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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

2014 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 demonstrating that the new intragenic transformation vector we constructed was functional. The central challenge in developing an intragenic vector is identification of plant-derived Agrobacterium border sequences (pDNA borders) from the target plant genome (native DNA). We had previously identified a highly functional citrus-derived left border sequence that efficiently terminated pDNA transfer. A new citrus intragenic binary vector was constructed with an alternative native right border and evaluated for functionality in Agrobacterium. These two pDNA borders functioned in Agrobacterium-mediated transformation with efficiencies similar to that of authentic Agrobacterium tDNA borders. This vector is ready for use in generating intragenic citrus. Progress on the second objective includes discovery of new control elements to promote high level transgene 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 related psyllid insect species. Both the ZC and HLB Liberibacter species proliferate specifically in the phloem. Over the past year we have identified two novel citrus genes with extraordinarily high levels of expression in phloem tissue. Together these genes support approximately 5% of the polyadenylated messenger RNAs (mRNAs) in this tissue, levels at least 20-fold higher than any other genes. The coding sequences of both genes are interrupted by a short, highly conserved intron. These genes, with 3 kilobase pairs (kbp) of flanking sequence, have been amplified from the genomes of Citrus ‘Clementina’ and ‘Carrizo’, and the Polymerase Chain Reaction (PCR) products sequenced. Marker gene (GUS) fusions transcribed from their promoter elements, including the first exon and single intron, have been constructed and inserted into a binary transformation vector. These fusions will be introduced into citrus and potato to evaluate their potential for expression of anti-microbial transgenes for ZC and HLB. The promoter of a related novel citrus gene with very high expression in fruit has been amplified by collaborators in our unit, and has been shown to direct efficient transcription in citrus and tomato fruits in transient assays. 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 evaluating 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. Thus, the actual number of these genes in the citrus genomes is approximately 10-fold higher than originally expected, greatly complicating identification and characterization of Poncirus-specific members. In addition, a field trial conducted during the 2013 growing season of transgenic potatoes containing a set of antimicrobial genes indicated they had no effect on ZC resistance. For these reasons, a search for alternative defense-related gene families was initiated. Over the past year, a previously unidentified, entirely novel citrus gene family encoding potential anti-microbial products has been identified. The members of this family, referred to as Small Citrus Amphipathic Peptides (SCAmpPs) genes, have a variety of features indicating that they function in plant defense. The SCAmpPs genes encode short amphipathic peptides with structural features similar to other anti-microbial gene products. The coding sequences of the active members of this gene family are evolutionarily unstable, with a remarkable level of sequence divergence between the Citrus and Poncirus species (4 million years). The expression profiles of these genes are also consistent with plant defense, with high level constitutive (potentially prophylactic) expression in tissues susceptible to microbial infection (abscission zones, phloem, roots). In terms of genomic architecture, the SCAmpPs genes are remarkably similar to the R-genes. Of the approximately 90 SCAmpPs genes identified in the citrus genomes, the majority (70%) appear to be pseudogenes. They exist largely in repetitive arrays in specific chromosomal locations, with actively expressed genes intermixed with pseudogenes. Finally, the SCAmpPs gene arrays are found in close proximity to arrays of potential R-genes (NB-ARC Leucine Rich Repeat Protein arrays). Sequences from SCAmpPs genes transcribed in different tissues (including roots, abscission zones and phloem) have been amplified by PCR, cloned and sequenced. The structure and abundance of the SCAmpPs gene-encoded proteins in plant tissues is currently being evaluated by Liquid Chromatography-Mass Spectrometry. In collaborative work under a Cooperative Research and Development Agreement (CRADA) held by a different scientist in the unit to develop a domestic source of natural rubber, progress was made in the sequencing of the guayule (Parthenium argentatum) genome. Approximately 8x coverage of the 2 gigabase genome was achieved by one high-throughput sequencing method and 40x coverage by another. Assembly and annotation of the sequence data are in progress. Genes encoding isoprenoid pathway components were assembled manually to accelerate characterization of key rubber biosynthesis genes.

1. Relationships among bacteria that cause potato Zebra Chip disease in Central America. Zebra Chip disease of potato is caused by species of the fastidious prokaryote Candidatus Liberibacter (Ca L.) solanacearum. The bacteria are spread by the phloem feeding of tomato potato psyllids (Bactericera Cockerelli), which can also feed on other crops within the same plant family. ARS scientists in Albany, California, and Wapato, Washington, have collaborated to catalogue the occurrence of Ca. L. solanacearum in a number of solanaceous crops in Central America. Through cloning and sequencing of diagnostic regions of the genome, the variation of the bacteria between geographic areas was established, providing insight into the spread of the disease between crops and informing potential mitigation measures. Knowledge of the differences among pathogenic bacteria in different parts of the world will aid in their identification and discrimination, and knowledge of their similarities will suggest possible targets for their control.

Review Publications
Munyaneza, J.E., Sengoda, V.G., Aguilar, E., Bextine, B.R., Mc Cue, K.F. 2013. First report of "Candidatus Liberibacter solanacearum" infecting eggplant in Honduras. Plant Disease. 97:1654
Munyaneza, J.E., Sengoda, V.G., Aguilar, E., Bextine, B., Mc Cue, K.F. 2014. First report of "Candidatus Liberibacter solanacearum" on pepper in Honduras. Plant Disease. 98:154.