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Research Project: Molecular Genetic Tools Advancing the Application of Biotechnology for Crop Improvement

Location: Crop Improvement and Genetics Research

2021 Annual Report

The long-term goal of this project is to develop useful biotechnology tools that enable the effective and precise genetic improvement of crop plants. Specifically, during the next five years we will focus on the three following objectives: Objective 1: Generate new molecular tools and new genetic strategies for effectively introducing and pyramiding multiple disease defense genes into citrus and potato to combat Huánglóngbìng and Zebra Chip diseases along with other priority traits. Objective 2: Identify and characterize new transcriptional control sequences (promoters and terminators) chosen for the precise control of gene transcription (tissue and/or developmental/environmental specificity) in crop plants containing single or multiple transgenes. Objective 3: Develop new methods that permit ARS biotechnology tools to be used for germplasm improvement in prioritized target crops and varieties. Subobjective 3A: Examine the capacity of the GAANTRY gene stacking system to enable the assembly and transfer of large arrays of sequences into transgenic plants. Subobjective 3B: Design and deploy a site-specific recombinase system that enables targeted transgene integration and marker removal in crop plants. Objective 4: Proposed research will create a new Objective 4: Understand the biochemical processes involved in smoke taint and apply plant biotechnology and genome editing to reduce smoke taint in commercial wine grape varietals. (NP301, C3, PS3A) Objective 4 will utilize existing resources for fast and efficient strategies to engineer wine grapes with reduced smoke-derived phenolic compounds and will coordinate findings with a growing network of smoke taint researchers in the U.S. Anticipated products include new transcriptomics and metabolomics studies as well as biotechnology-based approaches to genetically modify pathways involved.

Candidate plant defense response genes will be introduced into potato and citrus plants using established methods for Agrobacterium–mediated transformation. The defense genes will either be constitutively expressed throughout the plant or expressed specifically in the phloem, the site of infection. Ten or more independent events for each candidate defense gene will have their susceptibility to zebra chip (in potato) or Huánglóngbìng (in citrus) evaluated. Candidate promoters with useful cell-type/organ expression specificities will be identified from crop plants. The candidate promoters will be fused to a reporter gene and transformed into rice, using Agrobacterium and/or other established transformation methods. Novel transcription terminator sequences will also be isolated from crop plants and fused to a reporter gene. The functionality of these promoter and terminator testing constructs will be examined in transient expression assays and stably transformed transgenic plants. Reporter gene expression levels will be quantitatively measured in major organs and compared to identify the sequences that provide the highest levels of transgene products while preserving promoter expression specificity. Plant molecular biological techniques will be used to further develop sophisticated biotechnology tools and methods for the improvement of crops. Transformation constructs of various large sizes (greater than 20 kilo base pairs) will be assembled using the site-specific recombinase-based GAANTRY gene stacking system. These constructs will be evaluated for their stability in bacteria and used to generate transgenic plants. The resulting genetically engineered plants will be molecular characterized to determine the effective capacity of the gene stacking technology. In parallel, technology enabling targeted integration and precise marker removal in transgenic plants will be developed and evaluated. “Exchange” T-DNA vectors will be constructed and transformed into “target” transgenic plants. Selection and molecular screening will be used to identify plants in which the incoming DNA has replaced the original transgenic locus (Recombinase-Mediated Cassette Exchange or RMCE). The efficiencies of different combinations of the unidirectional recombinases in performing RMCE will be compared.

Progress Report
Limited progress was made on Objective 1 to generate new molecular tools and new genetic strategies for effectively introducing and pyramiding multiple disease defense genes into citrus and potato. This technology will be potentially useful in combatting Huanglongbing (HLB; citrus) and zebra chip (potato) diseases as well as the engineering of an array of other desirable priority traits like improved stress tolerance and/or improved end-use quality. Previously, multiple transformation vectors containing different candidate defense genes were assembled, validated, and transformed into potato and citrus plants. Recently, a series of five unique multi-gene transgenic stacks have been designed and assembled to increase pathogen detection in citrus and potato. These gene stacks are designed to increase the sensing of pathogen-derived molecules and trigger defense responses. Efforts to generate transgenic plants with these new constructs was delayed due to the pandemic, but will be initiated in the future. For Objective 2, five novel candidate rice promoters with callus and/or embryo-specific expression were previously selected, assembled into plant transformation vectors and introduced into rice and Brachypodium distachyon (a model grass). Molecular characterization of the transgenic plants has now been completed and multiple good quality transgenic events were identified for each candidate promoter. In addition, three candidate floral/reproductive-specific promoter candidates were isolated, assembled into test constructs and transformed into rice plants. The function of each of the promoters within various plant tissues has been examined and documented. The results confirm that several of the candidate promoters confer callus-specific or callus/embryo-preferential expression in the genetically engineered tissues. Further characterization of these transgenic plants is continuing to further document the strength and specificity of expression conferred by each promoter. Progress was also made on Objective 3. The overall goal is to develop new methods that enable biotechnology-based crop improvement. For Sub-objective 3A, previously a strategy was designed and successfully implemented to insert large cargo sequences into the Gene Assembly in Agrobacterium by Nucleic acid Transfer using Recombinase technologY (GAANTRY) gene stacking system. This new technology provides an efficient means of assembling large multigene constructs and transforming them into crop plants. Selected Bacterial Artificial Chromosome (BAC) vectors that carry between 16 kilobase pairs (kb) to more than 110 kb of cargo DNA sequence, were assembled into GAANTRY constructs. The transformation of these large assemblies into Arabidopsis plants and the molecular and phenotypic characterization of the resulting transgenic plants is now complete. The results demonstrate that the GAANTRY system can generate transgenic plants with large constructs up to 77 kb in size. Further technology development is needed to make the generation of biotech crops with constructs larger than 80 kb an efficient process. Some progress was also made on Sub-objective 3B to design and deploy a site-specific recombinase system that enables targeted transgene integration and marker removal in crop plants. Improved vectors were generated to increase efficiency of Recombinase-mediated DNA cassette exchange (RMCE) gene stacking and molecular detection. Efforts to create and characterize transgenic plants with these new constructs have been delayed due the pandemic. Experiments pursuing these goals is currently underway.

1. A versatile and robust gene stacking system for improved rice biotechnology. The genetic improvement of important crops like rice is one of the most effective ways to increase agricultural productivity. It has typically been difficult to genetically engineer improvements in complex traits like yield or disease resistance that require the action of multiple genes. ARS researchers in Albany, California, demonstrated that a novel technology called Gene Assembly in Agrobacterium by Nucleic acid Transfer using Recombinase technologY (GAANTRY), allows for the efficient assembly and introduction of multiple genes into rice. The system was shown to efficiently generate high-quality genetically engineered rice plants that carried all of the eleven introduced sequences and exhibited the desired traits. This technological breakthrough will enable the use of crop biotechnology to effectively improve complex traits in rice and related crop plants.

2. Development of novel ‘Lilac Limes’. Several unique promoters from citrus were identified and the expression patterns they confer in citrus and other plant species documented. ARS researchers in Albany, California, demonstrated that two of the promoters successfully expressed a citrus gene specifically within the fruit of Mexican lime trees which activated the accumulation of compounds turning the ripe fruit purple. These ‘Lilac Lime’ trees also rapidly flowered due to the introduction of a second trait that shortened the juvenile growth phase, making the trees yield fruit over a year earlier than normal. These Lilac Limes not only have a novel fruit color, but also contain higher levels of desirable anthocyanin compounds which are nutritious antioxidants. Field studies are underway to further document this novel fruit trait and to perform research for their potential future regulatory release.

Review Publications
Hathwaik, L.T., Thomson, J.G., Thilmony, R.L. 2021. Gene assembly in agrobacterium via nucleic acid transfer using recombinase technology (GAANTRY). In: Bandyopadhyay, A., Thilmony, R.L, editors. Rice Genome Engineering and Gene Editing. Methods in Molecular Biology. New York, NY: Humana. p. 3-17.
Hopper, J.V., Pratt, P.D., Reddy, A.M., McCue, K.F., Rivas, S.O., Grosholz, E.D. 2020. Abiotic and biotic influences on the performance of two biological control agents, Neochetina bruchi and N. eichhorniae, in the Sacramento-San Joaquin River Delta, California (USA). Biological Control. 153. Article 104495.
Hathwaik, L.T., Horstman, J.D., Thomson, J.G., Thilmony, R.L. 2021. Efficient gene stacking in rice using the GAANTRY system. Rice. 14. Article 17 (2021).