Sundaram Sathish, Brand Joshua H, Hymes Matthew J, Hiremath Shivanand, Podila Gopi K
Department of Biological Sciences, Michigan Tech University, Houghton, MI 49931, USA.
Present address: Vattikudi Urology Institute, Henry Ford Medical Center, Detroit, MI 48202, USA.
New Phytol. 2004 Feb;161(2):529-538. doi: 10.1046/j.1469-8137.2003.00935.x. Epub 2003 Nov 24.
• A yeast two-hybrid library prepared from Laccaria bicolor × Pinus resinosa mycorrhizas was screened using a LbRAS clone, previously characterized, as a bait to isolate LbRAS interacting signaling-related genes from L. bicolor. • Using this method, a novel line of Ras-interacting yeast two-hybrid mycorrhizal (Rythm) clones were isolated and analysed for their symbiosis-regulation. One such clone identified (RythmA) had homology to Ap180-like vesicular proteins. • Sequence homology and parsimony-based phylogenetic analysis showed its relatedness to Ap180-like proteins from other systems. DNA analysis suggested that L. bicolor had one or two copies of the RythmA gene. • An RNA analysis showed that the expression of RythmA could be detected 36 h after interaction with the host, which follows the expression of Lbras. Immunolocalization of LbRAS near dolipore septum of the fungal cells in the Hartig net area suggests that RythmA protein may be involved in the transport of signaling proteins such as LbRAS.
• 使用先前鉴定的 LbRAS 克隆作为诱饵，筛选由双色蜡蘑×树脂松菌根制备的酵母双杂交文库，以从双色蜡蘑中分离与 LbRAS 相互作用的信号相关基因。
• 利用该方法，分离出了一系列新的与 Ras 相互作用的酵母双杂交菌根（Rythm）克隆，并对其共生调节进行了分析。其中一个鉴定出的克隆（RythmA）与 Ap180 样囊泡蛋白具有同源性。
• 序列同源性和基于简约法的系统发育分析表明，它与其他系统中的 Ap180 样蛋白相关。DNA 分析表明，双色蜡蘑有一个或两个 RythmA 基因拷贝。
• RNA 分析表明，与宿主相互作用 36 小时后可检测到 RythmA 的表达，这与 Lbras 的表达一致。在哈蒂氏网区域真菌细胞的桶孔隔膜附近对 LbRAS 进行免疫定位表明，RythmA 蛋白可能参与 LbRAS 等信号蛋白的转运。
