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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

2015 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 included the development of a novel citrus transcriptional control element with the potential to obviate the need for a citrus intragenic selectable marker. In the past year, we characterized a root-specific promoter from the citrus Small Citrus Amphipathic Peptide (SCAmpPs) gene family. This promoter, referred to as the 302z promoter, directs extremely efficient and tissue-specific gene expression in roots of transgenic citrus and Arabidopsis. 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 characterization of new control elements to promote high level expression in phloem for ZC- and HLB-resistance transgenes. The causal agents of Huanglongbing (HLB) disease in citrus and of Zebra Chip (ZC) in potato are two related Liberibacter species introduced into the phloem by psyllid insect species. The SCAmpPs gene family, identified in citrus last year, contains members expressed at extraordinary levels in the citrus phloem. In the past year, reporter gene (GUS) fusions transcribed from a phloem-specific promoter element (the 396s) were introduced into citrus and Arabidopsis where they demonstrate appropriate expression profiles. In addition, over the past year, we constructed a variety of transgenes to evaluate specific components involved in the expression of two SCAmpPs genes, root-specific 302z and a fruit-specific AbSCAmpP gene. The level of expression of the SCAmpPs gene products in citrus is consistent with a translational enhancer, most probably associated with the initiator methionine codon. Transgenes designed to explore this possibility were constructed and introduced into Arabidopsis and citrus. The data obtained is consistent with a translational enhancer associated with the initiation codon of these two genes. Transgenes designed to evaluate the contribution of intron sequences to the expression profiles of both of these genes showed that tissue-specificity appears to be relaxed in the absence of the intron that interrupts the coding sequence. Progress on the third objective includes the use of the citrus DNA 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, computational analysis of the available citrus genomes over the past year revealed that the majority of these genes are not annotated by automated systems and the actual number of these genes in the citrus genomes is approximately 10-fold higher than originally estimated. This greatly complicates identification and characterization of Poncirus-specific R-gene members. Over the past year, we have shifted our search to species-specific members of the SCAmpPs gene family. The SCAmpPs genes and their products have a variety of features indicating that they function in plant defense. The encoded peptides are short, amphipathic and have structural features similar to other anti-microbial gene products. The coding sequences of the active members of this gene family evolve rapidly, with expression profiles and genomic architecture consistent with a role in defense. In the past year, we completed LC-MS characterization of a phloem SCAmpPs cyclic peptide and showed that it has valinomycin-like cation binding activity. In addition, we identified a phloem-specific SCAmpPs that is highly expressed in Poncirus, but absent (in the available databases) from the phloem of other citrus. A citrus sinensis SCAmpPs gene is currently being modified to produce the Poncirus-specific phloem SCAmpPs peptide. This will be transformed into potato to test its ability to confer resistance to ZC. In research funded by a USDA Biomass Research and Development Initiative (BRDI) grant, the complete guayule genome (as determined by read mapping) was computationally assembled from next generation DNA sequencing reads. Linkage of the short sequence reads into useful contigs required a High Performance Computer and development of customized software. The assembly includes approximately 1Gb of scaffolds containing more than 95% of the guayule genes, and has been verified via syntenic relationships to other dicotyledonous plant genomes. It contains a number of unexpected architectural features that will be explored further. The assembly and scaffolds have been uploaded for use by cooperators in the grant-funded consortium to improve guayule. Following publication within the next year, this genomic sequence will be made publicly available for guayule improvement by both public and private sector scientists. Two patents that resulted from project work more than a decade ago were granted this year: “Solanum bulbocastanum polyubiquitin BUL427 promoter and uses thereof” [U.S. Patent No. 8,921,656] and “Solanum bulbocastanum polyubiquitin BUL 409 promoter and uses thereof” [U.S. Patent No. 8,927,702].

1. Assembly of the guayule (Parthenium argentatum) genome sequence. Guayule is a desert shrub that can be grown in the United States on marginal lands and that could serve as a domestic source of high-quality rubber. Both genetic and biotechnological improvements of the plant would benefit from an accurate and complete genome sequence. In 2015, funded by a USDA Biomass Research and Development Initiative (BRDI) grant, ARS scientists in Albany, California, assembled short DNA sequences from the guayule genome into linear arrays that revealed the order and physical separation distances of more than 95% of its genes. The accuracy of the assembly was verified by comparison with previously obtained genome sequences from related plants. The data have been made available on the internet to the academic/industry/government research consortium funded by the grant. The assembled guayule genome sequence will soon be available to all public and private sector scientists for use in improving guayule as both a cultivated crop and a domestic source of rubber that can be made into tires, gloves and other industrial products.

Review Publications
Shepherd, L.V., Alexander, C.J., Hackett, C.A., Mcrae, D., Sungurtas, J.A., Verrall, S.R., Morris, J.A., Hedley, P.E., Belknap, W.R., Rockhold, D.R., Davies, H.V. 2015. Impacts on the metabolome of down-regulating polyphenol oxidase in transgenic potato tubers. Transgenic Research. 24:447-461.