The Conserved Guanyl‐Specific Ribonuclease T1 Effector EC19 Is Required for Full Virulence of the Vascular Wilt Fungus Fusarium oxysporum on Tomato

Plant fungal pathogens secrete a plethora of effectors into host cells to facilitate their infection by interfering with the normal physiological processes of the host plant. However, the role of guanyl-specific ribonuclease T1 effector protein in the virulence of fungal pathogens remains largely unexplored. Here, we show that an effector protein EC19, secreted by the soil-borne fungus Fusarium oxysporum f. sp. lycopersici (Fol) that causes tomato wilt disease, contributes to the full virulence of Fol. In-locus knockout of EC19 significantly reduced the virulence of Fol, while complementing EC19 in the knockout mutant restored the pathogenicity of Fol on tomato. EC19, which contains the ribonuclease T1-like domain, is highly conserved across fungal species and exhibited the ability to cleave single-stranded RNA isolated from tomato. EC19 was specifically induced during Fol infection. The AlphaFold3-predicted structure of EC19 shows a high degree of structural similarity with ribonuclease T1 from Aspergillus oryzae, with limited similarity to RNase T2. Using Agrobacterium-mediated transient assay in the leaves of Nicotiana benthamiana, we found that EC19 predominantly localised to the nucleus of plant cells. Stable transgenic susceptible tomato plants overexpressing EC19 exhibited increased susceptibility to Fol infection compared to wild-type plants. RNA-seq analysis of EC19-overexpressing tomato lines revealed that downregulated differentially expressed genes were predominantly enriched in plant hormone signal transduction, phenylpropanoid biosynthesis, and plant-pathogen interaction pathways. Taken together, these findings indicate that EC19 with ribonuclease activity promotes Fol infection, expanding our understanding of the molecular mechanisms underlying Fol virulence.