Making Security Viral: Shifting Engineering Biology Culture and Publishing.

The ability to construct, synthesize, and edit genes and genomes at scale and with speed enables, in synergy with other tools of engineering biology, breakthrough applications with far-reaching implications for society. As SARS-CoV-2 spread around the world in early spring of 2020, researchers rapidly mobilized, using these tools in the development of diagnostics, therapeutics, and vaccines for COVID-19. The sharing of knowledge was crucial to making rapid progress. Several publications described the use of reverse genetics for the de novo construction of SARS-CoV-2 in the laboratory, one in the form of a protocol. Given the demonstrable harm caused by the virus, the unequal distribution of mitigating vaccines and therapeutics, their unknown efficacy against variants, and the interest in this research by laboratories unaccustomed to working with highly transmissible pandemic pathogens, there are risks associated with such publications, particularly as protocols. We describe considerations and offer suggestions for enhancing security in the publication of synthetic biology research and techniques. We recommend: (1) that protocol manuscripts for the de novo synthesis of certain pathogenic viruses undergo a mandatory safety and security review; (2) that if published, such papers include descriptions of the discussions or review processes that occurred regarding security considerations in the main text; and (3) the development of a governance framework for the inclusion of basic security screening during the publication process of engineering biology/synthetic biology manuscripts to build and support a safe and secure research enterprise that is able to maximize its positive impacts and minimize any negative outcomes.

Agricultural Crop Security: Exploring US Federal Readiness and Response Capabilities.

A large-scale agroterrorism attack on the United States would likely have severe economic and social consequences. In particular, the destruction of crops with pests or pathogens could cause substantial damage to food, economic, and social stability, with relatively little health risk to the perpetrators. The tools of engineering biology could enable a well-trained, nefarious actor to amplify their desired impacts through the development of disease-intensifying traits. In the United States, plant health emergencies are handled first at the local and state levels, then escalated to include the support and leadership of the US Department of Agriculture (USDA) and other federal agencies. We used publicly available documents and interviews across government, academia, and industry to explore the strategic and tactical approaches of the US federal government to detect and respond to plant agroterrorism. In this article, we discuss the agroterrorism preparedness and response capabilities at 3 levels of federal response: (1) within the Plant Protection and Quarantine program of the Animal and Plant Health Inspection Service at the USDA, (2) between USDA components, and (3) between federal agencies. We outline the approaches currently taken and identify opportunities to strengthen these approaches.

Loss of function of a DMR6 ortholog in tomato confers broad-spectrum disease resistance.

Plant diseases are among the major causes of crop yield losses around the world. To confer disease resistance, conventional breeding relies on the deployment of single resistance (R) genes. However, this strategy has been easily overcome by constantly evolving pathogens. Disabling susceptibility (S) genes is a promising alternative to R genes in breeding programs, as it usually offers durable and broad-spectrum disease resistance. In Arabidopsis, the S gene DMR6 (AtDMR6) encodes an enzyme identified as a susceptibility factor to bacterial and oomycete pathogens. Here, we present a model-to-crop translational work in which we characterize two AtDMR6 orthologs in tomato, SlDMR6-1 and SlDMR6-2. We show that SlDMR6-1, but not SlDMR6-2, is up-regulated by pathogen infection. In agreement, Sldmr6-1 mutants display enhanced resistance against different classes of pathogens, such as bacteria, oomycete, and fungi. Notably, disease resistance correlates with increased salicylic acid (SA) levels and transcriptional activation of immune responses. Furthermore, we demonstrate that SlDMR6-1 and SlDMR6-2 display SA-5 hydroxylase activity, thus contributing to the elucidation of the enzymatic function of DMR6. We then propose that SlDMR6 duplication in tomato resulted in subsequent subfunctionalization, in which SlDMR6-2 specialized in balancing SA levels in flowers/fruits, while SlDMR6-1 conserved the ability to fine-tune SA levels during pathogen infection of the plant vegetative tissues. Overall, this work not only corroborates a mechanism underlying SA homeostasis in plants, but also presents a promising strategy for engineering broad-spectrum and durable disease resistance in crops.

Guiding Ethical Principles in Engineering Biology Research.

Engineering biology is being applied toward solving or mitigating some of the greatest challenges facing society. As with many other rapidly advancing technologies, the development of these powerful tools must be considered in the context of ethical uses for personal, societal, and/or environmental advancement. Researchers have a responsibility to consider the diverse outcomes that may result from the knowledge and innovation they contribute to the field. Together, we developed a Statement of Ethics in Engineering Biology Research to guide researchers as they incorporate the consideration of long-term ethical implications of their work into every phase of the research lifecycle. Herein, we present and contextualize this Statement of Ethics and its six guiding principles. Our goal is to facilitate ongoing reflection and collaboration among technical researchers, social scientists, policy makers, and other stakeholders to support best outcomes in engineering biology innovation and development.

Genetic Engineering and Editing of Plants: An Analysis of New and Persisting Questions.

Genetic engineering is a molecular biology technique that enables a gene or genes to be inserted into a plant's genome. The first genetically engineered plants were grown commercially in 1996, and the most common genetically engineered traits are herbicide and insect resistance. Questions and concerns have been raised about the effects of these traits on the environment and human health, many of which are addressed in a pair of 2008 and 2009 Annual Review of Plant Biology articles. As new science is published and new techniques like genome editing emerge, reanalysis of some of these issues, and a look at emerging issues, is warranted. Herein, an analysis of relevant scientific literature is used to present a scientific perspective on selected topics related to genetic engineering and genome editing.

Preference of Arabidopsis thaliana GH3.5 acyl amido synthetase for growth versus defense hormone acyl substrates is dictated by concentration of amino acid substrate aspartate.

The GH3 family of adenylating enzymes conjugate acyl substrates such as the growth hormone indole-3-acetic acid (IAA) to amino acids via a two-step reaction of acyl substrate adenylation followed by amino acid conjugation. Arabidopsis thaliana GH3.5 was previously shown to be unusual in that it could adenylate both IAA and the defense hormone salicylic acid (SA, 2-hydroxybenzoate). Our detailed studies of the kinetics of GH3.5 on a variety of auxin and benzoate substrates provides insight into the acyl preference and reaction mechanism of GH3.5. For example, we found GH3.5 activity on substituted benzoates is not defined by the substitution position as it is for GH3.12/PBS3. Most importantly, we show that GH3.5 strongly prefers Asp as the amino acid conjugate and that the concentration of Asp dictates the functional activity of GH3.5 on IAA vs. SA. Not only is Asp used in amino acid biosynthesis, but it also plays an important role in nitrogen mobilization and in the production of downstream metabolites, including pipecolic acid which propagates defense systemically. During active growth, [IAA] and [Asp] are high and the catalytic efficiency (kcat/Km) of GH3.5 for IAA is 360-fold higher than with SA. GH3.5 is expressed under these conditions and conversion of IAA to inactive IAA-Asp would provide fine spatial and temporal control over local auxin developmental responses. By contrast, [SA] is dramatically elevated in response to (hemi)-biotrophic pathogens which also induce GH3.5 expression. Under these conditions, [Asp] is low and GH3.5 has equal affinity (Km) for SA and IAA with similar catalytic efficiencies. However, the concentration of IAA tends to be very low, well below the Km for IAA. Therefore, GH3.5 catalyzed formation of SA-Asp would occur, fine-tuning localized defensive responses through conversion of active free SA to SA-Asp. Taken together, we show how GH3.5, with dual activity on IAA and SA, can integrate cellular metabolic status via Asp to provide fine control of growth vs. defense outcomes and hormone homeostasis.

Ventilation patterns of the songbird lung/air sac system during different behaviors.

Unidirectional, continuous airflow through the avian lung is achieved through an elaborate air sac system with a sequential, posterior to anterior ventilation pattern. This classical model was established through various approaches spanning passively ventilated systems to mass spectrometry analysis of tracer gas flow into various air sacs during spontaneous breathing in restrained ducks. Information on flow patterns in other bird taxa is missing, and these techniques do not permit direct tests of whether the basic flow pattern can change during different behaviors. Here we use thermistors implanted into various locations of the respiratory system to detect small pulses of tracer gas (helium) to reconstruct airflow patterns in quietly breathing and behaving (calling, wing flapping) songbirds (zebra finch and yellow-headed blackbird). The results illustrate that the basic pattern of airflow in these two species is largely consistent with the model. However, two notable differences emerged. First, some tracer gas arrived in the anterior set of air sacs during the inspiration during which it was inhaled, suggesting a more rapid throughput through the lung than previously assumed. Second, differences in ventilation between the two anterior air sacs emerged during calling and wing flapping, indicating that adjustments in the flow pattern occur during dynamic behaviors. It is unclear whether this modulation in ventilation pattern is passive or active. This technique for studying ventilation patterns during dynamic behaviors proves useful for establishing detailed timing of airflow and modulation of ventilation in the avian respiratory system.