2011 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.
The major problem areas being addressed are development of new molecular tools for potato and fruit tree improvement and construction of transgenic potatoes with improved postharvest and disease susceptibility properties. The project has three specific goals:.
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.
Transcriptional control elements developed as part of the first goal (molecular tools) are expected to have specific application in construction of intragenic potatoes and fruit trees, as well as broader applications in transgene construction for dicotyledonous crops. Progress in this goal in FY11 includes development of two polyubiquitin promoters from citrus, the Cit442 and Cit354 transcriptional control elements. These two elements, with unrelated 5’ control sequences, were selected for potential to direct high-level expression in citrus phloem tissue as a part of the effort to develop huanglongbing (HLB)-resistant citrus. This year functionality of both elements was demonstrated in transgenic plants, allowing construction of our first citrus promoter directed antibacterial transgenes, also completed in FY11. In addition, a potato SGT2 (from a solanidine glycosyltransferase gene) promoter construct was constructed and introduced into transgenic potato. Experiments carried out by collaborators at the ARS U. S. Horticultural Research Laboratory (Fort Pierce, FL) indicate functionality of a citrus intragenic vector, allowing “all native DNA” transformation of this commodity. Finally, transgenic potatoes expressing antibacterial transgenes previously developed in our laboratory have been entered into greenhouse evaluation for functionality in conferring resistance to the HLB-related disease Zebra Chip.
Progress in the second goal (reduction of glycoalkaloids) in FY11 includes demonstration that second generation siRNA construct assemblies failed to prevent glycoalkaloid accumulation in transgenic potatoes. A third generation of constructs, optimized for siRNA production, were completed in FY11 and introduced into transgenic potato for evaluation.
Progress on the third goal (to reduce susceptibility of commercial potato cultivars to potato viruses and Late Blight) includes initial field release in Idaho of transgenic Premier Russet, Norkotah Russet and Atlantic potatoes containing a novel 4-virus siRNA construct targeting Potato Virus Y, Potato Virus A, Potato Virus X and Potato Leaf Roll Virus. In addition, late blight-resistant potatoes (marker-free, Agrobacterium sequence-free) demonstrated resistance to Phytophthora infestans races currently affecting US production (US8, US11, as well as isolates from recent outbreaks).
Development of marker-free and “all native DNA” intragenic crops. The commercial introduction of crop plants improved using molecular genetic techniques has been limited by problems associated with public perceptions of transgenic foods. These limitations are being addressed in part by development of novel methods for genetic modification, referred to as "intragenic" technology. ARS scientists at the Western Regional Research Center (WRRC, Albany, California), in cooperation with ARS scientists from the U. S. Horticultural Research Laboratory (Fort Pierce, FL), the Vegetable Laboratory (Beltsville, MD) as well as the J.R. Simplot Company (Boise, ID) have successfully developed a number of technologies for construction of “intragenic” crops. We have successfully constructed marker-free potatoes, containing the RB gene from wild potatoes known to confer resistance to late blight, and shown that these potatoes are blight-resistant. We have also developed new citrus transcriptional control elements and used them to construct “intragenic” transgenes to confer resistance to citrus huanglongbing (HLB) disease.