Project description:When studying gene expression in microbe-animals symbioses collected in the field it is essential to quickly and efficiently preserve in situ symbiont and host gene expression patterns. One of the most commonly used sample preservation methods for samples targeted for proteomic analyses is flash freezing, however, liquid nitrogen or dry ice needed for flash freezing are often not available at remote field sites. We tested if RNAlater allows to preserve proteins in animal-microbe symbioses as efficiently as flash freezing and without introducing issues with downstream processing. We used the marine gutless oligochaete Olavius algarvensis as a model for testing. Olavius algarvensis lives in shallow water sediments off the coast of Elba, Italy. It has no digestive and excretory system and harbors five bacterial symbionts that fulfill its nutritional and waste recycling needs (Kleiner et al., 2012, PNAS 109(19):1173-82). We compared five RNAlater preserved and five flash frozen samples in terms of the number of identified proteins, abundances of individual proteins and potential biases against specific protein or taxonomic groups. Five worms were incubated in RNAlater for 24 hours. After incubation, RNAlater was removed and samples were stored at -80°C. The remaining five worms were preserved with liquid nitrogen and stored at -80 °C immediately after preservation.
Project description:When studying gene expression in microbe-animals symbioses collected in the field it is essential to quickly and efficiently preserve in situ symbiont and host protein abundance patterns. One of the most commonly used sample preservation methods for samples targeted for proteomic analyses is flash freezing, however, liquid nitrogen or dry ice needed for flash freezing are often not available at remote field sites. We replicated our experiment from PXD014591 to test if RNAlater allows preserving proteins in animal-microbe symbioses as efficiently as flash freezing and without introducing issues with downstream processing. We used the marine gutless oligochaete Olavius algarvensis as a model for testing. Olavius algarvensis lives in shallow water sediments off the coast of Elba, Italy. It has no digestive and excretory system and harbors five bacterial symbionts that fulfill its nutritional and waste recycling needs (Kleiner et al., 2012, PNAS 109(19):1173-82). We compared six RNAlater preserved and eight flash frozen samples in terms of the number of identified proteins, abundances of individual proteins and potential biases against specific protein or taxonomic groups. Six worms were incubated in RNAlater for 24 hours. After incubation, RNAlater was removed and samples were stored at -80°C. Eight worms were directly flash frozen in liquid nitrogen and stored at -80 °C immediately after preservation.
Project description:The identification of processes activated by specific microbes during microbiota colonization of plant roots has been hampered by technical constraints in metatranscriptomics. These include lack of reference genomes, high representation of host or microbial rRNA sequences in datasets, or difficulty to experimentally validate gene functions. Here, we recolonized germ-free Arabidopsis thaliana with a synthetic, yet representative root microbiota comprising 106 genome-sequenced bacterial and fungal isolates. We used multi-kingdom rRNA depletion, deep RNA-sequencing and read mapping against reference microbial genomes to analyse the in-planta metatranscriptome of abundant colonizers. We identified over 3,000 microbial genes that were differentially regulated at the soil-root interface. Translation and energy production processes were consistently activated in planta, and their induction correlated with bacterial strains’ abundance in roots. Finally, we used targeted mutagenesis to show that several genes consistently induced by multiple bacteria are required for root colonization in one of the abundant bacterial strains (a genetically tractable Rhodanobacter). Our results indicate that microbiota members activate strain-specific processes but also common gene sets to colonize plant roots.
Project description:When studying gene expression in microbe-animals symbioses collected in the field it is essential to quickly and efficiently preserve in situ symbiont and host gene expression patterns. One of the most commonly used sample preservation methods for samples targeted for proteomic analyses is flash freezing, however, liquid nitrogen or dry ice needed for flash freezing are often not available at remote field sites. We first tested if RNAlater allows to preserve proteins in animal-microbe symbioses as efficiently as flash freezing and without introducing issues with downstream processing (see PXD014591). Second, for the data in this PRIDE submission we tested if RNAlater preserves protein expression patterns over time at room temperature. We used the marine gutless oligochaete Olavius algarvensis as a test case. Olavius algarvensis lives in shallow water sediments off the coast of Elba, Italy. It has no digestive and excretory system and harbors five bacterial symbionts that fulfill its nutritional and waste recycling needs (Kleiner et al., 2012, PNAS 109(19):1173-82). For this dataset, we fixed a total of 33 worms and incubated them in RNAlater for up to 4 weeks. We then evaluated proteome preservation quality in terms of the number of identified proteins, abundances of individual proteins and potential biases against specific protein or taxonomic groups. Out of this 33 samples, eleven worms were incubated for 24 hours in RNAlater at 4°C (t0), while the other worms were incubated in RNAlater at room temperature (21-23°C) for additional 24 hours (t1, 6 worms), one week (t2, 8 worms), and four weeks (t3, 8 worms). We removed RNAlater from the worms after incubation and froze the samples at -80°C.