Bottleneck/Challenge: Detection technologies for soil-deployed biosensors are limiting.

Potential Solution: Research on reporter systems visible at a macro-scale.

Bottleneck/Challenge: Development of chassis with sensing and reporting capabilities that are suitable for environmental use.⁵Del Valle, I., Fulk, E. M., Kalvapalle, P., Silberg, J. J., Masiello, C. A., & Stadler, L. B. (2021). Translating New Synthetic Biology Advances for Biosensing Into the Earth and Environmental Sciences. Frontiers in Microbiology, 11. View Publication.

Potential Solution: Expanded engineering biology chassis, tools, and parts.

Bottleneck/Challenge: Most output reporters (fluorescent proteins, pigments, etc) require imaging, limiting their use in opaque environments like soils.

Potential Solution: Develop and refine macro-level reporters that can be inexpensively visualized or detected across space and time, and that do not negatively impact ecosystem members.

Bottleneck/Challenge: Unrefined capabilities to measure effectiveness and persistence in soil.

Potential Solution: High-throughput, field-deployable characterization capabilities for measuring effectiveness and persistence across space and time.

Bottleneck/Challenge: Application and continued function of sensors across agricultural fields throughout a growing season.

Potential Solution: Identify receptors/genes from plants known to respond to specific pathogens/pests that can be used as biosensors and engineer them into companion crops grown around a field or integrated at regular intervals into a field to provide lasting sensing capabilities.

Bottleneck/Challenge: Biosensor sensitivity needs to be improved to respond to physiologically relevant concentrations.

Potential Solution: Use automation and protein engineering capabilities to design sensors that function at low concentrations.

Bottleneck/Challenge: Controlling when, how, and at what level stress response pathways are activated (e.g., distinguishing between typical irrigation and flooding conditions).

Potential Solution: Characterize the extent of pathway activation and fine tune (using circuit controls like amplifier, band-pass filter, etc.) pathway activation to desired levels.

Bottleneck/Challenge: Data measuring many variables in situ are often very noisy, making it challenging to draw robust conclusions.

Potential Solution: Increase the number of variables for which information is collected (multiplex data collection and analysis) so the role that different variables, including gene expression level, temperature, soil composition, play can be better understood.

Bottleneck/Challenge: Because many crop plants are polyploid with significant genetic redundancy, pathways can be difficult to robustly and stably engineer.

Potential Solution: Plant whole-genome engineering approaches that enable specific editing of paralogs for precise control of expression and function (see Wang et al., 2014⁶Wang, Y., Cheng, X., Shan, Q., Zhang, Y., Liu, J., Gao, C., & Qiu, J.-L. (2014). Simultaneous editing of three homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nature Biotechnology, 32(9), 947–951. View Publication.; Lv, 2020⁷Lv, L., Zhang, W., Sun, L., Zhao, A., Zhang, Y., Wang, L., Liu, Y., Li, Z., Li, H., & Chen, X. (2020). Gene co-expression network analysis to identify critical modules and candidate genes of drought-resistance in wheat. PLOS ONE, 15(8), e0236186. View Publication.).

Bottleneck/Challenge: Delivery of gene editing machinery through plant cell walls can be challenging and limit uptake; plant transformation efficiencies are still often low despite decades of research.⁸Ramkumar, T. R., Lenka, S. K., Arya, S. S., & Bansal, K. C. (2020). A Short History and Perspectives on Plant Genetic Transformation. In S. Rustgi & H. Luo (Eds.), Biolistic DNA Delivery in Plants: Methods and Protocols (pp. 39–68). Springer US. View Publication.^{, 9}Altpeter, F., Springer, N. M., Bartley, L. E., Blechl, A. E., Brutnell, T. P., Citovsky, V., Conrad, L. J., Gelvin, S. B., Jackson, D. P., Kausch, A. P., Lemaux, P. G., Medford, J. I., Orozco-Cárdenas, M. L., Tricoli, D. M., Van Eck, J., Voytas, D. F., Walbot, V., Wang, K., Zhang, Z. J., & Stewart, C. N., Jr. (2016). Advancing Crop Transformation in the Era of Genome Editing. The Plant Cell, 28(7), 1510–1520. View Publication.

Potential Solution: Advances in delivery into protoplasts and plant regeneration.

Potential Solution: Development of nanoparticle-mediated delivery mechanisms that reliably make heritable genetic changes (see Sirirungruang et al., 2022¹⁰Sirirungruang, S., Markel, K., & Shih, P. M. (2022). Plant-based engineering for production of high-valued natural products. Natural Product Reports, 39(7), 1492–1509. View Publication.).

Bottleneck/Challenge: Plant life cycles and regeneration times after genome editing or engineering are significant, which slows the pace of research and development compared to microbes.

Potential Solution: Engineer broadly useful approaches to speeding post-transformation regeneration.¹¹Aregawi, K., Shen, J., Pierroz, G., Sharma, M. K., Dahlberg, J., Owiti, J., & Lemaux, P. G. (2022). Morphogene-assisted transformation of Sorghum bicolor allows more efficient genome editing. Plant Biotechnology Journal, 20(4), 748–760. View Publication.

Bottleneck/Challenge: Nutrient capture from already-deficient soils can further deplete those soils.

Potential Solution: Engineer microbiomes that capture needed atmospheric compounds and/or convert compounds to bioavailable forms.

Potential Solution: Enhance cover crop symbioses that replenish nutrients.

Bottleneck/Challenge: Nutrients must be in bioavailable forms for plant uptake.

Potential Solution: Microbiome engineering to convert nutrients to forms that enable plant uptake.¹²Deynze, A. V., Zamora, P., Delaux, P.-M., Heitmann, C., Jayaraman, D., Rajasekar, S., Graham, D., Maeda, J., Gibson, D., Schwartz, K. D., Berry, A. M., Bhatnagar, S., Jospin, G., Darling, A., Jeannotte, R., Lopez, J., Weimer, B. C., Eisen, J. A., Shapiro, H.-Y., … Bennett, A. B. (2018). Nitrogen fixation in a landrace of maize is supported by a mucilage-associated diazotrophic microbiota. PLOS Biology, 16(8), e2006352. View Publication.

Bottleneck/Challenge: Plant roots may be unable to penetrate soils sufficiently to reach necessary nutrients and water.

Potential Solution: Design soil microbial communities to concentrate minerals and other plant nutritive compounds for uptake.

Potential Solution: Incorporation of hygroscopic microbes into soil surface for atmospheric water capture.

Potential Solution: Design genetic circuits that predictably control root architecture in non-model plants.¹³Brophy, J. A. N., Magallon, K. J., Duan, L., Zhong, V., Ramachandran, P., Kniazev, K., & Dinneny, J. R. (2022). Synthetic genetic circuits as a means of reprogramming plant roots. Science, 377(6607), 747–751. View Publication.

Bottleneck/Challenge: Water and nutrient levels need to fall within preferred range to avoid over-abundance.

Potential Solution: Engineer rhizosphere microbes to modulate moisture levels in response to drought and/or flood conditions.

Bottleneck/Challenge: Ensuring microbial amendments are sufficiently present and persistent to have the desired impact without out-competing other valued community members.

Potential Solution: Develop feedback mechanisms that influence microbial reproduction (e.g., growth when concentration of antagonistic compounds are low).

Bottleneck/Challenge: Identification of pest-specific resistance traits that provide durable resistance.

Potential Solution: Use of artificial intelligence-/machine learning-based algorithms to design proteins with insect-specific toxicity.

Bottleneck/Challenge: Understanding of the genetic determinants of phenotypic diversity observed in plant varieties internationally and how/if research in a model variety can be useful for other varieties.

Potential Solution: Incorporate an understanding of relevant crop varieties earlier in research development, matching tolerance or resilience pathways to the plants most likely to be impacted.

Bottleneck/Challenge: Insufficient knowledge of the types of genetic diversity that might improve pest and disease resistance.

Potential Solution: Automated screening systems to rapidly advance knowledge of the genetic determinants of resistance to given pests.

Bottleneck/Challenge: Efficient expression and production of antimicrobial genes and compounds in response to pathogens that cannot easily be overcome by pathogen evolution.

Potential Solution: Better characterization of plant-pathogen interactions to identify and/or develop strategies for the durable production of pathogen-targeted antiviral molecules.

Bottleneck/Challenge: Some agriculturally valuable crops are perennials that do not produce a crop until several years of growth; thus engineered solutions cannot immediately be implemented to alleviate crop loss from intensifying pest situations.

Potential Solution: Engineer perennial crop varieties that reach maturity more quickly.

Bottleneck/Challenge: Engineering microbial communities that promote plant resilience in response to environmental stressors without compromising plant yield during ideal conditions.

Potential Solution: Design microbial communities whose growth and reproduction is induced by the environmental stressor for which they are engineered to promote resilience against.

Bottleneck/Challenge: Understanding and preventing displacement of necessary functions of wild-type leaf microbiomes.

Potential Solution: Ensure communities retain members that perform necessary functions, for example through hygroscopic bacteria.¹⁴Hernandez, M. N., & Lindow, S. E. (2019). Pseudomonas syringae Increases Water Availability in Leaf Microenvironments via Production of Hygroscopic Syringafactin. Applied and Environmental Microbiology, 85(18), e01014-19. View Publication.

Bottleneck/Challenge: Plant transformation and regeneration are slow processes with low efficiency (particularly in less-researched crops), so engineering entire pathways is slow and technically challenging.

Potential Solution: Additional research that supports more efficient transformation processes across crop species and varieties.

Bottleneck/Challenge: Environmental stressors can affect many plant pathways and processes; imbuing resilience may require not just the engineering of a desired pathway but precisely controlling other pathways.

Potential Solution: As minimal bacterial genomes research advances, advance a “minimal plant genome” project to better understand and engineer complex organismal systems and processes.¹⁵Hutchison, C. A., Chuang, R.-Y., Noskov, V. N., Assad-Garcia, N., Deerinck, T. J., Ellisman, M. H., Gill, J., Kannan, K., Karas, B. J., Ma, L., Pelletier, J. F., Qi, Z.-Q., Richter, R. A., Strychalski, E. A., Sun, L., Suzuki, Y., Tsvetanova, B., Wise, K. S., Smith, H. O., … Venter, J. C. (2016). Design and synthesis of a minimal bacterial genome. Science, 351(6280), aad6253. View Publication.

Bottleneck/Challenge: Every pathosystem is unique, and thus needs to be characterized to engineer resistance, especially as climate change impacts which pathogen species are present and lifecycle timing of both hosts and pathogens.

Potential Solution: Develop strategies for rapid characterization of host-pathogen relationships.

Bottleneck/Challenge: Plant transformation and regeneration are slow processes with low efficiency (particularly in less-researched crops), so engineering entire pathways is slow and technically challenging.

Potential Solution: Additional research that supports more efficient transformation processes across crop species and varieties.

Bottleneck/Challenge: Environmental stressors can affect many plant pathways and processes; imbuing resilience may require not just the engineering of a desired pathway but precisely controlling other pathways.

Potential Solution: As minimal bacterial genomes research advances, advance a “minimal plant genome” project to better understand and engineer complex organismal systems and processes.¹⁶Hutchison, C. A., Chuang, R.-Y., Noskov, V. N., Assad-Garcia, N., Deerinck, T. J., Ellisman, M. H., Gill, J., Kannan, K., Karas, B. J., Ma, L., Pelletier, J. F., Qi, Z.-Q., Richter, R. A., Strychalski, E. A., Sun, L., Suzuki, Y., Tsvetanova, B., Wise, K. S., Smith, H. O., … Venter, J. C. (2016). Design and synthesis of a minimal bacterial genome. Science, 351(6280), aad6253. View Publication.

Bottleneck/Challenge: Plant transformation and regeneration are slow processes with low efficiency (particularly in less-researched crops), so engineering entire pathways is slow and technically challenging.

Potential Solution: Additional research that supports more efficient transformation processes across crop species and varieties.

Bottleneck/Challenge: Difficult to provide agronomically-feasible, durable spatio-temporal control of engineered microbiomes.

Potential Solution: Identify new strategies for rapid, non-invasive induction of genetic pathways across organisms.

Bottleneck/Challenge: Identity of key biochemical molecules responsible for causing food spoilage along different points of the supply chain.

Potential Solution: Develop high-throughput methods to identify spoiling agents (e.g., metabolites) in commonly consumed food products.

Potential Solution: Develop biobased trackers (e.g., DNA barcoding) to enable tracking of food along the supply chain and identify timepoints where food spoilage is more likely to occur.

Bottleneck/Challenge: Biosensors for detecting food spoilage need to be more specific, sensitive, reproducible, and easy-to-read.

Potential Solution: Engineer and test food-safe biorecognition molecules (e.g., synthetic enzymes, aptamers) targeting key molecular indicators of food spoilage (e.g., mycotoxins).

Potential Solution: Couple biosensors to food-safe colorimetric or electrochemical reporters.

Bottleneck/Challenge: Need to develop food-safe biosensors that can be applied on food or food packaging.

Potential Solution: Develop consumable and/or washable biomaterials (e.g., hydrogels, paper, silk) embedded with food-safe biosensors (e.g., synthetic nucleic acid-based sensors).

Bottleneck/Challenge: Current biomaterial technologies do not contain the necessary dynamics or complexity.

Potential Solution: Incorporate microbes that naturally inhibit the growth of spoilage bacteria and fungi.

Potential Solution: Discover and expand the collection of enzymes and small molecules that protect food against spoilage.

Bottleneck/Challenge: Foods can lose natural defenses against pathogens once harvested; these defenses need to be replicated for post-harvest foods.

Potential Solution: Engineer food microbiomes to produce preservatives in response to specific environmental signals (e.g., time, temperature, pH).

Bottleneck/Challenge: It remains unclear how certain plant hormones, such as salicylic acid and jasmonic acid, are able to regulate plant ripening and drive ethylene production.

Potential Solution: Improve strategies for quantifying multiple plant hormones, measuring gene expression, and tying both to phenotypic outcomes.

Potential Solution: Develop and improve strategies to non-destructively measure plant hormone concentrations.