Community RNA-Seq: multi-kingdom responses to living versus decaying roots in soil

Roots are a primary source of organic carbon input in most soils. The consumption of living and detrital root inputs involves multi-trophic processes and multiple kingdoms of microbial life, but typical microbial ecology studies focus on only one or two major lineages. We used Illumina shotgun RNA sequencing to conduct PCR-independent SSU rRNA community analysis ("community RNA-Seq") and simultaneously assess the bacteria, archaea, fungi, and microfauna surrounding both living and decomposing roots of the annual grass, Avena fatua. Plants were grown in ¹³CO₂-labeled microcosms amended with ¹⁵N-root litter to identify the preferences of rhizosphere organisms for root exudates (¹³C) versus decaying root biomass (¹⁵N) using NanoSIMS microarray imaging (Chip-SIP). When litter was available, rhizosphere and bulk soil had significantly more Amoebozoa, which are potentially important yet often overlooked top-down drivers of detritusphere community dynamics and nutrient cycling. Bulk soil containing litter was depleted in Actinobacteria but had significantly more Bacteroidetes and Proteobacteria. While Actinobacteria were abundant in the rhizosphere, Chip-SIP showed Actinobacteria preferentially incorporated litter relative to root exudates, indicating this group's more prominent role in detritus elemental cycling in the rhizosphere. Our results emphasize that decomposition is a multi-trophic process involving complex interactions, and our methodology can be used to track the trajectory of carbon through multi-kingdom soil food webs.