Group I Intron as a Potential Target for Antifungal Compounds: Development of a Trans-Splicing High-Throughput Screening Strategy.

The search for safe and efficient new antifungal compounds for agriculture has led to more efforts in finding new modes of action. This involves the discovery of new molecular targets, including coding and non-coding RNA. Rarely found in plants and animals but present in fungi, group I introns are of interest as their complex tertiary structure may allow selective targeting using small molecules. In this work, we demonstrate that group I introns present in phytopathogenic fungi have a self-splicing activity in vitro that can be adapted in a high-throughput screening to find new antifungal compounds. Ten candidate introns from different filamentous fungi were tested and one group ID intron found in F. oxysporum showed high self-splicing efficiency in vitro. We designed the Fusarium intron to act as a trans-acting ribozyme and used a fluorescence-based reporter system to monitor its real time splicing activity. Together, these results are opening the way to study the druggability of such introns in crop pathogen and potentially discover small molecules selectively targeting group I introns in future high-throughput screenings.

Extracellular Vesicles of the Plant Pathogen Botrytis cinerea.

Fungal secretomes are known to contain a multitude of components involved in nutrition, cell growth or biotic interactions. Recently, extra-cellular vesicles have been identified in a few fungal species. Here, we used a multidisciplinary approach to identify and characterize extracellular vesicles produced by the plant necrotroph Botrytis cinerea. Transmission electron microscopy of infectious hyphae and hyphae grown in vitro revealed extracellular vesicles of various sizes and densities. Electron tomography showed the co-existence of ovoid and tubular vesicles and pointed to their release via the fusion of multi-vesicular bodies with the cell plasma membrane. The isolation of these vesicles and exploration of their protein content using mass spectrometry led to the identification of soluble and membrane proteins involved in transport, metabolism, cell wall synthesis and remodeling, proteostasis, oxidoreduction and traffic. Confocal microscopy highlighted the capacity of fluorescently labeled vesicles to target cells of B. cinerea, cells of the fungus Fusarium graminearum, and onion epidermal cells but not yeast cells. In addition, a specific positive effect of these vesicles on the growth of B. cinerea was quantified. Altogether, this study broadens our view on the secretion capacity of B. cinerea and its cell-to-cell communication.

The Analysis of the Editing Defects in the dyw2 Mutant Provides New Clues for the Prediction of RNA Targets of Arabidopsis E+-Class PPR Proteins.

C to U editing is one of the post-transcriptional steps which are required for the proper expression of chloroplast and mitochondrial genes in plants. It depends on several proteins acting together which include the PLS-class pentatricopeptide repeat proteins (PPR). DYW2 was recently shown to be required for the editing of many sites in both organelles. In particular almost all the sites associated with the E+ subfamily of PPR proteins are depending on DYW2, suggesting that DYW2 is required for the function of E+-type PPR proteins. Here we strengthened this link by identifying 16 major editing sites controlled by 3 PPR proteins: OTP90, a DYW-type PPR and PGN and MEF37, 2 E+-type PPR proteins. A re-analysis of the DYW2 editotype showed that the 49 sites known to be associated with the 18 characterized E+-type PPR proteins all depend on DYW2. Considering only the 288 DYW2-dependent editing sites as potential E+-type PPR sites, instead of the 795 known editing sites, improves the performances of binding predictions systems based on the PPR code for E+-type PPR proteins. However, it does not compensate for poor binding predictions.

Bioinformatic Analysis of Chloroplast Gene Expression and RNA Posttranscriptional Maturations Using RNA Sequencing.

Sequencing of total RNA enables the study of the whole plant transcriptome resulting from the simultaneous expression of the three genomes of plant cells (located in the nucleus, mitochondrion and chloroplast). While commonly used for the quantification of the nuclear gene expression, this method remains complex and challenging when applied to organellar genomes and/or when used to quantify posttranscriptional RNA maturations. Here we propose a complete bioinformatical and statistical pipeline to fully characterize the differences in the chloroplast transcriptome between two conditions. Experimental design as well as bioinformatics and statistical analyses are described in order to quantify both gene expression and RNA posttranscriptional maturations, i.e., RNA splicing, editing, and processing, and identify statistically significant differences.