Grau, C. | bioRxiv Science

bioRxiv2020

An Integrated Workflow for Enhanced Taxonomic and Functional Coverage of the Mouse Faecal Metaproteome

Intestinal microbiota plays a key role in shaping host homeostasis by regulating metabolism, immune responses and behaviour. Its dysregulation has been associated with metabolic, immune and neuropsychiatric disorders and is accompanied by changes in bacterial metabolic regulation. Although proteomics is well suited for analysis of individual microbes, metaproteomics of faecal samples is challenging due to the physical structure of the sample, presence of contaminating host proteins and coexistence of hundreds of microorganisms. Furthermore, there is a lack of consensus regarding preparation of faecal samples, as well as downstream bioinformatic analyses following metaproteomics data acquisition. Here we assess sample preparation and data analysis strategies applied to mouse faeces in a typical mass spectrometry-based metaproteomic experiment. We show that subtle changes in sample preparation protocols may influence interpretation of biological findings. Two-step database search strategies led to significant underestimation of false positive protein identifications. Unipept software provided the highest sensitivity and specificity in taxonomic annotation of the identified peptides of unknown origin. Comparison of matching metaproteome and metagenome data revealed a positive correlation between protein and gene abundances. Notably, nearly all functional categories of detected protein groups were differentially abundant in the metaproteome compared to what would be expected from the metagenome, highlighting the need to perform metaproteomics when studying complex microbiome samples.

microbiology↗

bioRxiv2018

Integrated soybean transcriptomics, metabolomics, and chemical genomics reveal the importance of the phenylpropanoid pathway and antifungal activity in resistance to the broad host range pathogen Sclerotinia sclerotiorum

Sclerotinia sclerotiorum, a predominately necrotrophic fungal pathogen with a broad host range, causes a significant yield limiting disease of soybean called Sclerotinia stem rot (SSR). Resistance mechanisms against SSR are poorly understood, thus hindering the commercial deployment of SSR resistant varieties. We used a multiomic approach utilizing RNA-sequencing, Gas chromatography-mass spectrometry-based metabolomics and chemical genomics in yeast to decipher the molecular mechanisms governing resistance to S. sclerotiorum in soybean. Transcripts and metabolites of two soybean recombinant inbred lines, one resistant, and one susceptible to S. sclerotiorum were analyzed in a time course experiment. The combined results show that resistance to S. sclerotiorum in soybean is associated in part with an early accumulation of JA-Ile ((+)-7-iso-Jasmonoyl-L-isoleucine), a bioactive jasmonate, increased ability to scavenge reactive oxygen species (ROS), and importantly, a reprogramming of the phenylpropanoid pathway leading to increased antifungal activities. Indeed, we noted that phenylpropanoid pathway intermediates such as, 4-hydroxybenzoate, ferulic acid and caffeic acid were highly accumulated in the resistant line. In vitro assays show that these metabolites and total stem extracts from the resistant line clearly affect S. sclerotiorum growth and development. Using chemical genomics in yeast, we further show that this antifungal activity targets ergosterol biosynthesis in the fungus, by disrupting enzymes involved in lipid and sterol biosynthesis. Overall, our results are consistent with a model where resistance to S. sclerotiorum in soybean coincides with an early recognition of the pathogen, leading to the modulation of the redox capacity of the host and the production of antifungal metabolites.\n\nAuthor SummaryResistance to plant fungal pathogens with predominately necrotrophic lifestyles is poorly understood. In this study, we use Sclerotinia sclerotiorum and soybean as a model system to identify key resistance components in this crop plant. We employed a variety of omics approaches in combination with functional studies to identify plant processes associated with resistance to S. sclerotiorum. Our results suggest that resistance to this pathogen is associated in part with an earlier induction of jasmonate signaling, increased ability to scavenge reactive oxygen species, and importantly, a reprogramming of the phenylpropanoid pathway resulting in increased antifungal activities. These findings provide specific plant targets that can exploited to confer resistance to S. sclerotiorum and potentially other pathogens with similar lifestyle.

plant biology↗