Understanding the biodiversity and genetics of the gut microbiome has important implications for host physiology. One underexplored and elusive group is ciliated protozoa, which play crucial roles in regulating gut microbial interactions. Integrating single-cell sequencing and an assembly-and-identification pipeline, we acquired 52 high-quality ciliate genomes of 22 rumen morphospecies for all major abundant clades. With these genomes, we firstly resolved the taxonomic and phylogenetic framework that reclassified them into 19 species spanning 13 genera and reassigned the genus Dasytricha from Isotrichidae to a new family Dasytrichidae. Via extensive horizontal gene transfer and gene family expansion, rumen ciliates possess a broad array of enzymes to synergistically degrade plant and microbial carbohydrates. In particular, ~80% of the degrading enzymes in Diplodiniinae and Ophryoscolecinae act on plant cell wall, and the high activities of their cellulase, xylanase and lysozyme reflect the potential of ciliate enzymes for biomass-conversion. Additionally, the new ciliate dataset greatly facilitated the rumen metagenomic analyses by allowing ~12% of reads to be classified.
In China, Zanthoxylum bungeanum Maxim, known as “Huajiao,” has a pleasant, fragrant flavor and several therapeutic properties. The nutritional content of plants is necessary for their defense response to insects. In this study, we analyzed the effects of soil fertilization treatments such as nitrogen (N), phosphorus (P), potassium (K), calcium (Ca), and special compound fertilizer for pepper (HZ) on the different growth parameters and expression of insect-defense-response genes in Z. bungeanum. The results show that the height and weight of prickly ash significantly differed after the application of fertilizers with different concentrations. Additionally, seedlings that were treated with low concentrations of nutrient fertilizers (N1, P1, K1, Ca1, and HZ1) were significantly higher, and they were selected for transcriptome analysis. According to transcriptomic analysis, a total of 65,566 unigenes were discovered, among which 61,379 corresponded to annotated protein-coding genes and 4187 to transcripts of novel protein-coding genes. A total of 294 unigenes were detected as candidate genes for regulating the defense response to insects, including 204 protease inhibitors, 29 plant lectins, and 61 other defense response genes. Additionally, trypsin inhibitors, cystatin, phytepsin, metalloproteinase, MMP, caffeic acid, resveratrol, and thiol proteinase inhibitors, ACA, TDC, and 28 BES1 were enriched in Z. bungeanum. Specifically, the leaves of Z. bungeanum that were treated with Ca and HZ fertilizations were dominated by the protease inhibitors. In addition, the type of fertilizer significantly affects gene expression in plants. The functional annotations were predicted by the number of differentially expressed genes and classified by GO and KEGG ontology enrichment analysis. Moreover, according to the GO database, biological processes were the largest group and contained a high frequency of differentially expressed genes. According to KEGG pathway results, significantly enriched genes belonged to the biosynthesis of secondary metabolisms, amino acid metabolism, and folding, sorting, and degradation. Overall, it was found that the type of fertilizer with low concentrations had an effect on Z. bungeanum’s primary and secondary metabolism, and these findings provided grounds for further research in forest protection science.
Rumen microorganisms can be used in in vitro anaerobic fermentation to encourage the sustainable exploitation of agricultural wastes. However, the understanding of active microbiota under in vitro ruminal fermentation conditions is still insufficient. To investigate how rumen microbes actively participate in the fermentation process in vitro, we resolved the metaproteome generated from ruminal fermentation broth after seven days of in vitro incubation. Herein, the sample-specific database for metaproteomic analysis was constructed according to the metagenomic data of in vitro ruminal fermentation. Based on the sample-specific database, we found in the metaproteome that Bacteroidetes and Firmicutes_A were the most active in protein expression, and over 50% of these proteins were assigned to gene categories involved in energy conversion and basic structures. On the other hand, a variety of bacteria-derived extracellular proteins, which contained carbohydrate-active enzyme domains, were found in the extracellular proteome of fermentation broth. Additionally, the bacterial intracellular/surface moonlighting proteins (ISMPs) and proteins of outer membrane vesicles were detected in the extracellular proteome, and these ISMPs were involved in maintaining microbial population size through potential adherence to substrates. The metaproteomic characterizations of microbial intracellular/extracellular proteins provide new insights into the ability of the rumen microbiome to maintain in vitro ruminal fermentation.
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