The Tomato <scp>PR</scp> ‐5 Proteins <scp>PR</scp> ‐5x and <scp>NP24</scp> Exert Antifungal Activity Against <i>Fusarium oxysporum</i>

ABSTRACT Vascular wilt diseases, caused by the soilborne pathogen Fusarium oxysporum (Fo), significantly impact agriculture and horticulture. Traditional management strategies are often environmentally harmful and not effective once the pathogen has entered the host. Consequently, understanding the molecular mechanisms underlying resistance to pathogenic Fo strains is crucial for developing novel control strategies. In tomato, resistance to F. oxysporum f. sp. lycopersici (Fol) can be mediated by R genes, such as I‐1 , I‐2 or I‐3 , or by Fo endophytes. Fol inoculation of resistant lines results in high accumulation of pathogenesis‐related 5 (PR‐5) proteins. However, the role of xylem‐localised PR‐5 proteins in Fol resistance in tomato plants remains unknown. Here, we demonstrate that apoplastic fluid enriched with recombinant PR‐5x or NP24 exhibits antifungal activity against Fol. Using CRISPR/Cas9‐mediated gene editing and overexpression studies, the role of PR‐5x and NP24 in disease resistance was studied. Loss‐of‐function mutations in either PR‐5 isoform did not compromise Fol resistance. However, overexpression of NP24 in tomato plants resulted in decreased susceptibility to Fol and surprisingly enhanced susceptibility to Ralstonia solanacearum . The role of PR‐5x in Fol resistance remains undetermined as lines overexpressing the transgene could not be identified. Our findings suggest that PR‐5 proteins are involved in restricting Fol proliferation in the xylem vasculature and thereby contribute to disease resistance.