外部施用雙股RNA防治蕪菁嵌紋病毒之研究

Abstract

蕪菁嵌紋病毒(turnip mosaic virus, TuMV)是一種嚴重危害作物產量與品質的植物病毒，由於其寄主範圍廣泛且主要透過蚜蟲進行非永續性傳播，使得防治TuMV是件相當艱難的任務。外部施用雙股 RNA (dsRNA)以誘導植物產生對病毒之抗性，已被證實是一種可有效抑制病毒感染且能取代轉基因植物的防治策略。然而，由於RNA分子的不穩定性，致使外部施用dsRNA通常僅能維持短暫的保護效果，如何提升dsRNA的穩定度是延長其誘發之保護作用的重要關鍵。本研究以大腸桿菌HT115 (DE3)表現細菌源dsRNA，將其萃取後外部施用於圓葉菸草(Nicotiana benthamiana)，之後再接種TuMV病毒，測試dsRNA對TuMV感染的保護效果。以I-ELISA分析接種葉和系統葉結果顯示，每片葉施用200 μg含TuMV hpNIa dsRNA之細菌全RNA，即可產生顯著抑制TuMV感染的效果。進一步測試作為靜默標的之TuMV序列，發現相較於TuMV 之HC或CP基因，以NIa基因所製備之細菌源dsRNA，經外部施用所誘發的保護效果最佳且最為穩定。此外，TuMV hpNIa dsRNA只能抑制TuMV感染，證實其保護作用具專一性。而經局部施用TuMV hpNIa dsRNA的植株，約有2/3能於未施用之上位葉產生保護效果，顯示外部施用後RNAi作用分子在植物中具有系統性移動的現象。另一方面，本研究發現單次施用以TRIzol萃取之含TuMV hpNIa dsRNA之細菌全RNA，保護效果大約能維持7天左右；且提高施用量或施用兩種不同dsRNA的混合物，皆無法延長保護效期。當改以製備方式較為簡便且成本較低的細菌包覆型態的TuMV hpNIa dsRNA進行測試，結果顯示其能誘發和萃取之dsRNA相似的保護效果，且單次施用也僅能有約7天之保護效期。當細菌包覆的dsRNA之外部施用量提高至3倍，結果發現並無法有效延長其保護效果。最後，嘗試將細菌包覆之TuMV hpNIa dsRNA 改為滑石粉劑型，然後以葉部噴灑方式施用；結果顯示滑石粉對保護效果無影響，而以低壓系統進行葉部噴灑，雖能抑制TuMV感染，但其效果不如機械接種穩定，保護效期也僅能維持約7天左右。綜合本研究結果，建議以簡單且低廉的方式製備細菌包覆的dsRNA，未來可配合高壓系統進行噴灑，藉此建立一套有效且實用的TuMV防治方法，若能有效解決延長保護效期的問題，將能使外部施用dsRNA技術對防治植物病毒病害有所助益。

Turnip mosaic virus (TuMV) is an important plant virus that seriously damages crop yield and quality. Due to its wide host range and non-persistent transmission mainly through aphids, the task to control TuMV is quite difficult. Exogenous application of double-stranded RNA (dsRNA) to induce virus resistance in plants has been shown to be an effective control strategy that can replace transgenic plants to inhibit viral infection. However, owing to the instability of RNA molecules, exogenous application of dsRNA usually can only maintain a short-term protective effect. How to improve the stability of dsRNA is an important crux to prolong the protection induced by this method. In this study, bacteria-derived dsRNAs were expressed in Escherichia coli HT115 (DE3) and then extracted dsRNAs were externally applied to Nicotiana benthamiana, which were later inoculated with TuMV to test the protective effect of dsRNA against the TuMV infection. After the inoculated and systemic leaves were analyzed by I-ELISA, the results showed that each leaf applied with 200 μg of TuMV hpNIa dsRNA-containing bacterial total RNA could significantly inhibit TuMV infection. The silencing targets of TuMV sequences were further examined, and compared to the HC-Pro or CP gene, the bacterial-derived dsRNA prepared from NIa gene was identified to induce the optimal and most stable protective effect by external application. In addition, TuMV hpNIa dsRNA could only inhibit TuMV infection, demonstrating that the above-mentioned protection is sequence-specific. However, around 2/3 of the plants that were topically applied with TuMV hpNIa dsRNA could generate protection on the upper untreated leaves, indicating that the RNAi molecules moved systemically in plants after exogenous application. On the other hand, a single external application of TuMV hpNIa dsRNA-containing bacterial total RNA extracted by TRIzol to plant was found to maintain the protective effect for approximately 7 days. Moreover, increasing the dosage or applying the mixture of two different dsRNAs could not extend the protection period. When the bacteria-encapsulated TuMV hpNIa dsRNA was prepared from a relatively simple and low-cost method, it was compared with the extracted dsRNA for the protection efficacy. The results showed that bacteria-encapsulated dsRNA could induce similar protective effect as the extracted dsRNA, and the protection only lasted for about 7 days from a single application. When tripled the dosage of bacterial-encapsulated dsRNA for external application, it turned out that the protective effect could not be successfully prolonged. Finally, an attempt was conducted to alter the bacteria-encapsulated TuMV hpNIa dsRNA into a talc powder-based formulation, and then apply it by foliar spraying. Although the results showed that talc powder made no difference on the protective effect, foliar spraying with low-pressure system could inhibit TuMV infection. However, the protective effect of foliar spraying was not as stable as mechanical inoculation, and its duration of protection could only last for 7 days. To summarize the results of this study, it is suggested to prepare bacteria-encapsulated dsRNA in a simple and cost-effective way, which can further combine with the high-pressured system to spray plant in the future, thereby establishing an effective and practical method to control TuMV. If the problem of extending protection period can be effectively solved, this may make the technique of exogenous application of dsRNA helpful to control plant viral diseases.