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

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.