# A non-classical PUF family protein in oomycetes functions as a pre-rRNA processing regulator and a target for RNAi-based disease control

## TL;DR

A new RNA-binding protein in oomycetes is found to regulate ribosome production and can be targeted to control plant diseases.

## Contribution

The discovery of Puf4 as a non-classical PUF protein regulating pre-rRNA processing and its use in RNAi-based disease control in oomycetes.

## Key findings

- Puf4 knockout in oomycetes leads to impaired growth and reduced pathogenicity.
- Puf4 binds to H68 of 25S rRNA and regulates rRNA processing intermediates.
- A nano-material-free dsRNA delivery system targeting Puf4 effectively reduces oomycete virulence.

## Abstract

Ribosome biogenesis is an essential and tightly regulated process linked to cell proliferation and growth. However, its regulatory mechanisms in oomycetes, a group of organisms with significant agricultural and ecological importance, remain unclear. In this study, we identify Puf4, a non-classical PUF (Pumilio and FBF) family RNA-binding protein that plays a conserved and crucial role in pre-rRNA processing in oomycetes. Knockout of PuPuf4 in Pythium ultimum or its ortholog PsPuf4 in Phytophthora sojae results in defective vegetative growth, impaired development, and reduced pathogenicity. Specifically, PuPuf4 binds to the H68 component of 25S rRNA, and its knockout leads to overaccumulation of rRNA processing intermediates, including 5′ETS, ITS1, and ITS2 precursors. Additionally, the AG-rich motif identified as the first binding motif of L-shaped PUF proteins, including PuPuf4, APUM24, and ScPuf6, may contribute to their specific RNA-binding affinity due to its unique structural features. Given the conserved role of Puf4 in oomycete pathogenicity, we developed the first nano-material-free dsRNA delivery system via zoospore-specific uptake, effectively attenuating virulence in Pythium aphanidermatum and Ph. sojae through RNAi targeting Puf4. This study presents novel findings on structural and functional conservation of Puf4 and offers a promising RNAi-based strategy for controlling oomycete plant diseases.

Ribosome biogenesis plays a crucial role in cell growth and proliferation. However, the regulation of this process in oomycetes, an ecologically and agriculturally important group of organisms, remains largely unknown. In this study, we identified Puf4, a non-classical PUF family RNA-binding protein of oomycetes, as a key factor promoting ribosome assembly in these pathogenic organisms. When we removed Puf4 from two harmful oomycetes, Pythium ultimum and Phytophthora sojae, their growth was inhibited, and their ability to infect plants was diminished. Specifically, PuPuf4 binds to the H68 component of 25S rRNA and regulates the degradation of rRNA maturation byproducts. Additionally, we discovered a novel AG-rich motif that serves as a binding site for L-shaped PUF proteins. Furthermore, we developed a nano-material-free dsRNA delivery system targeting Puf4, which effectively reduced the virulence of Pythium and Phytophthora pathogens. Our findings not only uncover a fundamental process in oomycetes but also provide a sustainable approach to protect crops from these destructive microbes.