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Transcription factors can promote gene expression through activation domains. Whole-genome screens have systematically mapped activation domains in transcription factors but not in non-transcription factor proteins (e.g., chromatin regulators and coactivators). To fill this knowledge gap, we employed the activation domain predictor PADDLE to analyze the proteomes of Arabidopsis thalianaand Saccharomyces cerevisiae. We screened 18,000 predicted activation domains from >800 non-transcription factor genes in both species, confirming that 89% of candidate proteins contain active fragments. Our work enables the annotation of hundreds of nuclear proteins as putative coactivators, many of which have never been ascribed any function in plants. Analysis of peptide sequence compositions reveals how the distribution of key amino acids dictates activity. Finally, we validated short, “universal” activation domains with comparable performance to state-of-the-art activation domains used for genome engineering. Our approach enables the genome-wide discovery and annotation of activation domains that can function across diverse eukaryotes.

RNA-guided CRISPR activation (CRISPRa) systems have been developed in plants. However, the simultaneous activation of multiple genes remains challenging. Here, we develop a highly robust CRISPRa system working in rice, Arabidopsisand tomato, CRISPR–Act3.0, through systematically exploring different effector recruitment strategies and various transcription activators based on deactivated Streptococcus pyogenesCas9 (dSpCas9). The CRISPR–Act3.0 system results in fourfold to sixfold higher activation than the state-of-the-art CRISPRa systems. We further develop a tRNA–gR2.0 (single guide RNA 2.0) expression system enabling CRISPR–Act3.0-based robust activation of up to seven genes for metabolic engineering in rice. In addition, CRISPR–Act3.0 allows the simultaneous modification of multiple traits in Arabidopsis, which are stably transmitted to the T3 generations. On the basis of CRISPR–Act3.0, we elucidate guide RNA targeting rules for effective transcriptional activation. To target T-rich protospacer adjacent motifs (PAMs), we transfer this activation strategy to CRISPR–dCas12b and further improve the dAaCas12b-based CRISPRa system. Moreover, we develop a potent near-PAM-less CRISPR–Act3.0 system on the basis of the SpRY dCas9 variant, which outperforms the dCas9–NG system in both activation potency and targeting scope. Altogether, our study has substantially improved the CRISPRa technology in plants and provided plant researchers a powerful toolbox for efficient gene activation in foundational and translational research.

Plants produce a wealth of biologically active compounds, many of which are used to defend themselves from various pests and pathogens. We explore the possibility of expanding upon the natural chemical diversity of plants and create molecules that have enhanced properties, by engineering metabolic pathways new to nature. We rationally broaden the set of primary metabolites that can be utilized by the core biosynthetic pathway of the natural biopesticide, brassinin, producing in plantaa novel class of compounds that we call crucifalexins. Two of our new-to-nature crucifalexins are more potent antifungals than brassinin and, in some instances, comparable to commercially used fungicides. Our findings highlight the potential to push the boundaries of plant metabolism for the biosynthesis of new biopesticides.

Agricultural biotechnology strategies often require the precise regulation of multiple genes to effectively modify complex plant traits. However, most efforts are hindered by a lack of characterized tools that allow for reliable and targeted expression of transgenes. We have successfully engineered a library of synthetic transcriptional regulators that modulate expression strength in planta. By leveraging orthogonal regulatory systems from Saccharomycesspp., we have developed a strategy for the design of synthetic activators, synthetic repressors, and synthetic promoters and have validated their use in Nicotiana benthamianaand Arabidopsis thaliana. This characterization of contributing genetic elements that dictate gene expression represents a foundation for the rational design of refined synthetic regulators. Our findings demonstrate that these tools provide variation in transcriptional output while enabling the concerted expression of multiple genes in a tissue-specific and environmentally responsive manner, providing a basis for generating complex genetic circuits that process endogenous and environmental stimuli.

The transcriptional effector domains of transcription factors play a key role in controlling gene expression; however, their functional nature is poorly understood, hampering our ability to explore this fundamental dimension of gene regulatory networks. To map the trans-regulatory landscape in a complex eukaryote, we systematically characterized the putative transcriptional effector domains of over 400 Arabidopsis thalianatranscription factors for their capacity to modulate transcription. We demonstrate that transcriptional effector activity can be integrated into gene regulatory networks capable of elucidating the functional dynamics underlying gene expression patterns. We further show how our characterized domains can enhance genome engineering efforts and reveal how plant transcriptional activators share regulatory features conserved across distantly related eukaryotes. Our results provide a framework to systematically characterize the regulatory role of transcription factors at a genome-scale in order to understand the transcriptional wiring of biological systems.

Glycosylation of metabolites serves multiple purposes. Adding sugars makes metabolites more water soluble and improves their biodistribution, stability, and detoxification. In plants, the increase in melting points enables storing otherwise volatile compounds that are released by hydrolysis when needed. Classically, glycosylated metabolites were identified by mass spectrometry (MS/MS) using [M-sugar] neutral losses. Herein, we studied 71 pairs of glycosides with their respective aglycones, including hexose, pentose, and glucuronide moieties. Using liquid chromatography (LC) coupled to electrospray ionization high-resolution mass spectrometry, we detected the classic [M-sugar] product ions for only 68% of glycosides. Instead, we found that most aglycone MS/MS product ions were conserved in the MS/MS spectra of their corresponding glycosides, even when no [M-sugar] neutral losses were observed. We added pentose and hexose units to the precursor masses of an MS/MS library of 3057 aglycones to enable rapid identification of glycosylated natural products with standard MS/MS search algorithms. When searching unknown compounds in untargeted LC-MS/MS metabolomics data of chocolate and tea, we structurally annotated 108 novel glycosides in standard MS-DIAL data processing. We uploaded this new in silico-glycosylated product MS/MS library to GitHub to enable users to detect natural product glycosides without authentic chemical standards.

Competing Interests: The authors declare no competing interests.