’Candidatus Phytoplasma mali’, is a bacterial pathogen associated with the so-called apple proliferation disease in Malus × domestica. The pathogen manipulates its host with a set of effector proteins, among them SAP11CaPm, which shares similarity to SAP11AYWB from ’Candidatus Phytoplasma asteris’. SAP11AYWB interacts and destabilizes the class II CIN transcription factors of Arabidopsis thaliana, namely AtTCP4 and AtTCP13 as well as the class II CYC/TB1 transcription factor AtTCP18, also known as BRANCHED1 being an important factor for shoot branching. It has been shown that SAP11CaPm interacts with the Malus × domestica orthologues of AtTCP4 (MdTCP25) and AtTCP13 (MdTCP24), but an interaction with MdTCP16, the orthologue of AtTCP18, has never been proven. The aim of this study was to investigate this potential interaction and close a knowledge gap regarding the function of SAP11CaPm. A Yeast two-hybrid test and Bimolecular Fluorescence Complementation in planta revealed that SAP11CaPm interacts with MdTCP16. MdTCP16 is known to play a role in the control of the seasonal growth of perennial plants and an increase of MdTCP16 gene expression has been detected in apple leaves in autumn. In addition to this, MdTCP16 is highly expressed during phytoplasma infection. Binding of MdTCP16 by SAP11CaPm might lead to the induction of shoot proliferation and early bud break, both of which are characteristic symptoms of apple proliferation disease.

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Atypical myopathy (AM) in horses is caused by ingestion of seeds of the Acer species (Sapindaceae family). Methylenecyclopropylacetyl-CoA (MCPA-CoA), derived from hypoglycin A (HGA), is currently the only active toxin in Acer pseudoplatanus or Acer negundo seeds related to AM outbreaks. However, seeds or arils of various Sapindaceae (e.g., ackee, lychee, mamoncillo, longan fruit) also contain methylenecyclopropylglycine (MCPG), which is a structural analogue of HGA that can cause hypoglycaemic encephalopathy in humans. The active poison formed from MCPG is methylenecyclopropylformyl-CoA (MCPF-CoA). MCPF-CoA and MCPA-CoA strongly inhibit enzymes that participate in β-oxidation and energy production from fat. The aim of our study was to investigate if MCPG is involved in Acer seed poisoning in horses. MCPG, as well as glycine and carnitine conjugates (MCPF-glycine, MCPF-carnitine), were quantified using high-performance liquid chromatography-tandem mass spectrometry of serum and urine from horses that had ingested Acer pseudoplatanus seeds and developed typical AM symptoms. The results were compared to those of healthy control horses. For comparison, HGA and its glycine and carnitine derivatives were also measured. Additionally, to assess the degree of enzyme inhibition of β-oxidation, several acyl glycines and acyl carnitines were included in the analysis. In addition to HGA and the specific toxic metabolites (MCPA-carnitine and MCPA-glycine), MCPG, MCPF-glycine and MCPF-carnitine were detected in the serum and urine of affected horses. Strong inhibition of β-oxidation was demonstrated by elevated concentrations of all acyl glycines and carnitines, but the highest correlations were observed between MCPF-carnitine and isobutyryl-carnitine (r = 0.93) as well as between MCPA- (and MCPF-) glycine and valeryl-glycine with r = 0.96 (and r = 0.87). As shown here, for biochemical analysis of atypical myopathy of horses, it is necessary to take MCPG and the corresponding metabolites into consideration.

Primary and secondary metabolites exuded by plant roots have mainly been studied under laboratory conditions, while knowledge of root exudate patterns of plants growing in natural communities is very limited. Focusing on ten common European grassland plant species, we asked to which degree exuded metabolite compositions are specific to species or growth forms (forbs and grasses), depend on environments and local neighbourhoods, and reflect traditional plant functional traits. Root exudates were collected under field conditions and analysed using a non-targeted gas chromatography coupled mass spectrometry (GC-MS) approach. In total, we annotated 153 compounds of which 36 were identified by structure and name as metabolites mainly derived from the primary metabolism. Here we show by using variance partitioning, that the composition of exuded polar metabolites was mostly explained by plot identity, followed by plant species identity while plant species composition of the local neighbourhood played no role. Total and root dry biomass explained the largest proportion of variance in exudate composition, with additional variance explained by traditional plant traits. Although the exudate composition was quite similar between the two growth forms, we found some metabolites that occurred only in one of the two growth forms. Our study demonstrated the feasibility of measuring polar exudates under non-sterile field conditions by mass spectrometry, which opens new avenues of research for functional plant ecology.

Standardized DNA assembly strategies facilitate the generation of multigene constructs from collections of building blocks in plant synthetic biology. A common syntax for hierarchical DNA assembly following the Golden Gate principle employing Type IIs restriction endonucleases was recently developed, and underlies the Modular Cloning and GoldenBraid systems. In these systems, transcriptional units and/or multigene constructs are assembled from libraries of standardized building blocks, also referred to as phytobricks, in several hierarchical levels and by iterative Golden Gate reactions. Here, a toolkit containing further modules for the novel DNA assembly standards was developed. Intended for use with Modular Cloning, most modules are also compatible with GoldenBraid. Firstly, a collection of approximately 80 additional phytobricks is provided, comprising e.g. modules for inducible expression systems, promoters or epitope tags. Furthermore, DNA modules were developed for connecting Modular Cloning and Gateway cloning, either for toggling between systems or for standardized Gateway destination vector assembly. Finally, first instances of a “peripheral infrastructure” around Modular Cloning are presented: While available toolkits are designed for the assembly of plant transformation constructs, vectors were created to also use coding sequence-containing phytobricks directly in yeast two hybrid interaction or bacterial infection assays. The presented material will further enhance versatility of hierarchical DNA assembly strategies.

Glucosinolates, a group of sulfur-rich thioglucosides found in plants of the order Brassicales, have attracted a lot of interest as chemical defenses of plants and health promoting substances in human diet. They are accumulated separately from their hydrolyzing enzymes, myrosinases, within the intact plant, but undergo myrosinase-catalyzed hydrolysis upon tissue disruption. This results in various biologically active products, e.g. isothiocyanates, simple nitriles, epithionitriles, and organic thiocyanates. While formation of isothiocyanates proceeds by a spontaneous rearrangement of the glucosinolate aglucone, aglucone conversion to the other products involves specifier proteins under physiological conditions. Specifier proteins appear to act with high specificity, but their exact roles and the structural bases of their specificity are presently unknown. Previous research identified the motif EXXXDXXXH as potential iron binding site required for activity, but crystal structures of recombinant specifier proteins lacked the iron cofactor. Here, we provide experimental evidence for the presence of iron (most likely Fe2+) in purified recombinant thiocyanate-forming protein from Thlaspi arvense (TaTFP) using a Ferene S-based photometric assay as well as Inductively Coupled Plasma-Mass Spectrometry. Iron binding and activity depend on E266, D270, and H274 suggesting a direct interaction of Fe2+ with these residues. Furthermore, we demonstrate presence of iron in epithiospecifier protein and nitrile-specifier protein 3 from Arabidopsis thaliana (AtESP and AtNSP3). We also present a homology model of AtNSP3. In agreement with this model, iron binding and activity of AtNSP3 depend on E386, D390, and H394. The homology model further suggests that the active site of AtNSP3 imposes fewer restrictions to the glucosinolate aglucone conformation than that of TaTFP and AtESP due to its larger size. This may explain why AtNSP3 does not support epithionitrile or thiocyanate formation, which likely requires exact positioning of the aglucone thiolate relative to the side chain.

Lipid identification is a major bottleneck in high-throughput lipidomics studies. However, tools for the analysis of lipid tandem MS spectra are rather limited. While the comparison against spectra in reference libraries is one of the preferred methods, these libraries are far from being complete. In order to improve identification rates, the in silico fragmentation tool MetFrag was combined with Lipid Maps and lipid-class specific classifiers which calculate probabilities for lipid class assignments. The resulting LipidFrag workflow was trained and evaluated on different commercially available lipid standard materials, measured with data dependent UPLC-Q-ToF-MS/MS acquisition. The automatic analysis was compared against manual MS/MS spectra interpretation. With the lipid class specific models, identification of the true positives was improved especially for cases where candidate lipids from different lipid classes had similar MetFrag scores by removing up to 56% of false positive results. This LipidFrag approach was then applied to MS/MS spectra of lipid extracts of the nematode Caenorhabditis elegans. Fragments explained by LipidFrag match known fragmentation pathways, e.g., neutral losses of lipid headgroups and fatty acid side chain fragments. Based on prediction models trained on standard lipid materials, high probabilities for correct annotations were achieved, which makes LipidFrag a good choice for automated lipid data analysis and reliability testing of lipid identifications.

The Ontology for Biomedical Investigations (OBI) is an ontology that provides terms with precisely defined meanings to describe all aspects of how investigations in the biological and medical domains are conducted. OBI re-uses ontologies that provide a representation of biomedical knowledge from the Open Biological and Biomedical Ontologies (OBO) project and adds the ability to describe how this knowledge was derived. We here describe the state of OBI and several applications that are using it, such as adding semantic expressivity to existing databases, building data entry forms, and enabling interoperability between knowledge resources. OBI covers all phases of the investigation process, such as planning, execution and reporting. It represents information and material entities that participate in these processes, as well as roles and functions. Prior to OBI, it was not possible to use a single internally consistent resource that could be applied to multiple types of experiments for these applications. OBI has made this possible by creating terms for entities involved in biological and medical investigations and by importing parts of other biomedical ontologies such as GO, Chemical Entities of Biological Interest (ChEBI) and Phenotype Attribute and Trait Ontology (PATO) without altering their meaning. OBI is being used in a wide range of projects covering genomics, multi-omics, immunology, and catalogs of services. OBI has also spawned other ontologies (Information Artifact Ontology) and methods for importing parts of ontologies (Minimum information to reference an external ontology term (MIREOT)). The OBI project is an open cross-disciplinary collaborative effort, encompassing multiple research communities from around the globe. To date, OBI has created 2366 classes and 40 relations along with textual and formal definitions. The OBI Consortium maintains a web resource (http://obi-ontology.org) providing details on the people, policies, and issues being addressed in association with OBI. The current release of OBI is available at http://purl.obolibrary.org/obo/obi.owl.

Oxylipins of the jasmonate family are active as signals in plant responses to biotic and abiotic stresses as well as in development. Jasmonic acid (JA), its precursor cis-12-oxo-phytodienoic acid (OPDA) and the isoleucine conjugate of JA (JA-Ile) are the most prominent members. OPDA and JA-Ile have individual signalling properties in several processes and differ in their pattern of gene expression. JA-Ile, but not OPDA, is perceived by the SCFCOI1-JAZ co-receptor complex. There are, however, numerous processes and genes specifically induced by OPDA. The recently identified OPDA-Ile suggests that OPDA specific responses might be mediated upon formation of OPDA-Ile. Here, we tested OPDA-Ile-induced gene expression in wild type and JA-deficient, JA-insensitive and JA-Ile-deficient mutant background. Tests on putative conversion of OPDA-Ile during treatments revealed only negligible conversion. Expression of two OPDA-inducible genes, GRX480 and ZAT10, by OPDA-Ile could be detected in a JA-independent manner in Arabidopsis seedlings but less in flowering plants. The data suggest a bioactivity in planta of OPDA-Ile.

Receptor-like protein kinases (RLKs) are the largest family of plant transmembrane signaling proteins. Here we present functional analysis of HAESA, an RLK that regulates floral organ abscission in Arabidopsis. Through in vitro and in vivo analysis of HAE phosphorylation, we provide evidence that a conserved phosphorylation site on a region of the HAE protein kinase domain known as the activation segment positively regulates HAE activity. Additional analysis has identified another putative activation segment phosphorylation site common to multiple RLKs that potentially modulates HAE activity. Comparative analysis suggests that phosphorylation of this second activation segment residue is an RLK specific adaptation that may regulate protein kinase activity and substrate specificity. A growing number of RLKs have been shown to exhibit biologically relevant dual specificity toward serine/threonine and tyrosine residues, but the mechanisms underlying dual specificity of RLKs are not well understood. We show that a phospho-mimetic mutant of both HAE activation segment residues exhibits enhanced tyrosine auto-phosphorylation in vitro, indicating phosphorylation of this residue may contribute to dual specificity of HAE. These results add to an emerging framework for understanding the mechanisms and evolution of regulation of RLK activity and substrate specificity.