Molecular Evolution of a Viral Non-Coding Sequence under the Selective Pressure of amiRNA-Mediated Silencing

Abstract

Plant microRNAs (miRNA) guide cleavage of target mRNAs by DICER-like proteins, thereby reducing mRNA abundance. Native precursor miRNAs can be redesigned to target RNAs of interest, and one application of such artificial microRNA (amiRNA) technology is to generate plants resistant to pathogenic viruses. Transgenic Arabidopsis plants expressing amiRNAs designed to target the genome of two unrelated viruses were resistant, in a highly specific manner, to the appropriate virus. Here, we pursued two different goals. First, we confirmed that the 21-nt target site of viral RNAs is both necessary and sufficient for resistance. Second, we studied the evolutionary stability of amiRNA-mediated resistance against a genetically plastic RNA virus, TuMV. To dissociate selective pressures acting upon protein function from those acting at the RNA level, we constructed a chimeric TuMV harboring a 21-nt, amiRNA target site in a non-essential region. In the first set of experiments designed to assess the likelihood of resistance breakdown, we explored the effect of single nucleotide mutation within the target 21-nt on the ability of mutant viruses to successfully infect amiRNA-expressing plants. We found non-equivalency of the target nucleotides, which can be divided into three categories depending on their impact in virus pathogenicity. In the second set of experiments, we investigated the evolution of the virus mutants in amiRNA-expressing plants. The most common outcome was the deletion of the target. However, when the 21-nt target was retained, viruses accumulated additional substitutions on it, further reducing the binding/cleavage ability of the amiRNA. The pattern of substitutions within the viral target was largely dominated by G to A and C to U transitions.

MicroRNA396-Targeted SHORT VEGETATIVE PHASE Is Required to Repress Flowering and Is Related to the Development of Abnormal Flower Symptoms by the Phyllody Symptoms1 Effector

“…The pairwise fourth and fifth positions are reportedly critical for the efficiency of low miRNA-guided cleavage of the target RNA (Lin et al, 2009). We assumed that the two mismatches at the fourth and fifth positions of the pairwise region will yield differences in cleavage between miR396/AtSVP and miR396/AtGRF1.…”

“…The 59 RACE data indicated that the bulge structure did not affect miR396-mediated AtGRF cleavage. However, interaction along the 59 portion of the miRNA is the feature most relevant to its activity and cleavage efficiency (Mallory et al, 2004;Schwab et al, 2005;Lin et al, 2009), and modifying the pairing status of the 59 portion of miR396 through mutagenesis alters the efficiency of AtGRF2 cleavage (Debernardi et al, 2012). Moreover, Li et al (2013) demonstrated that a 59 miRNA mismatch triggers miRNA-mediated target RNA translation inhibition in Arabidopsis.…”

Effect of citrus hosts on the generation, maintenance and evolutionary fate of genetic variability of citrus exocortis viroid

“…The heterogeneity of these CEVd populations, composed of different haplotypes adopting slightly different secondary structures, could aim to confer a certain level of resistance towards the plant RNA silencing machinery. In fact, it has been recently demonstrated for turnip mosaic virus that substitutions in the target of artificial micro RNAs allow the virus to escape from RNA silencing (Lin et al, 2009). Therefore, the fact that the P domain appears to be the most variable one in the sour orange populations suggests that it is involved in the viroid defence process.…”

Antiviral Silencing and Suppression of Gene Silencing in Plants

“…Great advances have been made to develop methods for viral disease control with the expression of artificial sRNAs/miRNAs targeting viral genomes in economically important plants (Kis et al 2016;Lin et al 2009;Niu et al 2006). Similar biotechnological approaches may be very useful to elaborate in the future for economically important crop protection.…”