Characterization of the novel Cercospora beticola necrosis-inducing effector CbNIP10

Cercospora Leaf Spot (CLS), caused by the hemibiotrophic fungus Cercospora beticola, is the most destructive foliar disease of sugar beet worldwide. During infection, plant pathogens secrete effectors that help establish disease. Although many effectors have been characterized in other pathosystems, no C. beticola effector proteins have been reported to date. To identify C. beticola proteins involved with virulence, we grew C. beticola in vitro under specific conditions and tested culture filtrate for necrosis-inducing activity by infiltration into sugar beet leaves. Culture filtrate from one growth condition reliably caused necrosis in sugar beet leaves within 24 h. Treatment of culture filtrate with a mixture of proteases abolished necrosis-inducing activity, confirming that the C. beticola effector(s) responsible for necrosis was proteinaceous in nature. Active culture filtrates were partially purified using liquid chromatography (LC). A single (LC) fraction was repeatedly identified that caused necrosis upon infiltration into host tissue. MS/MS analysis of this fraction identified three C. beticola proteins. Each protein exhibited classic effector characteristics, including secretion signal, high cysteine content and low molecular weight (6 to 11 kDa). Candidate effector proteins were produced in Pichia pastoris. Infiltration of the candidate effector CbNIP10 caused necrosis in sugar beet leaves, while the other two effector candidates did not. A detailed characterization of CbNIP10 will be presented. Gaining an understanding of C. beticola effector biology will give new insight in C. beticola pathology and may lead to novel CLS control measures.