Abstract
RNAi is a powerful method for generating loss of function mutants, especially for
targeting genes belonging to large gene families. We have recently shown that RNAi
functions in the moss Physcomitrella patens. We obtained stable lines that show constitutive silencing of a nuclearly localized
GFP:GUS fusion protein (NLS:GFP:GUS). However, lines that display silencing of the
protein do not necessarily have reduced transcript levels. Therefore, a system has
been developed that silences the NLS:GFP:GUS reporter construct at the same time as
it silences a gene of interest. Gateway® (Invitrogen) recombination cassettes were
incorporated into these vectors to facilitate cloning of many different cDNA sequences.
In addition, vectors were generated that contain genomic moss DNA sequence information
to increase the production of stable moss lines. Transformation with these constructs
results in strong silencing within 24 h and is stable for at least a month after transformation.
FtsZ2-1, whose loss of function phenotype is known, was incorporated as a test case
for analyzing phenotypes. One hundred per cent of regenerating colonies that have
silenced GFP exhibit a loss of function FtsZ2-1 phenotype, validating the use of this
system to assay phenotypes for plant genes of unknown function.
Key words
Rapid RNAi system -
Physcomitrella patens
- vectors - moss
References
- 1
Baulcombe D. C..
RNA silencing in plants.
Nature.
(2004);
431
356-363
- 2
Bezanilla M., Pan A., Quatrano R. S..
RNA interference in the moss Physcomitrella patens.
.
Plant Physiology.
(2003);
133
470-474
- 3
Hannon G. J..
RNA interference.
Nature.
(2002);
418
244-251
- 4
Jefferson R. A., Kavanagh T. A., Bevan M. W..
GUS fusions: betaglucuronidase as a sensitive and versatile gene fusion marker in
higher plants.
EMBO Journal.
(1987);
6
3901-3907
- 5
Marcotte Jr. W. R., Russell S. H., Quatrano R. S..
Abscisic acid-responsive sequences from the EM gene of wheat.
Plant Cell.
(1989);
1
969-976
- 6
Nishiyama T., Fujita T., Shin-I T., Seki M., Nishide H., Uchiyama I., Kamiya A., Carninci P.,
Hayashizaki Y., Shinozaki K., Kohara Y., Hasebe M..
Comparative genomics of Physcomitrella patens gametophytic transcriptome and Arabidopsis thaliana: implication for land plant evolution.
Proceedings of the National Academy of Sciences of the USA.
(2003);
100
8007-8012
- 7
Schaefer D. G., Zrӱd J.-P..
Efficient gene targeting in the moss Physcomitrella patens.
.
Plant Journal.
(1997);
11
1195-1206
- 8
Schaefer D. G., Zrӱd J.-P., Knight C. D., Cove D. J..
Stable transformation of the moss Physcomitrella patens.
.
Molecular and General Genetics.
(1991);
226
418-424
- 9
Sharp P. A..
RNA interference - 2001.
Genes and Development.
(2001);
15
485-490
- 10
Strepp R., Scholz S., Kruse S., Speth V., Reski R..
Plant nuclear gene knockout reveals a role in plastid division for the homolog of
the bacterial cell division protein FtsZ, an ancestral tubulin.
Proceedings of the National Academy of Sciences of the USA.
(1998);
95
4368-4373
- 11
Tijsterman M., Ketting R. F., Plasterk R. H..
The genetics of RNA silencing.
Annual Review of Genetics.
(2002);
36
489-519
M. Bezanilla
Department of Biology
Washington University
Campus Box 1229
1 Brookings Drive
Saint Louis, MO 63130
USA
Email: manena@biology.wustl.edu
Guest Editor: R. Reski
