A rapid and scalable approach to build synthetic repetitive hormone-responsive promoters.

Advancement of DNA-synthesis technologies has greatly facilitated the development of synthetic biology tools. However, high-complexity DNA sequences containing tandems of short repeats are still notoriously difficult to produce synthetically, with commercial DNA synthesis companies usually rejecting orders that exceed specific sequence complexity thresholds. To overcome this limitation, we developed a simple, single-tube reaction method that enables the generation of DNA sequences containing multiple repetitive elements. Our strategy involves commercial synthesis and PCR amplification of padded sequences that contain the repeats of interest, along with random intervening sequence stuffers that include type IIS restriction enzyme sites. GoldenBraid molecular cloning technology is then employed to remove the stuffers, rejoin the repeats together in a predefined order, and subclone the tandem(s) in a vector using a single-tube digestion-ligation reaction. In our hands, this new approach is much simpler, more versatile and efficient than previously developed solutions to this problem. As a proof of concept, two different phytohormone-responsive, synthetic, repetitive proximal promoters were generated and tested in planta in the context of transcriptional reporters. Analysis of transgenic lines carrying the synthetic ethylene-responsive promoter 10x2EBS-S10 fused to the GUS reporter gene uncovered several developmentally regulated ethylene response maxima, indicating the utility of this reporter for monitoring the involvement of ethylene in a variety of physiologically relevant processes. These encouraging results suggest that this reporter system can be leveraged to investigate the ethylene response to biotic and abiotic factors with high spatial and temporal resolution.

Loss of the Acetyltransferase NAA50 Induces Endoplasmic Reticulum Stress and Immune Responses and Suppresses Growth.

Stress signaling in plants is carefully regulated to ensure proper development and reproductive fitness. Overactive defense signaling can result in dwarfism as well as developmental defects. In addition to requiring a substantial amount of energy, plant stress responses place a burden upon the cellular machinery, which can result in the accumulation of misfolded proteins and endoplasmic reticulum (ER) stress. Negative regulators of stress signaling, such as ENHANCED DISEASE RESISTANCE 1 (EDR1), ensure that stress responses are properly suspended when they are not needed, thereby conserving energy for growth and development. Here, we describe the role of an uncharacterized N-terminal acetyltransferase, NAA50, in the regulation of plant development and stress responses in Arabidopsis (Arabidopsis thaliana). Our results demonstrate that NAA50, an interactor of EDR1, plays an important role in regulating the tradeoff between plant growth and defense. Plants lacking NAA50 display severe developmental defects as well as induced stress responses. Reduction of NAA50 expression results in arrested stem and root growth as well as senescence. Furthermore, our results demonstrate that the loss of NAA50 results in constitutive ER stress signaling, indicating that NAA50 may be required for the suppression of ER stress. This work establishes NAA50 as essential for plant development and the suppression of stress responses, potentially through the regulation of ER stress.

Arabidopsis EDR1 Protein Kinase Regulates the Association of EDS1 and PAD4 to Inhibit Cell Death.

ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) and PHYTOALEXIN DEFICIENT4 (PAD4) are sequence-related lipase-like proteins that function as a complex to regulate defense responses in Arabidopsis by both salicylic acid-dependent and independent pathways. Here, we describe a gain-of-function mutation in PAD4 (S135F) that enhances resistance and cell death in response to infection by the powdery mildew pathogen Golovinomyces cichoracearum. The mutant PAD4 protein accumulates to wild-type levels in Arabidopsis cells, thus these phenotypes are unlikely to be due to PAD4 over accumulation. The phenotypes are similar to loss-of-function mutations in the protein kinase EDR1 (Enhanced Disease Resistance1), and previous work has shown that loss of PAD4 or EDS1 suppresses edr1-mediated phenotypes, placing these proteins downstream of EDR1. Here, we show that EDR1 directly associates with EDS1 and PAD4 and inhibits their interaction in yeast and plant cells. We propose a model whereby EDR1 negatively regulates defense responses by interfering with the heteromeric association of EDS1 and PAD4. Our data indicate that the S135F mutation likely alters an EDS1-independent function of PAD4, potentially shedding light on a yet-unknown PAD4 signaling function.

Enhancing HR Frequency for Precise Genome Editing in Plants.

Gene-editing tools, such as Zinc-fingers, TALENs, and CRISPR-Cas, have fostered a new frontier in the genetic improvement of plants across the tree of life. In eukaryotes, genome editing occurs primarily through two DNA repair pathways: non-homologous end joining (NHEJ) and homologous recombination (HR). NHEJ is the primary mechanism in higher plants, but it is unpredictable and often results in undesired mutations, frameshift insertions, and deletions. Homology-directed repair (HDR), which proceeds through HR, is typically the preferred editing method by genetic engineers. HR-mediated gene editing can enable error-free editing by incorporating a sequence provided by a donor template. However, the low frequency of native HR in plants is a barrier to attaining efficient plant genome engineering. This review summarizes various strategies implemented to increase the frequency of HDR in plant cells. Such strategies include methods for targeting double-strand DNA breaks, optimizing donor sequences, altering plant DNA repair machinery, and environmental factors shown to influence HR frequency in plants. Through the use and further refinement of these methods, HR-based gene editing may one day be commonplace in plants, as it is in other systems.