利用LsGRP1轉基因阿拉伯芥探討其於植物免疫之角色

Abstract

植物第二型富含甘胺酸蛋白LsGRP1對於葵百合防禦病原菌上有所貢獻，本研究進一步檢視LsGRP1如何牽涉於植物防禦反應。於LsGRP1轉基因阿拉伯芥上接種十字花科炭疽病菌Colletotrichum higginsianum及番茄細菌性斑點病菌Pseudomonas syringae pv. tomato DC3000 (Pst DC3000)，顯示LsGRP1轉基因阿拉伯芥對病原菌具有比野生型植株較佳的防禦能力。利用第三型分泌系統缺失之Pst hrcC-突變株以及細菌病原相關分子模式flg22、真菌病原相關分子模式chitohexaose，證明LsGRP1轉基因阿拉伯芥顯著地誘發較高量的活性氧物質及癒傷葡聚醣(胼胝质)沉積，並增加呼吸爆發氧化酶基因表現；鈣離子依賴性蛋白激酶誘導性基因表現在LsGRP1轉基因阿拉伯芥上亦些微提升，顯示LsGRP1會使植株提升病原相關分子模式觸發免疫反應 (pathogen-associated molecular pattern-triggered immunity) 。除此之外，在Avr效應子 - AvrRpt2以及AvrRpm1的觸發下，LsGRP1轉基因阿拉伯芥具有比野生型植株更強的過敏性反應，顯示LsGRP1亦增強植物的效應子觸發免疫反應 (effector-triggered immunity)，證實LsGRP1可以透過多方面機制來提升植物免疫力。本研究中還觀察到LsGRP1轉基因阿拉伯芥的生長增強現象，並透過觀察LsGRP1-EGFP的次細胞定位，推測LsGRP1可能在葉綠體上參與植物生長和防禦的調控機制。

LsGRP1, a plant class II glycine-rich protein (GRP), contributes to the defense of Lilium cv. Star Gazer against pathogen attack. How LsGRP1 might involve in plant defense was investigated in the study. Firstly, the inoculation with Colletotrichum higginsianum and Pseudomonas syringae pv. tomato DC3000 showed that LsGRP1-transgenic Arabidopsis thaliana exhibited stronger defense against pathogens than the wild-type plants. By using Pst hrcC– mutant (type III secretion mutant), bacterial pathogen-associated molecular pattern (PAMP) flg22 and fungal PAMP chitohexaose, higher levels of reactive oxygen species (ROS), callose and respiratory burst oxidase homologs D (AtrbohD) gene expression were shown in LsGRP1-transgenic Arabidopsis. In addition, calcium-dependent protein kinase-induced gene expression was slightly enhanced in LsGRP1-transgenic Arabidopsis. These results reveal that the presence of LsGRP1 conducts stronger PAMP-triggered immunity in plants. Besides, the LsGRP1-transgenic Arabidopsis showed more severe hypersensitive responses than wild-type plants after triggered by Avr proteins, AvrRpt2 and AvrRpm1, demonstrating the enhancement of effector-triggered immunity mediated by LsGRP1. Thus, LsGRP1 would enhance plant immunity through different aspects. In addition, the enhanced growth phenomenon in LsGRP1-transgenic Arabidopsis and subcellular localization of LsGRP1-EGFP revealed that LsGRP1 might involve in the regulation of plant growth and defense occurring in chloroplasts.