CRISPR/LbCas12a-Mediated Genome Editing in Soybean.

Currently methods for generating soybean edited lines are time-consuming, inefficient, and limited to certain genotypes. Here we describe a fast and highly efficient genome editing method based on CRISPR-Cas12a nuclease system in soybean. The method uses Agrobacterium-mediated transformation to deliver editing constructs and uses aadA or ALS genes as selectable marker. It only takes about 45 days to obtain greenhouse-ready edited plants at higher than 30% transformation efficiency and 50% editing rate. The method is applicable to other selectable markers including EPSPS and has low transgene chimera rate. The method is also genotype-flexible and has been applied to genome editing of several elite soybean varieties.

Automated, High-Throughput Protoplast Transfection for Gene Editing and Transgene Expression Studies.

In an era of cost-efficient gene synthesis and high-throughput construct assembly, the onus of scientific experimentation is on the rate of in vivo testing for the identification of top performing candidates or designs. Assay platforms that are relevant to the species of interest and in the tissue of choice are highly desirable. A protoplast isolation and transfection method that is compatible with a large repertoire of species and tissues would be the platform of choice. A necessary aspect of this high-throughput screening approach is the need to handle many delicate protoplast samples at the same time, which is a bottleneck for manual operation. Such bottlenecks can be mitigated with the use of automated liquid handlers for the execution of protoplast transfection steps. The method described within this chapter utilizes a 96-well head for simultaneous, high-throughput initiation of transfection. While initially developed and optimized for use with etiolated maize leaf protoplasts, the automated protocol has also been demonstrated to be compatible with other established protoplast systems, such as soybean immature embryo derived protoplast, similarly described within. This chapter also includes instructions for a sample randomization design to reduce the impact of edge effects, which might be present when microplates are used for fluorescence readout following transfection. We also describe a streamlined, expedient, and cost-effective protocol for determining gene editing efficiencies using the T7E1 endonuclease cleavage assay with a publicly available image analysis tool.

Cellular Stress Upregulates Indole Signaling Metabolites in Escherichia coli.

Escherichia coli broadly colonize the intestinal tract of humans and produce a variety of small molecule signals. However, many of these small molecules remain unknown. Here, we describe a family of widely distributed bacterial metabolites termed the "indolokines." In E. coli, the indolokines are upregulated in response to a redox stressor via aspC and tyrB transaminases. Although indolokine 1 represents a previously unreported metabolite, four of the indolokines (2-5) were previously shown to be derived from indole-3-carbonyl nitrile (ICN) in the plant pathogen defense response. We show that the indolokines are produced in a convergent evolutionary manner relative to plants, enhance E. coli persister cell formation, outperform ICN protection in an Arabidopsis thaliana-Pseudomonas syringae infection model, trigger a hallmark plant innate immune response, and activate distinct immunological responses in primary human tissues. Our molecular studies link a family of cellular stress-induced metabolites to defensive responses across bacteria, plants, and humans.

Expansion of a core regulon by transposable elements promotes Arabidopsis chemical diversity and pathogen defense.

Plants synthesize numerous ecologically specialized, lineage-specific metabolites through biosynthetic gene duplication and functional specialization. However, it remains unclear how duplicated genes are wired into existing regulatory networks. We show that the duplicated gene CYP82C2 has been recruited into the WRKY33 regulon and indole-3-carbonylnitrile (ICN) biosynthetic pathway through exaptation of a retroduplicated LINE retrotransposon (EPCOT3) into an enhancer. The stepwise development of a chromatin-accessible WRKY33-binding site on EPCOT3 has potentiated the regulatory neofunctionalization of CYP82C2 and the evolution of inducible defense metabolite 4-hydroxy-ICN in Arabidopsis thaliana. Although transposable elements (TEs) have long been recognized to have the potential to rewire regulatory networks, these results establish a more complete understanding of how duplicated genes and TEs contribute in concert to chemical diversity and pathogen defense.

Evolution of Glucosinolate Diversity via Whole-Genome Duplications, Gene Rearrangements, and Substrate Promiscuity.

Over several decades, glucosinolates have become a model system for the study of specialized metabolic diversity in plants. The near-complete identification of biosynthetic enzymes, regulators, and transporters has provided support for the role of gene duplication and subsequent changes in gene expression, protein function, and substrate specificity as the evolutionary bases of glucosinolate diversity. Here, we provide examples of how whole-genome duplications, gene rearrangements, and substrate promiscuity potentiated the evolution of glucosinolate biosynthetic enzymes, regulators, and transporters by natural selection. This in turn may have led to the repeated evolution of glucosinolate metabolism and diversity in higher plants.

Hierarchical and Dynamic Regulation of Defense-Responsive Specialized Metabolism by WRKY and MYB Transcription Factors.

The plant kingdom produces hundreds of thousands of specialized bioactive metabolites, some with pharmaceutical and biotechnological importance. Their biosynthesis and function have been studied for decades, but comparatively less is known about how transcription factors with overlapping functions and contrasting regulatory activities coordinately control the dynamics and output of plant specialized metabolism. Here, we performed temporal studies on pathogen-infected intact host plants with perturbed transcription factors. We identified WRKY33 as the condition-dependent master regulator and MYB51 as the dual functional regulator in a hierarchical gene network likely responsible for the gene expression dynamics and metabolic fluxes in the camalexin and 4-hydroxy-indole-3-carbonylnitrile (4OH-ICN) pathways. This network may have also facilitated the regulatory capture of the newly evolved 4OH-ICN pathway in Arabidopsis thaliana by the more-conserved transcription factor MYB51. It has long been held that the plasticity of plant specialized metabolism and the canalization of development should be differently regulated; our findings imply a common hierarchical regulatory architecture orchestrated by transcription factors for specialized metabolism and development, making it an attractive target for metabolic engineering.

A new cyanogenic metabolite in Arabidopsis required for inducible pathogen defence.

Thousands of putative biosynthetic genes in Arabidopsis thaliana have no known function, which suggests that there are numerous molecules contributing to plant fitness that have not yet been discovered. Prime among these uncharacterized genes are cytochromes P450 upregulated in response to pathogens. Here we start with a single pathogen-induced P450 (ref. 5), CYP82C2, and use a combination of untargeted metabolomics and coexpression analysis to uncover the complete biosynthetic pathway to 4-hydroxyindole-3-carbonyl nitrile (4-OH-ICN), a previously unknown Arabidopsis metabolite. This metabolite harbours cyanogenic functionality that is unprecedented in plants and exceedingly rare in nature; furthermore, the aryl cyanohydrin intermediate in the 4-OH-ICN pathway reveals a latent capacity for cyanogenic glucoside biosynthesis in Arabidopsis. By expressing 4-OH-ICN biosynthetic enzymes in Saccharomyces cerevisiae and Nicotiana benthamiana, we reconstitute the complete pathway in vitro and in vivo and validate the functions of its enzymes. Arabidopsis 4-OH-ICN pathway mutants show increased susceptibility to the bacterial pathogen Pseudomonas syringae, consistent with a role in inducible pathogen defence. Arabidopsis has been the pre-eminent model system for studying the role of small molecules in plant innate immunity; our results uncover a new branch of indole metabolism distinct from the canonical camalexin pathway, and support a role for this pathway in the Arabidopsis defence response. These results establish a more complete framework for understanding how the model plant Arabidopsis uses small molecules in pathogen defence.

Seed after-ripening and dormancy determine adult life history independently of germination timing.

• Seed dormancy can affect life history through its effects on germination time. Here, we investigate its influence on life history beyond the timing of germination. • We used the response of Arabidopsis thaliana to chilling at the germination and flowering stages to test the following: how seed dormancy affects germination responses to the environment; whether variation in dormancy affects adult phenology independently of germination time; and whether environmental cues experienced by dormant seeds have an effect on adult life history. • Dormancy conditioned the germination response to low temperatures, such that prolonged periods of chilling induced dormancy in nondormant seeds, but stimulated germination in dormant seeds. The alleviation of dormancy through after-ripening was associated with earlier flowering, independent of germination date. Experimental dormancy manipulations showed that prolonged chilling at the seed stage always induced earlier flowering, regardless of seed dormancy. Surprisingly, this effect of seed chilling on flowering time was observed even when low temperatures did not induce germination. • In summary, seed dormancy influences flowering time and hence life history independent of its effects on germination timing. We conclude that the seed stage has a pronounced effect on life history, the influence of which goes well beyond the timing of germination.