A chemiluminescence-based heterogeneous asymmetric recombinase polymerase amplification assay for the molecular detection of mycotoxin producers.

The analysis of mold in indoor air is a prominent topic but it is hardly dealt with. The most affected fields of this issue are residential- and occupational safety since mold can have a number of impacts on human health. To date the most used methods for quantification of microorganism contamination in indoor air are culture- or microscopy-based and are not capable of translating the on-site situation to analytical data reliably. Here we present a chemiluminescence-based method to detect mycotoxin producers through isothermal amplification of mycotoxin biosynthesis genes using glass and polycarbonate carriers. In this proof-of-principle study, zearalenone producers were aimed to be detected by heterogeneous asymmetric recombinase polymerase amplification (haRPA). For this, an appropriate lysis method for fungal spores was developed allowing rapid access to DNA. A system calibration with spores of Fusarium culmorum as zearalenone-producing organism resulted in an LOD of 2.7 × 105 spores per ml. The system was shown to be specific for zearalenone producers. This work presents the first application of a heterogeneous isothermal amplification for rapid detection and quantification of mycotoxin producers. In the future, a multiplex detection can be possible by haRPA.

A unified multi-kingdom Golden Gate cloning platform.

Assembling composite DNA modules from custom DNA parts has become routine due to recent technological breakthroughs such as Golden Gate modular cloning. Using Golden Gate, one can efficiently assemble custom transcription units and piece units together to generate higher-order assemblies. Although Golden Gate cloning systems have been developed to assemble DNA plasmids required for experimental work in model species, they are not typically applicable to organisms from other kingdoms. Consequently, a typical molecular biology laboratory working across kingdoms must use multiple cloning strategies to assemble DNA constructs for experimental assays. To simplify the DNA assembly process, we developed a multi-kingdom (MK) Golden Gate assembly platform for experimental work in species from the kingdoms Fungi, Eubacteria, Protista, Plantae, and Animalia. Plasmid backbone and part overhangs are consistent across the platform, saving both time and resources in the laboratory. We demonstrate the functionality of the system by performing a variety of experiments across kingdoms including genome editing, fluorescence microscopy, and protein interaction assays. The versatile MK system therefore streamlines the assembly of modular DNA constructs for biological assays across a range of model organisms.

A quantitative hypermorphic CNGC allele confers ectopic calcium flux and impairs cellular development.

The coordinated control of Ca2+ signaling is essential for development in eukaryotes. Cyclic nucleotide-gated channel (CNGC) family members mediate Ca2+ influx from cellular stores in plants (Charpentier et al., 2016; Gao et al., 2016; Frietsch et al., 2007; Urquhart et al., 2007). Here, we report the unusual genetic behavior of a quantitative gain-of-function CNGC mutation (brush) in Lotus japonicus resulting in a leaky tetrameric channel. brush resides in a cluster of redundant CNGCs encoding subunits which resemble metazoan voltage-gated potassium (Kv1-Kv4) channels in assembly and gating properties. The recessive mongenic brush mutation impaired root development and infection by nitrogen-fixing rhizobia. The brush allele exhibited quantitative behavior since overexpression of the cluster subunits was required to suppress the brush phenotype. The results reveal a mechanism by which quantitative competition between channel subunits for tetramer assembly can impact the phenotype of the mutation carrier.