Characterization of Programmable Transcription Activators in the Model Monocot Setaria viridis Via Protoplast Transfection.

Recent advances in DNA synthesis and assembly allow for genetic constructs to be designed and constructed in high throughput. Characterizing large numbers of variant genetic designs is not feasible with low-throughput and time-consuming plant transformation workflows. Protoplast transformation offers a rapid, high-throughput compatible alternative for testing genetic constructs in plant-relevant molecular environments. Here, we describe a protocol for protoplast transformation using a recent experiment in genetic optimization of dCas9-based programmable transcription activators as an example.

Osmotic stress induces phosphorylation of histone H3 at threonine 3 in pericentromeric regions of Arabidopsis thaliana.

Histone phosphorylation plays key roles in stress-induced transcriptional reprogramming in metazoans but its function(s) in land plants has remained relatively unexplored. Here we report that an Arabidopsis mutant defective in At3g03940 and At5g18190, encoding closely related Ser/Thr protein kinases, shows pleiotropic phenotypes including dwarfism and hypersensitivity to osmotic/salt stress. The double mutant has reduced global levels of phosphorylated histone H3 threonine 3 (H3T3ph), which are not enhanced, unlike the response in the wild type, by drought-like treatments. Genome-wide analyses revealed increased H3T3ph, slight enhancement in trimethylated histone H3 lysine 4 (H3K4me3), and a modest decrease in histone H3 occupancy in pericentromeric/knob regions of wild-type plants under osmotic stress. However, despite these changes in heterochromatin, transposons and repeats remained transcriptionally repressed. In contrast, this reorganization of heterochromatin was mostly absent in the double mutant, which exhibited lower H3T3ph levels in pericentromeric regions even under normal environmental conditions. Interestingly, within actively transcribed protein-coding genes, H3T3ph density was minimal in 5' genic regions, coincidental with a peak of H3K4me3 accumulation. This pattern was not affected in the double mutant, implying the existence of additional H3T3 protein kinases in Arabidopsis. Our results suggest that At3g03940 and At5g18190 are involved in the phosphorylation of H3T3 in pericentromeric/knob regions and that this repressive epigenetic mark may be important for maintaining proper heterochromatic organization and, possibly, chromosome function(s).