Potato late blight, caused by the oomycete pathogen Phytophthora infestans, is one of the most destructive plant diseases. Despite decades of intensive breeding efforts, it remains a threat to potato production worldwide, because newly evolved pathogen strains have overcome major resistance genes quickly. The RB protein, from the diploid wild potato species Solanum bulbocastanum, confers partial resistance to most P. infestans strains through its recognition of members of the corresponding pathogen effector protein family IPI-O. IPI-O comprises a multigene family and while some variants are recognized by RB to elicit host resistance (e.g., IPI-O1 and IPI-O2), others are able to elude detection (e.g., IPI-O4). IPI-O1 is almost ubiquitous in global P. infestans strains while IPI-O4 is more rare. No direct experimental evidence has been shown to demonstrate the effect of IPI-O on pathogen virulence in the P. infestans-potato pathosystem. Here, our work has demonstrated that in planta expression of both IPI-O1 and IPI-O4 increases P. infestans aggressiveness resulting in enlarged lesions in potato leaflets. We have previously shown that IPI-O4 has gained the ability to suppress the hypersensitive response induced by IPI-O1 in the presence of RB. In this study, our work has shown that this gain-of-function of IPI-O4 does not compromise its virulence effect, as IPI-O4 overexpression results in larger lesions than IPI-O1. We have also found that higher expression of IPI-O effectors correlates with enlarged lesions, indicating that IPI-O can contribute to virulence quantitatively. In summary, this study has provided accurate and valuable information on IPI-O's virulence effect on the potato host.