2013 Annual Report
1a.Objectives (from AD-416):
1. Develop components for construction of intragenic citrus lines, products of direct genetic modification employing only native DNA sequences.
2. Use of potato Zebra Chip Disease as a model for evaluating a potential citrus Huanglongbing (HLB)-resistance transgene efficacy.
3. Develop and exploit extant molecular tools (15x genome of Carrizo that represents the best current citrus source of HLB tolerance) and Zebra Chip tolerant potato lines to identify potential Liberibacter disease tolerance/resistance genes with commercial applications.
1b.Approach (from AD-416):
In cooperation with the USDA/ARS U.S. Horticultural Research Lab (Fort Pierce, FL), identify and develop molecular tools for the construction of “intragenic” citrus. From citrus genome sequence data, identify sequences with homology to Agrobacterium T-DNA borders (P-DNA) and test them in a binary vector to determine efficiency in Agrobacterium-mediated transformation of citrus. Using expressed gene data, identify both constitutive and phloem specific promoters, fuse them to reporter gene coding sequences, and transform them into citrus and evaluate expression profiles. Make the promoters and P-DNA tools available to the citrus research community.
Isolate a set of of Carrizo-specific “R” (Nucleotide Binding-Leucine-Rich Repeat Proteins) candidate genes identified by genome sequencing and test their ability to provide HLB-tolerance by introducing them into HLB-sensitive citrus scions.
In cooperation with USDA/ARS Yakima Agricultural Research Lab (Wapato, WS), use potato ZC as a model system for identification of potential transgenic strategies for delivering HLB-resistance to citrus. Introduce candidate ZC-resistance transgenes into potato and evaluate their efficacy in controlling Liberibacter infection and development of ZC symptoms. Candidate resistance genes to be tested include coding regions for antimicrobial peptides and “R” genes identified from the ‘Carrizo’ citrus genome. Implement strategies shown to be successful in potato by introducing identical transgenes into citrus. Construct “citrus” versions of successful transgenes, employing molecular components from the citrus genome. Introduce these genes into citrus and evaluate HLB susceptibility. In parallel, identify homologues of successful citrus “R” genes in the Solanum genome. Fuse coding regions for those R-homolgues to the potato 409S promoter and polyadenylation signal and transform the constructions into potato. Evaluate ZC resistance of the resultant transgenic potato lines.
This report documents progress for this project, which started in May of 2013 and continues research from Project Number 5325-21420-004-00D, entitled “Molecular Genetic Tools for Potato and Fruit Tree Improvement". Progress was made on all three objectives, all of which fall under National Program 301, Component 3: Crop Biological and Molecular Processes; Problem Statement 3A: Fundamental knowledge of plant biological and molecular processes. The project has three objectives:.
1)to develop components for construction of intragenic citrus lines, products of direct genetic modification employing only native DNA sequences,.
2)Use of potato Zebra Chip (ZC) disease as a model for evaluating potential citrus Huanglongbing (HLB)-resistance transgene efficacy, and.
3)develop and exploit extant molecular tools (15x genome of Carrizo that represents the best current citrus source of HLB tolerance) and Zebra Chip tolerant potato lines to identify potential Liberibacter disease tolerance/resistance genes with commercial applications.
Progress on the first objective over this period includes introduction of intragenic transgenes into citrus for HLB resistance.
Progress on the second objective includes modification of the ZC model system to allow rapid identification of citrus-based transgenes for HLB resistance.
Progress on the third objective includes use of citrus databases to identify gene products with potential to impact ZC/HLB, utilize these sequences in construction of potato transgenes, screening of transgenic lines for potential ZC-resistance.
Identification of a potential citrus-based Zebra Chip-resistance transgene. The causal agents of Huanglongbing (HLB) disease in citrus and Zebra Chip (ZC) in potato are two related Liberibacter species. ARS scientists in Albany, California, in cooperation with ARS scientists in Wapato, Washington, have constructed transgenic potatoes expressing antibacterial citrus-based genes with the potential to confer ZC resistance. The introduced transgenes are based on endogenous citrus and potato proteins to allow construction of transgenic lines containing only native DNA sequences. The introduced genes encode proteins with antibacterial activity and have yielded positive results in initial greenhouse studies. Transgenic potato lines are currently (2013 growing season) in field evaluation in the state of Washington.