由病毒誘導基因靜默解析日日春NPR1與NPR3基因於抵抗日日春葉片黃化病植物菌質體所扮演之角色

Abstract

植物菌質體 (phytoplasma) 為一群缺乏細胞壁之絕對寄生菌，能在數百種經濟作物上造成數千種植物病害，其防治方式以控制媒介昆蟲與施用抗生素為主。近年來陸續在柑橘、葡萄與蓮霧發現疑似植物菌質體之新興病害，顯示目前防治策略仍有疏失。受日日春葉片黃化病植物菌質體感染之日日春 (Catharanthus roseus) 產生有病徵與無病徵枝條。於此二枝條中皆有CrPR1基因表現上升之現象，推測植物啟動系統性抗病 (systemic acquired resistance, SAR) 以抵禦植物菌質體入侵。本研究欲瞭解植物菌質體入侵後與寄主植物之交互作用，以釐清植物抵抗植物菌質體之系統性抗病路徑。採取策略乃利用病毒誘導基因靜默(virus-induced gene silencing, VIGS) 對植物系統性抗病路徑上重要基因NPR1與NPR3作靜默，觀察下游抗病基因表現量、病徵發展與菌量累積之改變，以得知它們在抵抗植物菌質體入侵上之重要性。首先需於日日春上建立有效VIGS系統；而後才可以此系統對CrNPR1與CrNPR3進行功能性分析。於第一階段，以Tobacco rattle virus (TRV) 做為病毒載體，PDS基因 (phytoene desaturase) 為測試基因。於篩選的四品系中，鐵達尼 ( Titan ) 具最佳的植物存活率與VIGS發生率，為最適日日春品系。在系統優化上，溫度對VIGS效力具顯著影響。溫度在22℃以下具較佳靜默效力。而所測試植物年齡（兩周、四周及六周）對VIGS效力無顯著影響。此外，在各器官中皆觀察到VIGS作用，然而以年輕葉及花之VIGS效應最好。從VIGS影響的研究中發現TRV2 含量與VIGS作用有密切關連性，其於植物中之分布不均似乎造成了VIGS效應於植物中的歧異。TRV的感染並未劇烈影響植物菌質體病徵發展，因此本系統應適用於植物--植物菌質體交互作用之研究。以此系統靜默CrNPR1及CrNPR3皆導致CrPR1於TRV感染植株中之表現量下降。然而僅CrNPR1靜默植株之植物菌質體病徵發展明顯加速，而病徵發展於CrNPR3靜默植株中反而較為遲緩。因此CrNPR1可能在植物抵抗植物菌質體入侵中扮演重要角色；CrNPR3於其中則是扮演負調控因子。本研究亦發現植物菌質體感染造成之CrPR1基因表現可能是藉由水楊酸 (salicylic acid, SA) 誘導之訊息傳導路徑。預期未來能藉由此研究進一步找出抗性網絡中，與CrNPR1及水楊酸相關之重要因子，對於植物菌質體病害能提供更有效之防治策略。

Phytoplasmas are prokaryotic plant pathogens causing considerable loss in many economical crops globally. Recently, an increasing number of phytoplasma diseases were reported on new plant hosts, indicating the traditional control strategies will need an improvement. Previously, we found that a systemic resistance is activated by PLY phytoplasma infection. Therefore, we aimed to investigate NPR1, a critical gene in SAR activation, to realize the effects of SAR on phytoplasma pathogenesis. Because a functional analysis system was lacking for periwinkle, a system based on virus-induced gene silencing (VIGS) was proposed for periwinkle. Tobacco rattle virus (TRV)-based VIGS system was tested in periwinkle and several potentially influential factors (plant cultivar, temperature, plant age, and plant organ) were analyzed to optimize the system. Based on silencing frequency and survival rate, Titan was the most appropriated cultivar for VIGS. In addition, high temperature showed negative effects on VIGS efficacy while VIGS was not significantly affected by plant age. Though VIGS reached to all plant organs, young leaves and flowers showed a more pronounced silencing effect than other organs. The differences of silencing efficacy were strongly associated with TRV2 concentration; therefore TRV2 should be critical for silencing. Moreover, because TRV infection did not greatly influence symptom development of phytoplasma, the system should be useful for plant-phytoplasma interaction studies. Using this system, CrNPR1 and CrNPR3 were respectively silenced to understand their roles in phytoplasma resistance. Silencing of either genes repressed induction of CrPR1 in TRV-infected plants. However, disease symptoms developed fastest in CrNPR1-silenced plants, and slowest in CrNPR3-silenced plants among all groups. CrNPR1 should be a critical element in defense against phytoplasma, and CrNPR3 may counteract its activity in the defense. Consistently, CrNPR1 but not CrNPR3 was induced by phytoplasma infection as well as SA treatment. The study highlights the importance of NPR1- and SA-mediated signaling pathway for resistance to phytoplasma in periwinkle, and gain more insights about plant-phytoplasma interactions to improve the disease control strategies.