RNA Silencing and its Suppressors in the Plant‐virus Interplay

Abstract

Viruses are obligate intracellular parasites that need to evade the host defences targeted against them. In plants, activation of ribonucleic acid (RNA) silencing is among the most powerful responses against viruses. To overcome this defence, both deoxyribonucleic acid (DNA) and RNA plant viruses make use of viral products able to interfere with silencing mechanisms, and known collectively as RNA silencing suppressors (RSSs). RSSs present an extraordinary diversity in modes of action, and therefore, their study is providing valuable information about the different silencing pathways in plants. This article reviews our knowledge on RSSs, including the experimental systems used to identify them, and the mechanisms by which they act. Finally, the additional effects that they cause in the network of silencing pathways, and hence the need of a tight control of their activity, are also considered. Key concepts: Argonaute: Family of proteins that contain certain conserved domains, such as PIWI domain (found in Piwi, or P‐element‐induced wimpy testis in Drosophila , and related proteins) and PAZ domain (found in Piwi, Argonaute and Zwille proteins). Proteins of this family are the main components of RNA silencing effector complexes. Dicer: Ribonuclease belonging to the RNase III family. Dicer‐like enzymes cleave hairpin or double‐strand RNA molecules into short RNA duplexes of 20–24 nucleotides with a 2‐nucleotides overhang at the 3′ ends, which are key components of the RNA silencing pathways. microRNA (miRNA): Endogenous single‐strand RNA of 20–21 nucleotides, which derives from a hairpin precursor. miRNAs are incorporated into RISC, guiding it, by base complementary, to silence endogenous mRNAs. RNA‐dependent RNA polymerase (RdRP, RDR): Enzyme able to synthesize RNA using RNA as template. These enzymes are involved in amplification steps of RNA silencing. RNA‐induced silencing complex (RISC): Multicomponent complex including an Argonaute protein and a short RNA. RISC can silence mRNAs by endonucleolytic cleavage, decay acceleration and translation inhibition. RNA‐induced transcriptional silencing complex (RITS): Multicomponent complex including an Argonaute protein and a short RNA that silence gene expression by inhibiting or downregulating the transcription activity. RNA silencing: General term used to describe different events triggered by small RNAs to induce transcriptional or posttranscriptional gene downregulation in a sequence‐homology manner. RNA silencing suppressor (RSS): Factor (usually a protein) with the capacity to interfere with the onset of RNA silencing or with its maintenance. The expression of viral RSSs is the most common strategy that plant viruses use to escape from RNA silencing. Short interfering RNA (siRNA): Small RNA of 20–24 nucleotides derived from Dicer‐mediated cleavage of a double‐stranded RNA precursor. One of the strands of the siRNA is incorporated in RISC or RITS to guide their RNA silencing effector activities.

El virus del manchado foliar de los cítricos: caracterización del promotor del RNA subgenómico del gen de la proteína de la cápsida y del supresor del silenciamiento de RNA

“…En los últimos años se han descubierto distintas funciones en las que está implicado el silenciamiento génico mediado por RNA y que consisten en: i) control de la expresión de genes en momentos clave del desarrollo y proliferación (Carthew, 2006;Chapman y Carrington, 2007;Jones-Rhoades et al, 2006;, ii) defensa frente a virus, transposones y transgenes (Voinnet, 2005a;Li y Ding, 2006;Valli et al, 2009), y iii) regulación de la estabilidad del genoma (Baulcombe, 2004;Lippman y Martienssen, 2004;Wassengger, 2005;Vaucheret, 2006;Rodríguez-Campos y Azorín, 2007). El silenciamiento puede ocurrir tanto en el núcleo como en el citoplasma de las células (Matzke y Birchler, 2005).…”

“…Supresores del silenciamiento de virus de plantas (adaptada de Valli et al, 2009). (Anandalakshmi et al, 2000;Anandalakshmi et al, 1998; y Carrington, 1998;Lakatos et al, 2006;Mérai et al, 2006) (Brigneti et al,1998;Goto et al, 2007; ( Chen et al, 2008ª;Li et al, 1999) (Chapman et al, 2004;Reed et al, 2003;Ye y Patel, 2005) CMV (Reed et al, 2003) (Yaegashi et al, 2007) ( Chiba et al, 2006;Zhou et al, 2006) ( Baumberger et al, 2007;Bortolamiol et al, 2007;Pazhouhandeh et al, 2006;Pfeffer et al, 2002) (Pfeffer et al, 2002) (Pfeffer et al, 2002) (Valli et al, 2009) (Brigneti et al,1998) Virus RNA de cadena positiva Bromoviridae (Anandalakshmi et al, 2000;Anandalakshmi et al, 1998; y Carrington, 1998;Lakatos et al, 2006;Mérai et al, 2006) (Brigneti et al,1998;Goto et al, 2007; ( Chen et al, 2008ª;Li et al, 1999) (Chapman et al, 2004;Reed et al, 2003;Ye y Patel, 2005) CMV (Reed et al, 2003) (Yaegashi et al, 2007) ( Chiba et al, 2006;Zhou et al, 2006) ( Baumberger et al, 2007;…”

“…El mecanismo molecular de la supresión del silenciamiento por proteínas se ha determinado en un número limitado de virus pero se sabe que es muy específico y que actúa a distintos niveles de la ruta del silenciamiento (Valli et al, 2009). Algunos supresores boquean el proceso en etapas clave, ya que afectan a la actividad de Dicer o RISC, sin embargo otros actúan en etapas menos importantes (Takeda et al, 2002;Ding y Voinnet, 2007).…”

“…Para determinar si las proteínas del virus expresadas directamente desde su genoma eran capaces de bloquear el silenciamiento intracelular y/o a larga distancia, se realizaron dos experimentos; el primero está basado en la agroinfiltración simultánea en una hoja con el gen de la GFP y el clon infeccioso del genoma completo de CLBV (IC-CLBV) (Vives et al, 2008) y el segundo está basado en la estrategia de la rotura del silenciamiento inverso (Roth et al, 2004, Zhou et al, 2006Valli et al, 2009). …”

“…Se han descrito diferentes métodos para identificar proteínas de virus que actúan como supresoras del silenciamiento génico postranscripcional del huésped (Roth et al, 2004;Valli et al, 2009). El método más utilizado es el ensayo de expresión transitoria mediante infiltración con Agrobacterium (Voinnet et al, 2000;Johansen y Carrington, 2001).…”