# Potato Virus Y NIb Multifunctional Protein Suppresses Antiviral Defense by Interacting with Several Protein Components of the RNA Silencing Pathway

## TL;DR

This study shows that the NIb protein from potato virus Y suppresses the plant's antiviral defenses by interacting with key proteins in the RNA silencing pathway.

## Contribution

The study identifies a new nuclear localization signal in NIb and shows its role in RNA silencing suppression.

## Key findings

- NIb suppresses RNA silencing and requires nuclear localization for this function.
- NIb interacts with four RNA silencing proteins: AGO4, HSP70, HSP90, and SGS3.
- Degradation products of AGO4, HSP70, and SGS3 are absent in NIb mutants that do not suppress RNA silencing.

## Abstract

Potyvirus genomes are expressed as a single large open reading frame, which is translated into a polyprotein that is post-translationally cleaved by three virus-encoded proteases into 10 functional proteins. Several of these potyviral proteins, including nuclear inclusion protein b (NIb), are multifunctional. Here, using the classic GFP silencing in Nicotiana benthamiana gfp-transgenic plants, we show that potato virus Y (PVY) NIb, in addition to its canonical role as the viral RNA-dependent RNA polymerase (RdRP), functions as a suppressor of RNA silencing. Mutational analyses reveal a previously unreported NIb nuclear localization signal (NLS) consisting of a triple-lysine motif. NIb suppression of RNA silencing activity was lost when the NLS was mutated, suggesting that nuclear localization is required for NIb suppression of RNA silencing activity. Analysis of sequenced GFP siRNAs revealed three reproducible hotspot regions at ≈175 nt, ≈320–330 nt, and a broader 3′-proximal region spanning ≈560–700 nt that contains multiple local maxima. These data show differences in the positional distribution of siRNAs between samples expressing NIb and those expressing NIbDel3×2, the NIb null mutant that does not suppress RNA silencing. However, the positional distribution of GFP-derived small RNAs across the transgene differed modestly between NIb and NIbDel3×2, while both treatments showed the same three reproducible hotspot regions. Furthermore, NIb was found to interact with four key RNA silencing pathway proteins—AGO4, HSP70, HSP90, and SGS3. Except for HSP90, each of these proteins showed degradation products that were absent in NIb mutants that did not suppress RNA silencing. These findings support a role for NIb in countering host defense during virus infection.