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ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Crop Improvement and Genetics Research » Research » Research Project #434430

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

Location: Crop Improvement and Genetics Research

2019 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.

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
In support of Objective 1, new molecular tools and genetic strategies were generated for effectively introducing and pyramiding multiple disease defense genes into citrus and potato. This technology is useful in combatting Huanglongbing (citrus) and Zebra chip (potato) diseases as well as the engineering of an array of other desirable priority traits. Multiple transformation vectors containing different candidate defense genes were assembled and validated. The transformation of these novel constructs into potato and citrus was initiated. In support of Objective 2, novel candidate rice promoters with callus and/or embryo-specific expression were selected and polymerase chain reaction (PCR) amplified from the rice genome. The cloning and sequence confirmation of six clones is underway. Progress was also made for Objective 2 with the selection and cloning of ten candidate dicot transcriptional terminators and ten candidate rice transcriptional terminators. Validated terminator sequences were inserted into a plant transformation vector designed for testing their function. A transient expression assay based on the leaf infiltration of Agrobacterium bacteria carrying control constructs successfully demonstrated that the research approach can provide functional data on the transcript termination activity of candidate sequences. Experiments evaluating and quantifying the performance of the selected candidate transcription terminator sequences has been initiated for Objective 2. In support of Sub-objectives 3A and 3B, new methods that permit ARS biotechnology tools to be used for germplasm improvement in prioritized target crops and varieties were developed and deployed. For Sub-objective 3A, a strategy was designed 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. A modified GAANTRY “Donor” plasmid vector was designed and constructed to successfully receive and stably maintain large fragments of deoxyribonucleic acid (DNA). Selected Bacterial Artificial Chromosome vectors that carry between ~10 kilobase pairs (kb) to more than 100 kb of plant genomic DNA sequence, were successfully inserted into the modified Donor vector to enable the GAANTRY-mediated assembly of these sequences into plant transformation constructs. The insertion of these large cargo sequences into the GAANTRY Agrobacterium recipient strain transformation construct was initiated. In support of Sub-objective 3B, a site-specific recombinase system that enables targeted transgene integration and marker removal in crop plants was designed and deployed. Transformation of potato and citrus lines was performed with a “Founder Line” construct. Methodologies were refined for the use of alternative selectable markers, which will be needed for use in sequential gene targeting in potato and citrus transformation. The EXCH-B and EXCH-P vector constructs were designed, assembled, and molecularly validated using DNA sequencing.

1. A versatile and robust gene stacking system for improved potato biotechnology. The genetic improvement of important crops, like potato, 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 the efficient assembly and introduction of multiple genes into potato. The system was shown to efficiently generate high-quality genetically engineered potato plants that carried all of the introduced genes and exhibited the desired traits. This technological breakthrough will enable the use of crop biotechnology to effectively improve complex traits in potato and related crop plants.

Review Publications
Hopper, J.V., McCue, K.F., Pratt, P.D., Duchesne, P., Grosholz, E.D., Hufbauer, R. 2019. Into the weeds: matching importation history to genetic consequences and pathways in two widely used biological control agents. Evolutionary Applications. 12(4):773-790.
Alabed, D.F., Huo, N., Gu, Y.Q., McCue, K.F., Thomson, J.G. 2019. Draft genome sequence of serratia sp. 1D1416. Microbiology Resource Announcements. 8(3):e01354-18.
Huo, N., Gu, Y.Q., McCue, K.F., Alabed, D., Thomson, J.G. 2019. Complete genome sequence of Agrobacterium fabrum strain 1D159. Microbiology Resource Announcements. 8(19):e00207-19.