Tremendous progress has been made over the last 10 years in small RNA-directed gene silencing and the development of related technologies in plants. To date, two general kinds of small RNAs have been discovered in plants: siRNAs and miRNAs. siRNAs can be further subdivided into three different types: trans-active siRNAs (tasiRNAs), natural antisense siRNAs, and repeat-associated siRNAs. Although all of these small RNAs can be used to silence genes in plants, tasiRNAs are a unique but intriguing population of small RNAs that are well conserved exclusively in various plant species. Production of tasiRNAs in plants requires both the miRNA and the RNA interference pathways, which makes gene silencing by tasiRNAs a potentially powerful system for gene function studies. Unlike other kinds of small RNAs that need pre-structured double-stranded RNAs (dsRNAs) as precursors for their production, tasiRNAs can use single-stranded RNA precursors to produce dsRNA through RNA-dependent RNA polymerase in cells. This unique quality makes it possible to introduce a randomized short sequence inside the tasiRNA precursors through genetic engineering. This EAGER project will explore the novel application of the plant tasiRNA pathway for generating plant mutant pools in the model plant Arabidopsis as proof-of-concept.
In addition to the broader impacts of a new tool for investigating gene function in crop plants, the project will provide research training opportunities for one postdoctoral fellow and nine undergraduate and high school students. Underrepresented minorities and women, as well as students from rural eastern Kentucky, will be especially targeted and recruited. All information regarding the method(s) developed and mutants generated will be available through publication and through the Arabidopsis Information Resource (TAIR). All vectors and transgenic seed will be available from the PI and from the Arabidopsis Biological Resource Center (ABRC).
(1) Describe the project outcomes or findings that address the intellectual merit and broader of impacts of your work as defined in the NSF merit review criteria. This explorative project was extended for additional year from 08/01/2013 to 07/31/2014 and came to its close. The basic findings or outcomes were the same as previous reports. Briefly, TasiRNA vector can be developed and improved for basic study, but has limited applications in gene silencing due to its lower silencing efficiency. TasiRNA vector can be used for generating a radomized mutant librray but the mutants were not as strong as amiRNA-generated mutants. (2) Briefly summarize the outcomes of your award (200 – 800 words) covering the entire life of the award: We generated a few modified tasiRNA vectors for gene silencing with an improved gene sielncing effciency. We proved the concept of tasiRNA vector for generating mutants, but the mutants generated by tasiRNA were weak. There was a recent publication on tasiRNA vector by other group, but its application is limited. The conclusion of this entire project indicates a ranomized tasiRNA mutant library is not a feasible approach to plant mutant production. We have trained several graduate students and post-docs over this project and published a series of publications during the practice of this project.
