印度梨形孢真菌與小白菜共生時之效應蛋白功能性探討

Abstract

印度梨形孢真菌(Piriformospora indica)為一宿主範圍極廣且具農業應用價值的共生真菌。共生性微生物能產生效應蛋白(effector protein)進而調控植物的生理機制，促進建立與宿主之共生關係。鑑於P. indica廣大的宿主範圍，對P. indica效應蛋白的研究有助於建立內共生菌(endophyte)與植物間共生機制的模型。本研究著重於進行效應蛋白的功能鑑定，根據實驗室過去研究成果，已由生物資訊方法篩選出兩個P. indica效應蛋白基因PIIN_09643與PIIN_11103，本研究利用建立小白菜轉殖毛狀根、小白菜葉部農桿菌注射的短暫過量表現與轉殖阿拉伯芥的方式，驗證水楊酸路徑指標基因PR-1之表現顯著下降。為闡明效應蛋白候選基因在共生階段的功能，本研究藉由酵母菌雙雜合篩選實驗得到與效應蛋白PIIN_11103互相作用的小白菜阿拉伯半乳聚醣蛋白Arabinogalactan protein 4 (AGP4) 。阿拉伯半乳聚醣蛋白為高度醣基化的醣蛋白，多分佈於植物細胞膜上，參與細胞增生、程序性細胞凋亡、花粉管生長、體胚再生等各種功能。本研究發現BrAGP4位於細胞膜與葉綠體的被膜，利用雙分子螢光互補實驗也證實PIIN_11103與BrAGP4可以交互作用，並且坐落於葉綠體被膜。以處理AGPs抑制劑的方式了解AGPs能夠影響生長素相關基因之表現、抑制根毛的發育，推論AGPs對於P. indica與小白菜建立共生之過程中也扮演著重要角色。

Piriformospora indica is a mutualistic endophyte with a broad host range and application potential in agriculture. Mutualistic microbes are able to produce effector proteins to interfere plant physiology for faciliting symbiosis with plants. In consideration of P. indica’s broad host-range, studies of P. indica effector proteins can establish a model for endophyte symbiosis with plants. This study focuses on functional characterization of effector proteins. According to lab’s previous studies, we already screen out two P. indica’s effector genes, PIIN_09643 and PIIN_11103 by bioinformatics approaches. We established overexpression lines of Chinese cabbage hairy root system and transgenic Arabidopsis thaliana. We also do agroinfiltrantion for transient overespression in Chinese cabbage leaves to confirm suppression of salicylic acid signal transduction. To investigate effectors function, we establish cDNA library to undertake yeast two-hybrid screening experiment. We found B. rapa’s Arabinogalactan protein 4 (AGP4) would interact with PIIN_11103. Arabinogalactan proteins are highy glycosylated glycoproteins mostly localized on plasma membrane, involved in cell proliferation, programmed cell death, pollen-tube development and somatic embryogenesis. This study found that BrAGP4 localized on plasma membrane and chloroplast envelope, and confirmed interaction of BrAGP4 and PIIN_11103 by BiFC. To characterize BrAGP4, we treated Chinese cabbage with AGPs inhibitor, found the inhibitory effects on root hair development but up-regulateing of auxin-relaed gene expressions. We also found that AGPs play a critical role in symbiosis process of P. indica and Chinese cabbage.