Antiviral ARGONAUTEs Against <em>Turnip Crinkle Virus</em> Revealed by Image-Based Trait Analysis^{<a href="#fn2" rid="fn2" class=" fn">1</a>,}^{<a href="#fn3" rid="fn3" class=" fn">[OPEN]</a>}

Abstract

Plants can protect themselves against invasive virus infection through RNA silencing by targeting viral RNA for degradation (Agius et al., 2012). This host silencing machinery is triggered by viral double-stranded RNAs, which are cleaved by Dicer-like (DCL) nucleases associated with double-stranded RNA binding proteins into 21–24-nucleotide RNA duplexes called “viral small interfering RNAs” (vsiRNAs). The vsiRNAs are then methylated and stabilized by HUA enhancer1. One strand of these stabilized vsiRNAs is recruited into the RNA-induced silencing complex containing ARGONAUTE (AGO) proteins, and then serves as the sequence-specific guide for specific AGOs to slice cognate viral RNAs (Bologna and Voinnet, 2014; Fang and Qi, 2016).

Most plant viruses have evolved to encode viral suppressors of RNA silencing (VSRs) that use varied mechanisms to target components in the silencing pathway (Incarbone and Dunoyer, 2013). One such mechanism is interference of AGOs that mediate antiviral silencing. Accumulating evidence indicates that various VSRs use diverse modes of action on AGO proteins, such as promoting AGO degradation (Pazhouhandeh et al., 2006; Baumberger et al., 2007; Bortolamiol et al., 2007; Chiu et al., 2010), inhibiting the slicing activities of AGOs (Zhang et al., 2006), or interfering with factors upstream of AGO activity such as RNA-dependent RNA polymerase-dependent silencing (Fang et al., 2016), obstructing siRNA-loaded RNA-induced silencing complex activity (Giner et al., 2010; Kenesi et al., 2017), or indirectly repressing AGO protein level (Várallyay et al., 2010). Because functional VSRs can mask host antiviral silencing effects, VSR-defective mutant viruses that can only successfully infect immunocompromised plants have been constructed to identify antiviral roles of key components in the silencing pathway during virus infection (Qu et al., 2008; Garcia-Ruiz et al., 2015).

The Arabidopsis (Arabidopsis thaliana) genome encodes 10 AGO proteins, several of which have demonstrated antiviral roles (Carbonell and Carrington, 2015). AGO1 (Qu et al., 2008; Wang et al., 2011; Dzianott et al., 2012; Kontra et al., 2016) and AGO2 (Harvey et al., 2011; Jaubert et al., 2011; Scholthof et al., 2011; Garcia-Ruiz et al., 2015) have been identified as prominent antiviral AGOs against several RNA viruses, whereas other AGOs, including AGO5, AGO7, and AGO10, have limited antiviral roles in some cases (Qu et al., 2008; Garcia-Ruiz et al., 2015). For DNA viruses, AGO4 is the main effector protein in the antiviral silencing machinery (Raja et al., 2008, 2014). The full complement of AGOs with roles in antiviral defense, and how distinct antiviral AGOs are coordinated to silence different viruses in different tissues, remains to be fully determined.

Turnip crinkle virus (TCV) is a positive single-stranded RNA virus belonging to the Carmovirus genus of the Tombusviridae family. The TCV genome encodes five proteins, including two replicase proteins (P28 and P88), two movement proteins (P8 and P9), and the coat protein (CP; P38). The CP is multifunctional, as it has roles in virus movement (Cohen et al., 2000; Cao et al., 2010), serves as a virulence factor (Donze et al., 2014), and functions as a VSR to suppress antiviral silencing (Thomas et al., 2003; Chen et al., 2014). Other virus groups in the Tombusviridae encode separate VSR proteins, such as P19 from Tomato bushy stunt virus (Kontra et al., 2016). TCV systemically infects the susceptible Arabidopsis ecotype Columbia-0 (Col-0), and causes disease symptoms that include severe chlorosis in leaves, stunted bolts, and reduced biomass. A previous study reported that replacement of a single amino acid residue in the TCV CP P38 (R130T) disrupts its VSR function without affecting other functions (Cao et al., 2010). The VSR-deficient TCV is unable to suppress the host antiviral silencing machinery, leading to a lack of disease symptoms in wild-type plants postinoculation.

In this study, the roles of Arabidopsis AGO proteins in anti-TCV silencing were analyzed using genetic and image-based quantitative phenotyping approaches. Most previous pathological studies have relied on qualitative and subjective visual scoring systems to identify and assess disease phenotypes in plants (Bock et al., 2010). A machine learning method (Abbasi and Fahlgren, 2016) and other analysis tools in the open-source Plant Computer Vision (PlantCV) platform (Fahlgren et al., 2015; Gehan et al., 2017) were used to detect subtle, reproducible differences in disease symptoms over time.

Acknowledgments

Footnotes