印度梨形孢真菌與小白菜共生時之效應蛋白的分離與功能性探討

Abstract

印度梨形孢真菌(Piriformospora indica, P. indica)為一宿主範圍極廣且極具農業應用價值的共生真菌。近年研究發現，共生性微生物能分泌效應蛋白(effector protein)來調節植物的生理，建立與宿主的共生關係，並且帶給宿主益生效應。然而，其種類與機制上的研究至今極少。對P. indica 效應蛋白的研究有助於建立菌根菌與植物間共生機制的良好模型。因此，本研究的研究目的著重於P. indica 效應蛋白基因之篩選與鑑定，並且探討這些基因在P. indica 與植物交互作用時扮演的角色。首先透過生物資訊方法、雙重扣減式cDNA 基因庫與現有研究文獻進行候選基因的篩選，並且透過檢測共生階段的基因表現樣態與細胞次定位分析驗證預測的結果。本研究進一步針對PIIN_09643 與PIIN_11103 這兩個受到共生現象誘導、並且座落於植物細胞核的效應蛋白候選基因進行分析。我們藉由建立小白菜毛狀根轉殖系統與大腸桿菌純化蛋白系統，從基因與蛋白層次探討候選基因對植物生理與免疫反應的影響。在毛狀根系統中，PIIN_09643 與PIIN_11103 會造成水楊酸(SA) 路徑指標基因PR1 之表現顯著下降，並且促進P. indica 與植物共生。而茉莉酸路徑指標基因PDF1.2 之表現則沒有明顯受調控的現象。利用外加純化蛋白處理分析所得的結果中，我們也發現PIIN_09643 與PIIN_11103 對於PR1 與PDF1.2 的影響與毛狀根系統中的結果一致。我們推論P. indica 可能藉由PIIN_09643 與PIIN_11103 抑制植物SA 防禦反應，以建立與植物的共生關係。另外，在毛狀根系統中也發現PIIN_09643 與PIIN_11103 可以造成氧化還原相關基因DHAR5 與GSTU 的表達量顯著提升，我們推論這亦是因為SA 路徑被壓抑所間接造成之現象。

Piriformospora indica is a mutualistic endophyte with a broad host range and great application potential in agriculture. Recent studies indicate that, like pathogenic microbes, mutualistic microbes use effector proteins to adjust plant physiology resulting a promotion of symbiosis with plant. The study of P.indica effector proteins and their mechanism can be a great model for endophyte symbiosis. Consequently, the objective of our study is to establish a examine system for identification and functional characterization of P.indica effector proteins. Based on previous studies, double subtractive cDNA library and bioinformatics methods, we picked nine genes to exam their expression pattern during symbiosis stage and the subcellular localization in plant cell. PIIN_09643 and PIIN_11103 are both induced during symbiosis stage, and their encoded proteins have the subcellular localization in nucleus. For functional study of these putative effector genes, we established transgenic hairy-root system of Chinese cabbage and recombinant protein system of Escherichia coli to investigate the role of these genes during symbiosis. In hairyroot system, we found PIIN_09643 and PIIN_11103 significantly suppress the expression level of SA pathway marker gene, PR1; and cause a promotion of symbiosis colonization. However, for JA pathway marker gene, PIIN_09643 and PIIN_11103 do not directly regulate the expression level of PDF1.2. As the supporting data, the treatment of PIIN_09643 and PIIN_11103 recombinant protein to Chinese cabbage leaves show a similar effect on expression level of PR1 and PDF1.2. It suggests that P. indica apply PIIN_09643 and PIIN_11103 to suppress SA response, and further facilitate the symbiosis process in planta. Moreover, we found PIIN_09643 and PIIN_11103 can enhance the expression level of redox status relative genes, DHAR5 and GSTU. It might be the indirect result of the downregulation of SA pathway.