GSE152914
Evaluating the Lettuce Metatranscriptome with MinION Sequencing for Future Spaceflight Food Production Application.
Healthy plants are vital for successful, long-duration missions in space, as they provide the crew with life support, food production, and psychological benefits. The microorganisms that associate with plant tissues play a critical role in improving plant growth, health, and production. To that end, it is necessary to develop methodologies that investigate the metabolic activities of the plant's microbiome in orbit to enable rapid responses regarding the care of plants in space. In this study, we developed a protocol to characterize the endophytic and epiphytic microbial metatranscriptome of red romaine lettuce, a key salad crop that was grown under International Space Station (ISS)-like conditions. Microbial transcripts enriched from host-microbe total RNA were sequenced using the Oxford Nanopore MinION sequencing platform. Results showed that this enrichment approach was highly reproducible and effective for rapid on-site detection of microbial transcriptional activity. Taxonomic analysis based on 16S and 18S rRNA transcripts identified that the top five most abundant phyla in the lettuce microbiome were Firmicutes, Proteobacteria, Actinobacteria, Bacteroidetes, and Ascomycota. The metatranscriptomic analysis identified the expression of genes involved in many metabolic pathways, including carbohydrate metabolism, energy metabolism, and signal transduction. Network analyses of the expression data show that, within the signal transduction pathway of the fungal community, the Mitogen-Activated Protein Kinase signaling pathway was tightly regulated across all samples and could be a potential driver for fungal proliferation. Our results demonstrated the feasibility of using MinION-based metatranscriptomics of enriched microbial RNA as a method for rapid, on-site monitoring of the transcriptional activity of crop microbiomes, thereby helping to facilitate and maintain plant health for on-orbit space food production. Overall design: Red romaine lettuce, Lactuca sativa cv 'Outredgeous' were grown in environmentally controlled growth chambers at Kennedy Space Center (KSC), FL, under conditions simulating those aboard the ISS (RH-50%, CO2-3000 ppm, and temperature 23 ºC). The growth cycle was established at 16h/8h light dark cycles with 200-300 µmol fluorescent lights and samples were harvested at day 28 for leafy greens.  Leaf tissue from each of the three pots (L93, L96, and L101) were placed immediately into RNAlater™  and stored at -80 ºC until ready for RNA extraction.Total RNA was extracted from each of the three lettuce leave replicates with the RNeasy Plant Mini Kit and was DNase-treated. A modified enrichment method was developed to remove plant mRNA and rRNA for compatibility with Nanopore library preparation protocols. Modified Invitrogen Dynabeads polyA enrichment protocol was performed to deplete the plant mRNA. Invitrogen RiboMinusTM plant kit was performed to removal of plant ribosomal RNA. Host depleted RNA form each sample was then A-tailed using Poly(A) polymerase.  Library preparation for cDNA-PCR sequencing was performed using the SQK-PCS109 sequencing kit following the manufacturer's instructions (Oxford Nanopore Technologies, Oxford, UK) with the following cycling conditions, an initial denaturing step at 95°C for 5 min, then 14 cycles of 15 s at 95°C, 15 s at 62°C, and 1 min at 65°C, followed by a final extension step at 65°C for 6 mins and a hold at 4°C.  cDNA library using 100 fmol of amplified cDNA with adapters was loaded onto a FLO-MIN106 flowcell and the RNA library was sequenced on a MinION device for 48?h. Basecalling was perform in real-time using the MinIT device. L93, L96 and L101 replicates were sequenced on individual flowcells to provide information about the process reproducibility and variability.