Abstract
A large body of evidence has lead to the suggestive proposal of a potential interplay between viroid-induced pathogenesis and RNA silencing regulatory mechanisms. A variety of techniques have been used to examine this interaction. This chapter outlines the use of a green fluorescent protein (GFP) sensor, containing viroid-specific sequence targets to study this phenomenon. In addition, a symptomatic transgenic line of Nicotiana benthamiana that expresses and processes Hop stunt viroid (HSVd) is used as stocks in grafting assays with the rdr6i-Nb lines, in which the RNA-dependent RNA polymerase 6 (NbRDR6) is constitutively silenced. The combination of the use of a viroid-specific GFP-sensor, the grafting assays with plants silenced for the RDR6 and the analysis of siRNAs allows to address how to monitor viroid-specific RNA silencing and how to associate this regulatory pathway with symptom expression.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Flores R, Delgado S, Gas ME, Carbonell A, Molina D, Gago S et al (2004) Viroids: the minimal non-coding RNAs with autonomous replication. FEBS Lett 567:42–48
Ding B, Itaya A (2007) Viroid: a useful model for studying the basic principles of infection and RNA biology. Mol Plant Microbe Interact 20:7–20
Tsagris EM, MartÃnez de Alba AE, Gozmanova M, Kalantidis K (2008) Viroids. Cell Microbiol 10:2168–2179
Flores R, Pallás V (2006) Viroids. In: Khan JA, Dijkstra J (eds) Handbook of plant virology. The Haworth Press Inc, New York, pp 93–104
Flores R, Hernández C, MartÃnez de Alba AE, Daròs JA, Di Serio F (2005) Viroids and viroid–host interactions. Annu Rev Phytopathol 43:117–139
Diener TO (1999) Viroids and the nature of viroid diseases. Arch Virol 15:203–220
Wang MB, Bian XY, Wu LM, Liu LX, Smith NA, Isenegger D et al (2004) On the role of RNA silencing in the pathogenicity and evolution of viroids and viral satellites. Proc Natl Acad Sci USA 101:3275–3280
Sänger H, Schiebel L, Riedel T, Pélissier T, Wassenegger M (1996) The possible link between RNA-directed DNA methylation, sense and antisense RNA, gene silencing, symptoms induction upon microbiological infection and RNA-dependent RNA polymerase. In: Stacey G et al (eds) Biology of plant–microbe interactions. pp 533–540
Wassenegger M, Heimes S, Riedel L, Sanger HL (1994) RNA-directed de novo methylation of genomics sequences in plants. Cell 76:567–576
Gómez G, Pallás V (2007) Mature monomeric forms of Hop stunt viroid resist RNA silencing in transgenic plants. Plant J 51:1041–1049
Itaya A, Folimonov A, Matsuda Y, Nelson RS, Ding B (2001) Potato spindle tuber viroid as inducer of RNA silencing in infected tomato. Mol Plant Microbe Interact 14:1332–1334
Itaya A, Zhong X, Bundschuh R, Qi Y, Wang Y, Takeda R et al (2007) A structured viroid RNA is substrate for Dicer-Like cleavage to produce biologically active small RNAs but is resistant to RISC-mediated degradation. J Virol 81:2980–2994
Markarian N, Li HW, Ding SW, Semancik JS (2004) RNA silencing as related to viroid induced symptom expression. Arch Virol 149:397–406
Matoušek J, Kozlová P, Orctová L, Schmitz A, Pesina K, Bannach O et al (2007) Accumulation of viroid-specific small RNAs and increase in nucleolytic activities linked to viroid-caused pathogenesis. Biol Chem 388:1–13
Sano T, Matsuura Y (2004) Accumulation of short interfering RNAs characteristics of RNA silencing precedes recovery of tomato plants from severe symptoms of Potato spindle tuber viroid infection. J Gen Plant Pathol 70:50–53
Gómez G, Martinez G, Pallas V (2009) Interplay between viroid-induced pathogenesis and RNA silencing pathways. Trends Plant Sci 14:264–269
Gómez G, Pallás V (2006) Hop stunt viroid is processed and translocated in transgenic Nicotiana benthamiana plants. Mol Plant Pathol 7:511–517
Martinez G, Pallás V, Gómez G (2008) Analysis of the symptoms developed in N. benthamiana plants expressing dimeric forms of the Hop stunt viroid. J. Plant Pathol 90:121–124
Schwach F, Vaistij FE, Jones L, Baulcombe DC (2005) An RNA-dependent RNA polymerase prevents meristem invasion by potato virus X and is required for the activity but not the production of a systemic silencing signal. Plant Physiol 138:1842–1852
Daròs JA, Flores R (2004) Arabidopsis thaliana has the enzymatic machinery for replicating representative viroid species of the family Pospiviroidae. Proc Natl Acad Sci USA 17:6792–6797
Hamilton CM, Frary A, Lewis C, Tanksley SD (1996) Stable transfer of intact high molecular weight DNA into plant chromosomes. Proc Natl Acad Sci USA 93:9975–9979
Acknowledgments
The work in our laboratory is supported by grant BIO2011-25018 from the Spanish granting agency Direccion General de Investigacion Cientifica y Tecnica and by grant PROMETEO 2001–003 from the Generalitat Valenciana. G.G. is the recipient of a contract from the Consejo Superior de Investigaciones Cientificas. G.M. is the recipient of a fellowship from the Ministry of Science and Innovation.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2012 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Pallas, V., Martinez, G., Gomez, G. (2012). The Interaction Between Plant Viroid-Induced Symptoms and RNA Silencing. In: Watson, J., Wang, MB. (eds) Antiviral Resistance in Plants. Methods in Molecular Biology, vol 894. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-882-5_22
Download citation
DOI: https://doi.org/10.1007/978-1-61779-882-5_22
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-61779-881-8
Online ISBN: 978-1-61779-882-5
eBook Packages: Springer Protocols