Establishment of an efficient transformation system and its application in regulatory mechanism analysis of biological macromolecules in tea plants.

Tea (Camellia sinensis), one of the most important beverage crops originated from China and is now cultivated worldwide, provides numerous secondary metabolites that account for its health benefits and rich flavor. However, the lack of an efficient and reliable genetic transformation system has seriously hindered the gene function investigation and precise breeding of C. sinensis. In this study, we established a highly efficient, labor-saving, and cost-effective Agrobacterium rhizogenes-mediated hairy roots genetic transformation system for C. sinensis, which can be used for gene overexpression and genome editing. The established transformation system was simple to operate, bypassing tissue culture and antibiotic screening, and only took two months to complete. We used this system to conduct function analysis of transcription factor CsMYB73 and found that CsMYB73 negatively regulates L-theanine synthesis in tea plant. Additionally, callus formation was successfully induced using transgenic roots, and the transgenic callus exhibited normal chlorophyll production, enabling the study of the corresponding biological functions. Furthermore, this genetic transformation system was effective for multiple C. sinensis varieties and other woody plant species. By overcoming technical obstacles such as low efficiency, long experimental periods, and high costs, this genetic transformation will be a valuable tool for routine gene investigation and precise breeding in tea plants.

Specific Enriched Acinetobacter in Camellia Weevil Gut Facilitate the Degradation of Tea Saponin: Inferred from Bacterial Genomic and Transcriptomic Analyses.

Beneficial gut bacteria can enhance herbivorous arthropod adaptation to plant secondary compounds (PSMs), and specialist herbivores provide excellent examples of this. Tea saponin (TS) of Camellia oleifera is triterpenoids toxic to seed-feeding weevil pest, Curculio chinensis (CW). Previous studies disclosed that Acinetobacter, which was specific enriched in the CW's gut, was involved in helping CW evade TS toxicity of C. oleifera. However, it is still not clear whether Acinetobacter is associated with other anti-insect compounds, and the molecular mechanism of Acinetobacter degradation of TS has not been clarified. To address these questions, we explored the relationship between host plant toxin content and Acinetobacter of CW gut bacteria. Results demonstrated that TS content significantly affected the CW gut microbiome structure and enriched bacteria functional for TS degradation. We further isolated Acinetobacter strain and conducted its genome and transcriptome analyses for bacterial characterization and investigation on its role in TS degradation. Biological tests were carried out to verify the ability of the functional bacterium within CW larvae to detoxify TS. Our results showed that TS-degrading bacteria strain (Acinetobacter sp. AS23) genome contains 47 genes relating to triterpenoids degradation. The AS23 strain improved the survival rate of CW larvae, and the steroid degradation pathway could be the key one for AS23 to degrade TS. This study provides the direct evidence that gut bacteria mediate adaptation of herbivorous insects to phytochemical resistance. IMPORTANCE Microorganism is directly exposed to the plant toxin environment and play a crucial third party in herbivores gut. Although previous studies have proved the existence of gut bacteria that help CWs degrade TS, the specific core flora and its function have not been explored. In this study, we investigated the correlation between the larva gut microbiome and plant secondary metabolites. Acinetobacter genus was the target flora related to TS degradation. There were many terpenoids genes in Acinetobacter sp. AS23 genome. Results of transcriptome analysis and biological tests suggested that steroid degradation pathway be the key pathway of AS23 to degrade TS. This study not only provides direct evidence that gut microbes mediate the rapid adaptation of herbivorous insects to phytochemical resistance, but also provides a theoretical basis for further research on the molecular mechanism of intestinal bacteria cooperating with pests to adapt to plant toxins.