Westfälische Wilhelms-Universität Münster, Institute of Biology and Biotechnology of Plants, Münster, Germany.
PLoS One. 2012;7(7):e41761. doi: 10.1371/journal.pone.0041761. Epub 2012 Jul 23.
In the gray mold fungus Botrytis cinerea the Gα subunit Bcg1 of a heterotrimeric G protein is an upstream activator of the Ca(2+)/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 ΔbccnA 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 Δbcrcn1 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 Ca(2+)/calcineurin-mediated signal transduction in B. cinerea, and that both proteins regulate fungal development and virulence.
在灰霉菌 Botrytis cinerea 中，三聚体 G 蛋白的 Gα亚基 Bcg1 是钙调蛋白依赖性磷酸酶钙调神经磷酸酶的上游激活剂。在这项研究中，我们专注于钙调神经磷酸酶的催化亚基（BcCnA）及其假定的调节蛋白 calcipressin（BcRcn1）的功能表征。我们删除了编码这两种蛋白质的基因，以研究它们在生长、分化和毒力方面的作用。ΔbccnA 突变体表现出严重的生长缺陷，不产生分生孢子且无毒力，而 BcRcn1 的缺失导致菌丝生长延迟和宿主植物感染延迟，但对分生孢子形成和菌核形成没有影响。几种钙调神经磷酸酶依赖性基因的表达和 bccnA 本身受到 BcRcn1 的正向影响。用 GFP-BcRcn1 融合构建体对 Δbcrcn1 突变体进行互补，表明 BcRcn1 定位于细胞质中，并在核周围积累。此外，我们表明 BcCnA 与 BcRcn1 和钙调神经磷酸酶的调节亚基 BcCnB 发生物理相互作用。我们研究了通过 BcRcn1 对 BcCnA 的调节和激活具有特征的几个蛋白质结构域的影响，例如磷酸化位点和钙调神经磷酸酶结合位点，通过 BcCnA 与野生型和突变型 BcRcn1 之间的物理相互作用研究。基于观察到的表型，我们得出结论，BcRcn1 是 B. cinerea 中 BcCnA 和 Ca2+/钙调神经磷酸酶介导的信号转导的正调节剂，并且这两种蛋白质调节真菌的发育和毒力。
