Phragmites australis has become a major problem in wetlands ecosystems in North America since the introduction of an aggressive genotype from Europe early in the twentieth century. This aggressive form has spread from the east coast to the Great Lakes and Pacific where it outcompetes native vegetation, leading to declines in plant species diversity and loss of critical habitats for fish, reptiles, and birds. While there is a plethora of papers to document its triumph, what is lacking is a mechanistic understanding of how Phragmites has achieved this daunting success and a cost effective means of combating this invasion. At the present, most common methods of Phragmites management such as flooding, burning, and spraying general herbicides lead to short-term environmental control without establishing a long-term solution. Researchers at Wayne State University have been exploring a novel approach that exploits gene-silencing technology to develop species-specific reduction in photosynthesis, root growth and seed set. Over the past 4 years (Phase 1), Wayne State had made significant progress on this technology. Researchers have focused on two areas: 1) The generation of an accessible genetic database for Phragmites and 2) The development and testing of multiple gene silencing approaches for Phragmites. The successful completion of the first area of research has resulted in the generation and partial annotation of a transcriptome library based on RNA extracted from field collected samples of rhizome, shoot meristem, leaf, and inflorescence tissue. The resultant database of processed contigs is searchable via common tools such as BLAST. Researchers at Wayne State have also begun to expand their scope to other invasive species to test whether gene silencing is a viable option for invasive plants with different growth forms than Phragmites. Lianas such as Celastrus orbiculatus (oriental bittersweet) and Lonicera japonica (Japanese honeysuckle) have invaded mesic woodland habitats and have negatively impacted forest composition and growth through alleopathy, girdling, light competition, and uprooting (Ladwig and Meiners, 2009; Leicht-Young et al., 2013). Management treatments to control invasives in these more complex local communities cannot rely on non-specific burning or herbicides as native, slow-growing perennials such as trees would not rapidly recover. WSU will explore whether invasive species management and restoration approaches can be developed to deal with large monotypic, herbaceous communities as well as with aggressive invasive species that establish within diverse and mosaic communities. We propose the continuing development of an extremely novel management approach to control Phragmites and other invasive species and to allow the gradual restoration of native vegetation through restoring native species competitive advantages. The long-term objective of this work is produce practical tools for the restoration of marsh, wetland, and mesic forest communities through the non-destructive control of Phragmites and Celastrus. Work thus far has focused on the genetics of Phragmites and on the development of silencing vectors using surrogate lab-species including maize (Zea mays) and sunflower (Helianths annua). Researchers at WSU have characterized and compared transcriptome data sets from rhizome, meristem, stem and inflorescence tissue from field collected Phragmites tissue. In doing so, they have established analytical protocols that will be used for future targeted species. They have developed and tested the efficacy of gene silencing vectors in Phragmites, maize, and sunflower, and have achieved some positive results in reducing growth. The proposed work will expand and further develop this approach. The specific objectives for the new cooperative agreement include two new objectives that either aim to expedite and thus expand screenings of silencing constructs for their efficacy in silencing (Objective 1) or directly expand the scope of our work so that we are working on targeting new species in addition to Phragmites (Objective 2). The remaining three objectives continue our work and progress established during our first cooperative agreement (Objectives 3, 4, and 5). Specifically, our objectives are: 1. Establishment of a rapid, protoplast-based screening system whereby panels of multiple amiR (artificial microRNA), VIGS (viral induced gene silencing) vectors, and hairpin RNA vectors can be tested efficiently and expeditiously. 2. Isolation, description, and analysis of transcriptome data from Celastrus orbiculatus and Lonicera japonica, thereby producing a database to identify and target specific genes in these species. 3. Extension and expansion of our laboratory trials to establish which silencing vectors produce the strongest (negative) effects on morphology and physiology for the target species in the laboratory. 4. Testing of effects of most efficacious silencing vectors in Phragmites, maize, and sunflower in laboratory competition experiments to establish concept of species-specific competitive modifications. 5. Coordination and cooperation with USGS to establish field trials on Phragmites and other targeted invasive species.