印度梨型孢真菌的效應蛋白對其宿主小白菜之功能性探討

Abstract

印度梨型孢真菌，為一內生性共生真菌且能與許多不同種類之植物建立共生，在實驗室過去的研究中發現，當小白菜與Piriformospora. indica建立共生後，會促進植物的生長及提高其對於生物與非生物性逆境之抵抗力。雖然已有許多報導指出P. indica與植物共生後對於植物具有多種效益，但對於其中的分子機制尚不瞭解，有鑑於此，本研究利用過去實驗室所建立之效應蛋白生物資訊篩選方法，從中鎖定了4個候選基因PIIN_04020, PIIN_04363, PIIN_07152及PIIN_08445，並利用小白菜毛狀根及過表現轉殖阿拉伯芥的方式，試圖找出效應蛋白可能之功能，另外亦透過酵母菌雙雜交試驗找到PIIN_04363及PIIN_07152的交互作用蛋白，分別為銅伴護蛋白(BrCCH)及穀胱苷肽S-轉移酶(BrGSTU24)。本研究發現，在P.indica與小白菜共生階段的早期便會大幅提高效應蛋白基因PIIN_04363之表現，並且將PIIN_04363過表現於毛狀根及阿拉伯芥中，可使水楊酸SA路徑的指標基因PR-1下降，且因而提高了P. indica與植物的共生效率。而在PIIN_07152方面，則在共生階段的晚期才提高表現，並且PIIN_07152於毛狀根及阿拉伯芥中過表現時， JA路徑指標基因LOX2及VSP2的表現量被提高，而氧化還原相關基因DHAR及MDAR之表現量亦有顯著的提升。最後透過觀察PIIN_04363、PIIN_07152及PIIN_08445的過表現轉殖阿拉伯芥7天大幼苗發現，PIIN_07152 OE的生長勢有較為遲緩的趨勢，推論可能是因為植株中JA路徑較高的表現抑制了植株的生長，另外在PIIN_08445 OE方面，發現其轉殖株幼苗時期的生長勢相較於EV control似乎有些許提升，推論該現象或許是因為生長素相關基因的提升導致。

Piriformospora indica, an endophytic root-colonizing fungus, interacts with various plants species. From our previous researches, Chinese cabbage root colonization by P. indica results in an increase in plant growth, root propagation and adaptation to abiotic stress. Although P. indica has been reported to colonize and cause various beneficial function on plants, little is known about its early colonizeal mechanism. Previously, we screened several putative effector genes of P. indica by the bioinformatics screening system established from previous researchers. Further to characterize the function of P. indica effector proteins, four candidate genes PIIN_04020, PIIN_04363, PIIN_07152 and PIIN_08445 were chosen to establish Chinese cabbage transgenic hairy-root system、transgenic Arabidopsis thaliana for further investigation. Yeast two hybrid screening were used to screen the target interacting proteins against the effector proteins. B. rapa’s copper chaperone (CCH) against PIIN_04363 and glutathione S-transferase U24 (GSTU24) against PIIN_07152 were found. In this study, we found that PIIN_04363 gene expression was upregulated in the early colonized stages. Transgenic hairy-root system and transgenic Arabidopsis thaliana showed that PIIN_04363 suppress the expression level of SA pathway marker gene, PR-1 and cause a promotion of colonization efficiency. PIIN_07152 enhances the expression level of JA pathway marker genes, LOX2 and VSP2. Redox status relative genes DHAR and MDAR are also upregulated. From the observation of 7days-old transgenic Arabidopsis thaliana, the growth rate of PIIN_07152 OE was retard. It suggests that it is caused by the up-regulation of JA pathway. On the other hand, 7days-old transgenic Arabidopsis thaliana of PIIN_08445 OE show the higher growth rate in seedling stage, suggesting the effects of the up-regulation of auxin-related genes.