Sponsoring Institution
Agricultural Research Service/USDA
Status
ACTIVE
Funding Source
USDA INHOUSE
Reporting Frequency
Annual
Project Director
THILMONY R L
Accession Number
434430
Project Number
2030-21220-002-000D
Dates
2018-03-26 - 2023-03-25
Recipient Organization
WESTERN REGIONAL RES CENTER
ALBANY,CA 94710
Keywords
biotechnology
chip
citrus
cpp-ata
disease
enhancer
expression
gene
greening
huanglongbing
insulator
potato
promoter
recombination
resistance
site-specific
stacking
terminator
transformation
transgene
zebra
Research Effort
Applied (25%)
Basic (75%)
Developmental (0%)
Classification Parameters
| Knowledge Area | Subject of Investigation | Field of Science | Percent |
|---|
| 212 - Pathogens and Nematodes Affecting Plants |
1131 - Wine grapes |
1040 - Molecular biology |
45% |
| 212 - Pathogens and Nematodes Affecting Plants |
999 - Citrus, general/other |
1040 - Molecular biology |
15% |
| 201 - Plant Genome, Genetics, and Genetic Mechanisms |
1310 - Potato |
1040 - Molecular biology |
10% |
| 201 - Plant Genome, Genetics, and Genetic Mechanisms |
1540 - Hard red winter wheat |
1040 - Molecular biology |
10% |
| 201 - Plant Genome, Genetics, and Genetic Mechanisms |
2410 - Cross-commodity research--multiple crops |
1040 - Molecular biology |
10% |
| 212 - Pathogens and Nematodes Affecting Plants |
1599 - Grain crops, general/other (includes buckwheat, millet, triticale) |
1040 - Molecular biology |
10% |
Goals / Objectives
- 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.
- 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.
- Develop new methods that permit ARS biotechnology tools to be used for germplasm improvement in prioritized target crops and varieties.
- Examine the capacity of the GAANTRY gene stacking system to enable the assembly and transfer of large arrays of sequences into transgenic plants.
- Design and deploy a site-specific recombinase system that enables targeted transgene integration and marker removal in crop plants.
Proposed research will create a new
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 5 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.
Methods (unparsed)
Candidate plant defense response genes will be introduced into potato and citrus plants using established methods for Agrobacteriumâ¿¿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. â¿¿Exchangeâ¿Â
Methods
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'