探討紅龍果X病毒鞘蛋白與寄主因子之交互作用

Abstract

紅龍果在分類上屬於仙人掌科(Cactaceae)三角柱屬(Hylocereus)，為現今重要經濟果樹。臺灣栽種的紅龍果大多受到紅龍果X病毒(Pitaya virus X, PiVX)、仙人掌X病毒(Cactus virus X, CVX)與蟹爪蘭X病毒(Zygocactus virus X, ZyVX)三種 Potexvirus 屬病毒的感染，然而紅龍果病毒在植物中的感染機制仍有許多未知的地方。曾有研究發現 Potexvirus 屬病毒鞘蛋白可和寄主蛋白產生交互作用，進而影響病毒的累積與移動。據前人研究，PiVX 的鞘蛋白突變株會影響病毒在圓葉菸草原生質體中的累積量，但尚不知道有哪些寄主因子參與在此過程中。本研究目標為找尋可和 PiVX 鞘蛋白產生交互作用之植物蛋白，並研究該蛋白對於病毒感染過程的影響。以接種PiVX的紅龍果與白藜作為材料，進行交聯免疫共沉澱，確立各項條件後，進行 LC-MS/MS 與資料分析。在四次獨立試驗中，共找到 64 個紅龍果蛋白可能和 PiVX 鞘蛋白產生交互作用，而這些僅出現一次的蛋白大致上可分為三大類群，分別是葉綠體與粒線體相關蛋白、核醣體相關蛋白，以及其他蛋白。以白藜作為植物材料之實驗中，共有8 個白藜蛋白於三次獨立試驗中都被發現可以和 PiVX 鞘蛋白產生交互作用。目前先挑選TOM1-like protein 9 (TOM1L-9)、reticulon like protein B8 (RTNLB8)以及 pathogenesis-related protein PRB1-3-like (PRB1-3L)三個蛋白進行後續實驗。為瞭解目標植物蛋白對於病毒感染過程的影響，先將目標白黎蛋白基因進行靜默，再接種 PiVX，而後分析在目標基因靜默時，植株中之 PiVX 累積量。使用 Tobacco rattle virus (TRV) 病毒載體進行病毒誘導基因靜默，以帶有目標基因片段的 TRV 植物汁液機械接種白黎，三天後白藜接種葉顯著表現出病毒誘導之基因靜默；反之，未接種之系統葉則無基因靜默表現。接種 PiVX 於目標基因靜默後的白藜接種葉，並與無靜默表現之對照組相比，結果發現三個目標基因無論靜默與否，都對於 PiVX 在白藜上的累積沒有影響。為驗證白藜蛋白與 PiVX 鞘蛋白在植物細胞內是否會產生交互作用，分別表現以 EGFP 與 mCherry標定之目標蛋白，觀察兩者在植物細胞中的坐落位置。於螢光顯微鏡與共軛焦雷射掃描顯微鏡下，觀察到 PRB1-3L 位於細胞膜與細胞核周圍，細胞質內亦有少量存在；而 PiVX 鞘蛋白則坐落在細胞膜和細胞核中，但不存在於核仁；並由螢光疊合結果推測兩蛋白可以在細胞膜產生交互作用。

Pitaya is a member of the genus Hylocereus in the family Cactaceae and recently becomes an important fruit crop. Most pitayas grown in Taiwan are infected by three potexviruses: Cactus virus X (CVX), Pitaya virus X (PiVX), and Zygocactus virus X (ZyVX). However, the infection mechanism of these viruses is still unknown. Coat proteins (CP) of potexviruses have been reported to interact with host proteins, and further affect viral accumulation and movement. According to previous research, CP mutant of PiVX reduced viral RNA accumulation in Nicotiana benthamiana protoplasts, but which host factor involving in this process is still unknown. The aim of this study is to find out which plant proteins can interact with PiVX CP, and to investigate the influence of these plant proteins on virus infection process. PiVX-infected pitaya (H. undatus) and quinoa (Chenopodium quinoa) were used for cross-linking co-immunoprecipitation (co-IP), and optimal experimental conditions were established before LC-MS/MS analysis of co-IP products. From four independent experiments, 67 pitaya proteins in total were found to interact with PiVX CP; however, every protein only appeared once. These pitaya proteins can be classified into chloroplast and mitochondria related proteins, ribosome related proteins and others. There were 8 quinoa proteins found to interact with PiVX CP in three independent experiments. Among these quinoa proteins, TOM1-like protein 9 (TOM1L-9), reticulon like protein B8 (RTNLB8) and pathogenesis-related protein PRB1-3-like (PRB1-3L) were chosen for further experiments. To investigate the effect of target protein on virus infection process, the quinoa target gene would be silenced before PiVX inoculation, and then virus RNA accumulation was quantified in silenced plants. Tobacco rattle virus (TRV)-based vector was used for virus-induced gene silencing on quinoa. Inoculated quinoa leaves expressed significant silencing at 3 days post inoculation with TRV-containing plant saps. However, there were no silencing effect on noninoculated systemic leaves of quinoa. PiVX inoculation was performed on these gene-silenced and unsilenced quinoa leaves. The results revealed whether the quinoa target genes were silenced or not, it had no effect on PiVX accumulation in quinoa. To confirm the interaction between quinoa target proteins and PiVX CP, these proteins were overexpressed with fused EGFP and mCherry tags in order to observe their co-localizations in plant cells. From the observation by fluorescence microscope and confocal laser scanning microscope, PRB1-3L was found at cell membrane, perinuclear region and cytoplasm. PiVX CP localized at cell membrane and within the nucleus but not in nucleolus. According to merged images, it suggested that these two proteins may interact with each other at cell membrane.