Strawberry mottle virus (SMoV) significantly compromises strawberry fruit quality and yield. The Pro2Glu protein of SMoV, an RNA silencing suppressor, is pivotal for viral infection
however, its interactions with host factors remain underexplored. This study identifies a critical interaction between SMoV Pro2Glu and strawberry NADH dehydrogenase (ubiquinone) 1 β subcomplex subunit 9 (FaNDUFB9). Using yeast two-hybrid assays and in vivo protein interaction analyses, we confirm that amino acid residues 31-45 of FaNDUFB9 are essential for binding Pro2Glu. Sequence analysis places FaNDUFB9 within the Complex1_LYR_superfamily, and subcellular localisation reveals its presence in both the mitochondria and nucleus. Silencing of NbNDUFB9 in Nicotiana benthamiana elevated Pro2Glu expression, whereas co-expression of FaNDUFB9 with Pro2Glu suppressed Pro2Glu levels and weakened its silencing activity. Furthermore, overexpression of FaNDUFB9 in transgenic N. benthamiana reduced transient SMoV RNA expression. These findings suggest that FaNDUFB9 inhibits expression of Pro2Glu, affecting its silencing activity, and thereby potentially further influencing viral infection.