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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

2018 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
This is the final report for this project which was replaced on March 26, 2018 by 2030-21220-002-00D, “Molecular Genetic Tools Advancing the Application of Biotechnology for Crop Improvement.” For additional information, see the report for the new project. To determine the copy number of transgenes in potato plants transformed with Agrobacterium, the digital drop Polymerase Chain Reaction (ddPCR) was developed and adopted in the fourth year of the project. This method enabled detection of single copy genes within the 850 billion base pairs that comprise the genome of tetraploid potato. Using the native StAAP2 the potato analog of the Arabidopsis amino Acid Permease gene (AtAAP2) as a tetrazygous (4-copy) gene reference, as few as 1 and as many as 14 transgenes were found in different transformation events. Collaborators in Albany, California, have also applied this technique to most of the major crop plants including citrus. Because of this work, ddPCR has replaced DNA blot analyses as an accurate and sensitive technique for measuring transgene copy number and eliminating the use of radioactive materials in the laboratory. Significant progress was made on all three objectives over the 58 months of the project despite the retirement of one of the project scientists in March of 2017. Emerging insect-transmitted bacterial diseases in citrus, potatoes and other crops are causing extensive losses in value and productivity in these crops. Currently, there is little or no genetic resistance or tolerance in the known crop or its relatives. This research has focused on the citrus greening disease (Huanglongbing) (HLB) and the Zebra Chip (ZC) disease of potato. Objective 1: The lack of HLB resistance in the available breeding stock combined with the limited time frame to develop it has increased industry interest in identifying transgenic solutions for HLB-resistance. The biotechnological approach to producing HLB-resistant citrus shows promise; however, there are concerns that consumers may be wary of accepting transgenic citrus due to the perceived negative aspects of transgenic plants containing “foreign” DNA. For this reason, a goal of this objective was to identify the components required for transfer and expression of disease resistance components into citrus without the introduction of foreign DNA or intragenics. The goals were achieved during the project period. The so called “P-DNA border’ sequences required for bacterial transfer of DNA during the process of transformation were identified in the citrus genome. This was accomplished in part through the sequencing and publication of a draft genome of the important citrus root stock Carrizo. This was followed by the accelerated release of additional genomic resources from other citrus species. These border sequences were demonstrated to substitute for their bacterial counterparts and enabled efficient transfer of DNA into plants (transformation). A group of small proteins were identified from gene expression databases that produce a family of Small Cyclic Amphipathic Peptides (SCAmpPs). The SCAmpPs genes were highly transcribed with different individuals expressed in different tissues within the plant. Genes were isolated for root, phloem and floral tissues. The gene promoters and terminator pairs were then tested with visual marker genes and verified to function as expected in the specific tissues. Also, the citrus Ubiquitin 6 promoter (CitUbi6) and transcription terminator sequences were isolated and demonstrated to drive high levels of constitutive expression in transgenic plants. These sequences provided the necessary variety of tools required for transfer, targeting and expression of DNA using only native DNA. Objective 2: Approaches were developed to target solutions to ZC and HLB diseases in potato and citrus. A strategy for quickly evaluating candidate disease resistance constructs in transgenic potato was utilized. This approach enables the rapid testing of a broader array of constructs than could be achieved through direct testing in citrus. This is due to the shorter time involved in generating, infecting and scoring the disease susceptibility in potato. In collaboration with ARS researchers in Wapato, Washington, and Fort Pierce, Florida, ARS researchers in Albany, California, tested transgenic lines carrying potential disease resistant genes. Thionin and Defensin genes from a variety of crop sources were tested for their ability to confer disease resistance, so far with inconsistent results. Additional disease-resistance candidate genes including the SCAmpPs genes, unique to the semi-tolerant Carrizo root stock, were combined with a phloem-specific SCAmpP4 promoter and transformed into plants. The resistance of these plants is currently being evaluated. Together, these novel citrus sequences, and the rapid disease screening in potato, provide useful tools for our continued efforts to introduce ZC and HLB disease resistance into crops. The successful application of disease resistance solutions is aimed to alleviate the millions of dollars of lost value in potatoes and citrus and to help reverse the decline of the multibillion dollar citrus industry in Florida. Objective 3: The focus of this objective is to develop molecular tools using information from the genome of Carrizo. Carrizo represents the best current citrus source of HLB tolerance, with research expanding to identify potential Liberibacter diseases tolerance/resistance genes with commercial applications. The initial genomic analysis of the R-gene family of resistance genes among the draft genomes of citrus did not provide conclusive differentiation between the large number of gene family members of the tolerant versus the resistant citrus varieties. Analysis of the citrus genome did facilitate the isolation of the SCAmpPs family of proteins which are the source of continued application for both providing expression control elements for targeted expression of transgenes as well further investigation into this family of small proteins with potential antimicrobial activity.


Review Publications
Belknap, W.R., Thomson, J.G., Thilmony, R.L., McCue, K.F., Hao, G., Stover, E.W. 2017. Small cyclic amphipathic peptides (SCAmpPs) genes in citrus provide promising tools for more effective tissue specific transgenic expression. Acta Horticulturae. 1172:85-90.