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ARS Home » Pacific West Area » Albany, California » Western Regional Research Center » Crop Improvement and Genetics Research » Research » Research Project #425037

Research Project: Host-Specific Molecular Genetic Tools for Development of Disease-Resistant Crops

Location: Crop Improvement and Genetics Research

2016 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 Report
Progress was made on all three objectives: developing 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, and developing and exploiting the genome sequence of variety ‘Carrizo’, which represents the best current citrus source of HLB tolerance. Progress on the first objective this year includes continuing evaluation of a root-specific promoter (302z) from the citrus Small Citrus Amphipathic Peptide (SCAmpPs) gene family. Previously shown to direct effective transcription in citrus and Arabidopsis roots, this control element was evaluated in potato in extensive greenhouse tests in cooperation with scientists at the J.R. Simplot Company (Boise, Idaho). This promoter offers the potential for high-level expression of an antibacterial gene in transgenic root stock, the product of which could function to confer disease-resistance to a non-transgenic scion. Thus, only the root stock would be transformed and citrus-derived intragenic marker genes would be less important for regulatory approval and public acceptance. Progress on the second objective included evaluation of control elements to promote high level expression for ZC- and HLB-resistance transgenes and introduction of candidate resistance genes into potato. The causal agents of HLB disease in citrus and of ZC in potato are two related Liberibacter species introduced into the phloem by psyllid insect species. This year we continued evaluation of promoters from the citrus SCAmpPs gene family. Transgenes using different SCAmpPs promoters modified to identify key potential transcriptional (intron) and translational (enhancer associated with initiator methionine) regulatory components have been evaluated in transgenic Arabidopsis plants. In addition, transgenes expressing citrus native and modified thionin genes, shown by collaborating ARS scientists in Fort Pierce, Florida, to influence HLB susceptibility in citrus, have been introduced into transgenic potatoes for ZC resistance evaluation in greenhouse and field tests. Progress on the third objective includes the use of the citrus DNA sequence databases to identify gene products with potential to impact ZC/HLB, addition of these sequences to potato via genetic transformation, and evaluation of the transgenic lines for potential ZC-resistance. While the original project plan called for identification and characterization of Poncirus-specific resistance (R)-genes, the unexpected abundance of these genes in the citrus genomic assemblies (10-fold higher than in original annotations) greatly complicated computational identification of Poncirus-specific R-genes. For this reason we have shifted our search to species-specific members of the SCAmpPs gene family that encode short, amphipathic peptides with structural features similar to other anti-microbial gene products. In the past year, we completed construction of a hybrid Citrus sinensis/Poncirus transgene to allow expression of a Poncirus-specific phloem SCAmpPs peptide in transgenic citrus. This phloem-specific SCAmpPs is the most highly expressed gene identified in Poncirus phloem, but is absent (in the available databases) from the phloem of other citrus. This hybrid transgene will be transformed into citrus to test its ability to confer resistance to HLB. In research funded by a USDA Biomass Research and Development Initiative (BRDI) grant, the guayule genome is being assembled and annotated. In the past year, acquisition and assembly of a compendium of expressed guayule genes, the “ transcriptome”, was completed, allowing more accurate assignment of gene identities in previous genomic DNA sequence assemblies. In addition, computational characterization of this genome over the past year has revealed a novel microsatellite-based targeting system (SaTar) that underlies modular assembly of arrays of diverse mobile DNA elements in discrete chromosomal loci. Similar SaTar-directed assemblies have been identified in the citrus, rice and sorghum genomes, indicating this system is a common element in determination of plant chromosome architecture. Upon publication, the guayule genomic sequence will become a resource for public and private sector scientists seeking to improve both the quality and quantity of rubber produced by this desert shrub.

1. Novel software allows rapid and inexpensive mapping of transgene locations in the genomes of transgenic crop plants. The precise determination of the DNA sequence of transgenes and their immediate genomic surroundings is critical for addressing concerns about unintended consequences of genetic modification of food crops. Thus, molecular characterization of transgene insertions is an integral part of the data packages required by government regulators as they consider granting permission for commercial release of transgenic products. ARS scientists in Albany, California, in collaboration with scientists at Okanagan Specialty Fruits in Summerland, British Columbia, Canada, and at the Universidad Autónoma de Nuevo León, Monterrey, Mexico, developed a computational method for easily extracting transgene/plant chromosome junction sequences from inexpensively obtained next generation DNA sequence data. This software employs open-source code and original scripts, requires only a laptop computer, and is currently used by companies that make food products from genetically engineered plants via biotechnology.


Review Publications
Shepherd, L., Hackett, C., Alexander, C., Mcnicol, J., Sungurtas, J., Mcrae, D., Mc Cue, K.F., Belknap, W.R., Davies, H. 2016. Impact of light-exposure on the metabolite balance of transgenic potato tubers with modified glycoalkaloid biosynthesis. Food Chemistry. 200:263-373.