# Sclerotinia sclerotiorum growth and aggressiveness are regulated by a mycoviral REP protein

## TL;DR

A viral protein from a mycovirus reduces the growth and harmfulness of a plant fungus, offering a new way to control fungal diseases.

## Contribution

The study identifies a viral replication-associated protein (REP) as a key driver of fungal hypovirulence and disease suppression.

## Key findings

- The SsHADV1 REP protein directly restricts fungal growth and disease development.
- Exogenous application of REP protects plants from fungal infection and enhances fungicide effectiveness.
- The REP's function is conserved across unrelated viruses and depends on a functional ATPase domain.

## Abstract

The mechanisms through which mycoviruses reduce fungal growth and aggressiveness remain unclear, particularly regarding the viral factors involved and their modes of action. In this work, we investigated the hypovirulence mechanism by which Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV1), of the species Gemycircularvirus sclero1, impaired the necrotrophic plant fungus Sclerotinia sclerotiorum growth. We first identified the replication-associated protein (REP) of SsHADV1 as the key factor of hypovirulence. Using different patho-systems, we demonstrated that the viral SsHADV1 REP outside the context of viral infection directly restricts fungal growth and disease development. This slow growth was associated with a reduction in fungal oxalic acid production, which is an essential factor for fungal pathogenicity, and with an increase in fungal susceptibility to sublethal concentration of commercial fungicide. Additionally, the exogenous application of SsHADV1 REP protected sunflower plants from basal stem rot by S. sclerotiorum. In tobacco plants, the overexpression of SsHADV1 REP primes plant stress-related and immune responses, likely enhancing the hypovirulence activity against S. sclerotiorum. Hypovirulence was replicated using the REP of an unrelated ssDNA virus of S. sclerotiorum despite their overall low sequence identity. This conserved function of REP is structurally dependent and requires a functional ATPase domain. This study provides the first molecular insight on the mode of action of an hypovirulent mycovirus. It further establishes the ecological role of mycoviruses and their encoded proteins as potential drivers of fungal disease outcome and severity.

Hypovirulent mycoviruses offer a promising potential for developing sustainable bio-fungicides. We identified the replication-associated protein (REP) of Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV1) as a natural anti-fungal agent against economically significant fungal plant pathogens, suppressing its growth and aggressiveness. To reveal its biological function, the non-pathogenic, gram-positive rhizobacterium Bacillus subtilis was engineered to serve as a delivery platform for SsHADV1 REP. Notably, pre-treating sunflower plants with culture of REP-secreting bacteria prevented root rot infection by Sclerotinia sclerotiorum. Furthermore, the application increased effectiveness of low dose of boscalid fungicide on the fungus. Such function was replicated with the REP of an unrelated ssDNA virus of S. sclerotiorum that shares similar structure and with a functional ATPase domain. This work highlights the molecular effect of mycoviruses on the plant-fungus interaction and their potential ability to regulate disease outcome.