Localized cell wall thickenings, so called papillae, are a common plant defense response to fungal attack at sites of penetration of the plant cell. The major constituent of papillae is callose, a (1,3)-β-glucan polymer, which contributes to slowing or blocking the invading fungal hyphae. In the model plant Arabidopsis thaliana, we could recently show that the overexpression of PMR4(POWDERY MILDEW RESITANT 4), which encodes a stress induced callose synthase, results in complete powdery mildew resistance. To evaluate if these findings are also transferable to monocot crops, we transiently expressed PMR4 under control of the 35S promoter in leaves of barley (Hordeum vulgare) seedlings, which were subsequently inoculated with the virulent powdery mildew Blumeria graminis f. sp. hordei. Fusion of the green fluorescent protein (GFP) to PMR4 allowed the identification of successfully transformed barley cells, which showed an increased penetration resistance to B. graminis compared to control cells that express only GFP.PMR4-GFP localized in a similar pattern at the site of attempted fungal penetration as observed inA. thaliana, which suggests that similar transport mechanisms of the callose synthase might exist in dicot and monocot plants.