RNA silencing
VIRUS INDUCED GENE SILENCING
References
Anandalakshmi, R., Pruss, G.J., Ge, X., Marathe, R., Mallory, A.C., Smith, T.G., Vance, V.B. A viral suppressor of gene silencing in plants PNAS 95, 13079-84 (1998)
Baulcombe, D. RNA silencing. Curr. Biol. 12, R82-84 (2002)
Baulcombe, D. Viral suppression of systemic silencing. Trends in Micro. 10, 306-8 (2002)
Kasschau1, K.D., Xie1, Z. , Allen, E., Llave, C., Chapman, E.J.,Krizan, K.A., Carrington, J.C. P1/HC-Pro, a Viral Suppressor of RNA Silencing, Interferes with Arabidopsis Development and miRNA Function, Dev. Cell 4, 205-217 (2003)
Llave, C., Kasschau, K.D., Rector MA, Carrington, J.C. Endogenous and Silencing-Associated Small RNAs in Plants. Plant Cell 14, 1605-1619 (2002)
Vance, V., Vaucheret, H. RNA silencing in plants - defense and counterdefense. Science 292, 2277-2280 (2001)
Voinnet, O. RNA silencing as a plant immune system against viruses. Trends in Genetics 17, 449-59 (2001)
Voinnet, O. RNA silencing, small RNAs as ubiquitious regulators of gene expression. Curr. Opinion in Plant Biol. 5, 444-451 (2002)
Waterhouse, P.M., Wang, M-B., and Finnegan, E.J. Role of short RNAs in gene silencing. Trends in Plant Sci. 6, 297-301 (2001)
Waterhouse, P.M., Wang, M-B., and Finnegan, E.J. Gene silencing as an adaptive defence against viruses. Nature 411, 834-42 (2001)
Outline
I- Early observations
1- Virus genes inserted into host will cross protect (reduce and slow infection)
2- Earliest transgenics contained capsid genes - thought to inhibit disencapsidation
3- Antisense RNA inserted into host interferes with virus replication
4- (+) sense "nongenic" RNA interferes with virus replication
5- (+) sense of nonviral protein inhibits replication of virus which contains that gene
5- Attempts by biotechnologists to overexpress genes leads to silencing
II- Features of silencing
1- A serious barrier to overexpressing genes in plants
2- (+), (-) and dsRNAs can all silence
3- Sequence specific, need at least 20 nucleotides of homology
4- Effective against exons, but not introns
5- dsRNA is an intermediate
6- RNA polymerases (including host RNA polymerases) play important roles
7- Small interfering (si)RNAs (~21, ~25 nucleotides) produced during silencing
8- Silencing spreads systemically via the larger class of siRNAs!
III- Role of viruses
1- PVX vector containing plant genes will silence
2- Phytoene desaturase or rubisco RNA will bleach leaves
3- PVX-GFP will silence GFP in GFP tobacco!
IV- Is silencing important for plant virus replication?
1- Plant viruses produce lots of (+) and dsRNA
2- Plants normally recover from virus infection (silencing?)
3- Plant viruses contain antisilencing genes
V- Discovery of plant virus antisilencing genes
1- PVX and TEV (tobacco etch, potyvirus) are synergistic in tobacco, increased symptoms, PVX replication
2- Insert TEV genes in tobacco, see which ones stimulate PVX replication
3- P1-HCPro is the active region (HC-Pro in particular)
4- Put PI-HCPro into PVX vector
A- Increased symptom severity
B- Increased replication, especially (-) strand
C- SiRNAs inhibited
5- PVX-HCPro relieves silencing! GFP-tobacco (silenced) is simplest tool.
6- Antisilencing now recognized as general feature of virus infection
A- 2b protein of CMV
B- p19 of TBSV
C- 25K (triple gene block) protein of PVX
VI- Antisilencing activity of HCPro
1- PVX-HCPro (which unsilences) a useful probe for silencing
2- PVX a useful vector for generalized silencing
3- rgsCaM (tobacco gene) interacts with HCPro by yeast 2-hybrid analysis
4- rgsCaM by itself has antisilencing activity
5- Plants transgenic for HCPro or rgsCaM develop abnormally
VII- General model for silencing
1- RNA polymerase (host or virus) makes dsRNA
2- RNase ("dicer") cuts dsRNA into discrete fragments
3- These fragments facilitate RNA degradation (RISC complex)
4- Higher organisms produce micro (mi)RNAs which interfere with gene expression
5- miRNAs are important in development.