In-locus gene silencing in plants using genome editing.

Gene silencing is crucial in crop breeding for desired trait development. RNA interference (RNAi) has been used widely but is limited by ectopic expression of transgenes and genetic instability. Introducing an upstream start codon (uATG) into the 5'untranslated region (5'UTR) of a target gene may 'silence' the target gene by inhibiting protein translation from the primary start codon (pATG). Here, we report an efficient gene silencing method by introducing a tailor-designed uATG-containing element (ATGE) into the 5'UTR of genes in plants, occupying the original start site to act as a new pATG. Using base editing to introduce new uATGs failed to silence two of the tested three rice genes, indicating complex regulatory mechanisms. Precisely inserting an ATGE adjacent to pATG achieved significant target protein downregulation. Through extensive optimization, we demonstrated this strategy substantially and consistently downregulated target protein expression. By designing a bidirectional multifunctional ATGE4, we enabled tunable knockdown from 19% to 89% and observed expected phenotypes. Introducing ATGE into Waxy, which regulates starch synthesis, generated grains with lower amylose, revealing the value for crop breeding. Together, we have developed a programmable and robust method to knock down gene expression in plants, with potential for biological mechanism exploration and crop enhancement.

Rapid and dynamic detection of endogenous proteins through in-locus tagging in rice.

Understanding the behavior of endogenous proteins is crucial for functional genomics, yet their dynamic characterization in plants presents substantial challenges. While mammalian studies have leveraged in-locus tagging with the luminescent HiBiT peptide and genome editing for rapid quantification of native proteins, this approach remained unexplored in plants. Here, we introduce the in-locus HiBiT tagging of rice proteins and demonstrate its feasibility in plants. We found that although traditional HiBiT blotting works in rice, it failed to detect two of the three tagged proteins, which is attributed to the low luminescence activity in plants. To overcome this limitation, we engaged in extensive optimization, culminating in a new luciferin substrate coupled with a refined reaction protocol that enhanced luminescence by up to 6.9-fold. This innovation led to the development of TagBIT (tagging with HiBiT), a robust method for high-sensitivity protein characterization in plants. Our application of TagBIT to seven rice genes illustrates its versatility on endogenous proteins, enabling antibody-free protein blotting, real-time protein quantification via luminescence, in-situ visualization using a cross-breeding strategy, and effective immunoprecipitation for protein interaction analysis. The heritable nature of this system, confirmed across T1 to T3 generations, positions TagBIT as a powerful tool for protein study in plant biology.

Complete plastome sequence of Dillenia indica linn. (Dilleniaceae): an Endangered (EN) species in South China.

Dillenia indica Linn. is an endangered plant species occurring in southern Guangxi province and Yunnan province of China, which has been grown in gardens as an ornamental plant and is widely used in the medical field. Here, we report and characterize the complete plastid genome sequence of D. indica to provide genomic resources useful for promoting its conservation. The complete plastome is 159,266 bp in length and contains the typical structure and gene content of angiosperm plastome, including two inverted repeat (IR) regions of 26,457 bp, a large single-copy (LSC) region of 88,305 bp, and a small single-copy (SSC) region of 18,047 bp. The plastome contains 115 genes, consisting of 81 unique protein-coding genes, 30 unique tRNA genes, and 4 unique rRNA genes (5S rRNA, 4.5S rRNA, 23S rRNA, and 16S rRNA). The overall A/T content in the plastome of D. indica is 63.40%. The complete plastome sequence of D. indica will provide a useful resource for the conservation genetics of this species as well as for the phylogenetic studies of Dilleniaceae.

Complete plastome sequence of Hoya pottsii Traill and Hoya liangii Tsiang (Apocynaceae).

Hoya is the largest genus (about 350-450 species) within Apocynaceae. It is a subshrub or liana, epiphytic or epilithic. Most species grow in tropical and subtropical South and Southeast Asia. Here we report and characterize the complete plastid genome sequence of Hoya pottsii Traill and Hoya liangii Tsiang in an effort to provide genomic resources useful for promoting its systematics research. The complete plastome of H. pottsii is 161,565 bp in length, including two Inverted Repeat (IR) regions of 24,657 bp, a Large Single-Copy (LSC) region of 92,532 bp, and a Small Single-Copy (SSC) region of 19,719 bp. The plastome contains 115 genes, consisting of 81 unique protein-coding genes, 30 unique tRNA genes, and 4 unique rRNA genes. The overall A/T content in the plastome of H. pottsii is 62.40%. The complete plastome of H. liangii is 162,989 bp in length, including two IR regions of 24,841 bp, a LSC region of 93,292 bp, and a SSC region of 20,015 bp. The plastome contains 115 genes, consisting of 81 unique protein-coding genes, 30 unique tRNA genes, and 4 unique rRNA genes. The overall A/T content in the plastome of H. pottsii is 62.30%. The complete plastome sequence of H. pottsii and H. liangii will provide a useful resource for the conservation genetics of the two species as well as for the phylogenetic studies of Hoya.

Complete plastome sequences of Diospyros maclurei Merr. and Diospyros hainanensis Merr. (Ebenaceae): two endemic species in Hainan Province, China.

Diospyros is the largest genus (about 485 species) in Ebenaceae. It is a deciduous or evergreen tree or shrub. It grows in pantropical and extending into temperate regions. Here, we report and characterize the complete plastid genome sequences of D. maclurei Merr. and D. hainanensis Merr. in an effort to provide genomic resources useful for promoting their systematics research and potential economic development. The complete plastome of D. maclurei is 157,946 bp in length, including two Inverted Repeat (IR) regions of 26,081 bp, a Large Single-Copy (LSC) region of 87,387 bp, and a Small Single-Copy (SSC) region of 18,397 bp. The plastome contains 114 genes, consisting of 80 unique protein-coding genes, 30 unique tRNA genes, and four unique rRNA genes. The overall A/T content in the plastome of D. maclurei is 62.60%. The complete plastome of D. hainanensis is 157,999 bp in length, including two Inverted Repeat (IR) regions of 26,077 bp, a Large Single-Copy (LSC) region of 87,523 bp, and a Small Single-Copy (SSC) region of 18,322 bp. The plastome contains 114 genes, consisting of 80 unique protein-coding genes, 30 unique tRNA genes, and four unique rRNA genes. The overall A/T content in the plastome of D. hainanensis is 62.60%. The complete plastome sequences of D. Maclurei and D. hainanensis will provide a useful resource for the conservation genetics of the two species as well as for the phylogenetic studies of Diospyros.