Improved Classical Dialysis, Optimized Expression And Functional Identification Of Blumeria Effectors

In present study, Blumeria effectors (BEs) defined as some small pathogenic proteins come from Blumeria graminis f. sp. Hordei, which causes powdery mildew in barley and other Hordeum species. Through researching host-pathogen (barley-Blumeria) interactions, some important BEs, namely BE1011, BE1054, BE1019and BE1005, were cloned and expressed for preliminary structural analysis by previous researchers. However, all heterologous recombinant BEs were only (or mostly) expressed in inclusion bodies, which is very difficult to work with. To solve this problem, we applied some protein refolding methods (e.g. dilution, dialysis, ultrafiltration, etc.) to obtain native refolded BEs. We, therefore, have successfully developed a protein refolding method called’ centrifugal ultrafiltration-dialysis’ which is capable of resolubilizing all insoluble BEs from inclusion bodies. Based on the results of this study, we have gained a preliminary structure of BE1054by1D-NMR. This study also shows that BE1011seem to be absorbed by the mesophyll cells of barley.