Pathogen recognition in compatible plant-microbe interactions

Microbial infections in plant leaves remain a major challenge in agriculture. Hence an understanding of disease mechanisms at the molecular level is of paramount importance for identifying possible intervention points for their control. Whole-transcriptome changes during early disease stages in susceptible plant species are less well-documented than those of resistant ones. This study focuses on the differential transcriptional changes at 24 hours post inoculation (hpi) in tomato leaflets affected by three pathogens: (1) Phytophthora infestans, (2) Botrytis cinerea, and (3) Oidium neolycopersici. Grey mould (B. cinerea) was the disease that had progressed the most by 24 hpi, both in terms of visible symptoms as well as differential gene expression. By means of RNA-seq, we identified  50 differentially expressed tomato genes induced by B. cinerea infection and 18 by P. infestans infection at 24 hpi. Additionally, a set of 63 genes were differentially expressed during all three diseases when compared by a Bayesian approach to their respective mock infections. And Gene expression patterns were found to also depend on the inoculation technique. These findings suggest a specific and distinct transcriptional response in plant leaf tissue in reaction to B. cinerea and P. infestans invasion at 24 hpi, indicating that plants may recognize the attacking pathogen

Kernel learning for ligand-based virtual screening: discovery of a new PPARgamma agonist

Poster presentation at 5th German Conference on Cheminformatics: 23. CIC-Workshop Goslar, Germany. 8-10 November 2009 We demonstrate the theoretical and practical application of modern kernel-based machine learning methods to ligand-based virtual screening by successful prospective screening for novel agonists of the peroxisome proliferator-activated receptor gamma (PPARgamma) [1]. PPARgamma is a nuclear receptor involved in lipid and glucose metabolism, and related to type-2 diabetes and dyslipidemia. Applied methods included a graph kernel designed for molecular similarity analysis [2], kernel principle component analysis [3], multiple kernel learning [4], and, Gaussian process regression [5]. In the machine learning approach to ligand-based virtual screening, one uses the similarity principle [6] to identify potentially active compounds based on their similarity to known reference ligands. Kernel-based machine learning [7] uses the "kernel trick", a systematic approach to the derivation of non-linear versions of linear algorithms like separating hyperplanes and regression. Prerequisites for kernel learning are similarity measures with the mathematical property of positive semidefiniteness (kernels). The iterative similarity optimal assignment graph kernel (ISOAK) [2] is defined directly on the annotated structure graph, and was designed specifically for the comparison of small molecules. In our virtual screening study, its use improved results, e.g., in principle component analysis-based visualization and Gaussian process regression. Following a thorough retrospective validation using a data set of 176 published PPARgamma agonists [8], we screened a vendor library for novel agonists. Subsequent testing of 15 compounds in a cell-based transactivation assay [9] yielded four active compounds. The most interesting hit, a natural product derivative with cyclobutane scaffold, is a full selective PPARgamma agonist (EC50 = 10 ± 0.2 microM, inactive on PPARalpha and PPARbeta/delta at 10 microM). We demonstrate how the interplay of several modern kernel-based machine learning approaches can successfully improve ligand-based virtual screening results

mRNA Inventory of Extracellular Vesicles from Ustilago maydis

Extracellular vesicles (EVs) can transfer diverse RNA cargo for intercellular communication. EV-associated RNAs have been found in diverse fungi and were proposed to be relevant for pathogenesis in animal hosts. In plant-pathogen interactions, small RNAs are exchanged in a cross-kingdom RNAi warfare and EVs were considered to be a delivery mechanism. To extend the search for EV-associated molecules involved in plant-pathogen communication, we have characterised the repertoire of EV-associated mRNAs secreted by the maize smut pathogen, Ustilago maydis. For this initial survey, we examined EV-enriched fractions from axenic filamentous cultures that mimic infectious hyphae. EV-associated RNAs were resistant to degradation by RNases and the presence of intact mRNAs was evident. The set of mRNAs enriched inside EVs relative to the fungal cells are functionally distinct from those that are depleted from EVs. mRNAs encoding metabolic enzymes are particularly enriched. Intriguingly, mRNAs of some known effectors and other proteins linked to virulence were also found in EVs. Furthermore, several mRNAs enriched in EVs are also upregulated during infection, suggesting that EV-associated mRNAs may participate in plant-pathogen interactions

MON-380 Tinnitus with Unexpected Spanish Roots: Head and Neck Paragangliomas Caused by SDHAF2 Mutation

Introduction: Head and neck paragangliomas (HNPGL) are a subtype of pheochromocytoma/paraganglioma (Pheo/PGL) that originate from the autonomuos nervous system. In contrast to abdominal and thoracic Pheo/PGL, HNPGL are usually non-secretory, of parasympathetic origin and metastasize only rarely. Although HNPGL may occur as sporadic tumors, it is estimated that up to 40% of all cases may have a hereditary background that impacts therapeutic strategies, follow-up of affected patients and diagnostic approaches of family members. The most common mutations are found in the succinate dehydrogenase (SDH) genes with the highest prevalence of mutations in SDH-D, followed by SDH-B and SDH-C. Clinical case: A 15 year old male patient presented with tinnitus and hearing loss of the left ear. Imaging revealed a left sided jugulotympanic tumor (33x34mm) and a tumor of the right carotid body (12x15mm). The patient was normotensive, did not report on spells and plasma free metanephrine/catecholamine were not elevated. The morphological suspicion of a paraganglioma was confirmed histologically following resection of the jugulotympanic lesion. Immunohistochemistry showed a loss of SDHB-expression and genetic testing (somatic and germline) revealed a mutation in the SDH assembly factor 2 (SDHAF2) gene (c.232G>A). The patient's father is of Spanish descent. There was no family history for tumors. The hereditary paraglioma syndrome 2 (PGL2) has first been described in 1982 in a Dutch and later in a Spanish family and was found to be caused by a mutation in the SDHAF2 gene. SDHAF2 is a highly conserved co-factor involved in the flavination of the SDH-A subunit. The inheritence is autosomal dominant with maternal imprinting, leading to tumorigenesis only by paternal transmission. This may explain the seemingly negative family history. As in our case, patients with PGL2 usually present at young age with multiple, benign and non-secretory HNPGL. The penetrance reaches 88-100% by the age of 50 years. Conclusion: Our findings emphasize the relevance of genetic testing in patients with HNPGL, also with negative family history, especially when the patients present at young age and with multiple lesions

The Viscum album Gene Space database

The hemiparasitic flowering plant Viscum album (European mistletoe) is known for its very special life cycle, extraordinary biochemical properties, and extremely large genome. The size of its genome is estimated to be 30 times larger than the human genome and 600 times larger than the genome of the model plant Arabidopsis thaliana. To achieve insights into the Gene Space of the genome, which is defined as the space including and surrounding protein-coding regions, a transcriptome project based on PacBio sequencing has recently been conducted. A database resulting from this project contains sequences of 39,092 different open reading frames encoding 32,064 distinct proteins. Based on ‘Benchmarking Universal Single-Copy Orthologs’ (BUSCO) analysis, the completeness of the database was estimated to be in the range of 78%. To further develop this database, we performed a transcriptome project of V. album organs harvested in summer and winter based on Illumina sequencing. Data from both sequencing strategies were combined. The new V. album Gene Space database II (VaGs II) contains 90,039 sequences and has a completeness of 93% as revealed by BUSCO analysis. Sequences from other organisms, particularly fungi, which are known to colonize mistletoe leaves, have been removed. To evaluate the quality of the new database, proteome data of a mitochondrial fraction of V. album were re-analyzed. Compared to the original evaluation published five years ago, nearly 1000 additional proteins could be identified in the mitochondrial fraction, providing new insights into the Oxidative Phosphorylation System of V. album. The VaGs II database is available at https://viscumalbum.pflanzenproteomik.de/. Furthermore, all V. album sequences have been uploaded at the European Nucleotide Archive (ENA)

De novo assembly of the dual transcriptomes of a polymorphic raptor species and its malarial parasite

Pauli M, Chakarov N, Rupp O, et al. De novo assembly of the dual transcriptomes of a polymorphic raptor species and its malarial parasite. BMC Genomics. 2015;16(1): 1038

Genotyping by sequencing and a newly developed mRNA-GBS approach to link population genetic and transcriptome analyses reveal pattern differences between sites and treatments in red clover (Trifolium pratense L.)

The important worldwide forage crop red clover (Trifolium pratense L.) is widely cultivated as cattle feed and for soil improvement. Wild populations and landraces have great natural diversity that could be used to improve cultivated red clover. However, to date, there is still insufficient knowledge about the natural genetic and phenotypic diversity of the species. Here, we developed a low-cost complexity reduced mRNA analysis (mRNA-GBS) and compared the results with population genetic (GBS) and previously published mRNA-Seq data, to assess whether analysis of intraspecific variation within and between populations and transcriptome responses is possible simultaneously. The mRNA-GBS approach was successful. SNP analyses from the mRNA-GBS approach revealed comparable patterns to the GBS results, but due to site-specific multifactorial influences of environmental responses as well as conceptual and methodological limitations of mRNA-GBS, it was not possible to link transcriptome analyses with reduced complexity and sequencing depth to previously published greenhouse and field expression studies. Nevertheless, the use of short sequences upstream of the poly(A) tail of mRNA to reduce complexity are promising approaches that combine population genetics and expression profiling to analyze many individuals with trait differences simultaneously and cost-effectively, even in non-model species. Nevertheless, our study design across different regions in Germany was also challenging. The use of reduced complexity differential expression analyses most likely overlays site-specific patterns due to highly complex plant responses under natural conditions

Evolution and development of fruits of Erycina pusilla and other orchid species

Fruits play a crucial role in seed dispersal. They open along dehiscence zones. Fruit dehiscence zone formation has been intensively studied in Arabidopsis thaliana. However, little is known about the mechanisms and genes involved in the formation of fruit dehiscence zones in species outside the Brassicaceae. The dehiscence zone of A. thaliana contains a lignified layer, while dehiscence zone tissues of the emerging orchid model Erycina pusilla include a lipid layer. Here we present an analysis of evolution and development of fruit dehiscence zones in orchids. We performed ancestral state reconstructions across the five orchid subfamilies to study the evolution of selected fruit traits and explored dehiscence zone developmental genes using RNA-seq and qPCR. We found that erect dehiscent fruits with non-lignified dehiscence zones and a short ripening period are ancestral characters in orchids. Lignified dehiscence zones in orchid fruits evolved multiple times from non-lignified zones. Furthermore, we carried out gene expression analysis of tissues from different developmental stages of E. pusilla fruits. We found that fruit dehiscence genes from the MADS-box gene family and other important regulators in E. pusilla differed in their expression pattern from their homologs in A. thaliana. This suggests that the current A. thaliana fruit dehiscence model requires adjustment for orchids. Additionally, we discovered that homologs of A. thaliana genes involved in the development of carpel, gynoecium and ovules, and genes involved in lipid biosynthesis were expressed in the fruit valves of E. pusilla, implying that these genes may play a novel role in formation of dehiscence zone tissues in orchids. Future functional analysis of developmental regulators, lipid identification and quantification can shed more light on lipid-layer based dehiscence of orchid fruits

Three-Dimensional Shapes of Spinning Helium Nanodroplets

A significant fraction of superfluid helium nanodroplets produced in a free-jet expansion have been observed to gain high angular momentum resulting in large centrifugal deformation. We measured single-shot diffraction patterns of individual rotating helium nanodroplets up to large scattering angles using intense extreme ultraviolet light pulses from the FERMI free-electron laser. Distinct asymmetric features in the wide-angle diffraction patterns enable the unique and systematic identification of the three-dimensional droplet shapes. The analysis of a large dataset allows us to follow the evolution from axisymmetric oblate to triaxial prolate and two-lobed droplets. We find that the shapes of spinning superfluid helium droplets exhibit the same stages as classical rotating droplets while the previously reported metastable, oblate shapes of quantum droplets are not observed. Our three-dimensional analysis represents a valuable landmark for clarifying the interrelation between morphology and superfluidity on the nanometer scale