2013 Annual Report
1a.Objectives (from AD-416):
1)Develop new promoter elements from potato that will allow refined expression profiles (tissue and/or developmental specificity) of transgenes to improve agronomic and quality properties of dicotyledonous crop species.
2)Discover and develop new molecular tools (promoters, terminators) from fruit trees. In particular, to isolate transcriptional control elements and polyadenylation signals from plum and apple.
3)Refine down-regulation technologies to improve general applications to metabolic regulation as well as improve design characteristics of pathogen-resistance transgenes.
1b.Approach (from AD-416):
Available EST, microarray and genomic DNA databases will serve as bioinformatics data sources to identify potato gene families, and specific family members, with requisite expression profiles to serve as sources of valuable transcriptional control elements. Putative elements will be isolated from a BAC library, assembled into marker-gene fusions, and function characterized in transgenic potatoes.
Polyubiquitin genes from apple and plum will serve as sources of transcriptional control elements for direction of commercial levels of transgene expression in these fruit trees. Appropriate polyubiquitin genes for these control sequences will be identified using EST databases to identify specific, highly constitutively transcribed family members. The molecular source of elements will include both BAC libraries and PCR-amplification. Putative promoter elements will be fused to standard marker and delivered to collaborators at the USDA/ARS Appalachian Fruit Research Station for introduction and characterization in apple.
Glycoalkaloids will be reduced in potatoes by suppression of both branches of the SGA pathway using small inverted hairpin structures of double stranded RNA-generating constructs. Small interfering RNAs (siRNAs) will be produced by these constructs specific for both the Sgt1 and Sgt2 gene family members responsible for the initial steps in each of the two SGA biosynthetic branches, resulting in gene inactivation. Suppression constructs will be tested for efficacy via standard genetic transformation, but will ultimately be adapted for intragenic transformation and eventual commercial application. Transgenic potato tubers producing siRNAs will be evaluated for transgene expression and SGA accumulation. Replacing 5325-21420-003-00D (08/2008). BL-1, certification 5/17/07.
This is the final report for this project, which has been replaced by new project 5325-21220-001-00D, "Host-Specific Molecular Genetic Tools for Development of Disease-Resistant Crops". For additional information, see the new project report. Progress was made on all three objectives, all of which fall under National Program 301, Component 3: Crop Biological and Molecular Processes; Problem Statement 3A: Fundamental knowledge of plant biological and molecular processes. The three objectives are:.
1)to discover and develop new molecular tools (promoters, terminators) for improvement of dicotyledonous crop species including intragenic modifications,.
2)to reduce levels of glycoalkaloid toxicants in potatoes, and.
3)to reduce susceptibility of commercial potato cultivars to potato viruses and Late Blight. Inclusion of development of molecular tools for citrus improvement was introduced into the research program at the direction of the National Program Leader.
Progress over the past year includes use of a 15X genome of the citrus rootstock variety Carrizo, which contains a source of resistance to Huanglongbing disease, to identify potential huanglongbing-resistance genes. Carrizo-specific NBS-LRRP (Nucleotide Binding Site-Leucine-Rich Repeat Proteins) were computationally identified by comparison to extant sweet orange genome sequences.
This year we constructed additional components for the citrus “intragenic” toolbox to allow direct genetic modification of these crops without introduction of non-native DNA. We completed construction of a novel citrus phloem-specific promoter for Liberibacter control that will be evaluated by ARS cooperators in Fort Pierce, Florida.
Progress in the second goal (reduction of glycoalkaloids) in FY12 includes final evaluation of siRNA constructs to down-regulate the glycoalkaloid biosynthetic pathway. Two novel constructs designed to down-regulate genes that carry out addition of glucose (solanidine glucosyltransferase SGT2) or glucose/galactose (solanidine galactosyltransferase SGT1 and SGT2) to the aglycone were evaluated. The final set of transgenes in these studies, which divert intermediates of the glycoalkaloid biosynthetic pathway to phytosterol nutrients, have been completed. These transgenes have been introduced into potato for metabolite analysis.
Progress on the third goal (to reduce crop disease susceptibility) included carrying out second year field evaluations of transgenic potatoes expressing a multiple virus-resistance transgene (PVY, PVA, PVX and PLRV) that showed successful conference of resistance to two of target pathogens (PVY and PLRV) in first year trials. Over the past year we, in cooperation with ARS scientists in Wapato, Washington, have developed a bacterial potato disease (Zebra Chip) for evaluating potential transgenic resistance strategies.
Successful development of a coordinated research program addressing Liberibacter-associated diseases in crop plants. Liberibacter species are associated with a number of emerging diseases in a variety of commodities. Two with significant economic impact are huanglongbing (HLB) in citrus and Zebra Chip (ZC) in potato. In the case of HLB, the disease is threatening a 10 billion dollar industry. No genetic source of resistance has been identified for either disease. ARS scientists in Albany, California, and Wapato, Washington, have developed ZC as model for expediting evaluation of approaches to the development of Liberibacter-resistant crops, as ZC symptoms appear in weeks as opposed to several years for HLB. This model has been employed to identify transgenic approaches for introduction of HLB/ZC resistance using citrus genes.
Mc Cue, K.F., Ponciano, G.P., Rockhold, D.R., Whitworth, J.L., Gray, S.M., Fofanov, Y., Belknap, W.R. 2012. Generation of PVY coat protein siRNAs in transgenic potatoes resistant to PVY. American Journal of Potato Research. 89(5):374-383. DOI: 10.1007/s12230-012-9257-0.
Benyon, L.S., Stover, E., Bowman, K.D., Niedz, R.P., Shatters, R.G., Zale, J.,M., Belknap, W.R. 2013. GUS expression driven by constitutive and vascular specific promoters in citrus hybrid US-802. In Vitro Cellular and Developmental Biology Plants. 49:255-265.
Munyaneza, J.E., Sengoda, V.G., Aguilar, E., Bextine, B., Mc Cue, K.F. 2013. First report of 'Candidatus Liberibacter solanacearum' associated with psyllid-affected tobacco in Nicaragua. Plant Disease. 97:1244.
Bextine, B., Aguilar, E., Rueda, A., Caceres, O., Sengoda, V.G., Mc Cue, K.F., Munyaneza, J.E. 2013. First report of 'Candidatus Liberibacter solanacearum' on tomato in El Salvador. Plant Disease. 97:1245.
Aguilar, E., Sengoda, V.G., Bextine, B., Mc Cue, K.F., Munyaneza, J.E. 2013. First report of 'Candidatus Liberibacter solanacearum' on tomato in Honduras. Plant Disease. 97:1375.
Aguilar, E., Sengoda, V.G., Bextine, B., Mc Cue, K.F., Munyaneza, J.E. 2013. First report of 'Candidatus Liberibacter solanacearum' associated with psylllid-affected tobacco in Honduras. Plant Disease. http://dx.doi.org/10.1094/PDIS-04-13-0453-PDN.