the ca2+calcineurin-dependent signaling pathway in the gray mold botrytis cinerea the role of calcipressin in modulating calcineurin activity灰霉病的ca2 + calcineurin-dependent信号通路葡萄孢菌calcipressin调制钙调磷酸酶活动的作用.pdfVIP

The Ca2+/Calcineurin-Dependent Signaling Pathway in the Gray Mold Botrytis cinerea: The Role of Calcipressin in Modulating Calcineurin Activity Karin Harren, Julia Schumacher, Bettina Tudzynski* ¨ ¨ ¨ ¨ Westfalische Wilhelms-Universitat Munster, Institute of Biology and Biotechnology of Plants, Munster, Germany Abstract In the gray mold fungus Botrytis cinerea the Ga subunit Bcg1 of a heterotrimeric G protein is an upstream activator of the Ca2+/calmodulin-dependent phosphatase calcineurin. In this study we focused on the functional characterization of the catalytic subunit of calcineurin (BcCnA) and its putative regulator calcipressin (BcRcn1). We deleted the genes encoding both proteins to examine their role concerning growth, differentiation and virulence. The DbccnA mutant shows a severe growth defect, does not produce conidia and is avirulent, while the loss of BcRcn1 caused retardation of hyphal growth and delayed infection of host plants, but had no impact on conidiation and sclerotia formation. Expression of several calcineurin- dependent genes and bccnA itself is positively affected by BcRcn1. Complementation of the Dbcrcn1 mutant with a GFP- BcRcn1 fusion construct revealed that BcRcn1 is localized in the cytoplasm and accumulates around the nuclei. Furthermore, we showed that BcCnA physically interacts with BcRcn1 and the regulatory subunit of calcineurin, BcCnB. We investigated the impact of several protein domains characteristic for modulation and activation of BcCnA via BcRcn1, such as the phosphorylation sites and the calcineurin-docking site, by physical interaction studies between BcCnA and wild-type and mutated copies of BcRcn1. Based on the observed phenotypes we conclude that BcRcn1 acts as a positive modulator of BcCnA and the