Adriana F. Fusaro

RNA interference‐inducing hairpin RNAs in plants act through the viral defence pathway

RNA interference (RNAi) is widely used to silence genes in plants and animals. It operates through the degradation of target mRNA by endonuclease complexes guided by approximately 21 nucleotide (nt) short interfering RNAs (siRNAs). A similar process regulates the expression of some developmental genes through approximately 21 nt microRNAs. Plants have four types of Dicer‐like (DCL) enzyme, each producing small RNAs with different functions. Here, we show that DCL2, DCL3 and DCL4 in Arabidopsis process both replicating viral RNAs and RNAi‐inducing hairpin RNAs (hpRNAs) into 22‐, 24‐ and 21 nt siRNAs, respectively, and that loss of both DCL2 and DCL4 activities is required to negate RNAi and to release the plants repression of viral replication. We also show that hpRNAs, similar to viral infection, can engender long‐distance silencing signals and that hpRNA‐induced silencing is suppressed by the expression of a virus‐derived suppressor protein. These findings indicate that hpRNA‐mediated RNAi in plants operates through the viral defence pathway.

The evolution and diversification of Dicers in plants.

Most multicellular organisms regulate developmental transitions by microRNAs, which are generated by an enzyme, Dicer. Insects and fungi have two Dicer‐like genes, and many animals have only one, yet the plant, Arabidopsis, has four. Examining the poplar and rice genomes revealed that they contain five and six Dicer‐like genes, respectively. Analysis of these genes suggests that plants require a basic set of four Dicer types which were present before the divergence of mono‐ and dicotyledonous plants (∼200 million years ago), but after the divergence of plants from green algae. A fifth type of Dicer seems to have evolved in monocots.

RNA silencing platforms in plants.

Since the discovery of RNAi, its mechanism in plants and animals has been intensively studied, widely exploited as a research tool, and used for a number of potential commercial applications. In this article, we discuss the platforms for delivering RNAi in plants. We provide a brief background to these platforms and concentrate on discussing the more recent advances, comparing the RNAi technologies used in plants with those used in animals, and trying to predict the ways in which RNAi technologies may further develop.

The Enamovirus P0 protein is a silencing suppressor which inhibits local and systemic RNA silencing through AGO1 degradation.

The P0 protein of poleroviruses and P1 protein of sobemoviruses suppress the plants RNA silencing machinery. Here we identified a silencing suppressor protein (SSP), P0(PE), in the Enamovirus Pea enation mosaic virus-1 (PEMV-1) and showed that it and the P0s of poleroviruses Potato leaf roll virus and Cereal yellow dwarf virus have strong local and systemic SSP activity, while the P1 of Sobemovirus Southern bean mosaic virus supresses systemic silencing. The nuclear localized P0(PE) has no discernable sequence conservation with known SSPs, but proved to be a strong suppressor of local silencing and a moderate suppressor of systemic silencing. Like the P0s from poleroviruses, P0(PE) destabilizes AGO1 and this action is mediated by an F-box-like domain. Therefore, despite the lack of any sequence similarity, the poleroviral and enamoviral SSPs have a conserved mode of action upon the RNA silencing machinery.

The Luteovirus P4 Movement Protein Is a Suppressor of Systemic RNA Silencing

The plant viral family Luteoviridae is divided into three genera: Luteovirus, Polerovirus and Enamovirus. Without assistance from another virus, members of the family are confined to the cells of the host plant’s vascular system. The first open reading frame (ORF) of poleroviruses and enamoviruses encodes P0 proteins which act as silencing suppressor proteins (VSRs) against the plant’s viral defense-mediating RNA silencing machinery. Luteoviruses, such as barley yellow dwarf virus-PAV (BYDV-PAV), however, have no P0 to carry out the VSR role, so we investigated whether other proteins or RNAs encoded by BYDV-PAV confer protection against the plant’s silencing machinery. Deep-sequencing of small RNAs from plants infected with BYDV-PAV revealed that the virus is subjected to RNA silencing in the phloem tissues and there was no evidence of protection afforded by a possible decoy effect of the highly abundant subgenomic RNA3. However, analysis of VSR activity among the BYDV-PAV ORFs revealed systemic silencing suppression by the P4 movement protein, and a similar, but weaker, activity by P6. The closely related BYDV-PAS P4, but not the polerovirus potato leafroll virus P4, also displayed systemic VSR activity. Both luteovirus and the polerovirus P4 proteins also showed transient, weak local silencing suppression. This suggests that systemic silencing suppression is the principal mechanism by which the luteoviruses BYDV-PAV and BYDV-PAS minimize the effects of the plant’s anti-viral defense.

Live Cell Imaging Reveals the Relocation of dsRNA Binding Proteins Upon Viral Infection

Viral infection triggers a range of plant responses such as the activation of the RNA interference (RNAi) pathway. The double-stranded RNA binding (DRB) proteins DRB3 and DRB4 are part of this pathway and aid in defending against DNA and RNA viruses, respectively. Using live cell imaging, we show that DRB2, DRB3, and DRB5 relocate from their uniform cytoplasmic distribution to concentrated accumulation in nascent viral replication complexes (VRC) that develop following cell invasion by viral RNA. Inactivation of the DRB3 gene in Arabidopsis by T-DNA insertion rendered these plants less able to repress RNA viral replication. We propose a model for the early stages of virus defense in which DRB2, DRB3, and DRB5 are invasion sensors that relocate to nascent VRC, where they bind to viral RNA and inhibit virus replication.