Location: Crop Improvement and Genetics Research2017 Annual Report
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.
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 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.
Progress was made on all three objectives, which fall under NP301 Component 3: Genetic Improvement of Crops, Problem Statement 3A: Genetic Theory and Methods of Crop Improvement. The goal of the project is to develop components for construction of intragenic citrus lines, using potato Zebra Chip (ZC) disease as a model for evaluating potential citrus Huanglongbing (HLB)-resistance transgene efficacy. Potential antimicrobial proteins have been tested in citrus and potato. Progress on the first objective included continued evaluation of the phloem-specific promoter (396SS) from the citrus Small Citrus Amphipathic Peptide (SCAmpPs) gene family. This promoter has been evaluated in Arabidopsis, citrus and potatoes and exhibited strong expression in vascular tissue of both potato and citrus. This promoter has potential for tissue-specific expression of antibacterial genes in phloem, the site of bacterial colonization in ZC and HLB infected potato and citrus plants. The coding region of the same citrus phloem-specific SCamPs gene has been modified to produce a potential antimicrobial peptide found in the tolerant Carrizo root stock. The gene has been transformed into citrus and potatoes to test its ability to confer resistance to HLB and ZC, respectively. Progress on the second objective this year included the construction of third generation antimicrobial peptides for evaluation in citrus and potato. This includes the modification of peptides from the second generation of constructs to optimize expression and cellular localization. Development of intragenic transgenes for potatoes will not be pursued until one or more effective resistance genes have been identified. Progress on the third objective this year continues with the development of new and modified potential resistance genes (R-genes). Due to the large numbers of R-genes in citrus, efforts continue to focus on the SCAmpPs and thionin gene families, members of which have been associated with HLB tolerance. A citrus thionin gene related to a potential resistance gene from spinach has been transformed into citrus and potato. The transgenic plants will be evaluated for HLB and ZC disease resistance.
Hopper, J.V., Pratt, P.D., McCue, K.F., Pitcairn, M.J., Moran, P.J., Madsen, J.D. 2017. Spatial and temporal variation of biological control agents associated with Eichhornia crassipes in the Sacramento-San Joaquin River Delta, California. Biological Control. 111:13-22. doi:10.1016/j.biocontrol.2017.05.005.
Nelson, W.R., Munyaneza, J.E., McCue, K.F., Bove, J.M. 2013. The Pangean origin of 'Candidatus Liberibacter' species. Journal of Plant Pathology. 95:455-461; doi: 10.4454/jpp.v9513.001.
Shepherd, L., Hacket, C., Alexander, C., McNicol, J., Sungurtas, J., Stewart, D., McCue, K.F., Belknap, W.R., Davies, H.V. 2015. Modifying glycoalkaloid content in transgenic potato – Metabolome impacts. Food Chemistry. 187:437-443.