome sequence of the oleaginous yeast Rhodotorula toruloides strain CGMCC 2.1609

This is the final version of the article. Available from the publisher via the DOI in this record.Most eukaryotic oleaginous species are yeasts and among them the basidiomycete red yeast, Rhodotorula (Rhodosporidium) toruloides (Pucciniomycotina) is known to produce high quantities of lipids when grown in nitrogen-limiting media, and has potential for biodiesel production. The genome of the CGMCC 2.1609 strain of this oleaginous red yeast was sequenced using a hybrid of Roche 454 and Illumina technology generating 13 × coverage. The de novo assembly was carried out using MIRA and scaffolded using MAQ and BAMBUS. The sequencing and assembly resulted in 365 scaffolds with total genome size of 33.4 Mb. The complete genome sequence of this strain was deposited in GenBank and the accession number is LKER00000000. The annotation is available on Figshare (doi:10.6084/m9.figshare.4754251).This research was funded by grants from Shell Global Solutions (UK). We gratefully acknowledge Liverpool Advanced Genomics Facility and Exeter Sequencing Service and computational core facilities at the University of Exeter supported by Wellcome Trust Institutional Strategic Support Fund (WT097835MF) and Wellcome Trust Multi User Equipment Award (WT101650MA)

Testing for hybridisation of the Critically Endangered Iguana delicatissima on Anguilla to inform conservation efforts

The Caribbean Island of Anguilla in the north-eastern Lesser Antilles is home to one of the last populations of the Critically Endangered Lesser Antillean iguana Iguana delicatissima. This population is highly threatened primarily because of hybridisation with non-native Iguana iguana. This study assesses the degree of hybridisation between Anguilla’s Iguana species firstly using morphological characteristics and then genetic analysis to validate the genetic integrity of morphologically identified I. delicatissima. We also examined the genetic diversity of Anguilla’s I. delicatissima population, and that of a population on the nearby island of Îlet Fourchue, St Barthélemy. Forty-five iguanas were captured in Anguilla and 10 in St Barthélemy, and sequences from 3 nuclear and 1 mtDNA genes were obtained for each. Of the 45 iguanas captured in Anguilla, 22 were morphologically identified as I. delicatissima, 12 as I. iguana and the remainder were identified as hybrids. Morphological assignments were all confirmed by genetic analyses except for one I. iguana and one hybrid individual. These two individuals appeared likely to have originated following ancestral hybridisation events several generations ago. A significant paucity of genetic diversity was found within Anguillan and St Barthélemy I. delicatissima populations, with a single haplotype being identified for each of the three nuclear genes and the mtDNA sequence. This study highlights the urgency for immediate action to conserve Anguilla’s remnant I. delicatissima population. Protection from hybridisation will require translocation to I. iguana-free offshore cays, with supplementary individuals being sourced from neighbouring islands to enhance the genetic diversity of the population

Who do we inform? The role of status and target in intergroup whistle-blowing

In two experiments (n = 87 and n = 90), we showed that strongly identifying members of a low status group are more likely to actively inform the ingroup rather than the outgroup about an outgroup transgression, and consider it as more loyal to the ingroup to do so. Moreover, strongly identifying members of a high status group are more likely to actively inform the outgroup rather than the ingroup about an outgroup transgression, and consider this to be more loyal to the ingroup. The results are in support of the notion that, depending on a group's existing status position, negative outgroup information can be used to enhance or confirm the ingroup's standing, affecting whether the ingroup or the outgroup will initially be informed about an outgroup transgression. Copyright © The Author(s), 2009

The Potential for pathogenicity was present in the ancestor of the Ascomycete subphylum Pezizomycotina

<p>Abstract</p> <p>Background</p> <p>Previous studies in Ascomycetes have shown that the function of gene families of which the size is considerably larger in extant pathogens than in non-pathogens could be related to pathogenicity traits. However, by only comparing gene inventories in extant species, no insights can be gained into the evolutionary process that gave rise to these larger family sizes in pathogens. Moreover, most studies which consider gene families in extant species only tend to explain observed differences in gene family sizes by gains rather than by losses, hereby largely underestimating the impact of gene loss during genome evolution.</p> <p>Results</p> <p>In our study we used a selection of recently published genomes of Ascomycetes to analyze how gene family gains, duplications and losses have affected the origin of pathogenic traits. By analyzing the evolutionary history of gene families we found that most gene families with an enlarged size in pathogens were present in an ancestor common to both pathogens and non-pathogens. The majority of these families were selectively maintained in pathogenic lineages, but disappeared in non-pathogens. Non-pathogen-specific losses largely outnumbered pathogen-specific losses.</p> <p>Conclusions</p> <p>We conclude that most of the proteins for pathogenicity were already present in the ancestor of the Ascomycete lineages we used in our study. Species that did not develop pathogenicity seemed to have reduced their genetic complexity compared to their ancestors. We further show that expansion of gained or already existing families in a species-specific way is important to fine-tune the specificities of the pathogenic host-fungus interaction.</p

The psychology of dynamic balance and peak performance in sport: correction theory

This article introduces a new approach to understanding peak performance and dysfunctional performance in sport, correction theory. Correction theory, based within a control theory and dynamical systems perspective, assumes that dynamic balance (a state in which a robust complex system will self-correct in response to imbalance) underwrites individual functioning. The central thesis presented in this article is that an interdependent relationship exists between peak performance and dysfunctional performance in sport. Peak performance is, in part, a (corrective) response to dysfunctional performance and vice versa. An overview of correction theory is presented, based on two propositions relating to balance. Implications of correction theory for understanding sporting performance are briefly considered.N/

Gene discovery in EST sequences from the wheat leaf rust fungus Puccinia triticina sexual spores, asexual spores and haustoria, compared to other rust and corn smut fungi

© 2011 Xu et al.; licensee BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.DOI: 10.1186/1471-2164-12-161Background.Rust fungi are biotrophic basidiomycete plant pathogens that cause major diseases on plants and trees world-wide, affecting agriculture and forestry. Their biotrophic nature precludes many established molecular genetic manipulations and lines of research. The generation of genomic resources for these microbes is leading to novel insights into biology such as interactions with the hosts and guiding directions for breakthrough research in plant pathology. Results. To support gene discovery and gene model verification in the genome of the wheat leaf rust fungus, Puccinia triticina (Pt), we have generated Expressed Sequence Tags (ESTs) by sampling several life cycle stages. We focused on several spore stages and isolated haustorial structures from infected wheat, generating 17,684 ESTs. We produced sequences from both the sexual (pycniospores, aeciospores and teliospores) and asexual (germinated urediniospores) stages of the life cycle. From pycniospores and aeciospores, produced by infecting the alternate host, meadow rue (Thalictrum speciosissimum), 4,869 and 1,292 reads were generated, respectively. We generated 3,703 ESTs from teliospores produced on the senescent primary wheat host. Finally, we generated 6,817 reads from haustoria isolated from infected wheat as well as 1,003 sequences from germinated urediniospores. Along with 25,558 previously generated ESTs, we compiled a database of 13,328 non-redundant sequences (4,506 singlets and 8,822 contigs). Fungal genes were predicted using the EST version of the self-training GeneMarkS algorithm. To refine the EST database, we compared EST sequences by BLASTN to a set of 454 pyrosequencing-generated contigs and Sanger BAC-end sequences derived both from the Pt genome, and to ESTs and genome reads from wheat. A collection of 6,308 fungal genes was identified and compared to sequences of the cereal rusts, Puccinia graminis f. sp. tritici (Pgt) and stripe rust, P. striiformis f. sp. tritici (Pst), and poplar leaf rust Melampsora species, and the corn smut fungus, Ustilago maydis (Um). While extensive homologies were found, many genes appeared novel and species-specific; over 40% of genes did not match any known sequence in existing databases. Focusing on spore stages, direct comparison to Um identified potential functional homologs, possibly allowing heterologous functional analysis in that model fungus. Many potentially secreted protein genes were identified by similarity searches against genes and proteins of Pgt and Melampsora spp., revealing apparent orthologs. Conclusions. The current set of Pt unigenes contributes to gene discovery in this major cereal pathogen and will be invaluable for gene model verification in the genome sequence