QS-21 is a potent vaccine adjuvant and remains the only saponin-based adjuvant that has been clinically approved for use in humans1,2. However, owing to the complex structure of QS-21, its availability is limited. Today, the supply depends on laborious extraction from the Chilean soapbark tree or on low-yielding total chemical synthesis3,4. Here we demonstrate the complete biosynthesis of QS-21 and its precursors, as well as structural derivatives, in engineered yeast strains. The successful biosynthesis in yeast requires fine-tuning of the host's native pathway fluxes, as well as the functional and balanced expression of 38 heterologous enzymes. The required biosynthetic pathway spans seven enzyme families-a terpene synthase, P450s, nucleotide sugar synthases, glycosyltransferases, a coenzyme A ligase, acyl transferases and polyketide synthases-from six organisms, and mimics in yeast the subcellular compartmentalization of plants from the endoplasmic reticulum membrane to the cytosol. Finally, by taking advantage of the promiscuity of certain pathway enzymes, we produced structural analogues of QS-21 using this biosynthetic platform. This microbial production scheme will allow for the future establishment of a structure-activity relationship, and will thus enable the rational design of potent vaccine adjuvants.

Cuscuta species (dodders) are agriculturally destructive, parasitic angiosperms. These parasitic plants use haustoria as physiological bridges to extract nutrients and water from hosts. Cuscuta campestris has a broad host range and wide geographical distribution. While some wild tomato relatives are resistant, cultivated tomatoes are generally susceptible to C. campestris infestations. However, some specific Heinz tomato (Solanum lycopersicum) hybrid cultivars exhibit resistance to dodders in the field, but their defense mechanism was previously unknown. Here, we discovered that the stem cortex in these resistant lines responds with local lignification upon C. campestris attachment, preventing parasite entry into the host. Lignin Induction Factor 1 (LIF1, an AP2-like transcription factor), SlMYB55, and Cuscuta R-gene for Lignin-based Resistance 1, a CC-NBS-LRR (CuRLR1) are identified as factors that confer host resistance by regulating lignification. SlWRKY16 is upregulated upon C. campestris infestation and potentially negatively regulates LIF1 function. Intriguingly, CuRLR1 may play a role in signaling or function as an intracellular receptor for receiving Cuscuta signals or effectors, thereby regulating lignification-based resistance. In summary, these four regulators control the lignin-based resistance response in specific Heinz tomato cultivars, preventing C. campestris from parasitizing resistant tomatoes. This discovery provides a foundation for investigating multilayer resistance against Cuscuta species and has potential for application in other essential crops attacked by parasitic plants.

Agrobacterium tumefaciens is unique in that it can facilitate the interkingdom transfer of DNA and genetically modify its plant host. While Agrobacterium has been coopted for use in the genetic modification of plants, it is also a major pathogen, causing crown gall disease in the nursery, orchard, and vineyard industries. Pathogenicity in Agrobacterium is the result of two components. First is the Ti plasmid, which carries virulence genes and the transferred T-DNA region. The second component is the chromosome of Agrobacterium, which comprises diverse bacterial lineages and multiple species-level groups. The Ti plasmid can be transferred from strain to strain, diversifying the pathogen and complicating efforts to understand its epidemiology. This system provides an opportunity to study transmission of plasmids and their impact on disease persistence and spread. However, the movement of plasmids, and diversity of chromosomal lineages, means that conventional methods of using whole genome SNPs to track outbreaks are not sufficient, and new techniques must be developed. Additionally, Ti plasmids, like Agrobacterium, are genetically diverse and represent multiple plasmid types. Using a framework of 200 sequenced Agrobacterium genomes isolated from around the world, and a previously developed model of Ti plasmid types, we modelled their epidemiology. Key to this study was that we first separately analyzed plasmids and strain. Combining results revealed links between nurseries, potential horizontal transfer of the plasmid between strains within nurseries, global spread of plasmids, and long-term persistence of plasmids in the agricultural system. Agricultural practices have the potential to promote the diversification of pathogens and the emergence of new pathogen lineages.

Rapid increases of energy consumption and human dependency on fossil fuels have led to the accumulation of greenhouse gases and consequently, climate change. As such, major efforts have been taken to develop, test, and adopt clean renewable fuel alternatives. Production of bioethanol and biodiesel from crops is well developed, while other feedstock resources and processes have also shown high potential to provide efficient and cost-effective alternatives, such as landfill and plastic waste conversion, algal photosynthesis, as well as electrochemical carbon fixation. In addition, the downstream microbial fermentation can be further engineered to not only increase the product yield but also expand the chemical space of biofuels through the rational design and fine-tuning of biosynthetic pathways toward the realization of "designer fuels" and diverse future applications.

Renewable fuels are needed to replace fossil fuels in the immediate future. Lignocellulosic bioenergy crops provide a renewable alternative that sequesters atmospheric carbon. To prevent displacement of food crops, it would be advantageous to grow biofuel crops on marginal lands. These lands will likely face more frequent and extreme drought conditions than conventional agricultural land, so it is crucial to see how proposed bioenergy crops fare under these conditions and how that may affect lignocellulosic biomass composition and saccharification properties. We found that while drought impacts the plant cell wall of Sorghum bicolor differently according to tissue and timing of drought induction, drought-induced cell wall compositional modifications are relatively minor and produce no negative effect on biomass conversion. This contrasts with the cell wall-related transcriptome, which had a varied range of highly variable genes (HVGs) within four cell wall-related GO categories, depending on the tissues surveyed and time of drought induction. Further, many HVGs had expression changes in which putative impacts were not seen in the physical cell wall or which were in opposition to their putative impacts. Interestingly, most pre-flowering drought-induced cell wall changes occurred in the leaf, with matrix and lignin compositional changes that did not persist after recovery from drought. Most measurable physical post-flowering cell wall changes occurred in the root, affecting mainly polysaccharide composition and cross-linking. This study couples transcriptomics to cell wall chemical analyses of a C4 grass experiencing progressive and differing drought stresses in the field. As such, we can analyze the cell wall-specific response to agriculturally relevant drought stresses on the transcriptomic level and see whether those changes translate to compositional or biomass conversion differences. Our results bolster the conclusion that drought stress does not substantially affect the cell wall composition of specific aerial and subterranean biomass nor impede enzymatic hydrolysis of leaf biomass, a positive result for biorefinery processes. Coupled with previously reported results on the root microbiome and rhizosphere and whole transcriptome analyses of this study, we can formulate and test hypotheses on individual gene candidates' function in mediating drought stress in the grass cell wall, as demonstrated in sorghum.

Terpenoids are a vast and diverse class of molecules with industrial and medicinal importance. The majority of these molecules are produced across kingdom Plantae via specialized metabolism. Microorganisms, mainly Escherichia coli and Saccharomyces cerevisiae, have become choice platforms for the biosynthesis of terpenoids due to recent advances in synthetic biology and metabolic engineering. New techniques for gene discovery have expanded our search space for novel terpene synthesis pathways and unlocked unrealized potential for the microbial production of more complex derivatives. Additionally, numerous advances in host and pathway engineering have allowed for the production of terpenoids requiring oxidation and glycosylation, effectively expanding the potential target space. These advances will lay the foundation for the microbial biosynthesis of a seemingly infinite domain of terpenoids with varying applications.

Despite intensive study, plant lysine catabolism beyond the 2-oxoadipate (2OA) intermediate remains unvalidated. Recently we described a missing step in the D-lysine catabolism of Pseudomonas putida in which 2OA is converted to D-2-hydroxyglutarate (2HG) via hydroxyglutarate synthase (HglS), a DUF1338 family protein. Here we solve the structure of HglS to 1.1 Å resolution in substrate-free form and in complex with 2OA. We propose a successive decarboxylation and intramolecular hydroxylation mechanism forming 2HG in a Fe(II)- and O 2 -dependent manner. Specificity is mediated by a single arginine, highly conserved across most DUF1338 proteins. An Arabidopsis thaliana HglS homolog coexpresses with known lysine catabolism enzymes, and mutants show phenotypes consistent with disrupted lysine catabolism. Structural and biochemical analysis of Oryza sativa homolog FLO7 reveals identical activity to HglS despite low sequence identity. Our results suggest DUF1338-containing enzymes catalyze the same biochemical reaction, exerting the same physiological function across bacteria and eukaryotes.

Ideotypes are theoretical archetypes of crops which serve as a practical framework for plant breeders to critically evaluate what traits they should be targeting for specific applications. With advances in plant biotechnology and a growing urgency to adopt more sustainable practices across our economy, new uses for crops as bioenergy feedstocks may pivot our definition of an ideal crop that is engineered for biomass and bioenergy production, in contrast to food production. Although there is a plethora of specific applications to which plant engineering efforts can contribute, here we highlight recent advances in two broad areas of research: increasing available plant biomass and engineering production of higher value co-products.

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 Saccharomyces spp., we have developed a strategy for the design of synthetic activators, synthetic repressors, and synthetic promoters and have validated their use in Nicotiana benthamiana and 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 accelerated evolution and spread of pathogens are threats to host species. Agrobacteria require an oncogenic Ti or Ri plasmid to transfer genes into plants and cause disease. We developed a strategy to characterize virulence plasmids and applied it to analyze hundreds of strains collected between 1927 and 2017, on six continents and from more than 50 host species. In consideration of prior evidence for prolific recombination, it was surprising that oncogenic plasmids are descended from a few conserved lineages. Characterization of a hierarchy of features that promote or constrain plasticity allowed inference of the evolutionary history across the plasmid lineages. We uncovered epidemiological patterns that highlight the importance of plasmid transmission in pathogen diversification as well as in long-term persistence and the global spread of disease.

Cuscuta species (dodders) are agriculturally destructive parasitic angiosperms. These parasitic plants use haustoria as physiological bridges to extract nutrients and water from hosts. Cuscuta campestris has a broad host range and wide geographical distribution. While some wild tomato relatives are resistant, cultivated tomatoes are generally susceptible to C. campestris infestations. However, some specific Heinz tomato hybrid cultivars exhibit resistance to dodders in the field, but their defense mechanism was unknown. Here, we discovered that the stem cortex in these resistant lines responds with local lignification upon C. campestris attachment, preventing parasite entry into the host. LIF1 ( Lignin Induction Factor 1 , an AP2 -like transcription factor), SlMYB55 , and CuRLR1 ( Cuscuta R-gene for Lignin-based Resistance 1 , a CC-NBS-LRR ) are identified as crucial factors conferring host resistance by regulating lignification. SlWRKY16 is upregulated upon C. campestris infestation and acts as a negative regulator of LIF1 function. Intriguingly, CuRLR1 may play a role in signaling or function as a receptor for receiving Cuscuta signals or effectors to regulate lignification-based resistance. In summary, these four regulators control the lignin-based resistance response, preventing C. campestris from parasitizing these resistant tomatoes. This discovery provides a foundation for investigating multilayer resistance against Cuscuta species and has potential for application in other essential crops attacked by parasitic plants.One-sentence summary Four key regulators confer lignin accumulation in the tomato stem cortex to block C. campestris host penetration upon infection.### Competing Interest StatementThe authors have declared no competing interest.

Rathayibacter toxicus is a species of Gram-positive, corynetoxin-producing bacteria that causes annual ryegrass toxicity, a disease often fatal to grazing animals. A phylogenomic approach was employed to model the evolution of R. toxicus to explain the low genetic diversity observed among isolates collected during a 30-year period of sampling in three regions of Australia, gain insight into the taxonomy of Rathayibacter, and provide a framework for studying these bacteria. Analyses of a data set of more than 100 sequenced Rathayibacter genomes indicated that Rathayibacter forms nine species-level groups. R. toxicus is the most genetically distant, and evidence suggested that this species experienced a dramatic event in its evolution. Its genome is significantly reduced in size but is colinear to those of sister species. Moreover, R. toxicus has low intergroup genomic diversity and almost no intragroup genomic diversity between ecologically separated isolates. R. toxicus is the only species of the genus that encodes a clustered regularly interspaced short palindromic repeat (CRISPR) locus and that is known to host a bacteriophage parasite. The spacers, which represent a chronological history of infections, were characterized for information on past events. We propose a three-stage process that emphasizes the importance of the bacteriophage and CRISPR in the genome reduction and low genetic diversity of the R. toxicus species. IMPORTANCE Rathayibacter toxicus is a toxin-producing species found in Australia and is often fatal to grazing animals. The threat of introduction of the species into the United States led to its inclusion in the Federal Select Agent Program, which makes R. toxicus a highly regulated species. This work provides novel insights into the evolution of R. toxicus. R. toxicus is the only species in the genus to have acquired a CRISPR adaptive immune system to protect against bacteriophages. Results suggest that coexistence with the bacteriophage NCPPB3778 led to the massive shrinkage of the R. toxicus genome, species divergence, and the maintenance of low genetic diversity in extant bacterial groups. This work contributes to an understanding of the evolution and ecology of an agriculturally important species of bacteria.