植物菌質體 PHYL1 效應蛋白干擾微型核酸調控引發花青素累積及 PHYL1 相互作用蛋白之研究

Abstract

花生簇葉病菌質體 (peanut witches’ broom phytoplasma, PnWB) 感染日日春植物後，能導致植株葉片黃化、枝葉叢生，並藉由 Phyllody Symptoms1 (PHYL1) 效應蛋白引發花器葉化 (phyllody)。本實驗室在罹病日日春或 GFP-PHYL1 轉基因阿拉伯芥 (GFP-PHYL1 plant) 之葉化花 (leafy flower) 中，發現 microRNA396 (miR396) 的表現受到抑制，而受 miR396 調控的 SHORT VEGETATIVE PHASE (SVP) 則大量表現並引發花器葉化。本研究則發現 GFP-PHYL1 plant 在六週大植株中的葉化花有花青素累積的情形，同時即時反轉錄聚合酶連鎖反應 (real time RT-PCR) 結果顯示葉化花中的花青素生合成之酵素基因的表現有明顯上調。我們推測 PHYL1 能夠誘發植物的花青素累積。我們發現 GFP-PHYL1 plant中的 miR156 表現有上升，而受 miR156 調控的 SQUAMOSA PROMOTER INDING PROTEIN-LIKE (SPL) 基因家族表現則顯著下降。SPL9 在花青素生合成中扮演負調控的角色，葉化花中 miR156 表現上升所導致的 SPL9 下降是花青素累積的成因之一。此外吉貝素 (gibberellic acids; GAs) 能藉由活化 SPLs 來促進開花，然而 SVP 會抑制 GAs 的生合成。在 GFP-PHYL1 plants 中，miR396 下降所導致的 SVP 過表現，使植株無法產生足夠的 GAs 來活化 SPL9，進而導致花青素累積。除此之外，本研究發現花青素在葉化花的生成與醣類的累積有關，並且蔗糖能夠促進花青素生合成相關基因的表現。另外前人研究指出 PHYL1 蛋白在植物體中的表現不穩定，我們推測可能有其他的菌質體蛋白能與之結合並幫助其穩定結構。針對感染 PnWB 日日春之葉化花進行免疫沉澱 (immunoprecipitation)，發現 PHYL1 可以和 PnWB effector 2 (PnE2) 直接交互作用，並且藉由 PnE2 間接地與 PnWB effector 1 (PnE1) 結合。根據胺基酸序列比對以及 signal peptide 的預測，PnE1 是未被報導過的效應蛋白，PnE2 則是嵌在菌質體細胞膜表面上的優勢免疫膜蛋白 (immunodominant membrane protein)。本研究發現 PHYL1 能干擾微型核酸 (microRNA) 的表現，間接地導致花青素累積於葉化花，此外也發現 PHYL1 能與兩個菌質體蛋白結合。我們認為花青素與醣類在葉化花的累積可能有利於菌質體的生存。

Peanut witches’ broom phytoplasma (PnWB) infected-Catharanthus roseus plants showed leaf yellowing, witches’ broom, and phyllody (herein referred as leafy flower) symptoms. Our previous studies demonstrated that Phyllody Symptoms1 (PHYL1) of PnWB is the effector to trigger the microRNA396 (miR396) down-regulation, and subsequently up-regulate the miR396-targeted SHORT VEGETATIVE PHASE (SVP) for leafy flower formation in PnWB-infected C. roseus and transgenic Arabidopsis expressing green fluorescent protein (GFP)-PHYL1 fusion gene (GFP-PHYL1 plant). Moreover, we observed the anthocyanin accumulation in the leafy flowers and the gene expressions of anthocyanin pathway were up-regulated in 6-week-old GFP-PHYL1 plants. We speculate that PHYL1 have ability to induce anthocyanin accumulation. In addition, we found that the miR156 expression levels was up-regulated, and the gene expression levels of miR156-regulated SQUAMOSA PROMOTER BINDING PROTEIN LIKE (SPL) genes were down-regulated in the GFP-PHYL1 plant. SPL9 is a negative regulator of anthocyanin biosynthesis; therefore, the anthocyanin accumulation in leafy flowers is caused by the miR156-mediated SPL9 down-regulation. Furthermore, GAs activate the SPLs for flowering; however, SVP inhibit the GAs biosynthesis. Therefore, up-regulated SVP expression, which mediate by low miR396, represses gibberellic acids (GAs) biosynthesis in GFP-PHYL1 plants, resulting in inactivation of SPLs and consequence anthocyanin accumulation. In addition, previous study demonstrated that sucrose can induce expressions of anthocyanin biosynthetic genes. In addition, our results showed that sucrose was accumulated in leafy flower and triggered the anthocyanin biosynthesis. Moreover, our previous study indicated that PHYL1 is an unstable protein in vivo that might need the other bacterial protein(s) for stabilizing. The results of immunoprecipitation (IP) from leafy flowers of PnWB-infected C. roseus indicated that PnWB effector 2 (PnE2) directly interacts with PHYL1, whereas the PnWB effector 1 (PnE1) indirectly interacts with PHYL1 through the interaction with PnE2. According to the amino acid sequence alignment and signal peptide prediction, we found that PnE1 is a novel effector, and PnE2 is an immunodominant membrane protein which anchor on membrane of phytoplasma. In this study, we demonstrated that PHYL1-interfered miRNA expressions have indirectly effects on anthocyanin accumulation of leafy flowers and two bacterial proteins interact with PHYL1. We suggest that anthocyanin and the carbohydrate accumulate in leafy flower might benefit for phytoplasma survival.