Interference of barley host cell death signaling by powdery mildew AVR-A effectors

Plants have evolved a variety of immune mechanisms, including a localized host cell death, to defend against microbes. Localized plant cell death is thought to be particularly effective against the spread of biotrophic plant pathogens, which rely on living host cells for proliferation. More recently, small nucleotide-based molecules were shown to be central regulators in immune-related cell death and resistance. The obligate biotrophic fungus Blumeria graminis f. sp. hordei (Bgh) causes the powdery mildew disease on barley leaves. During host infection, Bgh secrets hundreds of effectors, including the so-called AVR_A effectors, which are catalytically inactive RNase-like proteins. AVR_A effectors likely interfere with host cell death and immune signaling to promote Bgh virulence. One hypothesis is that that AVR_A effectors target immune-related nucleotide-based molecules. To test our hypotheses, we transiently expressed AVR_A effectors in barley protoplasts and treated the cells with several known cell death inducers to determine inhibition of cell death by the fungal effectors. We relied on a similar approach in stably transformed Arabidopsis thaliana to determine the conservation of cell death and effector function between monocots and dicots. As a first step, we could establish assays to quantitatively measure the cell death mediated by 2′-deoxyadenosine (dAdo), Bcl-2 associated X-protein (BAX), and the TIR domain of Recognition of Peronospora parasitica 1 (RPP1-TIR) in barley protoplasts.  Additionally, we demonstrated that the two Bgh effectors, AVR_A7-1 and AVR_A13-1, attenuate the dAdo-mediated cell death response of barley protoplasts. Furthermore, stable transgenic A. thaliana expressing AVR_a13-1 are less sensitive to dAdo treatment, indicating that the effector function is conserved between monocots and dicots. In the future, we will investigate whether the cell death inhibition is specific to AVR_A7-1 and AVR_A13-1 and if these results can be repeated in Nicotiana benthamiana.