Loss-of-function mutation of the MILDEW RESISTANCE LOCUS O (Mlo) gene confers durable and broad-spectrum resistance to powdery mildew fungi in various plants, including barley. In combination with the intracellular nucleotide-binding domain and leucine-rich repeat receptor (NLR) genes, which confer the race-specific resistance, the mlo alleles have long been used in barley breeding as genetic resources that confer robust non-race-specific resistance. However, a Japanese Blumeria graminis f. sp. hordei isolate, RACE1, has been reported to have the potential to overcome partially the mlo-mediated penetration resistance, although this is yet uncertain because the putative effects of NLR genes in the tested accessions have not been ruled out. In this study, we examined the reproducibility of the earlier report and found that the infectious ability of RACE1, which partially overcomes the mlo-mediated resistance, is only exerted in the absence of NLR genes recognizing RACE1. Furthermore, using the transient-induced gene silencing technique, we demonstrated that RACE1 can partially overcome the resistance in the host cells with suppressed MLO expression but not in plants possessing the null mutant allele mlo-5.