|
Plant Physiol, July 2001, Vol. 126, pp. 930-938
Silencing on the Spot. Induction and Suppression of RNA Silencing
in the Agrobacterium-Mediated Transient Expression
System1
Lisa K.
Johansen and
James C.
Carrington*
Institute of Biological Chemistry, Washington State University,
Pullman, Washington 99164-6340
The Agrobacterium-mediated transient expression
assay in intact tissues has emerged as a rapid and useful method to
analyze genes and gene products in plants. In many cases, high levels of active protein can be produced without the need to produce transgenic plants. In this study, a series of tools were developed to
enable strong or weak induction of RNA silencing and to suppress RNA
silencing in the absence of stable transgenes. Transient delivery of a
gene directing production of a double-stranded green fluorescent protein (GFP) transcript rapidly induced RNA silencing of a codelivered GFP reporter gene, effectively preventing accumulation of GFP protein
and mRNA. RNA silencing triggered by the strong dsGFP inducer was
partially inhibited by the tobacco etch virus silencing suppressor,
P1/HC-Pro. In the absence of the strong double-stranded GFP inducer,
the functional GFP gene served as a weak RNA silencing inducer in the
transient assay, severely limiting accumulation of the GFP mRNA over
time. The weak silencing induced by the GFP gene was suppressed by
P1/HC-Pro. These results indicate RNA silencing can be triggered by a
variety of inducers and analyzed entirely using transient gene delivery
systems. They also indicate that RNA silencing may be a significant
limitation to expression of genes in the
Agrobacterium-mediated transient assay but that this limitation can be overcome by using RNA silencing suppressors.
1
This work was supported by the National
Institutes of Health (grant nos. AI43288 and AI27832) and by the U.S.
Department of Agriculture (grant no. 98-35303-6485).
*
Corresponding author; e-mail carrington{at}wsu.edu; fax
509-335-2482.
© 2001 American Society of Plant Physiologists
This article has been cited by other articles:
|
|
|
|
 
V. Pantaleo and J. Burgyan
Cymbidium Ringspot Virus Harnesses RNA Silencing To Control the Accumulation of Virus Parasite Satellite RNA
J. Virol.,
December 1, 2008;
82(23):
11851 - 11858.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Torres-Barcelo, S. Martin, J.-A. Daros, and S. F. Elena
From Hypo- to Hypersuppression: Effect of Amino Acid Substitutions on the RNA-Silencing Suppressor Activity of the Tobacco etch potyvirus HC-Pro
Genetics,
October 1, 2008;
180(2):
1039 - 1049.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Avisar, A. I. Prokhnevsky, and V. V. Dolja
Class VIII Myosins Are Required for Plasmodesmatal Localization of a Closterovirus Hsp70 Homolog
J. Virol.,
March 15, 2008;
82(6):
2836 - 2843.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Avisar, A. I. Prokhnevsky, K. S. Makarova, E. V. Koonin, and V. V. Dolja
Myosin XI-K Is Required for Rapid Trafficking of Golgi Stacks, Peroxisomes, and Mitochondria in Leaf Cells of Nicotiana benthamiana
Plant Physiology,
March 1, 2008;
146(3):
1098 - 1108.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. A. Lindbo
TRBO: A High-Efficiency Tobacco Mosaic Virus RNA-Based Overexpression Vector
Plant Physiology,
December 1, 2007;
145(4):
1232 - 1240.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
X. Yang, S. Baliji, R. C. Buchmann, H. Wang, J. A. Lindbo, G. Sunter, and D. M. Bisaro
Functional Modulation of the Geminivirus AL2 Transcription Factor and Silencing Suppressor by Self-Interaction
J. Virol.,
November 1, 2007;
81(21):
11972 - 11981.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Gammelgard, M. Mohan, and J. P. T. Valkonen
Potyvirus-induced gene silencing: the dynamic process of systemic silencing and silencing suppression
J. Gen. Virol.,
August 1, 2007;
88(8):
2337 - 2346.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. V. Doukhanina, N. R. Apuya, H.-D. Yoo, C.-Y. Wu, P. Davidow, S. Krueger, R. B. Flavell, R. Hamilton, and S. C. Bobzin
Expression of Human Nuclear Receptors in Plants for the Discovery of Plant-Derived Ligands
J Biomol Screen,
April 1, 2007;
12(3):
385 - 395.
[Abstract]
[PDF]
|
|
|
|
|
|
|
A. Itaya, X. Zhong, R. Bundschuh, Y. Qi, Y. Wang, R. Takeda, A. R. Harris, C. Molina, R. S. Nelson, and B. Ding
A Structured Viroid RNA Serves as a Substrate for Dicer-Like Cleavage To Produce Biologically Active Small RNAs but Is Resistant to RNA-Induced Silencing Complex-Mediated Degradation
J. Virol.,
March 15, 2007;
81(6):
2980 - 2994.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Varrelmann, E. Maiss, R. Pilot, and L. Palkovics
Use of pentapeptide-insertion scanning mutagenesis for functional mapping of the plum pox virus helper component proteinase suppressor of gene silencing
J. Gen. Virol.,
March 1, 2007;
88(3):
1005 - 1015.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Goytia, L. Fernandez-Calvino, B. Martinez-Garcia, D. Lopez-Abella, and J. J. Lopez-Moya
Production of plum pox virus HC-Pro functionally active for aphid transmission in a transient-expression system.
J. Gen. Virol.,
November 1, 2006;
87(Pt 11):
3413 - 3423.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Valli, A. M. Martin-Hernandez, J. J. Lopez-Moya, and J. A. Garcia
RNA Silencing Suppression by a Second Copy of the P1 Serine Protease of Cucumber Vein Yellowing Ipomovirus, a Member of the Family Potyviridae That Lacks the Cysteine Protease HCPro.
J. Virol.,
October 1, 2006;
80(20):
10055 - 10063.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Z. Sh. Zhou, M. Dell'Orco, P. Saldarelli, C. Turturo, A. Minafra, and G. P. Martelli
Identification of an RNA-silencing suppressor in the genome of Grapevine virus A.
J. Gen. Virol.,
August 1, 2006;
87(Pt 8):
2387 - 2395.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
K. Gopinath, B. Dragnea, and C. Kao
Interaction between Brome Mosaic Virus Proteins and RNAs: Effects on RNA Replication, Protein Expression, and RNA Stability
J. Virol.,
November 15, 2005;
79(22):
14222 - 14234.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. I. Lukhovitskaya, N. E. Yelina, A. A. Zamyatnin Jr, M. V. Schepetilnikov, A. G. Solovyev, M. Sandgren, S. Yu. Morozov, J. P. T. Valkonen, and E. I. Savenkov
Expression, localization and effects on virulence of the cysteine-rich 8 kDa protein of Potato mop-top virus
J. Gen. Virol.,
October 1, 2005;
86(10):
2879 - 2889.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
X. Cui, G. Li, D. Wang, D. Hu, and X. Zhou
A Begomovirus DNA{beta}-Encoded Protein Binds DNA, Functions as a Suppressor of RNA Silencing, and Targets the Cell Nucleus
J. Virol.,
August 15, 2005;
79(16):
10764 - 10775.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Wang, K. J. Buckley, X. Yang, R. C. Buchmann, and D. M. Bisaro
Adenosine Kinase Inhibition and Suppression of RNA Silencing by Geminivirus AL2 and L2 Proteins
J. Virol.,
June 15, 2005;
79(12):
7410 - 7418.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
Z. Merai, Z. Kerenyi, A. Molnar, E. Barta, A. Valoczi, G. Bisztray, Z. Havelda, J. Burgyan, and D. Silhavy
Aureusvirus P14 Is an Efficient RNA Silencing Suppressor That Binds Double-Stranded RNAs without Size Specificity
J. Virol.,
June 1, 2005;
79(11):
7217 - 7226.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. F. Kreuze, E. I. Savenkov, W. Cuellar, X. Li, and J. P. T. Valkonen
Viral Class 1 RNase III Involved in Suppression of RNA Silencing
J. Virol.,
June 1, 2005;
79(11):
7227 - 7238.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. E. Yelina, T. N. Erokhina, N. I. Lukhovitskaya, E. A. Minina, M. V. Schepetilnikov, D.-E. Lesemann, J. Schiemann, A. G. Solovyev, and S. Yu. Morozov
Localization of Poa semilatent virus cysteine-rich protein in peroxisomes is dispensable for its ability to suppress RNA silencing
J. Gen. Virol.,
February 1, 2005;
86(2):
479 - 489.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Kawai-Toyooka, C. Kuramoto, K. Orui, K. Motoyama, K. Kikuchi, T. Kanegae, and M. Wada
DNA Interference: a Simple and Efficient Gene-Silencing System for High-Throughput Functional Analysis in the Fern Adiantum
Plant Cell Physiol.,
November 15, 2004;
45(11):
1648 - 1657.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. C. Canizares, K. M. Taylor, and G. P. Lomonossoff
Surface-exposed C-terminal amino acids of the small coat protein of Cowpea mosaic virus are required for suppression of silencing
J. Gen. Virol.,
November 1, 2004;
85(11):
3431 - 3435.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. J. Chapman, A. I. Prokhnevsky, K. Gopinath, V. V. Dolja, and J. C. Carrington
Viral RNA silencing suppressors inhibit the microRNA pathway at an intermediate step
Genes & Dev.,
May 15, 2004;
18(10):
1179 - 1186.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Limpens, J. Ramos, C. Franken, V. Raz, B. Compaan, H. Franssen, T. Bisseling, and R. Geurts
RNA interference in Agrobacterium rhizogenes-transformed roots of Arabidopsis and Medicago truncatula
J. Exp. Bot.,
May 1, 2004;
55(399):
983 - 992.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Miki and K. Shimamoto
Simple RNAi Vectors for Stable and Transient Suppression of Gene Function in Rice
Plant Cell Physiol.,
April 15, 2004;
45(4):
490 - 495.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Bucher, H. Hemmes, P. de Haan, R. Goldbach, and M. Prins
The influenza A virus NS1 protein binds small interfering RNAs and suppresses RNA silencing in plants
J. Gen. Virol.,
April 1, 2004;
85(4):
983 - 991.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Senda, C. Masuta, S. Ohnishi, K. Goto, A. Kasai, T. Sano, J.-S. Hong, and S. MacFarlane
Patterning of Virus-Infected Glycine max Seed Coat Is Associated with Suppression of Endogenous Silencing of Chalcone Synthase Genes
PLANT CELL,
April 1, 2004;
16(4):
807 - 818.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. Tzfira and V. Citovsky
The Agrobacterium-Plant Cell Interaction. Taking Biology Lessons from a Bug
Plant Physiology,
November 1, 2003;
133(3):
943 - 947.
[Full Text]
[PDF]
|
|
|
|
|
|
|
Z. Lichner, D. Silhavy, and J. Burgyan
Double-stranded RNA-binding proteins could suppress RNA interference-mediated antiviral defences
J. Gen. Virol.,
April 1, 2003;
84(4):
975 - 980.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Bucher, T. Sijen, P. de Haan, R. Goldbach, and M. Prins
Negative-Strand Tospoviruses and Tenuiviruses Carry a Gene for a Suppressor of Gene Silencing at Analogous Genomic Positions
J. Virol.,
December 20, 2002;
77(2):
1329 - 1336.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
F. Qu, T. Ren, and T. J. Morris
The Coat Protein of Turnip Crinkle Virus Suppresses Posttranscriptional Gene Silencing at an Early Initiation Step
J. Virol.,
December 6, 2002;
77(1):
511 - 522.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. E. Yelina, E. I. Savenkov, A. G. Solovyev, S. Y. Morozov, and J. P. T. Valkonen
Long-Distance Movement, Virulence, and RNA Silencing Suppression Controlled by a Single Protein in Hordei- and Potyviruses: Complementary Functions between Virus Families
J. Virol.,
November 13, 2002;
76(24):
12981 - 12991.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. C. Mallory, B. J. Reinhart, D. Bartel, V. B. Vance, and L. H. Bowman
From the Cover: A viral suppressor of RNA silencing differentially regulates the accumulation of short interfering RNAs and micro-RNAs in tobacco
PNAS,
November 12, 2002;
99(23):
15228 - 15233.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. I. Prokhnevsky, V. V. Peremyslov, A. J. Napuli, and V. V. Dolja
Interaction between Long-Distance Transport Factor and Hsp70-Related Movement Protein of Beet Yellows Virus
J. Virol.,
October 2, 2002;
76(21):
11003 - 11011.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. Llave, K. D. Kasschau, M. A. Rector, and J. C. Carrington
Endogenous and Silencing-Associated Small RNAs in Plants
PLANT CELL,
July 1, 2002;
14(7):
1605 - 1619.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
S. Pfeffer, P. Dunoyer, F. Heim, K. E. Richards, G. Jonard, and V. Ziegler-Graff
P0 of Beet Western Yellows Virus Is a Suppressor of Posttranscriptional Gene Silencing
J. Virol.,
June 5, 2002;
76(13):
6815 - 6824.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
