Exploring sagebrush leaf microbial metagenomes from deep, host-derived sequencing

Advanced sequencing technologies and improvements in bioinformatics have provided a new way to study plant-associated microbial communities, including the use of host genomic sequencing. Our study focuses on the leaf microbiome of basin big sagebrush (Artemisia tridentata subsp. tridentata), a foundational shrub of western North America. We analyzed Illumina shotgun sequences from sagebrush leaves to investigate the metagenomes of leaf-associated microbes that were sequenced alongside their plant hosts. We aimed to profile the leaf microbiome across different sample sources (magenta box, greenhouse, and field/wild), reconstruct metagenome-assembled genomes (MAGs) where possible, and investigate functional gene annotations of the resulting MAGs, specifically with regard to the potential metabolism of sagebrush chemicals. To achieve this, Illumina shotgun sequence reads (containing both host and associated microbial reads) were mapped to the reference genomes of Artemisia tridentata, Artemisia annua, and the human reference genome to remove plant host and human-associated sequences. Host-cleaned reads were then analyzed using microbial metagenomics techniques. Taxonomic profiling revealed that Phyllobacterium and Sphingomonas were the most abundant microbial genera in greenhouse-grown plants, with very little variation among the samples. Wild, field-collected samples were much more variable and were dominated by Klebsiella and Aureobasidium species. From the co-assembly of greenhouse samples, we reconstructed two high-quality MAGs (a Phyllobacterium species and a Sphingomonas species) with >98% completion and <1% contamination. Functional annotation of these MAGs uncovered genes associated with the degradation and metabolism of camphor and other essential oils such as pinene, geraniol, and limonene, which are part of sagebrush leaf chemistry.IMPORTANCEBig sagebrush (Artemisia tridentata), the foundation species of the sagebrush steppe, has broad ecological importance because its evergreen leaves offer nutrients and shade that facilitate the establishment of diverse understory plants in arid environments. Sagebrush leaves contain various secondary metabolites, including terpenoids, flavonoids, and phenolic compounds. These chemicals contribute to the plant's defense mechanisms against herbivores and pathogens. Despite this, sagebrush hosts diverse bacterial and fungal communities. We found that the microbial metagenome-assembled genomes (MAGs) we recovered contained genes that have the potential to degrade some of the chemical compounds in sagebrush leaves that could inhibit the growth of other microbes. This is the first study to mine plant genome data using host-derived sequences to generate microbial MAGs. Our results showed that MAGs can be recovered from plant host-derived sequence data, providing a new way to explore the identity and functional capabilities of difficult-to-culture microbes.