A Viral Long Non-Coding RNA Modulates Viral RNA Silencing Suppressor and DCL4-Associated DRB4 Protein Interaction

Area of Science:

• Plant virology
• Molecular biology
• RNA silencing

Background:

• The Beet necrotic yellow vein virus (BNYVV) utilizes a viral silencing suppressor (VSR) protein, p14, and a viral non-coding RNA (ncRNA3) to facilitate long-distance movement within its host, Beta species.
• RNA silencing pathways are crucial for plant defense against viruses, and VSRs are key viral factors that interfere with these mechanisms.

Purpose of the Study:

• To elucidate the molecular mechanisms by which the BNYVV p14 VSR and ncRNA3 mediate viral long-distance movement and RNA silencing suppression.
• To identify cellular partners of the p14 protein involved in these processes.

Main Methods:

• Immunoprecipitation of Flag-p14 and TurboID-p14 expression followed by mass spectrometry to identify p14 interacting proteins.
• Analysis of p14-DRB4 interactions using a hypomorphic p14 mutant (p14BA2) under different conditions (presence/absence of ncRNA3).
• Subcellular fractionation of infected plant tissues to determine the localization of ncRNA3 and genomic RNA3.

Main Results:

• Several p14 interacting proteins were identified, including SGS3 (involved in siRNA transitivity) and DRB4 (a DCL4 cofactor).
• The interaction between p14 and DRB4 was observed in nucleoli and disrupted by ncRNA3, correlating with viral movement.
• ncRNA3 was found to accumulate in the nucleus via p14 interaction, while genomic RNA3 remained cytoplasmic.

Conclusions:

• BNYVV ncRNA3 is actively transported to the nucleus through interaction with p14, where it contributes to silencing suppression by destabilizing the p14-DRB4 complex.
• The interaction between p14 and SGS3 provides a mechanistic basis for the VSR's role in promoting RNA silencing transitivity, enhancing viral spread.