LsGRP1C誘導百合灰黴病菌之計畫性細胞死亡現象

Abstract

植物演化出多種防禦機制，如產生抗菌物質以抵禦病原菌感染。本研究室以水楊酸誘發葵百合對灰黴病菌 (Botrytis elliptica) 之抗病性，並觀察到一解碼為富含甘胺酸蛋白之基因LsGRP1 (Lilium ‘Star Gazer’ glycine-rich protein 1) 表現量增加。生體外抑菌試驗指出LsGRP1C (LsGRP1 C端富含半胱胺酸區段) 合成胜肽能夠引起多種真菌的細胞膜透化、活性氧物質 (reactive oxygen species, ROS) 累積以及染色質凝集 (chromatin condensation) 之現象。據此，本研究推測B. elliptica感染過程中，葵百合可能透過LsGRP1C誘發B. elliptica之計畫性細胞死亡 (programmed cell death, PCD) 以增進百合對B. elliptica的抗病性。利用LsGRP1衍生性合成胜肽與大腸桿菌表現之融合蛋白於生體外處理B. elliptica的孢子，確認LsGRP1C具有抑制B. elliptica孢子發芽的活性；續以大腸桿菌蛋白表現系統生產LsGRP1、LsGRP1C與LsGRP1ΔC融合蛋白，處理B. elliptica之接種結果則指出， LsGRP1C不僅能夠抑制B. elliptica孢子發芽，亦可阻止百合上的病斑擴展。使用4',6-diamidino-2-phenylindole (DAPI) 與terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) 染色分析的結果指出，含有LsGRP1C的融合蛋白會於生體外誘發B. elliptica染色質凝集、細胞核DNA斷裂等PCD現象。經水楊酸預處理之百合，有較多的LsGRP1累積與較少的B. elliptica感染所造成之植物細胞死亡，且B. elliptica的PCD提早發生及發生率增加，暗示植物體上真菌PCD現象與LsGRP1的作用有關。進一步將LsGRP1衍生性融合蛋白分別注入葵百合葉片後接種B. elliptica，以trypan blue 染色和即時定量聚合酶連鎖反應 (real-time quantitative polymerase chain reaction) 進行檢測的結果，含有LsGRP1C之融合蛋白的葉片中病原菌族群量較低，且B. elliptica的PCD發生時間點較早，持續時間較久且發生率較高。因此，LsGPR1之C端區段應可透過誘發真菌PCD以減少B. elliptica對百合的侵染，此為百合上未曾被報導之防禦機制。

Plants evolve various defense mechanisms against pathogen infection, such as production of antimicrobial compounds. Our previous studies demonstrated that salicylic acid pretreatment was able to induce disease resistance against pathogenic fungus Botrytis elliptica, accompanying with an enhanced expression of LsGRP1, a glycine-rich protein encoding gene. The C’-terminal region of LsGRP1, LsGRP1C, was proved to inhibit several plant fungal pathogens in vitro via a mechanism involving permeabilization of fungal membrane, reactive oxygen species accumulation and chromatin condensation. Accordingly, function of LsGRP1 was proposed to increase lily resistance to B. elliptica via inducing fungal programmed cell death (PCD) in planta, through the antimicrobial activity offered by LsGRP1C. In this study, in vitro antifungal assay of the synthetic peptides and Escherichia coli-expressed fusion proteins derived from LsGRP1 confirmed that LsGRP1C exhibited inhibition effect on spore germination of B. elliptica. Furthermore, coinoculation of B. elliptica with E. coli-expressed LsGRP1, LsGRP1C and LsGRP1ΔC fusion proteins conducted reduction of spore germination and lesion development in lily. Using 4',6-diamidino-2-phenylindole staining (DAPI) and terminal deoxynucleotidyl transferase dUTP nick end labeling assay (TUNEL), LsGRP1C-containing fusion proteins were proved to cause PCD phenomena, including chromatin condensation and DNA strand breaks in B. elliptica. The PCD phenomena of B. elliptica occurred earlier in lily leaves pre-treated with salicylic acid, in which the amount of LsGRP1 increased and plant cell death became less, suggesting the phenomena of fungal PCD in planta were related to the action of LsGRP1. In planta assay showed that less B. elliptica biomass was present in lily leaves pre-infiltrated with LsGRP1C-containing fusion proteins as investigated by trypan blue staining and real-time quantitative polymerase chain reaction; meanwhile, the PCD phenomena of B. elliptica occurred earlier and lasted longer at a higher level. Thus, the C-terminal region of LsGRP1 capable of protecting lily from B. elliptica infection via inducing fungal PCD was presumed, which is a defense mechanism firstly reported in lily.