Ming-Bo Wang and Eric Huttner
The session was focused on
posttranscriptional gene silencing (PTGS) and its potential application in
functional genomic studies in plants. There were three speakers, Drs. Peter
Waterhouse and Chris Helliwell from CSIRO Plant Industry in
Dr. Waterhouse first gave a brief overview on gene silencing research in a historical perspective, from the early discovery of the sequence-specific RNA degradation phenomenon in plants by William Dougherty and colleagues, the first demonstration of double-stranded (ds) RNA being the trigger of PTGS or RNA interference (RNAi) by the CSIRO Plant Industry group and Andy Fire’s group, to the finding by Phil Zamore’s group that dsRNA is processed by DICER into 21-25 siRNAs that guide mRNA degradation. Dr. Waterhouse then presented examples of inverted-repeat (i/r) transgene-induced gene silencing in plants and successful applications of the i/r transgene technology in modifying agronomical traits in cotton and barley. He also summarized the optimizations made to the i/r transgene technology by the CSIRO PI group.
Dr. Shimamoto presented an excellent example showing how the i/r transgene approach can be used to study the functions of individual members of a large gene family. He showed that, through targeting of the divergent 3’ UTR regions, individual members of the rice OsRac gene family were specifically silenced. He also presented their preliminary data suggesting that it may not be easy to simultaneously silence all members of a gene family by targeting the conserved coding regions. In addition, Dr. Shimamoto made a suggestion that transitive silencing (or spreading) does not occur in the case of OsRac silencing in rice. During the discussion session, Dr. Waterhouse noted there may be differences between species in term of transitive silencing.
Dr. Helliwell’s talk was focused on the potential application of the i/r transgene technology in functional genomic studies. He first discussed the advantages of using the i/r transgene approach over other reverse genetic approaches, and then described the Gateway recombination system-based vectors developed in CSIRO Plant Industry for high-throughput preparation of i/r transgene constructs. He also described the current efforts in CSIRO Plant Industry to develop strategies for multiple silencing and inducible silencing in plants using i/r transgenes.
During the end-of-session discussion, Dr. Eric Huttner of CAMBIA raised a question as to whether promoter strength of an i/r transgene affects the efficiency of silencing. Dr. Helliwell commented that the strong 35S promoter is usually used and is effective for silencing a range of genes in dicots. Dr. Shimomoto suggested that the level of siRNAs may not be the limitation but step(s) downstream of siRNA production is the key in determining the efficiency of silencing. Dr. Waterhouse added that the oil research group in CSIRO Plant Industry has successfully silenced fatty acid biosynthetic genes using seed-specific promoters.
Dr. Shimamoto asked about Dr. Waterhouse’s view on the importance of including an intron loop in the i/r transgene constructs. He said that most of their i/r constructs do not contain an intron loop and yet most of these constructs gave near 100% silencing efficiency in rice. The main point made by Dr. Waterhouse in answering Dr. Shimamoto’s question was that it may be that the splicing of intron, rather than the position of intron in the i/r construct, is important for high efficiency of silencing.
Finally, Dr.
Bill Crosby of