Summary of Research Project Results under JSPS FY2004
"Research for the Future Program"

1. Research Institution   Kyoto University
2. Research Area Life Sciences
3. Research Field Plant Genetics
4. Term of Project FY 2000 - FY 2004
5. Project Number 00L01606
6. Title of Project RNA Silencing in Higher Plants and Functional Genomics

7. Project Leader

Name Institution, Department Title of Position
Fumihiko, Sato Kyoto University, Graduate School of Biostudies Professor

8. Core Members

Names Institution, Department Title of Position
Ei-ichiro, Fukusaki Osaka University, Graduate School of Engineering Associate Professor
Kimiko, Itoh Niigata University, Graduate School of Science and Technology Associate Professor

9. Summary of Research Results

Development of practical systems to identify gene functions based on RNA silencing (RNA interference; RNAi) and its applications for the characterization of genome network have been investigated. Molecular bases of RNAi in plant cells also have been investigated for the establishment of next generation of genetic engineering with more controlled gene expression. Research achievements are following; 1) The general methodology for the gene specific silencing among family genes has been established using specific double stranded (ds)RNA, as well as for the comprehensive silencing of whole family genes in stable transformants. Using specific gene silencing, practical molecular substitution of target gene has been developed. These methods have been successfully applied to characterize the functions of important photosynthetic PsbP of tobacco, rice Rac genes in signal transduction of pathogenesis and so on. 2) Transitive RNAi method with artificially synthesized dsRNA has been developed and used for the identification of several trans-factors in medicinally important isoquinoline alkaloid biosynthesis in Coptis japonica cells. 3) Several RNAi vectors, such as those for practical RNAi vector (pANDA) in rice based on Gateway system, for the quantitative control of RNAi using splicing mutation, for cell specific silencing in endosperm and so on have been constructed. Using these vectors, Wx mRNA function was efficiently and quantitatively disrupted in rice. 4) RNAi has been successfully applied for metabolic engineering to produce intermediate accumulators and also to create new biosynthetic pathway. 5) Molecular characterization of DNA methylation revealed the existence of novel system of gene regulation/recognition in plant cells. 6) RNAi in plants is known as a defense system against virus infection. On the other hand, viruses have RNAi suppressors to counteract RNAi. The NSs protein of Tomato spotted wilt virus has been characterized to be as the first RNAi suppressor in negative strand RNA viruses and found that NSs could interfere with multiple steps in the RNAi pathway. Also, a novel mechanism of RNAi suppression by Red clover necrotic mosaic virus, which needs multiple viral components, has been identified.

10. Key Words

( 1 ) RNA interference (RNAi) ( 2 ) Specific gene silencing ( 3 ) Functional genomics
( 4 ) Silencing of family genes ( 5 ) Quantitative regulation of gene silencing ( 6 ) transient RNAi
( 7 ) Metabolic engineering with RNAi ( 8 ) Genome network analysis with RNAi ( 9 ) DNA methylation